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Development of a sensitive, quantitative high-performance liquid chromatographic assay for the measurement… Embree, Leanne 1989

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DEVELOPMENT OF A S E N S I T I V E , QUANTITATIVE HIGH-PERFORMANCE L I Q U I D CHROMATOGRAPHIC ASSAY FOR THE MEASUREMENT  OF DIGOXIN I N PATIENT GROUPS  WITH HIGH LEVELS OF D I G O X I N - L I K E IMMUNOREACTIVE SUBSTANCES  LEANNE EMBREE B. S c . ( P h a r m ) , U n i v e r s i t y o f B.C., V a n c o u v e r , B.C., C a n a d a , 1982 M. S c . , U n i v e r s i t y o f B.C., V a n c o u v e r , B.C., C a n a d a , 1985 A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in  THE FACULTY OF GRADUATE STUDIES F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s D i v i s i o n of P h a r m a c e u t i c a l Chemistry We a c c e p t  this  t h e s i s as  to the required  conforming  standard  THE UNIVERSITY OF B R I T I S H COLUMBIA January  1989  L e a n n e Embree  In  presenting  degree  this  at the  thesis  in  University of  partial  fulfilment  British Columbia,  freely available for reference and study. copying  of  department publication  this or of  thesis by  this  for scholarly  his thesis  or  her  of  the  I agree  I further agree  purposes  may  representatives.  It  be is  requirements  Pha-r/rta C e is- frctx /  DE-6G/81)  tf  2^  S 9 P  ?  advanced make it  that permission  for extensive  granted  head  by the  understood  that  for financial gain shall not be allowed without  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date  an  that the Library shall  permission.  Department of  for  S~c ie yt c e J  of  my  copying  or  my written  i i Abstract  Digoxin f o r the  is  treatment of  disturbances observed digoxin  the most  of  for  commonly  congestive  heart  c a r d i a c rhythm. digoxin  therapy  and  have  used d i g i t a l i s  glycoside  f a i l u r e and c e r t a i n  The low t h e r a p e u t i c  the  clinical  necessitated  index  significance  of  development  of  the  s e n s i t i v e a n a l y t i c a l methods f o r the q u a n t i t a t i o n of d i g o x i n in b i o l o g i c a l  samples.  methods i n c l u d i n g and  Digoxin  may be analysed  immunoassays, chromatographic  v a r i o u s b i o l o g i c a l and chemical Immunoassays,  both  the c l i n i c a l  radioimmunoassay  r e a c t i o n of methods with  and  of  their  low c o s t .  speed, However,  a n t i b o d i e s used i n the immunoassay  digoxin metabolites,  as d i g o x i n - l i k e drugs that  and r e l a t i v e l y  the d i g o x i n  (RIA)  (FPIA) procedures, are  l a b o r a t o r y because  precision, sensitivity  procedures  methods.  f l u o r e s c e n c e p o l a r i z a t i o n immunoassay used i n  by s e v e r a l  endogenous compounds  immunoreactive substances (DLIS),  may be co-administered  such  and other  with d i g o x i n continues  to  be a major problem. The lack d i g o x i n has values. have  of s p e c i f i c i t y of the immunoassay methods f o r l e d to  i n i n t e r p r e t a t i o n of assay  Attempts t o compensate f o r t h i s l a c k of s p e c i f i c i t y  included  elaborate  difficulties  sample  However, s i n c e  the  use  handling  of  chromatographic  methods  an immunoassay  prior  was used  to for  systems  as  immunoassay. detection  of  digoxin  in  these  techniques,  the s p e c i f i c i t y  may s t i l l  be  quest ionable. A  sensitive  physico-chemical needed.  A  and  specific  methods method  derivatization  of  dinitrobenzoyl  of  was  developed  d i g o x i n and  chloride  Although  dual  resolution  followed A  metabolites  was  obtained,  d e r i v a t i v e and samples were  by  the  administered derivatization a c i d and  (0.394  with  3,5-  analysis  with  ng  o f 0.883  digoxin)  low  digoxin  yield  of  of m e t a b o l i t e s  was  t h i s method  and i t s  the  digoxin  when s m a l l ( n g ) unsuitable  for  samples. l i q u i d chromatographic  using post-column d e r i v a t i z a t i o n from  HPLC  derivatized  d e r i v a t i z e d made  A high-performance  digoxin  pre-column  d e t e c t i o n i n t h e r e d o x mode.  the formation  evaluating patient  using  using  therefore  maximum s e n s i t i v i t y  electrode between  is  i t s metabolites  3,5-dinitrobenzoyl digoxin  observed using  for digoxin  f o r measurement  electrochemical detection. ng  assay  i t s metabolites drugs, of  was  digoxin  dehydroascorbic  detection, allowed  of d i g o x i n , which and  some  developed. with acid,  (HPLC) a s s a y separated  commonly  Post-column  co(PC)  concentrated  hydrochloric  followed  fluorescence  forquantitation  by  within the therapeutic  range of d i g o x i n . S t e r o i d s which  h a v e been  digoxin antisera  were  developed  study.  in this  assayed  reported using  The s t e r o i d  to cross-react the  HPLC-PC  with  method  samples e i t h e r d i d not  iv e l u t e from  the HPLC system or d i d not produce a f l u o r e s c e n t  product under these c o n d i t i o n s . Serum samples u s i n g both to the  from d i g i t a l i z e d p a t i e n t s were e v a l u a t e d  the HPLC-PC and the FPIA methods.  HPLC procedure,  average, higher  the FPIA  assay  digoxin l e v e l s .  When compared  results  T h i s may  gave,  on  have been due to  the i n c l u s i o n of d i g o x i n m e t a b o l i t e s or endogenous  compounds  with the FPIA assay. Serum samples high DLIS  from u n d i g i t a l i z e d  l e v e l s have  been r e p o r t e d  p a t i e n t groups where were a l s o  evaluated.  These i n c l u d e d u m b i l i c a l c o r d blood samples and samples from hypertensive patients, failure patients.  renal f a i l u r e  Comparison  of  p a t i e n t s and the  HPLC-PC  hepatic and  FPIA  methods demonstrated t h a t the HPLC-PC assay gave fewer  false  p o s i t i v e r e s u l t s than the FPIA. The HPLC-PC assay developed f o r a n a l y s i s of d i g o x i n u n a f f e c t e d by  the presence of d i g o x i n m e t a b o l i t e s , numerous  s t e r o i d s , co-administered most of  was  which have  drugs and  been r e p o r t e d  r e s u l t s with the FPIA.  endogenous to give  false  compounds, positive  V  TABLE OF CONTENTS  Chapter  Page Abstract  i i  List  of Tables  List  of F i g u r e s  xii xiv  Symbols and A b b r e v i a t i o n s  xvi  Acknowledgement  xix  Introduction 1 .  1  L i t e r a t u r e Survey  1.1  4  Pharmacodynamic P r o p e r t i e s  4  1.1.1  4  Cardiovascular  Actions  1.1.1.1  Contractility  4  1.1.1.2  Conduction  5  1.1.2  Mechanism of A c t i o n  5  1.1.3  Toxicity  6  1.1.4  Pharmacokinetics  8  1.1.4.1  Absorption  9  1.1.4.2  Distribution  11  1.1.4.3  Metabolism And E l i m i n a t i o n  13  1.1.5  Chemistry  15  1.1.6  Structure-Activity Relationships  16  1.2  Digoxin  Therapy  1.3  Digoxin-Like  1.4  Digoxin-Drug I n t e r a c t i o n s  27  1.4.1  27  Immunoreactive Substance(s)  Digoxin-Quinidine  19 20  vi 1.4.2 1.5  1.6 2.  Other Drug I n t e r a c t i o n s  29  Digoxin Analysis  32  1.5.1  Immunoassays  33  1.5.2  B i o l o g i c a l Assays  37  1.5.3  Chemical Methods  38  1.5.4  Chromatographic Techniques  40  1.5.5  HPLC-RIA  43  HPLC-EC  44  EXPERIMENTAL 2.1  Supplies  49  2.1.1  Chemicals  49  2.1.2  Solvents  50  2.1.3  Extraction Supplies  50  2.1.4  Post-Column D e r i v a t i z a t i o n S u p p l i e s  51  2.1.5  Commercial Radioimmunoassay  52  2.1.6  Commercial F l u r o e s c e n c e  2.1.7 2.2  49  Kits  Polarization  Immunoassay K i t  53  Filtration  53  of HPLC Mobile Phase  Equipment  53  2.2.1  HPLC Equipment  53  2.2.2  E l e c t r o c h e m i c a l Detector  54  2.2.3  F l u o r e s c e n c e Detector Flow C e l l  56  2.2.4  Post-Column Reactor and Pressure Chamber  56  2.2.5  E x t r a c t i o n Equipment  56  2.2.6  Radioimmunoassay  59  Equipment  vi i 2.3  Pre-column 3 , 5 - D i n i t r o b e n z o y l C h l o r i d e D e r i v a t i z a t i o n Procedures  59  2.3.1  For Gram Q u a n t i t i e s of Digoxin  59  2.3.2  For M i l l i g r a m Q u a n t i t i e s of C a r d i a c  2.3.3 2.4  Glycosides  59  For Nanogram Q u a n t i t i e s of D i g o x i n  60  P r e p a r a t i o n of S o l u t i o n s  60  2.4.1  61  Reagents  For 3,5-DNBCl D e r i v a t i z a t i o n  2.4.1.1  3,5-DNBCl i n P y r i d i n e  61  2.4.1.2  Sodium Bicarbonate  61  2.4.1.3  Dilute Hydrochloric Acid  2.4.2  (5%) with 4-DMAP  61  Reagents For F l u o r o g e n i c D e r i v a t i z a t i o n  2.4.2.1  Ascorbic Acid Solution  2.4.2.2  D i l u t e Hydrogen Peroxide  2.4.2.3  Dehydroascorbic  2.4.2.4  Hydrogen Peroxide  61 61  Solution  62  Acid  62  with Phosphoric  Acid  62  2.4.3  Sodium Acetate B u f f e r (0.1 M, pH 4.6)  62  2.4.4  Mobile  Phase  62  2.4.5  Standard S o l u t i o n s  63  2.4.5.1  Digoxin  i n Ethanol  2.4.5.2  3 , 5 - D i n i t r o b e n z o y l Digoxin  2.4.5.2  D i g i t o x i g e n i n i n Ethanol  63  2.4.5.3  Dihydrodigoxin  64  2.4.5.4  S t e r o i d Samples i n Methanol  2.4.5.5  P r e p a r a t i o n of M e x i l e t i n e H y d r o c h l o r i d e in 50% E t h a n o l  63 i n Methanol  i n Ethanol  63  64  65  viii 2.4.5.6  P r e p a r a t i o n of Plasma Samples f o r RIA Analysis  2.4.5.7  65  P r e p a r a t i o n o f Serum S a m p l e s S p i k e d Digoxin Metabolites  2.5  P r e p a r a t i o n of S o l v e n t s  with  f o r FPIA A n a l y s i s  f o r Serum E x t r a c t i o n  2.5.1  Isooctane/Dichloromethane  2.5.2  E x t r a c t i o n Solvent  2.5.3  Preparation  (20/5)  66 66 66 66  of P r o t e i n  Precipitation  Reagents  66  2.5.3.1  Zinc S u l f a t e Heptahydrate  (5%)  2.5.3.2  Barium Hydroxide Octahydrate  2.5.3.3  Cupric  2.5.3.4  Sodium H y d r o x i d e  S u l f a t e Pentahydrate  66  ( 0 . 3 N) (10%)  (12%)  67 67 67  2.6  RIA A n a l y s i s and C r o s s - R e a c t i v i t y C a l c u l a t i o n s  67  2.7  HPLC W i t h E l e c t r o c h e m i c a l D e t e c t i o n  68  2.7.1  Optimization  68  2.7.2  Mobile  of E l e c t r o c h e m i c a l C o n d i t i o n s  P h a s e s f o r HPLC w i t h  Electrochemical  Detection 2.8  69  HPLC P o s t - C o l u m n D e r i v a t i z a t i o n A s s a y  69  2.8.1  A s s e m b l y o f S t e e l a n d PVC P r e s s u r e  2.8.2  Optimization  Vessels  o f HPLC P o s t - C o l u m n  Derivatization 2.8.2.1  69  Fluorogenic  70 Derivatization  Hydrogen P e r o x i d e , Concentrated  HC1  with  Phosphoric  A c i d and 70  ix 2.8.2.2  E v a l u a t i o n of the F l u o r e s c e n c e F i l t e r s with Dehydroascorbic  Emission  A c i d and  HC1 D e r i v a t i z a t i o n 2.8.3 2.9  71  F i n a l HPLC-PC Assay Procedure  71  Serum E x t r a c t i o n Procedure  71  2.9.1  P r o t e i n P r e c i p i t a t i o n Methods  71  2.9.2  C e n t r i f r e e and C e n t r i f l o F i l t r a t i o n  73  2.9.3  Solvent-solvent Extraction  73  2.9.4  F i n a l Procedure f o r E x t r a c t i o n of Digoxin in Serum  73  2.10 Recovery and P r e c i s i o n of E x t r a c t i o n  74  2.11 C a l i b r a t i o n Curve  76  2.12 S p e c i f i c i t y  76  2.12.1  Steroids  76  2.12.2  Co-Administered  Drugs  76  2.13 Q u a l i t y C o n t r o l Procedure  77  2.14 A n a l y s i s of D i g i t a l i z e d P a t i e n t Serum Samples  77  2.15 A n a l y s i s of Serum from U n d i g i t a l i z e d P a t i e n t Groups  3.  Where High DLIS L e v e l s have been Reported  77  2.15.1  Hypertension  77  2.15.2  Renal F a i l u r e  77  2.15.3  Hepatic  78  2.15.4  U m b i l i c a l Cord Blood Samples  Failure  78  R e s u l t s and D i s c u s s i o n s  79  3.1  Radioimmunoassay of Digoxin and I t s M e t a b o l i t e s  3.2  Fluorescence Metabolites  P o l a r i z a t i o n Immunoassay i n Serum  79  of Digoxin 82  3.3  Pre-Column  3,5-Dinitrobenzoyl  D e r i v a t i z a t i o n of  C a r d i a c G l y c o s i d e s with HPLC-EC A n a l y s i s  84  3.3.1  U l t r a v i o l e t Detection  84  3.3.2  D e t e c t i o n With a S i n g l e Glassy Carbon Electrode  3.3.3 3.4  87  Dual E l e c t r o d e D e t e c t i o n  v  Post-Column F l u o r o g e n i c HPLC Assay  97  3.4.1  Pressure V e s s e l  97  3.4.2  O p t i m i z a t i o n of Post-Column Reactor Conditions  3.5  98  3.4.3  Maximum S e n s i t i v i t y  101  3.4.4  E v a l u a t i o n of D i g o x i n and i t s M e t a b o l i t e s  103  A n a l y s i s of B i o l o g i c a l Samples Using The HPLC F l u o r o g e n i c Assay  105  3.5.1  105  O p t i m i z a t i o n of Serum E x t r a c t i o n Method  3.5.1.1  P r o t e i n P r e c i p i t a t i o n Methods  105  3.5.1.2  Removal of Endogenous  107  Interference  3.5.2  Recovery and P r e c i s i o n  109  3.5.3  C a l i b r a t i o n Curve  112  3.5.4  Comparison of HPLC-PC and FPIA Methods i n Spiked  3.5.5  3.6  90  Serum Samples  Specificity  114 119  3.5.5.1  Steroids  119  3.5.5.2  Co-administered Drugs  119  A n a l y s i s of D i g o x i n  i n D i g i t a l i z e d P a t i e n t Serum  120  xi 3.7  A n a l y s i s of Serum from U n d i g i t a l i z e d P a t i e n t Groups Where High DLIS L e v e l s Have Been Reported  122  3.7.1  Hypertensive P a t i e n t s  122  3.7.2  Renal F a i l u r e P a t i e n t s  124  3.7.3  Hepatic F a i l u r e P a t i e n t s  125  3.7.4  U m b i l i c a l Cord Blood  128  4.  Summary and Conclusions  130  5.  References  133  xii List  of T a b l e s  Table  Page  I  Summary Of D i g o x i n Pharmacokinetics  II  Percent C r o s s - R e a c t i v i t y on a Molar  13 Basis f o r  D i g o x i n M e t a b o l i t e s With Commercial D i g o x i n RIA Kits III  80  E v a l u a t i o n of D i g o x i n M e t a b o l i t e s i n Serum Using the FPIA f o r D i g o x i n A n a l y s i s  83  IV  HPLC R e t e n t i o n Times of 3,5-DNB D e r i v a t i v e s  85  V  R e s o l u t i o n of 3,5-DNB D i g o x i n and 3,5-DNB Dihydrod i g o x i g e n i n Using HPLC-EC with V a r i o u s Mobile Phases  VI  Effect  88  of V a r y i n g the Aqueous P o r t i o n of the Mobile  Phase on Peak Height Using HPLC-PC F l u o r e s c e n c e Assay VII  99  E f f e c t of Emission F i l t e r s on Peak Height of Digoxin Using HPLC-PC F l u o r e s c e n c e Assay  100  VIII E f f e c t of HC1 Flow Rate on Peak Height of Digoxin Using HPLC-PC F l u o r e s c e n c e Assay  101  IX  Recovery of D i g o x i n from Spiked Serum Samples  111  X  P r e c i s i o n of D i g o x i n Assay  112  XI  Comparison of the HPLC-PC and FPIA Methods f o r Digoxin A n a l y s i s i n D i g i t a l i z e d P a t i e n t s  XII  121  Comparison of U n d i g i t a l i z e d Hypertensive P a t i e n t Samples E v a l u a t e d by the HPLC-PC and FPIA Methods 123  xi i i XIII Hepatic F a i l u r e P a t i e n t Samples E v a l u a t e d by the HPLC-PC and FPIA Methods XIV  126  Mixed U m b i l i c a l Cord Blood Samples E v a l u a t e d by the HPLC-PC and FPIA Methods  128  xiv  List  of F i g u r e s  Figure  Page  1  The C h e m i c a l  2  Reaction  3  Schematic  of N o i s e Reducer  55  4  Schematic  o f HPLC-EC S y s t e m  57  5  Schematic  o f HPLC-PC F l u o r o g e n i c  6  D i a g r a m o f HPLC-PC P r e s s u r e  7  F l o w D i a g r a m f o r Serum E x t r a c t i o n P r o c e d u r e  75  8  S t r u c t u r e o f 3,5-DNB D i g o x i n  85  9  Chromatogram o f 3,5-DNB D i g o x i n  Involved  Milligram 10  S t r u c t u r e of D i g o x i n i n Immunoassay  16 Procedures  33  System  58  V e s s e l With F i t t i n g s  72  Derivatized in  Quantities  86  Chromatogram o f D i g o x i n  and i t s M e t a b o l i t e s a s T h e i r  3,5-DNB D e r i v a t i v e s u s i n g E l e c t r o c h e m i c a l D e t e c t i o n 89 11  Chromatogram o f 125 ng o f 3,5-DNB Purified  12  by D o u b l e R e c r y s t a l l i z a t i o n  Chromatogram o f 80 ng o f D i g o x i n Milligram  13  Digoxin 92  Derivatized in  Quantities  -  Chromatogram o f D i g o x i n  a n d I t s M e t a b o l i t e s by  HPLC-PC 14  Ethanol  Interfering 15  104  Chromatograms o f D i g o x i n in  and Blank  Serum  Containing 108  Chromatogram o f B l a n k  Calibration  and I t s M e t a b o l i t e s  Peak  1.5 ng D i g o x i n 16  93  Serum a n d Serum S p i k e d  and I n t e r n a l Standard  Curve  f o r HPLC-PC D i g o x i n  with 110  Assay  113  XV  C o r r e l a t i o n Between HPLC-PC and FPIA Methods For Digoxin  Analysis  xvi Symbols and A b b r e v i a t i o n s  3,5-DNBCl 4-DMAP  3,5-Dinitrobenzoyl chloride 4-Dimethylaminopyridine  ACN  HPLC grade a c e t o n i t r i l e  ANP  Atrial natriuretic  ACU  U.B.C. H e a l t h S c i e n c e s Centre Acute Care U n i t  ATP  Adenosine  B. C.  B r i t i s h Columbia  C  Centigrade  CA  California  Cl^j.  Creatinine clearance  cm  Centimeter  C. V.  C o e f f i c i e n t of V a r i a t i o n  Da  Daltons  DLIS  D i g o x i n - l i k e immunoreactive  EC  Electrochemical  EMIT  Enzyme-multiplied  EtOH  Absolute  FlA  Fluorescence  FPIA  Fluorescence p o l a r i z a t i o n  g  gram  GFR  Glomerular  GLC  G a s - l i q u i d chromatography  HC1  Concentrated  HPLC  High-performance l i q u i d chromatography  peptide  Triphosphate  substance(s)  immunoassay  ethanol immunoassay  filtration  immunoassay  rate  hydrochloric acid  xv i i HPLC-EC  High-performance electrochemical  HPLC-PC  liquid  chromatography-  detection  High-performance  liquid  chromatography  column d e r i v a t i z a t i o n I.D.  I n t e r n a l diameter  IL  Illinois  in.  Inch  I PA  HPLC grade  lb.  Pound  M  Molar  MA  Massachusetts  MD  Maryland  MeOH  HPLC grade  MI  Michigan  min.  Minutes  MO  Missouri  isopropanol  methanol  Na ,K -ATPase  Sodium-potassium  ng  Nanogram  NJ  New  NV  Nevada  NY  New  Ont.  Ontario  PC  Post-column  PTFE  Polytetrafluoroethylene  PVC  Polyvinylchloride  Que.  Quebec  r  Correlation coefficient  +  +  dependent  Jersey  York  ATPase  post-  xvi i i r .c. f .  Relative c e n t r i f u g a l force ( g r a v i t i e s )  RIA  Radioimmunoassay  S.D.  Standard  TLC  Thin-layer  TX  Texas  U.S.A.  U n i t e d S t a t e s of America  V  Volts  WI  Wisconsin  UL  Microliter  Mm  Micrometer  deviation chromatography  xix Acknowledgement  I wish  to thank my  his valuable I am  s u p e r v i s o r , Dr. K e i t h McErlane, f o r  guidance throughout  also grateful  to the  the course of t h i s study.  other members  committee,  Drs.  James Orr  for their helpful advice.  Axelson i s for  Frank Abbott,  acknowledged  for  of  my  graduate  Helen Burt, C h a r l e s Kerr and Furthermore, Dr. James  c o n t r i b u t i n g s i g n i f i c a n t ideas  consideration. I  am  grateful  S t e i n b r e c h e r , G.  to  Sandor and  in o b t a i n i n g c l i n i c a l Financial  Drs.  C.  Kerr,  S. Stordy f o r t h e i r  support  from  I would p a r t i c u l a r l y  technical I  am  Radana  Price,  U.  assistance  samples. the  Canadian  Columbia Heart Foundations i s g r a t e f u l l y  Henriksen,  J.  and  British  acknowledged.  l i k e t o thank Roland Burton, T r i n e  Vaughan  and  Dale  Embree  for  their  assistance. indebted  to  Dr.  Allan  Goodeve  for  numerous  d i s c u s s i o n s and p r o o f r e a d i n g of t h i s document. Finally,  I  would  like  to thank  h i s d e v o t i o n and encouragement.  my husband, Dale, f o r  1 INTRODUCTION  D i g i t a l i s glycosides  form one  of the most b e n e f i c i a l  group of drugs a v a i l a b l e t o a i d the f a i l i n g h e a r t . the drugs  of c h o i c e  failure  and  Although  digoxin  certain  for c l i n i c a l  low  disturbances  i s the  therapeutic  g r e a t e r than  with therapy  index.  At  i n response  t h i s value.  an  In  t o x i c i t y while d i g o x i n serum  effort  maintaining  These  Digoxin has  fluorescence  may l e a d to  the  avoid  and  inter-  to t o x i c i t y  below  manifestations  desired therapeutic  s e n s i t i v e assay  been analysed  polarization  immunoassays  r o u t i n e l y used  effect,  techniques.  by s e v e r a l methods i n c l u d i n g  cost;  immunoassay,  immunoassay,  and v a r i o u s  in  terms  therefore  in c l i n i c a l  high-performance  b i o l o g i c a l and chemical  None of the methods developed  s e n s i t i v i t y and  of  between 0.5 and  enzyme-multiplied  l i q u i d chromatography,  the major  e x i s t due to  low c o n c e n t r a t i o n s have n e c e s s i t a t e d the  radioimmunoassay,  the  rhythm.  concentrations  conditions  c o n c e n t r a t i o n s a r e maintained  development of extremely  with  still  digoxin  physiological  patient variation  methods.  in cardiac  heart  3 ng/mL i n serum, there i s a high i n c i d e n c e of However,  2.0 ng/mL.  of c o n g e s t i v e  only d i g i t a l i s g l y c o s i d e a v a i l a b l e  1937, problems  toxicity.  treatment  use i n Canada and has been employed m e d i c a l l y  s i n c e about its  f o r the  They are  of  t o date can compete speed,  immunoassay  laboratories.  precision, methods  are  However, one of  problems with the immunoassay methods f o r d i g o x i n  2  analysis  is  that  of  cross-reactivity  a n t i b o d i e s with d i g o x i n m e t a b o l i t e s , co-administered  with  d i g o x i n and  as d i g o x i n - l i k e immunoactive The for  analysis  has  reliability.  In order  to a v o i d  estimation  d i g o x i n due  these methods protocols  still  questions  be  p r i o r to the  of over-  introduced  immunoassay.  elaborate  antibodies  its  from endogenous  i n v e s t i g a t o r s have  use  of  the p o s s i b i l i t y  to i n t e r f e r e n c e  generally require  and  drugs that may  immunoassay methods used  prompted  other drugs,  chromatographic methods  digoxin  substance(s).  digoxin  substances and  other  the  endogenous compounds such  l a c k of s p e c i f i c i t y of the  of  of  sample  with  However, handling  questionable  specificity. A sensitive  and  specific  assay f o r d i g o x i n using more  r e l i a b l e methods f o r measurement i s t h e r e f o r e needed. performance l i q u i d successful  in  metabolites  but  chromatographic  separating  samples (Bockbrader 1977;  Nachtmann  have s u f f i c i e n t for  1984;  Desta  1976a, 1976b). r e s o l u t i o n of  q u a n t i t a t i o n (Reh  (HPLC) methods have been  digoxin  lack the s e n s i t i v i t y  from  most  plasma  without  using  1987; Other digoxin  Fujii  its  Gfeller  from  not  dihydrodigoxin  1985).  post-column  the m e t a b o l i t e s  (Kwong 1986a, 1986b).  1983;  HPLC methods do  fluorogenic  i n t e r f e r e n c e from a number of commonly  drugs and  of  required for b i o l o g i c a l  An HPLC assay f o r d i g o x i n at t h e r a p e u t i c in  High-  of  digoxin  has  concentrations derivatization co-prescribed been  reported  3 The  specific  1.  a i m s of  to a s c e r t a i n  metabolites  of d i g o x i n  the  the  p r o j e c t were as  amount  of  follows:  i n t e r f e r e n c e from  i n radioimmunoassay k i t s  the  commercially  available. 2.  to  sensitivity 1986a,  investigate of  the  1986b)  the  by  derivatization  analysis  electrochemical  detection.  column  to improve the fluorogenic  1986b) t o possible  allow  4.  (Fujii  faster  of  increasing assay  digoxin  1983)  sample from  method  processing  endogenous  prior  (Kwong  compounds  to with  HPLC  and  the  (Kwong  coupled  e x t r a c t i o n p r o c e d u r e and  derivatization  interference  prescribed  of  HPLC p o s t - c o l u m n f l u o r o g e n i c  chromatographic  3.  possibility  post1986a,  eliminate or  co-  medication. to  pathological  evaluate conditions  serum  samples  where  from  patients  endogenous  i m m u n o r e a c t i v e s u b s t a n c e ( s ) h a v e been  reported.  with  digoxin-like  4 1.  Digitalis available  glycosides  Since  e f f e c t i v e n e s s of  SURVEY  are  f o r the c l i n i c a l  failure.  certain  LITERATURE  the  most  management o f  Withering digitalis  (1937)  valuable  drugs  congestive  heart  first  preparations  documented  i n the  the  therapy of  f o r m s o f d r o p s y , a t t e n t i o n h a s been d i r e c t e d t o w a r d s  elucidation  of  mechanisms t h a t efforts,  their  mechanism  affect  their  serious  Digoxin, the  problems  cardiac  of  action  efficacy. with  glycoside  and  cellular  In s p i t e of these  therapy  still  available  in  w i d e l y used i n t h e treatment of c o n g e s t i v e h e a r t  exist.  Canada,  is  f a i l u r e and  c e r t a i n d i s t u r b a n c e s of c a r d i a c rhythm.  1.1  Pharmacodynamic  1.1.1  Cardiovascular Actions  The  main  glycosides of t h e  Properties  action  of  i s the a b i l i t y  beating heart  conduction,  and  other  digitalis  to increase the c o n t r a c t i l e  (Haustein  force  1983; H o f f m a n 1 9 8 0 ) .  The  r e f r a c t o r i n e s s and a u t o m a t i c i t y of t h e h e a r t a r e  also affected.  Therapeutic  changes i n c o n t r a c t i l i t y  1.1.1.1 The  digoxin  clinical  treatment of  use of  digoxin  i s based  on  and c o n d u c t i o n .  Contractility a n d hemodynamic  congestive heart  changes observed  failure are  i n the  the r e s u l t of a  5 direct  positive  myocardium This  (Blaustein  increase  output,  inotropic  volume,  heart  failure  heart  The are  on  the  1975; H a u s t e i n  1983).  t o an i n c r e a s e d  cardiac  s i z e , decreased venous pressure  (Haustein  direct  digoxin  o f edema i n p a t i e n t s  1983; H o f f m a n  and with  1980).  Conduction e f f e c t s of  d i g o x i n on  electrical  activity  s t r o n g l y d e p e n d e n t on t h e p h y s i o l o g i c a l c o n d i t i o n o f t h e  heart. were  Also, differences i n electrical found  Hoffman  between  1980).  muscle  Generally,  to depress the conduction atrioventricular of d i g o x i n  nodal  indirectly  impulses can the  leads  d i u r e s i s and r e l i e f  1.1.1.2  of  1985; D o h e r t y  in contractility  decreased  blood  effect  types  high digoxin  (Haustein  conduction decrease  (Endou the rate  to the  1983;  l e v e l s are thought  v e l o c i t y and t o d e l a y  be t r a n s m i t t e d  refractory  fiber  response t o d i g o x i n  or block the  1982). at  Low d o s e s  which  atrial  v e n t r i c l e s and  prolong  p e r i o d of the a t r i o v e n t r i c u l a r  node  (Hoffman  1980) .  1.1.2  Mechanism of A c t i o n  Therapeutic selectively  inhibit  Na ,K -ATPase) i n +  and  +  vascular  cells. an  This  increase  concentrations the plasma  of  membrane s o d i u m  a v a r i e t y of c e l l  smooth muscle inhibition i n the  digitalis  cells,  glycosides pump  (the  types i n c l u d i n g cardiac neurons and r e n a l  of outward sodium t r a n s p o r t  sodium c o n c e n t r a t i o n  tubule  leads t o  i n s i d e the  cell.  6 These c e l l s  a l s o have  sodium-calcium exchanger  which decrease  the i n t r a c e l l u l a r  calcium  or reducing  influx  resultant results  rise  in  in  more c a l c i u m  Blaustein  1984a). activity  being  1985;  (Doherty  The  calcium  concentration  account  Repke  1984;  f o r the  effects  been n o t e d  on  cardiotonic  the  15  1975).  At  of such  to  20%  of  hospitalized  (Aronson However,  1983),  i s a high  physiological  conditions  this value.  an  toxicity,  the  maintained  between  effort  therapeutic  p a r a m e t e r s , such  greater  there  i n response  In  p a t i e n t s i s from 7 t o 50%  digoxin concentrations  patient variation  may l e a d to  avoid  as r e n a l  and  inter-  to toxicity  below  manifestations  concentration  0.5 a n d 2.0 ng/mL. function  cardiac have  Haustein  and non-cardiac  been r e c e n t l y  in  1983).  Disorders  of  plasma  of is  D e p e n d i n g on p a t i e n t and  age,  manifestations  reviewed  than 3  i n c i d e n c e of  d o s e s o f d i g o x i n a r e u s u a l l y 0.125 mg t o 0.50 mg  1983;  kidneys,  smooth m u s c l e .  that  mortality rate  toxicity.  toxicity  Smith  r e c e i v i n g d i g o x i n e x h i b i t symptoms o f t o x i c i t y , a n d  ng/mL p l a s m a  The  (Akera  Toxicity  I t has  that the  or both.  efflux  1980;  w e l l as  nervous t i s s u e and v a s c u l a r  patients  the  a v a i l a b l e t o the c o n t r a c t i l e  Hoffman can  d i g o x i n as  1.1.3  enhancing  to a positive inotropic effect  These e f f e c t s of  the calcium  intracellular  elements and t h e r e f o r e 1985;  s o d i u m by  mechanisms  maintenance daily. of digoxin  (Antman 1985; A r o n s o n the g a s t r o i n t e s t i n a l  7 tract  due  to  vomiting,  central  anorexia  disorders are  and  system  diarrhea)  u s u a l l y the  (Antman 1985; A r o n s o n central  nervous  earliest  effects  and side  (nausea,  cardiac  rhythm  effects  observed  1983; H a u s t e i n 1983; Mason 1 9 8 1 ) .  n e r v o u s s y s t e m e f f e c t s o f d i g i t a l i s a r e numerous  include  headache,  dizziness,  weakness,  vertigo,  ataxia,  psychosis, neuralgia, Haustein  1983).  flickering  or  earliest  Visual  of  t h e r a p e u t i c plasma in  memory ( T u c k e r  hallucinations,  symptoms s u c h a s b l u r r i n g , lights,  dimness,  a n d may  (Closson  l e v e l s , d i g o x i n may auditory verbal Various  1983)  produce a  learning  Takayanagi  1986).  m a n i f e s t a t i o n s of (Smith  1984a,  digitalis  digitalis  toxicity  diagnosis  difficult.  are  Digoxin-specific successfully  used  intoxication  (Cohen  Rozkovec  1982;  a b s o r p t i o n of  toxicity  1984b, 1 9 8 4 c ) .  short-term blockage  the  1982;  from  been  1983;  Agents  and  reviewed  symptoms  fragments treatment  Mason  mechanisms  have  been  digitalis  Murphy that  the g a s t r o i n t e s t i n a l  of  making  have of  in  evidence  non-specific,  Friedman  1982).  the  s i g n s and  usually  for  Zucker digoxin  The  antibody  At  1983; H a u s t e i n 1983;  Recently,  the  progressive  and  arrhythmias,  (Antman 1985; A r o n s o n  be  1983).  c o n d u c t i o n , and w o r s e n i n g h e a r t f a i l u r e a r e c a r d i a c of t o x i c i t y  1985;  and c o l o r d i s t u r b a n c e s w i t h  toxicity  1983).  and  depression,  s t u p o r and coma (Antman  a r e most common ( A r o n s o n  symptoms  deterioration  drowsiness, confusion,  seizure,  flashing  yellow v i s i o n  1981;  The  1982;  decrease tract  such  8 as c h o l e s t y r a m i n e  and a c t i v a t e d  c h a r c o a l , have  used i n t r e a t i n g d i g o x i n t o x i c i t y Tissue digoxin determination levels  of  i n cases  ng/mL w h i l e  toxicity.  of d i g i t a l i s  those  from  were  c a s e s where  cause of  death  Andersson  1975; Margot 1983).  elevated digoxin (Aderjan  found  must t a k e  of  uptake  1.1.4  assay  methods f o r t o have  antemortem  data 1983;  r e s u l t s h a v e been  (Andersson blood  t h e time site  1979;  1975; M a r g o t  postmortem d i g o x i n  intersubject  of  1975).  The  concentrations  of blood  (Margot  collection  1983) s i n c e  these  observed.  variation  d i g o x i n (Aronson  1974;  Luchi  interference with  method  o t h e r compounds 1974;  1979; L i n d e n b a u m  Kramer  pharmacokinetic  1977; R i e t b r o c k  t h e most (Butler  1978;  1983b;  the  1983; C l a r k 1974; G a u l t 1979;  common d i g o x i n a s s a y  1974a,  1982; M u l l e r  Ravel  1981,  1978, 1979; D i P i r o 1980;  Kubasik  1975; M a l i n i  1983a,  in  1968; M o v s e l l i  1985) a n d  1982; Pudek  not the  Pharmacokinetics  Large  by  43 t o 283  samples appear  Similar  the concentrations  digoxin  was  immunoassay  1983; K i m  1978).  from  i n the  (Aderjan  compared t o  and t h e sampling  factors affect  ng/mL  Using  into consideration  a f t e r death  Holtzman  t o 463  l e v e l s when  a rubidium  interpretation  Kramer  0  ranged toxicity  postmortem b l o o d  1986; V o r p a h l  using  behavior  from  1979; H a s t r e i t e r  McKercher  Postmortem  toxicity  been  1980).  c o n c e n t r a t i o n s may n o t be u s e f u l digoxin  digoxin analysis,  (Hoffman  also  1974b;  Lichey  1978; O s t e r l o h  1975; S c h e r r m a n n  1980;  9 Schreiber it Due  1981a, 1981b, 1 9 8 1 c ; S i l b e r 1979;  difficult to  the  to g e n e r a l i z e intrapatient  p h a r m a c o k i n e t i c s as of d i g o x i n , have not  w e l l as  the  dosage  are  partially  eliminated  reduced r e n a l  by  the  gastric  mucosa, h y p o x i a  the  cardiac  patient  (Gault  1 9 8 2 a , 1982b;  (Smith  1977;  and  adjusting drugs  that  in patients  with  of  and  diarrhea  1984a). 1981;  t h a t may  Loo  and  the  it 1977;  be  be  the  gastric  this  these  has  been  compounds  leads  degraded  to-  acid  by  bis-  in  the  from  those  I t has lower  assumed t o be to  Magnusson  regarding  been e s t a b l i s h e d . pH  been  degradation  differentiated  Thus, f i r m c o n c l u s i o n s have not  has  occur  of  of  exist in  i t s mono- and  may  levels  from  congestion  While  this  cannot  a lowered  levels  in vascular  t h a t d i g o x i n can  tract,  plasma  i s adequately absorbed  and  that  degradation  sensitivity  useful for  kidneys  digoxigenin  m e t a b o l i c a l l y formed.  digoxin  the  Lindenbaum  enzymes t o  gastrointestinal  found that  be  regimen  suggested  cardiovascular  t r a c t , even  Sonobe 1980)  digitoxosides  vivo  may  digoxin  gastrointestinal  in  of a d o s a g e  Absorpt ion  administered  a c i d and/or  immunoassays  h o w e v e r , i t was  other  the  products in  the  the  function.  1.1.4.1  suggested  pharmacokinetics.  problems with  1986)  and  make  variation in  Recently,  of d i g o x i n  1973)  interpatient  nomograms f o r d e t e r m i n a t i o n  t h a t a nomogram ( B j o r n s s o n  Orally  about d i g o x i n  and  been p o p u l a r .  Smith  due  (Hossie  the been  plasma to  the 1977;  10 Sternson 1976)  1978).  regard  the lowering  hyperacidity  to  p a t i e n t s more intestinal  Some i n v e s t i g a t o r s (Brown 1978; G r e e n b l a t t  be  sufficient  closely.  flora  may be  The  of food  (Malini  (Dobkin  (Greenblatt  1976;  indicating  of  was a b o u t  that  Soft  at.  (1986) a l s o  digoxin  trough  inverse  was c o n s i d e r e d  capsule  than that  form  of t a b l e t s ,  o f d i g o x i n may  in  also  of d i g o x i n as a  (Johnson  formulations.  of d i g o x i n  1986).  Johnson  urinary  in  excretion  They f u r t h e r n o t e d This this  h a v e a l s o been f o u n d t o v a r y  substances are administered  t o be  Arterial  blood  directly  at steady s t a t e  ( C a t e n a z z o 1985) of d i g o x i n  a  in detail.  1981).  was f o u n d  plasma l e v e l s  to result in  metabolism with b i o a v a i l a b i l i t y .  drugs and  Manninen  t o 7.50  has a l s o  t o be due t o c o l o n i c b a c t e r i a , a l t h o u g h  Plasma l e v e l s  1983;  nor  of  1981).  significant variability  concentrations  a s p e c t was n o t s t u d i e d  when o t h e r  in  formulation  n o t i c e d no  r a t i o of  the conversion  g e l a t i n capsules  between t a b l e t and c a p s u l e an  these  levels.  digoxin  s o l u t i o n g a v e 90 t o 100% a b s o r p t i o n et  for  Wagner 1974)  20% h i g h e r  t h e dosage  affect absorption.  with  monitor  t o drug therapy  i n d i g o x i n plasma  bioavailability 1983)  to  levels  1983; L i n d e n b a u m  ingestion prior  large discrepancies The  reason  responsible  dihydrodigoxin  been r e p o r t e d  digoxin  I t h a s a l s o been s u g g e s t e d t h a t t h e  digoxin to effect  of plasma  may e x p l a i n  (Haustein  pH i n t h e r a n g e  correlated  (Catenazzo  with  1985).  i n patients with respiratory acidosis.  digoxin  These  the s t r o n g l y reduced  7.25  data  activity  11 Permeation (Cairncross that  of  digoxin  1985) s t u d i e d u s i n g  i t may be  possible to  transdermal therapeutic  1.1.4.2 After  to  central  signs  disorientation, (Aronson  1980;  Digoxin 65%  is  and  1.5%  binding  is also  develop a c l i n i c a l l y  efficient  the  tissue  is  thought  toxicity  of  produce  as  the  drowsiness,  visual  disturbances  1981).  digoxin  t o other  k i d n e y s (Aronson  by  may  I t has a l s o  involve  the  tissues:  4% i n t h e h e a r t ,  about  3% i n t h e b r a i n  1980).  Serum  protein  t o a l b u m i n , v a r i e s from  1977; S m i t h  concentration  In in  1980).  1976) b u t  comparison  the  /3-lipoprotein  concentration  blood  the  concentration  of l i p o p r o t e i n - b o u n d  10  1984a) a n d a p p e a r s  (Aronson  ^ - l i p o p r o t e i n s (Brock  than d i g i t o x i n .  bound i s m i n i m a l .  of d i g o x i n  1982).  1974; M o v s e l l i  bound t o  to  such and  digoxin, principally  unaffected  digoxin  t h e r e s i s t a n c e t o t h e a r r h y t h m o g e n i c and  effects  i n the  serum  The d i s t r i b u t i o n  i s also distributed  lower a f f i n i t y  vitro  H a u s t e i n 1983; R i e t b r o c k  (Otsuka  t o 4 0 % (Kramer t o be  indicates  1980).  found i n the l i v e r ,  of  skin  a peak b e t w e e n 45 m i n u t e s a n d 3 h o u r s  of  been s u g g e s t e d t h a t  hypothalamus  human  system  hallucinations  vasoconstrictor  and  system.  Hoffman  nervous  neurological  an in  administration,  reaches  1975;  mouse  Distribution  oral  concentration (Doherty  through  is  digoxin  Digoxin with  a  t o albumin,  so s m a l l  that  to the t o t a l  T h u s , i t h a s been s u g g e s t e d , t h e f r a c t i o n  12 o f unbound d i g o x i n i n s e r u m c a n be p r e d i c t e d s o l e l y albumin concentration A  model  with  compartments can (Keys  1980;  a n d pH at  (Brock  least  be u s e d  1976).  two  kinetically  to describe digoxin's  N y b e r g 1974;  Rietbrock  represents  f l u i d s and  t i s s u e and t h e p e r i p h e r a l compartment  slowly-perfused  that the with a  data  and  best  compartment  significant  1982;  and  A  well-perfused  recent  body  represents  study  suggests  (Kramer 1979).  biliary  excretion  circulation  Schafer  1985).  or  Digoxin  (Reissell  intestinal  This area  model may 1982)  secretion  is still  disputed  the l i t e r a t u r e . During  from  the d i s t r i b u t i o n  the  central  distribution steady  of  state  digoxin in volume  the  1975).  The r e p o r t e d  (Rietbrock  is  observed.  tissue i s  Extensive  i n d i c a t e d by a l a r g e  distribution.  Using  tritiated  h a l f - l i f e was 60 m i n u t e s  steady-state  volume of d i s t r i b u t i o n  5.0 L / k g i n p a t i e n t s w i t h c a r d i a c  failure  3.3 a n d 4.4 L / k g i n p a t i e n t s w i t h r e n a l  failure  cardiac disease  Morphological may  alter  change i n  t h e volume  volume of  distribution  b e t w e e n 5.1  of  L/kg i n  1981).  variable:  (Doherty  a n d 8.1  subjects,  between  of  distribution  d i g o x i n was e x t r e m e l y healthy  phase of d i g o x i n , r a p i d removal  compartment  digoxin,  and  disposition  f i t a n o n - l i n e a r , two-compartment  enterohepatic  (Reissell in  body s p a c e .  deep t i s s u e  a l s o undergo  blood  distinct  1981) w h e r e t h e c e n t r a l  compartment  the  the  from the  changes i n the h e a r t  digoxin binding  of d i s t r i b u t i o n appears  (Keys  t o vary with  and  due t o  produce  1980).  a The  renal function  13 but  large  renal  variation exists  function  digitalis  (Keys 1980).  receptors  patients  (Malini  activity  of  A summary  in individuals  has  A  been  digitalis of t h e  decrease i n observed  1985), which  with  may  glycosides  in  explain i n chronic  pharmacokinetic  data  comparable  t h e number o f renal  failure  the decrease i n renal  failure.  f o r d i g o x i n i s shown  in Table I .  Table  I.  Summary Of  Pharmacokinetic  Digoxin  Pharmacokinetics  Parameter  A v a i l a b i l i t y (Oral) Urinary Excretion Bound i n P l a s m a Volume o f D i s t r i b u t i o n ( L / K g ) ' H a l f - l i f e (Hours) Time t o Maximum C o n c e n t r a t i o n ( H o u r s ) Time t o Maximum E f f e c t ( H o u r s )  70 + 13% 60 + 11% 25 + 5% 7 + 30% 39+13 2 to 3 4 to 6  2  3  4  4  From G i l m a n et al. ( 1988). Decreases with uremia. Decreases with h y p o t h y r o i d i s m and i n c r e a s e s w i t h h y p e r thyroidism. D e c r e a s e s w i t h h y p e r t h y r o i d i s m and i n c r e a s e s w i t h u r e m i a , c o n g e s t i v e h e a r t f a i l u r e and h y p o t h y r o d i s m . 1  2  3  4  1.1.4.3  M e t a b o l i s m And E l i m i n a t i o n  M e t a b o l i s m of  digoxin occurs  1965) a n d  the drug  glomerular  filtration  Although  t h i s area  investigators  i n the l i v e r  and t u b u l a r s e c r e t i o n ( S t e i n e s s  is still have  (Abel  l a r g e l y by t h e k i d n e y s v i a  many r e p o r t s h a v e e v a l u a t e d  of d i g o x i n , Some  i s excreted  mainly  the metabolic  one o f c o n s i d e r a b l e  suggested  that  only  1982).  turn-over dispute. a  small  14 proportion 1972;  of  d i g o x i n was  Doherty  h a v e shown excreted recent  1970;  that  as  variation of t h e  57  1974;  excretion in  the drug  Luchi  a  large  such t h a t a few  80%  et  al.  digoxigenin  as d i h y d r o d i g o x i n .  Subjects  w i t h minimum  (Gault  1980)  metabolites. variability specificity complexity The  in of  the  only  (1974)  metabolism  RIA  for  o f e v a l u a t i n g RIA  digoxin results  trace  produces  an  conjugation  reactions  decrease.  L a g e and  c l e a v a g e of  the  increase  lipid  would cause Spratt  the  (1966)  Reduction  s u b s t a n t i a l l y decreased of  (Rietbrock  1981).  lipid  54.5%  0.8%  as  and  0.3%  function of  that  the  lack  of  increase  the  samples. digitoxose  solubility  reported  while  solubility  to  that  stepwise  progressively  decreased  of the  the a c t i v i t y  dihydrodigoxin  orally  levels  the  for patient  digitoxose residues  the c a r d i o a c t i v i t y .  cardioactivity  in  the  reported  speculated  would  55%  but  renal  and  A  to  excreted  m e t a b o l i s m of d i g o x i n by c l e a v a g e o f t h e  residues  digoxin  Kalman  digoxin  f r o m 20  as d i g o x i g e n i n  excreted  C l a r k and  was  interpatient  bisdigitoxoside,  0.25%  Gibson  1968)  ( 1979)  digoxigenin monodigitoxoside,  1979;  others  Using  t h a t , w i t h n o r m a l r e n a l f u n c t i o n , d i g o x i n was as  Beerman  patients,  unchanged.  t r i t i a t e d d i g o x i n , Gault  u n c h a n g e d , 2.0%  1958;  largely dihydrodigoxin.  noted  metabolized  excreted  administered  (Clark  1980)  (Ashley  Marcus 1966), w h i l e  product,  metabolite  d r u g was  1980;  t o 60%  (Aronson  in  majority  Gibson  a metabolic  report  metabolized  lactone ( K e y s 1980)  being  Epimerization  ring  also  with  the  that  of  of d i g o x i g e n i n  and  only  1/20  15 conjugation in a  reactions  with s u l f a t e and glucuronide r e s u l t e d  l o s s of almost a l l a c t i v i t y  male  mice,  the  potency  ratios  bisdigitoxoside, digoxigenin and  3-epidigoxigenin  1/1.33,  1/1.47,  respect  1/46)  1.1.5  1/4.84  monodigitoxoside,  digoxigenin  a n d 1/6.45  has  f o u n d t o be  respectively.  a l s o been  reported.  Chemi s t r y  a steroid  naturally  nucleus  occurring  cardioactive  and a five-membered  Removal o f  aglycone. foxglove  the  species  gitoxin,  Digitalis  diginatin glycosides  unsaturated  lactone  groups a  addition,  nucleus  group  digitalis  aglycone  of  (digitoxin,  a a  of  cyclopentanofive-membered  i n t h e 17-/3 p o s i t i o n .  a t carbon  10  a n d 13  (0  14 (/3 p o s i t i o n ) a n d  (0 p o s i t i o n ) i s a t t a c h e d a t  aglycones  f o r digoxin,  h y d r o x y l g r o u p a t c a r b o n 12.  1983) which  The a g l y c o n e  a t carbons  lactone ring  lactone  leaves of the  glycosides  with  ring attached  hydroxyl  in a l l the  consists  residues,  the genin o r  (Haustein  Ianata  are attached  five-membered 17  i n the  and g i t a l o x i n ) .  perhydrophenanthrene  position),  i s found  steroid,  unsaturated  residues leaves  number o f o t h e r d i g i t a l i s  digitalis  carbon  sugar  This glycoside  contain a  Methyl  With  for dihydrodigoxigenin  t h r e e s t r u c t u r a l components; a s e r i e s o f sugar  ring.  adult  digoxigenin  c o m p a r e d t o d i g o x i n were  (Brown 1962)  Digoxin, a  the  Using  of  t o d i g o x i n , the potency r a t i o  ( l e s s than  has  (Keys 1980).  (Haustein  1983).  digoxigenin,  In  has a  16 Naturally  occurring  component a t t a c h e d (Myerson  1967).  d i g i t a l i s glycosides  at position  have a sugar  3 v i a a glycosidic linkage  These s u g a r s a r e u s u a l l y six-membered  i n t h e p r e f e r r e d c h a i r c o n f o r m a t i o n and a r e l i n k e d by 0 - g l y c o s i d i c chain  of  position Figure  1,4-linkages.  digitoxose 3.  The  residues  For digoxin, i sjoined  rings  together  a three  sugar  t o the aglycone a t  chemical s t r u c t u r e of d i g o x i n  i s shown i n  1.  OH  Figure  1.1.6  The C h e m i c a l  Structure  Despite been  1.  and  Digoxin  Relationships glycosides  due t o t h e low t h e r a p e u t i c  intra-patient  Numerous s t u d i e s activity  of  t h e length of time the cardiac  i n use, problems  inter-  Activity  Structure  variability  are s t i l l  have examined t h e c h e m i s t r y  relationships  of  these  have  index and present.  and s t r u c t u r e -  glycosides.  For  17 cardioactivity following i)  to  be p r e s e n t ,  (Guntert  1981;  Haustein  the  1983):  a c i s - c i s j u n c t i o n of the C and D r i n g s of the genin moiety, an u n s a t u r a t e d l a c t o n e m o i e t y a t t a c h e d a t c a r b o n 17 i n t h e j3 c o n f i g u r a t i o n , a s u g a r component a t t a c h e d t o t h e 3 - / 3 - h y d r o x y l on t h e genin v i a a g l y c o s i d i c l i n k a g e .  ii) iii)  The  C  and  fused c i s  D  with  position.  r i n g s of the  l o s t when Rings  C  and  hydrophobic 1980)  are  members  Smith  1981;  0  the  1981)  i n the t r a n s  with is  position.  der Waals and/or (Thomas  1982; steric  the carbon  17 s i d e - c h a i n . t h a t t h e r e was  composed of  the genin  this  17  five  i n the  for cardiac a c t i v i t y . side-chain yields Tamm 1 9 6 3 ) .  analogues  electronic  an a d d i t i o n a l 16-/3  1976;  indicated  requirements al.  and  for  (1986)  receptor binding formate  A  Replacing  1974b)  et  0  inactive  (Fullerton  Recently, G r i f f i n  glycosides since  f o u r or  at carbon  Thomas 1974a, and  in  in activity.  S a i t o 1970;  are both  for d i g i t a l i s  (Guntert  t o u n d e r g o Van  essential  that there  suggested  fused  r i n g w i t h open-chain  1978;  in  indicate that a c t i v i t y  lactone ring  c o n f i g u r a t i o n of  lactone  14  the c o n f i g u r a t i o n leads to a decrease  almost  compounds ( G u n t e r t  are u s u a l l y  w i t h the proposed r e c e p t o r  attached to is  position  aglycones  t h e r e f o r e a decrease  configuration  Gelbart  D  are thought  unsaturated  change i n  at  compounds  and  changing  b i n d i n g and  carbon  D  14-a  interactions  and  An  14-0  rings C  cardiac glycosides  hydroxyl  Comparison of  the corresponding  the  t h e g l y c o s i d e s must h a v e  site  acetate  18 e s t e r s showed  increased a c t i v i t y  with respect t o the parent  compound. Pharmacological a c t i v i t y the  sugars attached at p o s i t i o n  solubility 1963).  and  potency of  greatly  in activity  reduced a c t i v i t y  Thomas 1 9 8 0 ) .  gastrointestinal  Formation  of  the  h y d r o g e n a t i o n ' may 3  (Luchi  1965;  o b s e r v e d when g e n i n may which  is  almost  Yoda  suggest  function at  3  (Gault  1977; Repke  followed  the 3  o f one  1963).  by  're-  0-hydroxyl-genin or 3 is  sugar r e s i d u e  i s present  Part of the decrease  in activity  sugar r e s i d u e  i s removed  ( G u n t e r t 1981).  that  both  the  to binding.  concerning the  c a r b o n 3.  preparation, Saito not r e q u i r e d  lead to the aglycone with a  from t h e  Binding studies  ( A k e r a 1 9 8 1 ; T a k i u r a 1974; W a l l i c k 1974;  component c o n t r i b u t e been r e p o r t e d  sugar  T h i s e p i m e r i z a t i o n a t carbon  inactive  +  1974)  a  t o e p i m e r i z a t i o n t o t h e 3 a - h y d r o x y l form  with Na ,K -ATPase +  can  either  1963).  the l a s t  be due  Removal o f t h e l a s t  3-dehydro-genin  a minimum Repke  (Tamm  b e f o r e and a f t e r a b s o r p t i o n from  a-hydroxyl-genin.  p r e v e n t e d when  glycoside  1980; L u c h i 1965; Repke 1963;  carbon  yield  but  of the sugar m o i e t i e s produced  (Keys  tract  group a t  the aglycone  resulting  1980).  Metabolism  free hydroxyl  the  (Keys  in  3 m o d i f y t h e water and l i p i d  the  P r o g r e s s i v e removal  decrease  the  resides  et  Using al.  foractivity.  genin  and  Conflicting  isolated  sugar  results  requirements f o r an  the  have  the  oxygen  frog's  heart  ( 1 970) f o u n d t h a t t h e o x y g e n was Inhibition  studies with  Na ,K +  +  19 ATPase ( W i t t y position  1975),  however,  3 i s required  Replacement of branched groups  for full  (Siebeneick  the  oxygen  at  of the aglycone.  residues  with  various  1978) g a v e compounds w i t h  activity.  +  that  activity  the digitoxose  Na ,K -ATPase i n h i b i t o r y +  show  lower  Aminosugar d e r i v a t i v e s  were a p p r o x i m a t e l y t e n t i m e s more p o t e n t i n h i b i t o r s t h a n t h e parent  glycoside  (Caldwell  1978).  amino-deoxyglyco-cardenolides  Naturally  (Choay  occurring  1978) h a v e  also  been  identified.  1.2  Digoxin  Therapy  Digitalis  glycosides  a r r h y t h m i a s and were  1985). in  congestive  considered  ventricular  (Klein  the  rate  the  1986).  verapamil  heart  best  in atrial  The c a l c i u m  slowing  a r e i n d i c a t e d f o r t h e treatment of  agents  antagonist  ventricular  drugs of  both  choice  fibrillation.  For congestive  glycosides  useful  are  ventricular  failure  in  therapy with that  30%  cardiac  latter  in a group  the  (Doherty  fibrillation  glycosides  the treatment  heart  failure,  and  of a t r i a l  the d i g i t a l i s  left  or  combined  1985). clinical  response  plasma l e v e l s ,  patients studied  efficiency  Whether t h e  the  i t s associated  of the  atrial  digitalis in  of  was a l s o e f f e c t i v e  either right,  (Doherty  Reports comparing  in  control  or f l u t t e r  verapamil rate  T h e s e compounds  for  fibrillation  Presently,  are  failure.  did  of  have  digoxin indicated  not  improve  their  p r e d i c t a b l e manner  (Kramer  1979).  was u n i m p r o v e d  due t o i n a d e q u a t e  20 therapy  or  due  administration digoxin  to  was  an  inability  not c l e a r .  to  Also,  titrate  the  drug  problems  with  the  p l a s m a a s s a y h a v e been s u g g e s t e d i n t h i s  There i s great of c o n g e s t i v e digitalis  controversy  heart  failure  glycosides  study.  over the p r o l o n g e d  treatment  i n specif-ic p a t i e n t groups  (Applefeld  1986;  Doherty  1985;  Yusuf  1986).  Long-term therapy  with  digoxin  (Spector  1979)  only  a s m a l l number o f p a t i e n t s ,  but  at present  the  drug  to benefit  i t i s not considered  once a d e q u a t e  e f f e c t s of d i g o x i n a study risk of  patients  only  digoxin  area  A more  cardiac  While only  indicated  that  stopping  digoxin  heart  failure  in  14%  suggested  indication  for  f o r d i s c o n t i n u a t i o n of  1986)  function  role  little  of the  Digoxin-Like  a  had  be c o n s i d e r e d  recent  caution  therapy  and t h a t  literature  in  in this  should  those  be  patients  close monitoring  of  i s essential.  Immunoreactive  Endogenous f a c t o r s , play  who  review  (Applefeld  w i t h moderate  1.3  should  with  The  w i t h d r a w a l a r e u n d e r s c o r e d by  studied deteriorated, the authors  therapy  exercised  achieved.  therapy discontinued.  those p a t i e n t s  therapy.  been  1983) i n w h i c h p a t i e n t s who were a t r e d u c e d  had t h e i r d i g o x i n  the  that  (Bowman  reported  good t h e r a p y t o w i t h d r a w  c o n t r o l has  therapeutic  h a s been  with  distinct  sodium e x c r e t i o n  regulation,  have  been  factors are  secreted  Substance(s)  reported  from and  aldosterone, extracellular  (Wilkins  1985).  i n response t o hypervolemia,  that fluid These  circulate  21 i n plasma  and s t i m u l a t e  sodium and peptide  water b a l a n c e .  (ANP),  been f o u n d Wilkins  i n the  enzyme t o  +  renal tubules a f f e c t e d the and t h i s  block  (De W a r d e n e r s o d i u m and  second f a c t o r  to  1977,  natriuretic  activity,  factor that  sodium  inhibited  factor  1982).  was  termed  the a c t i v i t y  Hnatowich  1984;  digoxin-like The compared  of Na ,K -ATPase +  Wilkins  properties (Wilkins  have  o f ANP 1985).  and The  DLIS  Na ,K -ATPase +  (ANP  +  vasoconstriction  (ANP  and may in cardiac  1985;  called  factor  have  recently  been  i n m o l e c u l a r mass. does  not),  DLIS causes  i s excreted in  i n the hypothalamus  (ANP i s  atria).  normal a d u l t  s u b j e c t s who  1984; C l e r i c o  1985; D i a m a n d i s  1982; V a l d e s  1983a; V i n g e  umbilical cord  ( B e s c h 1976;  1985;  this  D L I S h a s been f o u n d i n t i s s u e s and b i o l o g i c a l  found i n  Schreiber  DLIS.  causes v a s o d i l a t i o n ) ,  be p r o d u c e d  digitalis-  s t r u c t u r e o f D L I S i s unknown  t o be l e s s t h a n 500 Da  inhibits  produced  1985)  +  1982a; D i a m a n d i s  immunoreactive s u b s t a n c e ( s ) or  but thought  the u r i n e  (De W a r d e n e r  also  Since both digoxin  endoxin (Diamandis  Some r e s e a r c h e r s  i n the  1985)  and r e a c t w i t h d i g o x i n a n t i b o d i e s , t h e e n d o g e n o u s like  has 1984;  transport  1982a; W i l k i n s  water b a l a n c e .  inhibit  restore  ( L a n g 1985; M i l l s  second endogenous  the Na ,K -ATPase  order  potent n a t r i u r e t i c  mammalian a t r i a  A  in  R e c e n t l y , an a t r i a l  which has  1985).  +  natriuresis  Diamandis  never r e c e i v e d  blood 1985;  digoxin  1985; Hamlyn 1982;  1988). and Kelly  fluids  (Balzan  Klingmuller  T h i s s u b s t a n c e has placental 1981;  Ng  of  been  homogenates 1985;  Pudek  22 1983a,  1983b;  Scherrmann  n e o n a t a l serum Pudek  1983a,  (Clerico  (Barbarash  Gusdon  1984; L o n g e r i c h  (Drost  1977; V a l d e s  cord  blood  significantly  1984; B o i n k  1988; P h e l p s  venous  greater  and  t h e r e was  with  still  have  and  i n umbilical  pregnancies  were  (Koren  significantly  1988).  P h e l p s and  increasing  g e s t a t i o n a l age  but that  d i f f e r e n c e between DLIS l e v e l s i n  DLIS and  A l t h o u g h no c a u s a l  p r e e c l a m p s i a h a s been  an e t i o l o g i c  role  found,  i n development of t h i s  (Phelps 1988).  DLIS has  been r e p o r t e d  1988; Pudek  1984) a n d  (Bourgoignie  1972; C r a v e r  1983b; Kramer  inbile  i n patients  1985,  from p a t i e n t s to contain  1986; Yang  with  renal  loaded  1988).  (DiPiro  impairment  1984) a n d i n  (Kramer 1985a).  with hepatic  DLIS  (Kieval  1984; G r a v e s 1983a,  1985; Y a t s c o f f  s u b j e c t s who h a v e been s a l t  been r e p o r t e d  a n d meconium  1983; D'Arcy  1985b; O l d f i e l d  The serum  increase  arterial  and without p r e e c l a m p s i a .  r e l a t i o n s h i p between  Nanji  (1987)  Also,  no s i g n i f i c a n t  patients with  healthy  al.  (1988) found DLIS t o a p p e a r i n m a t e r n a l serum and  increased  condition  et  fluid  than i n m a t e r n a l venous b l o o d .  i n normal pregnancies  may  1988) a n d a m n i o t i c  were  higher than  it  1984;  blood)  i n high-risk  levels  1984), sera from  DLIS l e v e l s  DLIS l e v e l s  co-workers  1984),  1977; G r a v e s  Gonzalez  report that  (both  Yatscoff  1983b; Y a t s c o f f  1983b).  (1988)  al.  1986b;  1985; H e a z l e w o o d 1984; K o r e n 1984;  1983b; V a l d e s  p r e g n a n t women  K o r e n et  1986a,  f a i l u r e has a l s o  1980; Greenway 1985;  Initially,  a  2 to  i n D L I S l e v e l s was f o u n d i n p a t i e n t s w i t h  3 fold  alcoholic  23 cirrhosis reported liver  (DiPiro in  of  1980).  patients with infectious hepatitis,  pregnancy  and  1985; N a n j i 1 9 8 5 ) . DLIS l e v e l s diseases  D L I S l e v e l s up t o 0.8  metastatic liver  (Frewin  suggest  be t h e  hypertension  (Buckalew  that  natriuretic  1984; W i l k i n s  in normotensive  subjects (Cloix  Peak serum  DLIS l e v e l s  correlated with  the  indicating  D L I S may  that  The s o u r c e ,  adrenal cortex  i n pre-term  (Pudek 1 9 8 3 b ) .  identification  of  and t e r m  infants  (Ebara  1986a),  Longerich  (Pernollet  pregnant  1986;  1987) a s h a s t h e f e t a l manuscript  (Diamandis  and c o r t i s o n e a r e p r e s e n t i n  elution  reactive et  al.  and m a t e r n a l  blood  volumes  these  w i t h RIA (1988)  C o r t i s o l and p r o g e s t e r o n e  DLIS i n  factors  The a d r e n a l g l a n d s  of DLIS  One  chromatographic  digoxin analysis.  1982).  infants.  blood, placenta  particularly  1982a,  a l s o be p r e s e n t  sodium  1981b, 1981c; S h i l p  and  f r a c t i o n s were  in  c o m p o s i t i o n of DLIS a r e the  a source  of c o r d  the plasma  of  i n pre-term  that progesterone  the  involved  be among t h e p a t h o l o g i c a l  HPLC f r a c t i o n s that  fraction  1987; De W a r d e n e r  a number o f i n v e s t i g a t i o n s .  1981a,  a  and  1987; Hamlyn  s t r u c t u r e and  suggested as  or  1985) and may  excretion  i n hyponatremia  1985) n o t e d  (Greenway  to find elevated  DLIS,  hormone  1984; C l o i x  1982b; G r a n t h a m  Schreiber  fatty  1986).  t h e r e o f , may  h a v e been  disease  O t h e r s , however, f a i l e d  reports  s u b j e c t s of  acute  i n p a t i e n t s w i t h a l c o h o l i c and m e t a s t a t i c l i v e r  Recent  involved  ng/mL were  women.  While  of  methods reported  of the  i n a p o r t i o n of these  substances  24 in themselves high  are u n l i k e l y  reactivity  validity  of  causes  the  RIA  t o be  t h e DLIS m a t e r i a l ,  further  difficulties  procedure. Cortisol,  dione, progesterone  and g l y c o c h e n o d e o x y c h o l i c  similar  digoxin antisera  p a r t of  and  co-workers  t h e DLIS  phospholipid. compound  found i n  F a s t atom  (DLIS-2)  a phosphoserine group  (Matthewson  (1987,  1988)  bile acids  and  1988)  times  1988).  patients  bombardment mass  that  was  s p e c t r a of  a this  s u p p o r t e d t h e p r e s e n c e of  i n the m o l e c u l e .  liver  cross-  established  Another group,  mass s p e c t r o m e t r y o f HPLC f r a c t i o n s o f p l a s m a with renal  acid  and had HPLC r e t e n t i o n  haemodialysis  (Dasgupta  the  deoxycortisone, A^androstene-  to DLIS-containing fractions  Dasgupta  with  Dehydroepiandrosterone-  sulfate, cortisone,  reacted with  their  impairment, reported  from a  using  patient  the p r e s e n c e of  i n t h e f r a c t i o n s c o n t a i n i n g DLIS ( T o s e l a n d 1988).  U s i n g r e v e r s e d - p h a s e HPLC, D L I S h a s been s e p a r a t e d i n t o several  immunoreactive  i s not  a single  suggested  to  manuscripts  substance (Diamandis be  however, t h i s  h a s been  e n t e r o l a c t o n e which o f d i g o x i n , may  implicated Valdes  indicated that 1985).  dehydroepiandrosterone  (Braquet  these samples  f r a c t i o n s which  disputed  1986a,  (Pudek  1986b;  DLIS has (Vasdev  contains a lactone ring  a n d mono  and  (Soldin  1986a).  and  co-workers  diglycerides  (1985a,  Recent noted  similar  c o n t r i b u t e to the d i g i t a l i s - l i k e have  1985b)  been  1985),  1983a).  F a g o o 1986)  DLIS  that  to that  activity also  reported  of  been  the  p r o t e i n b i n d i n g of endogenous d i g o x i n - i m m u n o r e a c t i v e f a c t o r s  25 i n human  serum a n d u r i n e .  heat s t a b l e and n e u t r a l an a p p a r e n t  a  i n molecular charge.  m o l e c u l a r mass  appears t o has  These f a c t o r s a r e water s o l u b l e ,  be 400  higher  Da  o f 200 Da w h i l e t h a t f r o m u r i n e  (Valdes  affinity  1985a, 1 9 8 5 b ) .  for  digoxin  D L I S may  represent a  found i n serum. to  Normally over  90% o f  serum  therefore  not  conventional  RIA  the t o t a l  1985a, 1 9 8 5 b ) .  the i n c r e a s e  i n DLIS  n e o n a t e s and  increase  in  t h e amount  an  increase  indicated that  (Valdes  in  1985b).  immunoreactivity  bound t o p r o t e i n a n d i s measurement  1985a,  weakly p r o t e i n  with  1985b).  bound  or  The unbound  ( 1 9 8 5 a , 1985b) s u g g e s t e d  levels,  in  factor  1985a,  endogenous  direct  Valdes  failure,  than  by  DLIS i s  seen  in patients  p r e g n a n t women,  was  with due  an i n c r e a s e  DLIS. i n serum  Other  that renal  to  of w e a k l y p r o t e i n bound DLIS total  less  from serum.  of the  (Valdes  but r e v e r s i b l y  methods  and  serum D L I S a p p e a r s t o be bound  protein  detected  r e m a i n i n g serum (Valdes  conjugated metabolite  serum  i s tightly  isolated  DLIS  1985a, 1985b) t h a t t h e u r i n a r y  In a d d i t i o n ,  noncovalently  in  (Valdes  Urinary  antisera  r e s i s t a n c e t o a c i d h y d r o l y s i s than that I t h a s been s u g g e s t e d  Serum D L I S h a s  an  rather  reports  have  D L I S l e v e l s may  be a t  l e a s t p a r t l y due t o a d e c r e a s e i n i t s r e m o v a l v i a t h e k i d n e y (Clerico  1988a)  positively  and  that  the  urinary  excretion  correlated with physical a c t i v i t y  can  (Clerico  be  1988a,  1988b). The p r e s e n c e particularly  of s u f f i c i e n t  neonates,  DLIS i n  patient  s e r i o u s l y compromises  the  samples, accuracy  26 and  interpretation  Several Pudek  investigators  1985;  commercial having  kits  lower  effected  1986a; W i t h e r s p o o n  and h a v e  conditions  1985; Ng  1987)  to  with  DLIS.  the apparent d i g o x i n  levels  Yannakou 1987).  RIA  tested  brands  Alterations  commercial  1985;  have  recommended p a r t i c u l a r  reactivity  1987;  f r o m immunoassay m e t h o d s .  ( G o r t n e r 1985; M c C a r t h y  Scherrmann  incubation  (Smith  of the r e s u l t s  in  methods  as the  also  i n the p r e s e n c e of DLIS  Ultrafiltration  of  serum,  w h i c h h a s been shown t o remove a p p r o x i m a t e l y 90% o f t h e D L I S present  (Christenson  minimize the use of may  1987; G r a v e s  interference with  reduce  While the  interference  from  i t entirely  In a d d i t i o n immunoreactive  to the  was  to  (Clerico  but  Soldin  will  1986b).  i n immunoassay  to  kits  digitoxin-like  detected in amniotic f l u i d  A significant correlation and  digitoxin-like  r o l e of 1987).  excess production the  digoxin  and  between  immunoreactive  found.  physiological  e f f e c t s on similar  DLIS  also  1984)  The  t o be a s o l u t i o n  presence of DLIS, a  substance  of  s u b s t a n c e was  due  1986;  appear  to  s o l u t i o n over time.  c o r d b l o o d (Ebara 1986b). levels  used  immunoassays.  (Yatscoff  batch-to-batch variances  make t h i s an u n r e l i a b l e  investigated  DLIS  may  be  immunoassays f o r  (Bianchi  above p r o c e d u r e s  the problem,  The  digoxin  fluorescence polarization  not e l i m i n a t e  the  1 9 8 6 ) , may  heart  and  D L I S has y e t t o be  entirely  H i g h l e v e l s of DLIS,  whether  o r r e d u c e d e x c r e t i o n , may  produce  the  autonomic  t o those seen w i t h d i g i t a l i s  toxicity  nervous (Kieval  system 1988).  27 Using  DLIS  isolated et  al.  fibers,  Kieval  similar  t o ouabain.  1.4  Digoxin-Drug The  and  by  problems  human b i l e  (1988)  management  with the  of  index  h a s been  therapeutic  range  being  interaction  of  digoxin with  plasma  to  and u n t r e a t e d  arrhythmias  may  be  p a t i e n t s be  carefully  fatal,  ng/mL  r e g i m e n be  a low  serum.  The  a g e n t s w h i c h may p r o d u c e lead to toxic or  concentrations. congestive  heart  i t i s essential  observed  is  pharmacokinetics  Furthermore,  d i s p o s i t i o n may digoxin  digoxin  f o r digoxin, with the  2.0  other  digoxin toxicity  on  of d i g o x i n  reported  0.5  i n digoxin  their digoxin  Purkinje  cardiotoxicity  patients  assay methods.  therapeutic  sub-therapeutic  canine  demonstrated  the v a r i a b i l i t y  s m a l l changes  and  Interactions  clinical  complicated  from  f a i l u r e or that  and a p p r o p r i a t e  made when  other  Since  these  changes i n  drugs  are co-  administered.  1.4.1  Digoxin-Quinidine  M u l t i p l e drug therapy arrhythmias  that  Unfortunately, between d i g o x i n noted drug widely of a  used  i s o f t e n u s e d t o c o n t r o l edema o r  may accompany many  drug  and o t h e r  interaction  of  antiarrhythmic  problem with  congestive  interactions co-therapeutic digoxin agent.  heart have  been  agents.  i s with  failure. found  The most  quinidine,  One o f t h e f i r s t  a  reports  t h i s c o m b i n a t i o n was i n 1932 ( G o l d 1932)  28 when an  increased  was  noticed.  the  distribution  both the and  risk  of d r u g - i n d u c e d a r r h y t h m i a s i n dogs  In g u i n e a - p i g s ,  g u i n i d i n e was  of d i g o x i n  (Okudaira  ATP-dependent b i n d i n g  the  (Schafer  the  intestinal  studies  volunteers patients  (one (at  t h a t more  of  Also,  liver  quinidine  i n guinea  one total  body  inhibited  renal tubular  Gustafsson  pigs  serum d i g o x i n  irrespective relative  of  levels the  clearances,  and  of  skeletal  patients i n the  Steady-state  al.  have  (1981b)  muscle to  clearance  and  of et  renal  same amount  and  to  the  present  time.  a  reduction  serum d i g o x i n  concentrations  The  non-renal  digoxin  reported  to the  reported  impairment.  renal of  (Schenck-  (1983)  to about the  also  producing  digoxin al.  cardiac It  levels  of in  quinidine-induced  a p p a r e n t volume of d i s t r i b u t i o n digoxin  suggested Quinidine  1981)  unknown a t t h e  which c o n t r i b u t e d  serum  cardiac  1982).  impaired  q u i n i d i n e are  S c h e n c k - G u s t a f s s o n et ratio  of  re-distribution  interaction with  and  i n p a t i e n t s thus  increased  of  healthy  non-renal  (Ochs  Fichtl  degree  contributions  and  clearance  Recently,  in  involved.  1981a,  secretion  renal  1982).  was  volunteers  dose)  levels)  clearance  (Schenck-Gustafsson  i n the  digoxin  mechanism  in healthy  a decrease  interaction  digoxin  patients  decrease  in heart.  this  intravenous  than  of d i g o x i n  cardiac  decreasing  m u s c l e and  s e c r e t i o n of d i g o x i n  steady state  d e c r e a s e d the  the  by  inhibit  1985).  Further  that  1986)  in heart,  ATP-independent b i n d i n g  inhibited  found to  for  after  digoxin. quinidine  29 administration  correlated  concentrations (Friedman  before  1982),  1.4.2  with  quinidine therapy  allowing  l e v e l s that w i l l  well  f o r the  Many o t h e r  estimation  drugs i n t e r a c t  observed  a  and  food  inotropic food  reported caused 1985a).  and  t o impair  in  Crane  positive  digoxin  together.  plasma  Diazepam produced  in  (1986)  volunteers.  digoxin clearance  (Ochs  Cholestyramine, a by t h e  digoxin  and  not  has  been which  (Pedersen  i n plasma  1980)  did  digoxin  of d i g o x i n levels  an i n c r e a s e  alprazolam,  a  inotropic  Quinine  digoxin  (Castillo-Ferrando  benzodiazepine,  to bind  similar  and  the extrarenal clearance  increase  absorbed  t o produce  r e s p o n s e e x h i b i t e d p o t e n t i a t i o n when  concentrations  not  digoxin  or a l t e r a t i o n s of i t s  Burgess  intravenous  were a d m i n i s t e r e d  an  of  administration.  with digoxin  humans.  quantitatively  response f o r This  in  patients  Interactions  change i n t h e plasma l e v e l s o f d i g o x i n effect  digoxin  i n cardiac  be r e a c h e d a f t e r q u i n i d i n e  Other Drug  inotropic  serum  digoxin  while  another  significantly  alter  1985). basic anion  exchange r e s i n  that i s  g a s t r o i n t e s t i n a l t r a c t , h a s been prevent  absorption  (Hoffman  noted 1980),  thereby reducing  plasma d i g o x i n  l e v e l s by 6 9 % f r o m t h o s e o f  control  (Brown 1 9 7 6 ) .  Activated charcoal  subjects  been o b s e r v e d t o d e c r e a s e t h e a b s o r p t i o n gastrointestinal gastric motility  tract  (Reissell  m o d i f i e r , has  of d i g o x i n  1982). been found  has a l s o from t h e  Metoclopramide, a by one g r o u p o f  30 investigators  to  lower plasma  1971), w h i l e  a more  recent  metoclopramide reduced l e v e l s and used.  study  both the  the absorption  levels  (Johnson time t o  when  tablet  (Lindenbaum  1984) n o t e d r e a c h peak  that  plasma  formulations  were  H o w e v e r , t h e o v e r a l l e f f e c t on d i g o x i n a b s o r p t i o n  metoclopramide digoxin (Johnson The  in  was  minimized  solution) rather  been  serum  shown t o  Some  (thiazides, digoxin 1980).  capsules  (containing  t h a n t a b l e t s were  concentration  of  result in  administered  due t o  The r e n a l  a f f e c t e d by  the  to  digoxin.  Hypokalemia when  serum  range  (Keys  that  cause  hypokalemia  ethacrynic  acid)  may  this electrolytic  e x c r e t i o n of  furosemide  electrolytes  the therapeutic  diuretics and  certain  carciac toxicity  were i n  furosemide  toxicity  of  sensitivity  digoxin concentrations 1980).  when  by  1984).  influence myocardial has  digoxin  (Brown  affect  effect  (Hoffman  d i g o x i n , however,  was n o t  1976; M a l c o l m  1977; T i l s t o n e  1 977). Spironolactone  reduces  renal tubular  s e c r e t i o n and  (Bussey  Waldorff  1982;  when c o m b i n i n g P h e n y t o i n was  digoxin clearance the d i g o x i n  volume  Caution with  distribution,  was a l s o  digoxin  significantly  suggesting  of  non-renal clearance  1978).  trimethoprim found t o  the  serum c o n c e n t r a t i o n s  should  n e c e s s a r y , t h e d i g o x i n dose i n c r e a s e d  suggested  (D'Arcy  increase  that, during  of d i g o x i n  the  1985). total  co-administration, be m o n i t o r e d a n d i f  (Rameis  1985).  31 The  antianginal  n i f e d i p i n e and digoxin  Pedersen  the  amiodarone  concentration  volunteers  calcium  and a n t i a r r h y t h m i c  (Belz  1981,  serum  1986).  in  1981a, 1982,  antagonist,  cause  an  1985b; bepridil,  also  amiodarone  intoxication  h a s been r e p o r t e d  In r a t s , p r o d u c e d no  Koren change  suggested  that  occurred.  The  an  al.  in  tissue  serum healthy  Klein  1982;  1986).  The  p r o d u c e d an  increase in  in healthy  subjects  (Belz  patients  treated  with  in  (1983)  1985).  found  that  uptake  of  of  digoxin  inhibition  tissue/serum  1985,  (Ben-Chetrit  et  and  1982;  Venkatesh  verapamil,  i n the  patients  George  digoxin concentrations Digoxin  increase  cardiac  1983;  agents  verapamil  digoxin,  r a t i o s of d i g o x i n  which  elimination concentrations  i n r a t m y o c a r d i u m , s k e l e t a l m u s c l e , and b r a i n w e r e d e c r e a s e d when a m i o d a r o n e were a d d e d  t o the regimen  amiodarone digoxin  had no e f f e c t  effect on  serum  conflicting Wellens  1980)  concentration  levels  ( V e n k a t e s h 1985,  1986).  However,  digoxin  another  levels  Some  have f o u n d  of  1985).  disopyramide,  results.  has  antiarrhythmic  been  investigators  no c h a n g e  i n the  studied (Leahey serum  with 1980;  digoxin  i n c a r d i a c p a t i e n t s when t h e r a p e u t i c l e v e l s o f  d i s o p y r a m i d e were high  of  desethylamiodarone,  on t h e k i n e t i c s o f a s i n g l e d o s e  i n the r a b b i t (Buss  The agent,  or i t s major m e t a b o l i t e ,  co-administered  with digoxin.  o f d i s o p y r a m i d e (mean o f 5.05  jig/mL c o m p a r e d t o Mg/mL f o r a t r i a l  therapeutic concentrations  o f 2.8  arrhythmias)  p r o d u c e d a 15% i n c r e a s e  (Gilman  1980)  t o 3.2  Relatively  i n serum  32 digoxin concentration  b u t was  thought  to  clinical  (Manolas  1980).  The  significance  ethmozine produced  no s t a t i s t i c a l l y  be  of  limited  antiarrhythmic  s i g n i f i c a n t change  serum d i g o x i n c o n c e n t r a t i o n s o f c a r d i a c p a t i e n t s w i t h renal  1.5  function  Digoxin The  plasma  have prompted  1986).  levels  of d i g o x i n  t h e development  procedures.  d i g o x i n has  for  (Butler  1978,  (Brunk  1977;  Rosenthal  the intact  imperative  Butler Sun  1978;  Eriksen  1976),  (FPIA) ( B u t l e r  1982;  Kwong 1986a,  Desta  (Haustein 1981).  compete w i t h precision,  t h e immunoassay  sensitivity  have  replaced  1987;  polarization 1983),  Eriksson  1965;  methods i n  and  1981a; chemical  Simson  terms  As a r e s u l t  in clinical RIA  1983;  1962;  methods d e v e l o p e d t o d a t e can  and c o s t .  f r e q u e n t l y used  Linday  (HPLC) ( B e a s l e y 1983;  Lowenstein  of the  be  (EMIT)  1984; R a w a l  various biological  1983;  None  1978;  fluorescence  1982b,  may  immunoassay  1978; E r i c k s o n  1982a,  1986b), and  Digoxin  radioimmunoassay (RIA)  high-performance l i q u i d chromatography  methods  i tis  drug substance.  1979), e n z y m e - m u l t i p l i e d  1976;  immunoassay  a r e most  i n t h e m e t a b o l i s m and  been r e p o r t e d ,  a n a l y s e d by s e v e r a l m e t h o d s i n c l u d i n g  Stewart  assay  a s s a y p r o c e d u r e s f o r t h e measurement o f d i g o x i n be  specific  methods  and i t s m e t a b o l i t e s  of extremely s e n s i t i v e  Since a large v a r i a b i l i t y  e x c r e t i o n of  Davydov  normal  Analysis  low  t h a t any  (Kennedy  in  procedures  of  speed,  t h e s e methods  laboratories. in  many  FPIA  clinical  33 laboratories since analysis  (20  digoxin,  the  radioactive  1.5.1 The  min.),  system  greater  reagents  provides  precision  have  a  and  longer  more  rapid  recovery  shelf-life  isotopes a r e not r e q u i r e d (Erickson  of and  1984).  Immunoassays analytical  an a n t i g e n  a n d an  immunoassay. is  t h e FPIA  p r o c e d u r e b a s e d on t h e r e a c t i o n b e t w e e n antibody  to  the antigen  i s termed  an  T h i s r e a c t i o n o b e y s t h e Law o f Mass A c t i o n a n d  shown i n F i g u r e 2.  Antigen  +  Antibody  ^  (Free F r a c t i o n )  Figure  2.  Immunoassays sensitivity  Reaction  Involved  use  labelled  of  a  Antigen-Antibody  —  the assay.  (Bound C o m p l e x )  i n Immunoassay  antigen Competitive  l a b e l l e d and  non-labelled antigen  l a b e l l e d and  n o n - l a b e l l e d bound  antigen then  i n either  known c o n c e n t r a t i o n the c o n c e n t r a t i o n  By  of analyte  binding  complexes.  The  the  of the form  labelled  o r t h e bound f r a c t i o n i s  comparison t o of analyte  increase  with the antibody  the free fraction  determined.  to  Procedures  a s e r i e s of samples of  (assayed i n an  a t t h e same t i m e ) ,  unknown s a m p l e  c a n be  determined. Radioisotopes, latex  particles  enzymes, and  metals  coenzymes, as  well  r e d blood as  cells,  fluorescent,  34 bioluminescent for  a n d c h e m i l u m i n e s c e n t m o l e c u l e s h a v e been u s e d  labelling  radioactivity,  antigens. enzymes  For  and f l u o r e s c e n t  most common l a b e l l i n g m e t h o d s ( B u t l e r One o f  t h e major  for digoxin antibodies  the  e n d o g e n o u s compounds. particularly  in  many p a t i e n t  although  groups and  at  (Aronson  also exhibit  (Butler  1979;  digoxin  and  t h a t DLIS  that digoxigenin known  to  lower  have  other  results,  i s present i n  a n d i t s mono- a n d cardiac  potencies  activity,  than  digoxin  antibodies  relatively  short  immunization w i t h t h e d i g o x i n c o n j u g a t e a r e used Pudek 1983a;  Schreiber  1981a,  1981b,  1981c;  1970, 1 9 7 3 ) .  label,  have  serum o r Butler  techniques,  been d e v e l o p e d  plasma.  the  metabolites  have  Other c a r d i a c  of d i g o x i n  a l s o been  The d e t e r m i n a t i o n  a  radioisotope  a n a l y s i s of d i g o x i n i n  glycosides 1982; W e i l e r  (Besch  1975;  1980) a n d some  ( B u t l e r 1978, 1 9 8 2 ; E i c h h o r s t  a s s a y e d by  R I A h a s a l s o been r e p o r t e d  using  f o r the  1978; L e n z 1975; R e i s s e l l  techniques. by  procedures  N a t u r a l l y o c c u r r i n g and s y n t h e t i c s t e r o i d s  Radioimmunoassay  1981)  of  f r o m t h e s e r a o f a n i m a l s when o n l y of  of  immunoassay  some c r o s s - r e a c t i v i t y w i t h d i g o x i n  periods  Smith  are  are the  may l e a d t o e r r o n e o u s  the fact  substantially  1980).  isolated  This  molecules  c r o s s - r e a c t i v i t y of the  metabolites  view of  bis-digitoxosides  been t h e  analysis,  1978).  problems w i t h  a n a l y s i s has with  digoxin  similar of digoxin  (Christenson  radioimmunoassay i n urine 1982).  samples  35 Antibody s p e c i f i c i t y for digoxin. has  produced  Lichey  a synthetic  interference with  some RIA  1 979;  Schreiber  than canrenone)  not  other  1981c;  may  be  Ravel  with 1975). the  method  1979).  endogenous  RIA  determination  Lindenbaum  1975)  and  such  as  not  (other  interference.  1979; been  albumin  DLIS (Valdes  found  using  to  the  RIA  substances,  (Holtzman  1974; well  s a m p l e s (Boone 1977) values  did  Muller  1983a, 1 9 8 3 b ) , a s  in apparent d i g o x i n  al.  spironolactone  of d i g o x i n  b a c t e r i a l c o n t a m i n a t i o n of p a t i e n t caused a l t e r a t i o n s  this  In a d d i t i o n to exogenous  substances  1980;  S i l b e r et  (Hansell has  RIA  (DiPiro  spironolactone  for  method  the  s t e r o i d analogue, kits  indicated that  Furosemide  interfere with (Hansell  of  responsible  the  with  S i l b e r 1 979).  that metabolites  r e p o r t s have  interfere  1978;  major problem  Spironolactone,  (1979) s u g g e s t e d  Still  is a  using  as  have  the  RIA  procedure. Variations  in c r o s s - r e a c t i v i t y with digoxin  and  endogenous  s u b s t a n c e s has  the  commercial  sources  Kubasik  1974a,  MacKinney tritium  1975; or  1980).  the  1974b; K u c z a l a  radioiodine  RIA  Larson  Comparing  labelled  less variable  However,  to d i f f e r e n c e s  kits  1976;  Smith 1976).  t r i t i u m method was Vemuri  of  been t r a c e d  radioiodine  advantages.  Iodine-125 i s  f a s t e r , more  convenient  and  less  scintillation  counting  which  is  1979;  1977;  1977;  Loo kits  show  1975;  Pippin  and  expensive necessary  using  that  labelling  a gamma e m i t t e r  in  (Hansell  RIA  digoxin  (Kubasik  metabolites  has  the 1976; some  counting than  with  is  liquid tritium  36 (Butler  1978).  iodine-125 FPIA tracer  (Kroening is  light  (Landon  fluorescence  (Dandliker The  1981).  Jolley  precipitation  of  (Erickson  the  for  1987).  total  assay.  The  1984)  increased protein  a  random excited  of the  emitted  and  free  separation  step  1981). f o r d i g o x i n from Abbott This  assay  requires  with trichloroacetic  acid  or  acid  5-sulfosalicylic  (Porter  decreases the  measured  i n f l u e n c e d by  1984)  such  digoxin  that  levels.  use o f a c i d f o r p r o t e i n p r e c i p i t a t i o n , h o w e v e r , h a s  suggested to r e s u l t metabolites 1977;  to  Sonobe  method,  1980;  digoxin  metabolites  Sternson can  present  been r e p o r t e d DLIS as  i n the t r a n s f o r m a t i o n  d i g o x i g e n i n and/or  i n the  t o be  compared t o  H o w e v e r , numerous 1987;  be  subject the  RIA  reports in  Kanan 1987;  Therefore  to  The  TDx  FPIA  (Yatscoff  the l i t e r a t u r e  S k o g e n 1987;  this  from  minimal i n t e r f e r e n c e procedure  (Gault  with  differentiated  serum s a m p l e .  been  o f d i g o x i n and i t s  dihydrodigoxigenin  1978).  not  of  their  r e s u l t s are s i g n i f i c a n t l y  protein concentration  of  antibody-bound  1981; L a n d o n  Porter  extent  during  require  serum p r o t e i n s  1984;  polarization  the p o l a r i z a t i o n  not  TDx  The  the  c o m m e r c i a l l y a v a i l a b l e FPIA is  Frye  Since  different  1964;  on  molecules  m e t h o d s do  Laboratories  The  depend  of the  is  a n t i g e n , FPIA  (Skogen  will  rotation  with  ( F I A ) where a f l u o r e s c e n t  i s excited with polarized l i g h t .  Brownian  the  i s not observed  1976).  a fluoroimmunoassay  the emitted  state  In a d d i t i o n , quenching  (Bianchi  S o l d i n 1986b;  its has from  1984). 1986; Weiner  37 1987)  have  shown t h a t  apparent d i g o x i n addition,  it  precipitation  levels has  free  in  been  step  i n t e r f e r e n c e by allowing  t h e TDx  assay  the  indicated  presence  suggested  used w i t h  the  of  that  FPIA  DLIS t o  be  detected  DLIS. the  may  d i s r u p t i n g the D L I S - p r o t e i n  increased In  protein  enhance  DLIS  i n t e r a c t i o n and  (Skogen  1987;  Soldin  1986b).  1.5.2  Biological  Digoxin binding brain  to  inhibits the  enzyme  t i s s u e s has  f o r the  determination  inhibit  having  ten  +  of  Na ,K -ATPase +  isolated  +  from  heart  of  cardiac glycoside  binding a f f i n i t y  Marcus 1975). the  As  with  immunoassay  of  assays  digoxin  dihydrodigoxin  of d i g o x i n  a result,  and  concentrations  the m e t a b o l i t e s  activity,  +  by  (Gundert-  the ATPase  methods, l a c k  assay  specificity  digoxin. The  of  ATPase  Na ,K -ATPase  procedures, l i k e for  and  activity  However,  times the  Remy 1981;  the  been u s e d t o d e v e l o p enzyme b i n d i n g  (Gundert-Remy 1981). also  Assays  rubidium-86 uptake assay  rubidium  digitalis  method i s  into  glycosides  Determination technique  uptake  of  requires  red  (Belz  digitalis  i s b a s e d on blood  1981b;  glycosides  approximately  8  cells  the in  1981b).  by  vitro  Lowenstein  1965).  i n plasma u s i n g  h o u r s , hence t h i s  more t i m e c o n s u m i n g t h a n c o n v e n t i o n a l  methods ( B e l z  inhibition  this assay  immunoassay  38 A number o f t e s t a n i m a l s , i n c l u d i n g g u i n e a p i g s , cats  and  pigeons,  digitalis  (Tyler  have  been  1976; USP are  diluted digitalis  preparation  dies  with this  effect.  In general,  potency  specificity  of  (Simson  sensitivity, digitalized  1962; T y l e r  1976).  (Simson  toxicity  and  lack  1976).  Chemical Methods h a s an u l t r a v i o l e t a b s o r p t i o n  absorption  and  have been  reported  glycosides  alter  the  have  not  been s p e c i f i c Flasch  o f 16595 ( F l a s c h  been  biological  Rowson  1952a,  sufficiently samples  digitalis  carbonyl glycosides  absorbance, of d i g i t a l i s  1952b).  sensitive  These f o r the  ( F l a s c h 1981) a n d h a v e n o t  fora particular d i g i t a l i s 1 9 8 1 ; Rowson 1952a,  1981).  both the i n t e n s i t y of  w a v e l e n g t h o f maximum  1981;  unsaturated of  maximum a t 217 nm  for quantitative analysis  (Flasch  e v a l u a t i o n of  residues  Problems  predict the  tested  1962; T y l e r  molar e x t i n c t i o n c o e f f i c i e n t  The  a  Along w i t h a lack of  D e r i v a t i z a t i o n procedures, t o increase  1952;  of  therapeutic  unable t o  being  for  intervals until  i t assumes t h a t  glycoside  of  b i o a s s a y methods a r e i m p r a c t i c a l f o r e v a l u a t i n g  Digoxin  methods  volumes  (USP 1 9 8 5 ) .  bioassays are  the  USP a s s a y  fixed  a t f i v e minute  bioassay  proportional t o , the  p a t i e n t samples  1.5.3  with a  with  cardiac arrest  w i t h , and  the  In the  b i o a s s a y a r e found s i n c e  is associated  oral  injected  from  in  1985).  digoxin, pigeons  the pigeon  used  frogs,  glycoside  (Eastland  1952b). and  the  c a n be  digitoxose derivatized.  sugar In  39 alkaline  solution,  dinitrobenzoic  picric  acid  (Kedde  (Rabitzsch reaction) compounds r e a c t ring.  and  w i t h the  Reviews i n the  1952b) h a v e reasonably  (Baljet  reaction),  reaction), tetranitrobiphenyl  several  other  carbonyl  function  literature  indicated that, specific  acid  aromatic  of the  ( F l a s c h 1981;  lactone  Rowson  although  these  reagents  lactone  ring,  they  1952a, were  did  not  d i f f e r e n t i a t e between the v a r i o u s g l y c o s i d e s , a g l y c o n e s ,  and  metabolites  with unsaturated  Colored formed  derivatives  by  xanthydrol  the  chloride,  Kilani  process  residues with product  nm).  would not  sulfuric  by  nm).  of  t h e r e f o r e the detected limit  of  p r o c e d u r e s was  i n the  ng  specific  a  sugars of a  (Flasch  red  f o r the  to  colored at  digitoxose  of the  molecule  Rowson 1 9 5 2 a ) .  detection with  nm.  digitoxose  (maximum a b s o r b a n c e  aglycone portion  range  590  thiobarbituric  formation  ( F l a s c h 1981;  lower  produces  to  digitoxose  s u g a r and  r e a c t i o n s are  the  The  Keller-  and  derivatization  maximum a t 520 oxidation  nm  and Using  a c i d , the  a t 470  2-thiobarbituric acid  be  acid  Rowson 1 9 5 2 a ) .  reaction hydrolyses  o p e n i n g of the  These  s u g a r s and  reported  xanthydrol  acid, followed  complex w i t h 532  ( F l a s c h 1981;  a b s o r p t i o n maxima  involves  dialdehydes,  thiobarbituric  removes t h e d i g i t o x o s e r e s i d u e s and  (absorption  reaction  moieties.  of t h e d i g i t o x o s e s u g a r s h a v e been  a c e t i c a c i d and  derivatives with the  lactone  Keller-Kilani,  reactions  ferric  Similarly,  f o r the  dinitro  The  these c o l o r i m e t r i c  1981).  40 Fluorescent glycosides example,  lower  the  for  the detection  r e a c t i o n of  phosphoric,  and  1981;  methods a r e  specific  metabolites  analysis  limit  t o t h e ng r a n g e .  or  hydrolysis  Jensen  the  digoxin  hydrochloric  dehydration (Flasch  methods  for  strong  acids  the  methods t o  allow  glycosides.  the  in  14,16-dianhydrogenin  steroid  part  n o t be  combination  isolation  of  w e l l as  the  1.5.4  digoxin,  techniques (Flasch  Chromatographic  have  Formation  (Watson  1972) d e t e c t o r digoxin. d i g o x i n and  systems  The GLC  individual  carbonyl  color  d e r i v a t i v e s , have liquid  1981).  i n the l i t e r a t u r e (Jelliffe  1972) d e r i v a t i v e s  a n a l y s i s h a v e been r e p o r t e d . mass s p e c t r o m e t r y ( F l a s c h  and  of  (GLC) methods o f a n a l y s i s o f  trimethylsilyl  heptafluorobutyryl  chromatographic  Techniques  been r e p o r t e d of  but have  w i t h p a p e r , t h i n - l a y e r and  G a s - l i q u i d chromatographic digoxin  with  the fluorescent  i n combination  chromatographic  sugar  assay  differentiated.  and p u r i f i c a t i o n  For example,  d e r i v a t i v e s , as been u s e d  used  such as  Since these f l u o r e s c e n t  of the a g l y c o n e would  been  For  causes  C h e m i c a l methods o f a n a l y s i s a r e n o t s p e c i f i c , frequently  cardiac  trichloroacetic,  to  1952).  with  of  Flame i o n i z a t i o n  (Flasch  1981).  1963)  and  prior  to  (Kibbe  GLC  1973),  1981) a n d e l e c t r o n c a p t u r e (Watson  h a v e been u s e d  a s s a y methods  i t s metabolites  i n t h e GLC a n a l y s i s o f  lacked  specificity,  since  were c o n v e r t e d t o t h e a g l y c o n e  41 portion  of  the molecule during  (Flasch  1981; J e l l i f f e  Thin-layer digoxin, plates Sabatka  1963; K i b b e  chromatographic  using  have  silica  been  1976).  reported  sensitivity  was  ( T L C ) methods o f a n a l y s i s o f  (Flasch  and  1981;  reversed-phase Jelliffe  glycosides  s a m p l e v o l u m e s were r e q u i r e d  inadequate  reports  by  appeared  on  for  therapeutic  in  the  analysis  literature.  i t s metabolites  required.  the  high-performance l i q u i d  attractive alternative  since, unlike  and t h e  monitoring  are  not  Various types  degraded  1976a,  Nachtmann  (Flasch  1981)  and  Davydov  1982;  Desta  1981b; G a u l t  1986a, 1986b; column and  by  1982a,  mode 1982b,  the  phases  Pekic  et  al.  applications glycosides  of  (1981) HPLC  of  reviewed for  the  and r e l a t e d s t e r o i d s .  conditions have  been  phases  (Beasley  1983;  1987;  Diamandis  1985;  1983; P l u m 1 9 8 6 ) .  sensitivity  digoxin  phases  1980; Kwong Both p r e -  t e c h n i q u e s h a v e been  used f o r the a n a l y s i s of d i g i t a l i s g l y c o s i d e s the  an  ion-exchange  post-column d e r i v a t i z a t i o n  increase  offer  1981b;  1982; G f e l l e r 1977; G i b s o n  Loo 1981;  have  1984; E r i k s s o n  1976b),  partition  digitalis  methods  of s t a t i o n a r y  Loo  1977;  of  GLC m e t h o d s ,  g e l (Bockbrader  Seiber  using  chromatography  These  used, i n c l u d i n g s i l i c a  to  1969;  1981).  Numerous  Eriksson  1972).  B i o l o g i c a l s a m p l e s have been e v a l u a t e d  but large  and  1973; Watson  gel, cellulose  TLC methods  (Flasch  d e r i v a t i z a t i o n or e x t r a c t i o n  HPLC  i n an a t t e m p t  assay  the analysis  techniques.  techniques of  and  digitalis  42 Pre-column  derivatization  dinitrobenzoyl chloride been r e p o r t e d . glycosides  from  The  detection digoxin and  analysed  formed  at  254 nm.  developed at  was  on  (1976a,  with  With t h i s  digoxin  with  was  a silica  et  al.  derivatization  latter  of  ng.  pre-column followed  by  p h a s e , Nachtmann f o rdigoxin  el  o f 11  a t 254 nm. have  of d i g o x i n  C  with  been  and i t s  formed  by  metabolites.  reagents.  an a i r - s e g m e n t a t i o n A detection with  1977).  modification  of  Further  method digoxin  lanatoside  hydrochloric  was f o u n d  of  Using  limit  C  resolution  chloride  ultraviolet  chloride  desacetyllanatoside  segmentation  derivatized  procedure, the l i m i t  digoxin, digoxigenin,  dehydroascorbic acid using the  with  (1977) d e v e l o p e d a p r o c e d u r e f o r p o s t - c o l u m n  desacetyllanatoside  delivering  plasma  system w i t h  derivatives  post-column d e r i v a t i z a t i o n  in  (1983)  gel stationary  fluorescent  gel stationary  3,5-dinitrobenzoyl  1976b) f o u n d a d e t e c t i o n  Highly  1983) h a s  3,5-dinitrobenzoyl  al .  2  3,5-  (1984) e x t r a c t e d t h e  a silica  4-nitrobenzoyl  ng/mL i n p l a s m a w i t h d e t e c t i o n  Gfeller  et  using  Fujii  ng/mL  HPLC a s s a y  230 nm. of  100  Fujii  a micro  detection  separation  1984;  the  these using  sensitivity  derivatization  al.  urine,  and i t s m e t a b o l i t e s  detection of  (Bockbrader  Bockbrader and Reuning  d e r i v a t i v e and phase.  techniques  (Desta from  1987)  this  acid  and and  procedure f o r  limit this  C  o f 0.5 ng f o r  system  (Gfeller  HPLC-PC  produced  dihydrodigoxin  air  complete but  the  43 sensitivity  (10  development  ng  of  of a r o u t i n e c l i n i c a l  Recent m a n u s c r i p t s described  digoxin)  the  therapeutic  development  of  (1986a)  of d i g o x i n for  a  height  detectable quantity (Kwong  found  1986b). was  s e p a r a t i o n of  interference  from  was  also  post-column by Kwong  several  drugs  Partial resolution which  allowed  assay.  The  minimum  0.5  ng  digoxin/mL i n  post-column d e v e l o p e d by  d i g o x i n from  possible  at  achieved  t o be A  have  digoxin  using  with digoxin.  was  for  1985)  procedure reported  quantitative  d e r i v a t i z a t i o n method (1985) but  plasma  for  d i g o x i n from  from d i h y d r o d i g o x i n  peak  plasma  The  separated  frequently co-prescribed  1986b; Reh  HPLC a s s a y s in  fluorogenic derivatization.  inadequate  assay.  (Kwong 1986a,  concentrations  and M c E r l a n e  was  fluorogenic Reh  and  Jork  dihydrodigoxin  co-prescribed  drugs  and  were  not  i t s metabolites  and  evaluated.  1.5.5 The  HPLC-RIA  separation  of d i g o x i n  endogenous s t e r o i d s digoxin Margot 1983)  fraction 1983;  separated  u s i n g HPLC p r i o r  u s i n g RIA  M o r a i s 1981;  increased  from  (Gibson Plum  specificity.  digoxin  from  three  t o the a n a l y s i s of the 1980;  1986; The  Loo  Vasdev  1977,  1981;  1985;  HPLC p r o c e d u r e s  metabolites;  Wagner reported  digoxigenin,  d i g o x i g e n i n m o n o d i g i t o x o s i d e and d i g o x i g e n i n b i s d i g i t o x o s i d e (Gibson have  1980;  been  Plum  shown  1986; to  Wagner 1 9 8 3 ) .  exhibit  high  These  metabolites  cross-reactivity  with  44 d i g o x i n RIA HPLC-RIA  procedures.  methods  concentrations eluate  sufficient  of d i g o x i n  fractions  m e a s u r e m e n t may 1.6  was  with  introduce  to  monitor  of  these  therapeutic  i n plasma,  the c o l l e c t i o n  of  subsequent  evaporation  and  timed RIA  methodological errors.  (EC)  electroactive analyte Generally, flow can  a  can  be  constant p o t e n t i a l  groups  i n c l u d e amines,  as a that  groups  that  esters,  ketones,  groups  have  ( F l e e t 1974;  or  time  oxidative  has  Volke  (Brooks  1986;  1983;  Stewart  Reviews of  Hu  1981,  EC d e t e c t i o n  current 1974).  electroactivity  1983).  Functional  diazo  include  compounds  and  1983).  to very Kreuzig  1986;  the  electroactivity  ethers,  Volke  led  reduced.  (Fleet  c o m b i n a t i o n o f HPLC w i t h EC d e t e c t i o n  d r u g measurement  an  phenols, aromatic hydroxyls  reductive  aldehydes,  require  oxidized  f u n c t i o n of  ( F l e e t 1974;  exhibit  methods  i s a p p l i e d and  phenothiazines,  and c a t e c h o l a m i n e s  The  detection  that  be m e a s u r e d  Functional  1979;  sensitivity  HPLC-EC  Electrochemical  nitro  While the  Volke  s e n s i t i v e assay  1981;  Leelavathi  1983;  methods and  HPLC h a v e been p r e p a r e d by W h i t e  (HPLC-EC) f o r  Wang  methods  1986;  1986,  flow c e l l  Lund 1987).  geometry f o r  (1984) and F l e e t and  Little  (1974). Numerous d r u g s been  evaluated  Biogenic assayed  amines  and  using and  biologically  a c t i v e compounds h a v e  HPLC-EC  the  in  catecholamines  by HPLC-EC ( B a u e r s f e l d  1986;  have Davis  oxidative frequently 1981;  Holly  mode. been 1983;  45 Krstulovic  1982;  Zaczek  1982).  Drugs  o x i d a t i v e HPLC-EC  include theophylline  (Greenberg  dopazinol  1979),  [trimeprazine (Tjaden  (Hu  1976),  1986) ] ,  1986),  antidepressants (Wang 1 9 8 6 ) ] ,  [desipramine, guanethidine  1987),  (Krause  r e d u c t i o n of  use  and  1986),  trimipramine  atropine  codeine  anthracycline  (Shah  1986),  (Humbert  and  1987)  (Leroy 1987),  antibiotics  (Croteau  HPLC-EC  poses  numerous  oxygen and  trace  metals  (Caudill  of continuous  h e l i u m gas (Caudill  tricyclic  1987), 0-receptor  and  mepindolol  1980)].  Reductive  analysis  1986),  (Stewart  (Stewart  [bopindolol  fluphenazine (Svendsen  erythromycin  hydrochlorothiazide  and  imipramine  1987),  phenothiazines  thioridazine  ( I s a k s s o n 1987),  (Shibanoki  antagonists  1986),  (Leelavathi  1987) , p h y s o s t i g m i n e  (Riley  and  using  and i t s d e r i v a t i v e s  perphenazine  chlorpromazine  indoramin  pentazocine  (Mazzo  evaluated  in  At  to  maintain  can  The  with  EC  This n e c e s s i t a t e s the  a  p o t e n t i a l s more  d i s s o l v e d oxygen  baseline noise.  1979).  interfere  s p a r g i n g of t h e mobile  order  1985).  reduction of  1985; L u n d  problems.  phase w i t h argon or de-oxygenated  state  n e g a t i v e t h a n -0.4 V, significantly  increase  P a s s i v a t i o n o f t h e HPLC pump, i n j e c t o r a n d  associated tubing  w i t h 20% n i t r i c  a c i d removes t r a c e  t h a t c a n be washed o f f by t h e m o b i l e  metals  phase c r e a t i n g b a s e l i n e  noise. R e d u c t i v e mode of  benzodiazepines  HPLC-EC h a s such  as  been u s e d f o r t h e a n a l y s i s nitrazepam,  diazepam  and  46 chlordiazepoxide  (Lund  1979).  The d e t e c t i o n  limits  were  f o u n d t o d e p e n d s t r o n g l y on t h e p o t e n t i a l u s e d ; s u c h t h a t a s the p o t e n t i a l increased  became  (Lund  1979).  d e t e r m i n a t i o n of o x i d a t i v e and  more  negative  Brooks  a large  (1983)  from b r a i n  r e v i e w e d t h e HPLC-EC  i n series,  tissue  HPLC a n d r e d u c t i v e EC, M e e r i n g et of  limit  Using  b o t h modes o f  used t o observe c h l o r p r o m a z i n e , t h i o r i d a z i n e  and m e t a b o l i t e s  limits  detection  number o f b e n z o d i a z e p i n e s .  reductive electrodes  d e t e c t i o n were  the  2  to  4  pg  (Svendsen  al.  (1984)  for misonidazole  1986). found and  With  detection desmethyl-  misonidazole. The  electrochemical  established (Fleet  by  1974;  a number Morales  p a r t i c u l a r aromatic acidic  solutions et  Mousty  r e d u c t i o n o f n i t r o g r o u p s h a s been of i n v e s t i g a t o r s  1987;  ( M o r a l e s 1987;  al.  (1986)  in  N i t r o groups, i n easily  Plambeck  reported  that  reduced  1982; Ryan  U s i n g column Brilmyer in  et  EC h a v e  on t h e not used  chromatography al.  (1975)  75% aqueous analysed  1984).  p-nitrophenoxyalkyl  d e r i v a t i v e s of  flow-through  a n d EC d e t e c t i o n  buffer at  pH 6.0.  trinitrobenzene  7-aminobutyric a c i d  w i t h a m o b i l e phase  a n a l y s i s of n i t r o  Many groups  detectors.  (0 t o -1.15 V ) ,  s e p a r a t e d o r t h o and p a r a  the  in  i n two s u c c e s s i v e s t e p s i n a c i d i c  the l i t e r a t u r e  using reductive  (1985)  are  years  (pH 2.7) u s i n g p o t e n t i a l s f r o m -0.25 t o -1.0 V.  reports  acids  1984).  n i t r o groups,  ammonium s a l t s a r e r e d u c e d medium  White  f o r many  nitrobenzoic  Caudill sulfonic  et  al. acid  w i t h HPLC-EC a t -0.55 V  o f 5 0 % a q u e o u s b u f f e r a t pH 3.0.  47 HPLC w i t h  electrochemical  used f o r the a n a l y s i s compounds.  suggested  for  use  1984;  d e r i v a t i v e s of evaluated  by r e d u c t i v e  a s s a y methods  or  aromatic  HPLC-EC  other  (Nachtmann  nitro  has  nitro  1976a)  been  digoxin containing  have  not  been  EC o r HPLC-EC.  low This  levels  of  necessitates  sensitivity  aromatic n i t r o  widely  3,5-dinitrobenzoyl  1983)  for evaluating  expected  of  reductive  1979),  digoxin  therapeutically.  utility  with  Fujii  Exceptionally  The  the  (Kissinger  (Bockbrader  i s becoming  of t r a c e q u a n t i t i e s of e l e c t r o a c t i v e  Although  derivatives  detection  digoxin  using  patient  of  samples  i n v e s t i g a t i o n of  f o r a n a l y s i s of the 3 , 5 - d i n i t r o b e n z o y l  used  t h e most s e n s i t i v e  electrochemical  g r o u p s make  are  for digoxin. detectors using  to  HPLC-EC  d e r i v a t i v e of d i g o x i n  imperat i v e . et  Gfeller  al.  (1977)  d e v e l o p e d an HPLC p r o c e d u r e f o r  post-column  derivatization  hydrochloric  acid.  either  the  digoxin specific  sensitive  reliable delivery method  (Kwong  investigation  of  of  reagents i n  glycosides  1987) o r (Reh  digoxin  the  1986a) h a v e  using  1985)  required  assay.  post-column also  the r e s o l u t i o n  been  observed.  r e s o l u t i o n from  digoxin  lack of  for a  Problems  reagents  post-column d e r i v a t i z a t i o n  combine s e n s i t i v i t y , specificity  (Desta  i t s metabolites  and  cardiac  M o d i f i c a t i o n s o f t h i s HPLC-PC a s s a y  sensitivity  from  of  with  with one  Further  of d i g o x i n  to  metabolites,  f o r d i g o x i n and r e l i a b l e d e l i v e r y of post-column one HPLC  assay  is  required.  Samples  from  48 patient  groups  should a l s o specificity  where h i g h l e v e l s o f D L I S h a v e been  be e v a l u a t e d for  digoxin i s  e n d o g e n o u s compounds. thesis.  by t h e  These  HPLC a s s a y  maintained  in  reported  t o ensure that  the presence  were t h e p r i m a r y  of  aims of t h i s  49 2.  2.1  EXPERIMENTAL  Supplies  2.1.1  Chemicals  Digoxin,  digoxigenin  monodigitoxoside,  bisdigitoxoside,  digoxigenin,  digoxigenin, digitoxigenin Boehringer  (Mannheim,  was p u r c h a s e d Ont.,  peroxide  (Aristar (30%),  chloride  acid  (3,5-DNBCl)  octahydrate  sulfate  purchased Sodium  acetic  Company, I n c .  (Gold  estradiol,  were p u r c h a s e d  and  from  hydrogen sulfate  (Toronto,  (Gold  label),  3,5-dinitrobenzoyl  from A l d r i c h  J.  sodium  zinc  Chemicals  U.S.A.).  Barium T.  Chemical hydroxide  Baker  Chemical  Cupric sulfate pentahydrate  A l l i e d Chemical  estrone,  (4-DMAP),  ( M o r r i s t o w n , N J , U.S.A.). norethindrone,  4-  6a-methyl-17o-hydroxyl-progesterone  17a-methyl-testosterone,  androstane-3,17-dione,  was  I7a-ethynyl  acetate, estradiol, estrone-3-sulfate, testosterone, testosterone,  from  (Burlington,  (HCl),  and  trihydrate  label)  purchased  Dimethylaminopyridine  BDH  acetate  ( M i l w a u k e e , WI,  was  anhydrous  from  ( P h i l l i p s b u r g , N J , U.S.A.). o b t a i n e d from  Ingelheim L t d .  grade), hydrochloric acid  sodium  Canada).  glacial  were o b t a i n e d  Sodium b i c a r b o n a t e , L - a s c o r b i c a c i d ,  h e p t a h y d r a t e were Ont.,  dihydro-  R,S,-Mexiletinehydrochloride  from B o e h r i n g e r  Canada).  hydroxide  dihydrodigoxin,  and g i t o x i n  GFR).  digoxigenin  adrenosterone,  A -androstene-3,17-dione,  androsten-11/3-ol-3,1 7-dione,  19-nor-  4  deoxycorticosterone,  5aA 4  21-  50 deoxycortisone,  estriol,  hydroxypregnenolone, 3/3,20a-diol,  17a-hydroxyprogesterone,  samples  progesterone  were o b t a i n e d  L o u i s , MO,  U.S.A.).  from  Commercial  grade  absolute  Alcohols  and  f r o m BDH  Sigma  Reichstein's  Chemical  Co. ( S t .  1 7 / 3 - e s t r a d i o l were  ( M o n t r e a l , Que.,  Canada).  Solvents  Reagent  methanol  and  1 7 a - E s t r a d i o l and  from A y e r s t L a b o r a t o r i e s  2.1.2  cortisone,  dehydroisoandrosterone-3-sulfate,  5/3-pregnane-3a, 2 0 a - d i o l , S  17a-  A^-pregnene-  5-pregnen-3j3-ol-20-one,  dehydroisoandrosterone,  Substance  hydrocortisone,  ethanol  was  purchased  L t d . (Toronto, Ont., Canada).  p r o p a n - 1 - o l were  Chemicals  reagent grade  (Toronto,  Ont.,  Acetone,  and  obtained  Canada).  Isooctane  ( 2 , 2 , 4 - t r i m e t h y l p e n t a n e ) was  glass  purchased from  (Toronto, Ont., Canada).  grade water  BDH C h e m i c a l s  was p r o d u c e d  ( M i l l i p o r e Corp., r e m a i n i n g HPLC Chemicals  M i l f o r d , MA,  grade  (Toronto,  was d i s t i l l e d  2.1.3 A Volac  using the  solvents  distilled  Milli-Q  U.S.A.).  Prior  quality  Water  and HPLC System  P y r i d i n e and t h e  were O m n i s o l v  Ont., Canada).  from  grade  from  BDH  t o use, p y r i d i n e  and s t o r e d over sodium h y d r o x i d e .  Extraction  Supplies  pipette controller  ( S c i e n c e w a r e , Pequannock, (Labindustries,  Berkeley,  f o r volumes  o f 1 t o 20 mL  N J , U.S.A.) a n d R e p i p e t D i s p e n s e r CA,  U.S.A.)  were  dispensing organic solvents during the extraction  used  for  procedure.  51 The (0.45 Co.,  filtration  um  13 mm  r  Inc.,  filter  Woburn, MA,  (Becton  to  a 5  of  using  and  Centriflo  serum s a m p l e s  t h e Amicon  Two  15.2 80  drilling)  and  (Schedule  80  were used  cm A-53  (6  to  extraction  membrane  A-53  i n . ) diameter  Grade  two 15.2  f o r the  Two 5.1  A  cones  80  flange d r i l l i n g ) (Schedule  C a n a d a ) were  neck  150  with  was  (CF25)  pressure T M  blind  sealant  B.C., The  Canada) gasket  (W.L. G o r e a n d  U.S.A.). PVC s o c k e t  weld  150 l b . f l a n g e d r i l l i n g ) , flanges  flanges  l b . flange  vessel.  joint  flanges  l b . flange  150  Vancouver,  in.) diameter  PVC w i t h  with  steel  Sales Ltd., steel  weld  in.) diameter  A  Teflon Gore-Tex  cm (2  steel  cm (6  Grade  (2 i n . ) d i a m e t e r b l i n d  vessel.  syringe  C e n t r i f r e e m i c r o p a r t i t i o n system  A s s o c i a t e s , I n c . , E l k t o n , MD,  pipe  glass  G r a c e a n d C o . , D a n v e r s , MA, U . S . A . ) .  (Grinnell  m a t e r i a l was  (Schedule  U.S.A.)  Post-Column D e r i v a t i z a t i o n S u p p l i e s  (Schedule  drilling)  B-D  MA,  Ont., Canada).  prior  ultrafiltration  ( A m i c o n D i v . , W.R.  2.1.4  Multifit  Instrument 13 M i l l i p o r e  Milford,  Canada, M i s s i s s a u g a ,  achieved the  disc (Rainin  Corp.,  mL L u e r - L o c k  Dickinson  N y l o n 66 membrane  U.S.A.) w i t h a S w i n n e x  (Millipore  Filtration  of a  diameter) f i l t e r  holder  attached  unit consisted  (Schedule  flanges  two 5.1 cm  80 PVC w i t h  150 l b .  a n d 36.8 cm o f 5.1 cm (2 i n . ) d i a m e t e r PVC 80) ( S c e p t e r M f g . Co. L t d . , V a n c o u v e r , B.C.,  used  in  construction  Two 5.1 cm (2 i n . ) d i a m e t e r  of full  the face  PVC  pressure  0.32 cm ( 1 / 8  52 in.)  Hypalon  gaskets  Canada) were blind  (Custom  used between  flanges.  For both  the non-metric  Gaskets,  Vancouver,  t h e p i p e s e c t i o n f l a n g e s and t h e t h e s t e e l a n d PVC p r e s s u r e  e q u i v a l e n t component  p a r t s were  F i g u r e 6 f o r d i a g r a m o f PVC p r e s s u r e v e s s e l w i t h Polytetrafluoroethylene I.D.)  fittings. (0.16  and  reactor.  The  The  s i x c o m m e r c i a l RIA  were  England).  Kits  k i t s used f o r d i g o x i n a n a l y s i s  D i g i - T a b RIA ( N u c l e a r - M e d i c a l L a b o r a t o r i e s Inc., Irving,  Bio-RIA  of Bio-Endocrinology  Digoxin  M o n t r e a l , Que.,  RIA  (Becton  (Institute  Canada), Amerlex  Corp., A r l i n g t o n  column  obtained  D i v . , Warner-Lambert T e c h n o l o g i e s 1-125  and  Omnifit L t d . ,  PTFE t u b i n g a n d c o n n e c t o r s  Commercial Radioimmunoassay  were a s f o l l o w s :  1102  was b e t w e e n t h e p r e s s u r e v e s s e l ,  from O m n i f i t L t d . (Cambridge,  2.1.5  number  reactor  cm  o f t h e column and p r e s s u r e v e s s e l t o t h e r e a c t o r .  Cambridge, England)  f o r the  See  connection  (part  used  tubing  used.  0.3  PTFE v a l v e  was  (PTFE)  vessels  diameter,  A three-way  mm  B.C.,  Heights,  Dickinson  U.S.A.),  GammaCoat  Travenol  Laboratories  TX, U . S . A . ) ,  D i g o x i n R I A K i t (Amersham  I L , U.S.A.), D i g o x i n S o l i d  Immunodiagnostics, Digoxin  RIA  Inc.,  Orangeburg,  (Clinical  I n c . , Cambridge,  MA,  Phase NY,  Assays D i v . , U.S.A.)  and  D i g o x i n 1-125  Imusay ( A b b o t t L a b o r a t o r i e s D i a g n o s t i c s D i v . ,  North Chicago,  I L , U.S.A.).  53 2.1.6  Commercial Fluorescence  Polarization  Immunoassay  Kit The A b b o t t immunoassay  L a b o r a t o r i e s TDx  for  digoxin  Diagnostics D i v . , North  2.1.7  Filtration  FP V e r i c e l Sciences Millipore  o f HPLC M o b i l e 0.45  Ann A r b o r ,  all-glass  Mm  (Abbott  Laboratories  MA,  Phase  membrane  M I , U.S.A.)  filter  Associates, Milford,  used  polarization  C h i c a g o , I L , U.S.A.).  47 mm  Inc.,  was  fluorescence  (Gelman  were u s e d  apparatus  U.S.A.)  filters  with the  (Millipore  for  filtration  Waters of  the  mobile phase.  2.2  Equipment  2.2.1  HPLC E q u i p m e n t  A Beckman system  t h e HPLC pump.  of e i t h e r  a m o d e l U6K  U.S.A.)  or  a  Instrument,  Inc.,  fluorescence  detector  (1 mm  A dual  (Beckman I n s t r u m e n t ,  used as  MA,  M o d e l 100  I.D. by  A l t e x CR1A  40 mm  solvent  Inc., Fullerton,  The r e m a i n i n g injector model  m o d e l 420 quartz  CA, U . S . A . ) .  metering  CA, U.S.A.) was  HPLC s y s t e m c o n s i s t e d  (Waters A s s o c i a t e s ,  210  Fullerton,  Chromatopac Data  Inc., Fullerton,  piston  Altex CA,  injector  U.S.A.),  (Beckman a  AC w i t h a q u a r t z  tubing)  (section  Processor The  Waters flow  cell  2.2.3) a n d an  (Beckman  noise  Milford,  Instrument,  reducer  shown i n  54 Figure  3  was  fluorescent A  constructed  wavelength  model  153  " F u l l e r t o n , CA,  U.S.A.) was  A NewGuard  was  cartridge used  Spherisorb  holder  the  as  a  ODS  I I (3M)  guard  (Alltech Associates,  nm)  Waters  ultraviolet  Beckman  420  detector  Instrument  Inc.,  3,5-dinitrobenzoyl  i t s metabolites.  e q u i p p e d w i t h a 1.5  cm x 3.2  mm  I n c . , S a n t a C l a r a , CA,  column  and  15 cm X 4.6  mm  Deerfield, IL,  i n j e c t o r and g u a r d column,  placed I.D.  to  analytical  the  column  Between the  a d i r e c t c o n n e c t column  Deerfield, IL,  U.S.A.)  prior  U.S.A.).  I.D.  U.S.A.) was  prefilter used as  an  filter.  2.2.2  Electrochemical  The  Coulochem  d e t e c t o r model cell  5012  a gold  with  (Bedford,  MA,  before the  directly  dual  electrode  m o d e l 5010  and  The  electrochemical  model 5020, d u a l  electrode  screened w a l l j e t c e l l  e l e c t r o d e were  U.S.A.).  HPLC  p u r c h a s e d from  guard c e l l  model  ESA,  e l e c t r o d e was  Inc. placed  i n j e c t o r and used t o e l e c t r o c h e m i c a l l y c l e a n  m o b i l e phase analytical  Detector  5100A, g u a r d c e l l  analytical  4.  with  used t o assay  (Brownlee Labs  (Alltech Associates,  inline  (254  detector,  d e r i v a t i v e s o f d i g o x i n and  ODS  used  detector.  fixed  (Beckman  and  before  cell  after  the  (model  introduction  5010  or model  of 5012)  t h e a n a l y t i c a l HPLC c o l u m n  samples. was  the The  connected  a s shown i n F i g u r e  55  100,000 1  OHM  470  MICROFARAD/25  Figure  +  VOLT 2700  ELECTROLYTIC  OHM  -wv  -AAA-  VOLT  100,000  OHM  CAPACITORS  3.  Schematic  of Noise  Reducer  56 2.2.3 All cell  Fluorescence D e t e c t o r Flow  stainless  were  steel  removed.  positioned  in  fittings  A 40  mm  the flow c e l l  Cell  i n the  quartz  fluorometer  tube  (1  NY,  I.D.)  Technicon  Instruments  tubing (part  Corp.,  Tarrytown,  U.S.A.).  2.2.4  Post-Column Reactor  A schematic  diagram  (HPLC-PC) f l u o r o g e n i c Beckman  model  Instrument,  system  110 A  Inc.,  of  and P r e s s u r e the  is  final  HPLC  post-column  in  Figure  s i n g l e piston metering  system  F u l l e r t o n , CA,  given  Chamber  U.S.A.) was  5.  A  (Beckman  u s e d t o pump  hexane i n t o t h e p r e s s u r e v e s s e l t o d i s p l a c e c o n c e n t r a t e d from a  s t e e l o r PVC p r e s s u r e chamber  post-column (0.3 mm  reactor  I.D.).  r e a c t i o n bath temperature NJ,  was  b l o c k , h e l d i n p l a c e w i t h epoxy  g l u e a n d j o i n e d t o t h e PTFE t u b i n g v i a a c i d f l e x number 116-0538-09,  mm  flow  which  consisted  The r e a c t o r thermostated  circulator  ( s e c t i o n 2.1.4) t o t h e o f k n i t t e d PTFE t u b i n g  was m a i n t a i n e d  using  (Fisher  HC1  a t 79°C  a Haake m o d e l D1  Scientific  by a  constant  Co., F a i r  Lawn,  U.S.A.).  2.2.5  E x t r a c t i o n Equipment  A Vortex-Genie U.S.A.), a U.S.A.) a n d  Labquake Shaker an IEC  Western S c i e n t i f i c , extraction  (Fisher S c i e n t i f i c  Co., F a i r Lawn, N J ,  (Labindustries, Berkeley,  HN-SII C e n t r i f u g e  (Damon/IEC  CA,  Division,  V a n c o u v e r , BC, C a n a d a ) were u s e d i n t h e  of b i o l o g i c a l  samples.  57  GUARD CELL RECORDER  o  HPLC PUMP  INJECTOR ELECTROCHEMICAL DETECTOR  HPLC COLUMN  u  ANALYTICAL FLOW CELL  HPLC MOBILE PHASE TO WASTE  Figure  4.  S c h e m a t i c o f HPLC-EC S y s t e m  58  TO WASTE  0.4 mL/min  Q  INJECTOR RECORDER HPLC COLUMN  HPLC MOBILE PHASE THREE WAY VALVE  u  FLUOROMETER (360/425 nm) REACTOR (20 m)  HEATER  WATER BATH (79° C)  PVC PRESSURE VESSEL CONCENTRATED HCI  HPLC HEXANE  Figure  5.  S c h e m a t i c o f HPLC-PC F l u o r o g e n i c  System  59 2.2.6  Radioimmunoassay Equipment  A Nuclear-Chicago (Searle  Co.,  determining  2.3  Des  1185 s e r i e s Plaines,  iodine-125  automatic  I L , U.S.A.)  gamma was  counter  used f o r  decay w i t h t h e c o m m e r c i a l RIA k i t s .  Pre-column 3 , 5 - D i n i t r o b e n z o y l  Chloride Derivatization  Procedures  2.3.1  F o r Gram Q u a n t i t i e s o f D i g o x i n  A s o l u t i o n " o f 3,5-DNBCl was  added  pyridine mixture  s o l u t i o n of  (molar was  mL c o l d was  to a  ( 3 . 2 g i n 30 mL d r y p y r i d i n e )  r a t i o of  stirred  for  digoxin  ( 0 . 5 g) i n 20 mL d r y  3,5-DNBCl/digoxin  a n d washed  with water.  and t h e p r e c i p i t a t e The c r u d e p r o d u c t  dissolved  i n 1 L of e t h y l acetate/hexane  w i t h 250  mL o f  times  with  layer with vacuo,  5% s o d i u m b i c a r b o n a t e  HPLC w a t e r anhydrous  (300 mL). sodium  2.3.2  (reagent  glycoside  vortexed  DNBCl/digoxin  twice  w i t h 4-DMAP a n d t h r e e  After drying the organic  sulfate  and  in  evaporation  recrystallization  For M i l l i g r a m Q u a n t i t i e s of Cardiac  (3.0  was  g r a d e ) (mp 199-204°C).  3,5-DNBCl ( 1 5 mg) was  was  ( 1 / 1 ) , washed  t h e r e s i d u e was p u r i f i e d by d o u b l e  in methanol  The  25 m i n u t e s a t 50°C, p o u r e d i n t o 60  d i l u t e HC1 ( 1 % o f c o n c e n t r a t e d )  filtered  (22/1)).  added t o  Glycosides  a s o l u t i o n of c a r d i a c  mg) i n 0.2 mL o f d r y p y r i d i n e a n d t h e m i x t u r e f o r 20 (20/1)).  seconds After  (molar  ratio  of  3,5-  r e a c t i n g f o r 3 h o u r s a t room  60  temperature, the  sample  was p l a c e d i n a 37°C w a t e r b a t h a n d  t h e p y r i d i n e was e v a p o r a t e d u n d e r dissolved  i n HPLC g r a d e  four times  with  w i t h 4-DMAP. with  nitrogen.  ethyl acetate  1 mL o f a 5% s o l u t i o n  The  The r e s i d u e was  ( 1 . 5 mL) a n d washed  of sodium b i c a r b o n a t e  o r g a n i c l a y e r was t h e n washed f o u r  times  1 mL o f 1% o f c o n c e n t r a t e d HC1 a n d t h e n f o u r t i m e s w i t h  HPLC g r a d e acetate  water  (1  with  mL).  nitrogen  After  evaporation  gas,  the  r e c o n s t i t u t e d w i t h t h e m o b i l e phase  2.3.3 To ng o f  derivatives  were  a n d a n a l y z e d by HPLC.  F o r Nanogram Q u a n t i t i e s o f D i g o x i n  0.5  t o 10 ng o f d i g o x i n  i n 0.5 mL d r y p y r i d i n e , 100  3,5-DNBCl i n 10 ML o f p y r i d i n e was a d d e d  of 3 , 5 - D N B C l / d i g o x i n digoxin,  of the e t h y l  f r o m 34/1 t o 6 7 7 / 1 ) .  f r o m 34/1  t o 54/1).  ratio  To 50 t o 80 ng o f  800 ng o f 3,5-DNBCl i n 80 txL was a d d e d  of 3 , 5 - D N B C l / d i g o x i n  (molar  (molar  ratio  The s a m p l e s  a l l o w e d t o r e a c t a t room t e m p e r a t u r e f o r 2 h o u r s a f t e r the p y r i d i n e  was e v a p o r a t e d  n i t r o g e n gas.  After  e t h y l acetate/hexane four times 1% o f  with 2  reconstitution (1/1), the  under  a n d HPLC  nitrogen.  r e c o n s t i t u t e d w i t h m o b i l e phase  2.4  Preparation  of  Solutions  37°C w a t e r of  the  which  bath  residue  organic layer  mL o f 5% s o d i u m  c o n c e n t r a t e d HC1  evaporation  using a  were  and with  was  washed  bicarbonate with  4-DMAP,  grade water The  just prior to  derivative  was  a n d a n a l y s e d by HPLC.  then  61 2.4.1  Reagents  2.4.1.1  F o r 3,5-DNBCl  3,5-DNBCl i n P y r i d i n e  3,5-DNBCl (1  mg) was d i s s o l v e d  v o l u m e o f 100 mL w i t h was  stirred  drypyridine  final  ( 1 0 ng/uL).  Sodium B i c a r b o n a t e  Sodium b i c a r b o n a t e  a  i n and taken t o a  final  The s o l u t i o n  f o r 20 m i n u t e s p r i o r t o u s e .  2.4.1.2  dissolved  Derivatization  ( 5 g)  and  (5%)  with  4-DMAP  4-DMAP  ( 2 5 0 mg)  were  i n HPLC g r a d e w a t e r a n d t h e s o l u t i o n was t a k e n t o volume o f  100 mL  s o l u t i o n was s t i r r e d  2.4.1.3  with  HPLC  grade  water.  The  f o r 20 m i n u t e s .  Dilute Hydrochloric  Acid  C o n c e n t r a t e d HC1 ( 1 0 mL) was a d d e d t o 600 mL HPLC g r a d e water, taken  to a  final  volume  of 1  L and s t i r r e d  f o r 20  minutes.  2.4.2  Reagents F o r F l u o r o g e n i c  2.4.2.1  Ascorbic Acid  L-Ascorbic acid taken t o a f i n a l stirred  (250  mg)  Derivatization  Solution was w e i g h e d  v o l u m e o f 500 mL w i t h  f o r 20 m i n u t e s p r i o r t o u s e .  out,  dissolved,  HPLC g r a d e w a t e r , a n d  62 2.4.2.2  D i l u t e Hydrogen P e r o x i d e  Hydrogen p e r o x i d e to  200  used  mL w i t h HPLC g r a d e  water,  Dehydroascorbic  D i l u t e hydrogen ascorbic acid  peroxide  solution  dehydroascorbic  use.  stirred  f o r 20 m i n u t e s a n d  (500  (12.5  mL) was  mL) a n d  stirred  to the  acetic acid  The  Acid  in a  phosphoric  acid  1/1 r a t i o .  This  daily.  trihydrate  f o r 20  New b u f f e r  Mobile  HPLC  individually  e v e r y week.  Concentrated  (6.8  i n 500  taken t o a f i n a l  stirred  2.4.4  water.  were d i s s o l v e d  s o l u t i o n was  use.  until  S o d i u m A c e t a t e B u f f e r (0.1 M, pH 4.6)  Sodium a c e t a t e  water,  2 hours.  (40 uL o f 30%) was d i l u t e d a n d t a k e n  hydrogen p e r o x i d e  m i x t u r e was p r e p a r e d  2.4.3  for  Hydrogen P e r o x i d e w i t h P h o s p h o r i c  5 mL w i t h HPLC g r a d e  was a d d e d  added t o t h e  a c i d was s t o r e d i n a r e f r i g e r a t o r  Hydrogen p e r o x i d e  until  Acid  New d e h y d r o a s c o r b i c a c i d was p r e p a r e d  2.4.2.4  to  o f 30%) was d i l u t e d a n d t a k e n  immediately.  2.4.2.3  The  (1 mL  Solution  g) a n d mL  3  HPLC  mL  glacial  water.  v o l u m e o f 1 L w i t h HPLC  minutes  The grade  and s t o r e d i n a r e f r i g e r a t o r  s o l u t i o n was p r e p a r e d e v e r y week.  Phase  mobile measured  phase  was  solvents  prepared and  then  by  mixing  the  degassing  the  63 mixture prior  by  to  stirring use.  rapidly  The m o b i l e  f o r 30 m i n u t e s a n d was phases used  are  filtered  described  in  s e c t i o n s 3.3 a n d 3.4.  2.4.5  Standard  2.4.5.1 A stock  o f d i g o x i n was p r e p a r e d  The s t o c k  of  stock  s o l u t i o n was f u r t h e r  solution  respectively). diluted  to  25 mL  ng/10 uL  3  serum samples solutions  methanol  to  10  i n e t h a n o l (1  d i l u t e d t o give  mL  with  ethanol  ( 1 . 2 5 mL s t o c k  solution  was u s e d t o p r e p a r e 4 and  6  mL  t o 10 mL w i t h e t h a n o l ) . with  10  of  stock (1  mg/100 mL  taken  the  of  t o 10 mL w i t h  in  diluted  ( 0 . 5 mL o f s t o c k  i n Ethanol  f l u o r o g e n i c HPLC a s s a y , A stock  digoxin  methanol).  Diqitoxigenin  i n t e r n a l standard.  digoxin  serum).  was f u r t h e r  c o n c e n t r a t i o n o f 5 ng/10 uL  curve  i n Methanol  3,5-dinitrobenzoyl  o r 100 ng/10 uD  ng/jiL  0.5  uL o f t h e f i n a l  3,5-Dinitrobenzoyl Digoxin  solution  1.5, 2  Calibration  ( 1 . 5 , 2, 3, 5, 7 a n d 10 ng d i g o x i n / 3 mL  2.4.5.2 For  (3,  were s p i k e d  to give a f i n a l solution  with ethanol) solutions  2.4.5.2 A  taken  A 0.5 n g / y L s o l u t i o n  solution diluted  as  i n Ethanol  c o n c e n t r a t i o n s o f 5, 7 a n d 10 ng/10 uL ( 0 . 5 , 0.7 a n d 1  final  and  Digoxin  solution  mg/100 m L ) .  mL  Solutions  digitoxigenin  solution  was u s e d  o f d i g i t o x i g e n i n was  64 prepared  in  ethanol  further diluted (10 mL  (4  s o l u t i o n was  o f 80 ng/10 uL  t o 50 mL w i t h e t h a n o l ) .  Serum s a m p l e s  uL o f  w i t h 20  The s t o c k  concentration  to give a f i n a l  stock d i l u t e d  were s p i k e d  mg/100 m L ) .  this  internal  standard  solution  (160 n g ) .  2.4.5.3  Dihydrodigoxin  One m i l l i g r a m and  made  up t o  solution ML).  of d i h y d r o d i g o x i n  100 mL  the  was a c c u r a t e l y w e i g h e d  with ethanol.  ( 0 . 5 mL) was d i l u t e d  To t e s t  i n Ethanol  2.4.5.4  a l i q u o t of t h i s  t o 10 mL w i t h e t h a n o l  specificity  of  the  10 uL o f t h e f i n a l  fluorogenic assay,  An  HPLC  (5 ng/10  post-column  s o l u t i o n was a s s a y e d .  S t e r o i d Samples i n Methanol  One m i l l i g r a m  of each  of the  following steroids  was  w e i g h e d a c c u r a t e l y , s e p a r a t e l y made up t o 100 mL i n m e t h a n o l and  the  i n d i c a t e d amount  fluorogenic  assay  to  fluorogenic assay: estradiol ng),  (18  (55  (133  methyltestosterone m i l l i g r a m of mL  the  post-column  specificity  (680  ng),  of the  17a-ethynyl  ng),  6a-methyl-17a-hydroxylprogesterone (55 n g ) , e s t r o n e - 3 - s u l f a t e  ng), 19-nortestosterone (60  (30 n g ) ,  (67 n g ) a n d 1 7 a -  n g ) . F o r t h e f o l l o w i n g s a m p l e s , one  e a c h was w e i g h e d a c c u r a t e l y , a n d made up t o 10  i n methanol  evaluated  the  norethindrone  (25 n g ) , e s t r i o l  testosterone  evaluate  by  n g ) , 1 7 a - e s t r a d i o l ( 9 6 n g ) , 1 7 0 - e s t r a d i o l (37  estrone  acetate  was a s s a y e d  using  and  approximately  t h e HPLC-PC  assay:  500  ng  of  each  was  andrenosterone,  5a-  65 androstane-3,17-dione,  A -androstene-3,17-dione,  a n d r o s t e n - 1 1 /3-ol-3,1 7-dione, estriol,  hydroxypregnendone,  17a-hydroxyprogesterone,  cortisone,  dehydroisoandrosterone, ate,  5/3-pregnene-3a, 2 0 a - d i o l ,  a n d R e i c h s t e i n ' s S u b s t a n c e S.  2.4.5.5  P r e p a r a t i o n of  Mexiletine  Hydrochloride  50% E t h a n o l One  milligram  of  R,S-mexiletine  weighed a c c u r a t e l y ,  dissolved i n  water and  a final  was  17a-  A^-pregnene-  5-pregnen-3/3-ol-20-one,  dehydroidoandrosterone-3-sulf  in  21-  hydrocortisone,  dehydroisoandrosterone,  progesterone  4  deoxycorticosterone,  deoxycortisone,  3/3,20a-diol,  A -  4  then  taken  to  evaluated  hydrochloride  50% e t h a n o l  v o l u m e o f 1 mL.  by t h e HPLC-PC f l u o r o g e n i c  2.4.5.6  Preparation of  was  i n HPLC g r a d e This solution assay.  Plasma Samples  f o r RIA  Analysis S a m p l e s were  prepared  individual metabolites Serial dilutions made a n d  and  3.0  of the  added t o  volunteer, to  of  s a m p l e s were a l s o  2 mL  dissolving  digoxin  stock  plasma from  give f i n a l  ng/mL i n  by  in  solution  20%  of plasma.  or the  ethanol-water.  (1 mg/100 mL) were  a healthy,  concentrations  prepared.  digoxin  non-digitalized  o f 0, 0.5, 1.0, 2.0  F o r d i g o x i n , 5.0 ng/mL  66 2.4.5.7 Digoxin  P r e p a r a t i o n o f Serum S a m p l e s S p i k e d  Metabolites  Samples  f o r FPIA A n a l y s i s  were  prepared  metabolites  of  and  the volume t o  taking  this  stock  digoxin  (1  solution  concentration  was  by  mg), 100  mL  were  1 ng/10  prepared ML.  s o l u t i o n s ) and  2.5  t h e ACU  the m i x t u r e  2.5.2  was  Zinc taken  ethanol  Dilutions  that  the  metabolite the  serum  (from  of  final mL)  5 to  samples  10  were  f o r Serum E x t r a c t i o n  mL)  stirred  (2  mL)  was  Preparation  (20/5)  added t o i s o o c t a n e  f o r 20  mL)  minutes.  and  dichloromethane to  (98  of P r o t e i n P r e c i p i t a t i o n  Zinc S u l f a t e Heptahydrate  sulfate  heptahydrate volume of  (5 g) was 100  mL  mL)  were  use.  2.5.3.1  to a f i n a l  (20  Solvent  f o r 20 m i n u t e s j u s t p r i o r  2.5.3  and  (5  Extraction  Propan-1-ol stirred  one  Isooctane/Dichloromethane  Dichloromethane and  them i n  FPIA.  P r e p a r a t i o n of S o l v e n t s  2.5.1  so  individual  B l a n k serum s a m p l e s (1  ML  a s s a y e d by  the  with ethanol.  spiked with  the d i l u t e  weighing dissolving  were i n d i v i d u a l l y of  with  Reagents  (5%)  weighed, d i s s o l v e d  with d i s t i l l e d  water.  67 2.5.3.2  Barium Hydroxide Octahydrate  Barium hydroxide d i s s o l v e d and distilled  taken  octahydrate to  Cupric in  distilled  water.  pentahydrate  and t a k e n  2.5.3.4  taken t o a f i n a l  to a  (10  final  samples  (12 g)  were a s s a y e d  t h e s i x RIA k i t s .  samples  was  procedures. determine  assayed was  mL  with  weighed,  o f 100  was w e i g h e d ,  mL  with  dissolved  evaluated A blank  i n and  water.  Calculations  i n d u p l i c a t e u s i n g one l o t o f The p r e s e n c e o f e t h a n o l i n t h e  for  plasma  interference sample  i f any c r o s s - r e a c t i n g  plasma  was  v o l u m e o f 100 mL w i t h d i s t i l l e d  each of  Each  50  g)  volume  R I A A n a l y s i s and C r o s s - R e a c t i v i t y All  of  weighed,  Sodium H y d r o x i d e (12%)  Sodium h y d r o x i d e  RIA  volume  was  C u p r i c S u l f a t e P e n t a h y d r a t e (10%)  sulfate  dissolved  the  final  g)  water.  2.5.3.3  2.6  a  (23.7  ( 0 . 3 N)  was  with  also  the  RIA  assayed  to  c o m p o n e n t s were p r e s e n t i n  used. of  in  the four duplicate.  c a l c u l a t e d as  d i g o x i n and  m e t a b o l i t e - s p i k e d plasma Cross-reactivity with the r a t i o  the quantity  of m e t a b o l i t e  displacement of the d i g o x i n t r a c e r . c r o s s - r e a c t i v i t y was  between t h e which  samples  was  the Digi-Tab quantity  of  caused  50%  F o r a l l o t h e r RIA  kits,  c a l c u l a t e d as the r a t i o of t h e d i g o x i n  68 concentration inhibition  to  of  procedure i s basis  the maximum  designed  and  these  molecular  metabolite binding.  for  Since  competitive  metabolites  weight,  concentration  the  binding  differ  percent  the  at  50%  RIA  assay  on a  molar  in  their  greatly  cross-reactivity  was  d e t e r m i n e d on a m o l a r b a s i s .  2.7  HPLC W i t h E l e c t r o c h e m i c a l  2.7.1  Optimization  Using  the  guard c e l l screened  wall  of E l e c t r o c h e m i c a l  HPLC s y s t e m  electrode  as  buffer  the  (20/7),  confirmed  using  m o d e l 153  carbon e l e c t r o d e the v o l t a g e g r e a t e s t peak experiments.  height  digoxin  and  5020  the  5012  electrode,  3,5-  and  dihydrodigoxin  a m o b i l e p h a s e of a c e t o n i t r i l e / a c e t a t e location  of  5012  t o -0.85 and  derivatized  detection  Recording  from the  from 0  4 w i t h the  injector  analytical  of  ultraviolet  detector).  the  the  dinitrobenzoyl derivatives With  Conditions  shown i n F i g u r e  before  jet cell  were a s s a y e d .  Detection  V  from cell),  at  nm  was  (Beckman  detector  1  gradually  decreasing  indicated  t h e r e f o r e was  254  digoxin  -0.80 used  (glassy  V gave for  the  further  69 2.7.2  Mobile  Phases  f o r HPLC  with  Electrochemical  Detection The  limit  of d e t e c t i o n  dihydrodigoxin mobile phases  2.8  and r e s o l u t i o n of  d i g o x i n was d e t e r m i n e d f o r a v a r i e t y o f  ( s e c t i o n 3.2.1) u s i n g -0.80 V f o r d e t e c t i o n .  HPLC P o s t - C o l u m n D e r i v a t i z a t i o n A s s a y  2.8.1 The  A s s e m b l y o f S t e e l a n d PVC P r e s s u r e steel  t o back. end  from  for digoxin  of  with  w e l d neck  Teflon  joint  cm ( 3 / 4  stainless The  solvent  were p r e p a r e d  followed  glued.  by  was a p p l i e d t o t h e e x p o s e d  was  surfaces. section  T h r e a d , 316  L t d . , Vancouver, B.C.).  s e c t i o n and t h e s o c k e t s  glue  sanding.  (Scepter  of the  PVC c l e a n i n g  Mfg.  w e l d f l a n g e s were p r e s s  and s e c u r e d  wiped  clean  and t h e  (National  (Indufast Fastners the flanges  Coarse  until  from  Hypalon gaskets  flanges  f l a n g e s w e r e mounted  ( N a t i o n a l Coarse  by l i g h t  PVC  back  Co., L t d . ,  C a n a d a ) were a p p l i e d t o a l l s u r f a c e s t o be  The s o c k e d  ends of t h e p i p e  secure  in.) bolts  e n d s o f t h e PVC p i p e  V a n c o u v e r , B.C.,  bolts  sealant  steel) (Indufast Fastners  PVC f l a n g e s  glue  f l a n g e s were w e l d e d t o g e t h e r  b o t h neck f l a n g e s and t h e b l i n d  1.9  Vessels  the  internal  and  between  Excess external  the  pipe  f l a n g e s a n d 1.3 cm ( 1 / 2 i n . )  Thread,  L t d . , Vancouver, together.  onto the  t h e g l u e was s e t .  were p l a c e d  blind  fitted  316  stainless  B.C.)  were  steel) used  to  70 On flanges 3/8  both  the  (to  in.  be  steel  the  National  Tube F i t t i n g s ,  thread.  were u s e d as  v e s s e l s , the  blind  threaded to  accept  Swagelok f i t t i n g s and  Fitting  t o produce the  shown i n F i g u r e  Optimization  analytical  pressure  w e r e d r i l l e d and  Co.,  flow  0.4  to  samples were e v a l u a t e d . concentrated vessel.  HC1  an ODS  pump a t a f l o w m reactors,  the  c o m p a r i n g peak various  mL/min.,  The  HPLC e f f l u e n t was  0.5  to  the  a steel  1.0  mL/min.  cardiac  or  from  9.1  ng  II  (3  mobile  (52/3/1/45) at glycoside  combined PVC  Using  of d i g o x i n  with  pressure  v e s s e l v i a an  p o s t - c o l u m n d e r i v a t i z a t i o n was heights  ODS  g u a r d c o l u m n and  added t o the p r e s s u r e  r a t e of  f o r hexane  Spherisorb  0.5  d e l i v e r e d from  Hexane was  North  6.  i n j e c t o r , the  column w i t h  r a t e of  Ltd.,  inlet  p h a s e of m e t h a n o l / e t h a n o l / i s o p r o p a n o l / a q u e o u s a  (Swagelok  o f HPLC P o s t - C o l u m n D e r i v a t i z a t i o n  U s i n g t h e W a t e r s U6K Mm)  PVC  Columbia Valve  o u t l e t f o r HC1  2.8.2  top)  pipe  V a n c o u v e r , B.C.) and  and  HPLC  10 m and  20  optimized  by  under  the  conditions.  2.8.2.1 Peroxide,  P h o s p h o r i c A c i d and  Using the m o b i l e phase  10 m  reactor  D e r i v a t i z a t i o n with  Concentrated and  (using  a c i d and  a c i d , peak h e i g h t 360  nm/425 nm  Hydrogen  HC1  the aqueous p o r t i o n of  as d e h y d r o a s c o r b i c  with phosphoric of d i g o x i n  Fluorogenic  was  hydrogen  peroxide  d e t e r m i n e d f o r 9.1  filters).  the  ng  71 2.8.2.2 Filters  E v a l u a t i o n of  with Dehydroascorbic  With a described  360  in  nm  A c i d and  excitation  s e c t i o n 2.7.3  nm)  by  i n j e c t i o n s of  2.8.3  The  mL/min. w i t h  2.9  (425,  phase  heights  440  and  460  from  13  ng  Procedure  o f t h e HPLC-PC a s s a y p r o c e d u r e final  a hexane  e x c i t a t i o n and  filters  c o m p a r i n g peak  F i n a l HPLC-PC A s s a y  5.  the mobile  digoxin.  A schematic Figure  and  w i t h a l l of the aqueous p o r t i o n  a c i d , emission  evaluated  Emission  HCl D e r i v a t i z a t i o n  filter  as d e h y d r o a s c o r b i c were  the Fluorescence  425  nm  assay  u s e d an  flow rate emission  i s shown i n  HPLC f l o w r a t e of  o f 0.5  mL/min. and  f i l t e r s w i t h the  0.4  360  nm  fluorometer.  Serum E x t r a c t i o n P r o c e d u r e  2.9.1  Protein Precipitation  B l a n k serum acetone  (3  mL  sulfate plus sulfate  (1  hydroxide)  and 0.6  mL  s a m p l e s (3  of  6 mL),  mL)  Methods were  zinc/barium  precipitated (0.6  mL  o f 0.3  10%  cupric s u l f a t e plus  f o l l o w e d by  mL  of  N barium hydroxide) 1 mL  e x t r a c t i o n (Kwong 1986a,  of  using  5%  zinc  and  cupric  12%  sodium  1986b).  72  Concentrated HC1  1 cm (3/8 in.) X 0.16 cm (1/16 i n . ) Stainless Steel Reducing Union (1 of 2).  0.16 cm (1/16 i n . ) PTFE Tubing (0.3 mm I.D.). One continuous length t o bottom o f pressure vessel. 1 cm (3/8 in.) Stainless Steel Union Tee.  Hexane  1 cm (3/8 in.) Diameter X 3.8 cm (1 1/2 i n . ) Stainless Steel Tube (1 of 3).  1 cm (3/8 in.) NPT X 1 cm (3/8 in.) Stainless Steel Tube Union (1 o f 2). 5.1 cm (2 in.) Diameter Schedule 80 150 l b . PVC Blind Flange (1 o f 2 ) . Top only d r i l l e d and tapped with 3/8 i n . NPT.  5.1 cm (2 in.) Diameter Schedule 80 150 l b . PVC Socket Weld Flange (1 o f 2). 5.1 cm (2 in.) Diameter Schedule 80 150 l b . PVC Pipe (36.8 cm long).  1.3 cm (1/2 in.) X 6.4 cm (2 1/2 i n . ) 316 Stainless Steel UNC Bolt complete with nut and two 1.3 cm (1/2 i n . ) Diameter X 0.16 cm (1/16 in.) 316 Stainless Flat Washers (1 sets). 0.32 cm (1/8 in.) Thick X 5.1 cm (2 i n . ) Diameter Full Face Hypalon Gasket (1 o f 2).  Figure  6.  Diagram  o f HPLC-PC P r e s s u r e  V e s s e l With  Fittings  73 2.9.2  C e n t r i f r e e and  Serum s a m p l e s  were f i l t e r e d  serum c e n t r i f u g e d Centriflo  (3  mL  at  extraction  1000  serum  minutes) systems  Centriflo  ng  for  an  samples  evaluated  and  the  1000  for  40  r.c.f. wash and  double  w a t e r s a m p l e s were  prepared  using  systems as d e s c r i b e d above. using  the  mL  and  isooctane  digoxin  Centriflo  C e n t r i f r e e (1  30 m i n u t e s )  B o t h serum and  of  C e n t r i f r e e and were  the  c e n t r i f u g e d at  f o l l o w e d by  9.1  by  r.c.f.  (Kwong 1 9 8 6 a ) .  spiked with  Filtration  final  the These  HPLC-PC  assay  procedure.  2.9.3  Solvent-solvent Extraction  Using (20/5) as ng  2  mL  isooctane  and  isooctane/dichloromethane  s o l v e n t w a s h , w a t e r s a m p l e s (3 mL)  o f d i g o x i n w e r e e x t r a c t e d and  assay.  Serum  serum samples an  of  (3 mL)  containing  were e x t r a c t e d  isooctane/dichloromethane  assayed using  2.9.4  t h e HPLC-PC  Final  evaluated 1.5  ng  spiked with  using  9.1  t h e HPLC-PC  of d i g o x i n and  blank  (Kwong 1986a, 1986b) f o l l o w i n g s o l v e n t wash (2 mL  o f 20/5)  and  assay.  Procedure  for Extraction  of D i g o x i n  in  Serum The  frozen  temperature just  serum prior  samples, 3  mL  digoxin  ethanol  curve  in  and  of  patient  blank (1.5  samples to  were  analysis.  serum to  samples,  was  10 ng/10 20  ML  thawed For  spiked ML). of  at  room  standard  curve  with For  10  uL  of  a l l standard  internal  standard  74 solution 3  mL  ( 1 6 0 ng) was a d d e d t o t h e s e r u m . acetone,  centrifuged  the  sample  vortexed  solution  was t r a n s f e r r e d  was  then  centrifuged  to a  washed  isooctane/dichloromethane  partially  bath f o r  20 m i n u t e s .  (20/5),  vortexed  twice  d r i e d under n i t r o g e n  with  p h a s e s were  filtered  nitrogen.  The  methanol/water  The  remaining  10  mL  (Nylon  residue  (50/50).  performed using  2.10  clean  with  of  ( d i c h l o r o m e t h a n e / p r o p a n - 1 - o l 98/2)  procedure  seconds,  tube.  This  2  mL  of  60  seconds,  5 m i n u t e s a t 1500 r . c . f . a n d t h e a q u e o u s / a c e t o n e  l a y e r was  Reno, NV,  20  5 m i n u t e s a t 1500 r . c . f . a n d t h e a q u e o u s / a c e t o n e  supernatant layer  extracted  was  A f t e r a d d i t i o n of  U.S.A.). i s given  was  A  i n Figure  layer  extraction  in  into the syringe  diagram  organic  and d r i e d  resuspended  of  was  solvent  and t h e combined  Injections  flow  a 37°C w a t e r  aqueous  66 membrane)  uL H a m i l t o n  a 100  using  100  under uL  of  HPLC-PC were  (Hamilton this  Co.,  extraction  7.  R e c o v e r y and P r e c i s i o n o f E x t r a c t i o n  The  recovery  of d i g o x i n  e x t r a c t i o n procedure 3 and  was e v a l u a t e d .  10 ng o f d i g o x i n  standard  solution  prior  to analysis.  peak  height  extracted  f r o m serum  using  the  Serum s p i k e d  were e x t r a c t e d  final  with  1.5,  a n d 10 uL o f e x t e r n a l  ( d i g i t o x i g e n i n i n e t h a n o l ) was a d d e d j u s t  ratio  R e c o v e r y was c a l c u l a t e d by c o m p a r i n g t h e (digoxin  serum s a m p l e s t o t h a t  to  external  f o r unextracted  standard) samples.  for  75  1  3 mL Serum 3 mL Acetone  I  Vortex 20 seconds. Centrifuge 5 minutes (1500 r . c . f . ) .  Wash with 2 mL isooctane/dichloromethane (20/5) Vortex 60 seconds. Centrifuge 5 minutes (1500 r . c . f . ) . Acetone/aqueous layer  Isooctane layer ^_ (discard)  I  Evaporate (20 minutes)  Add 10 mL Extraction solvent (Dichloromethane/ propan-l-ol 98/2) Mix 10 minutes. Centrifuge 5 minutes (1500 r . c . f . )  1X  Organic layer  Aqueous layer*  1  Filter. Evaporate.  Reconstitute in 0.1 mL methanol/water (50/50).  F i g u r e 7. F l o w D i a g r a m  f o r Serum E x t r a c t i o n  Procedure  76  Precision  of t h e e x t r a c t i o n procedure  repeated extraction spiked with for  of f i v e  blank  f i v e consecutive days.  Calibration  The c o e f f i c i e n t  HPLC-PC  (section  2.12  (3  mL)  standard  of v a r i a t i o n f o r  calculated.  mL)  2.4.5.1),  fluorogenic  concentration  (3  Curve  Serum s a m p l e s ethanol  samples  o f d i g o x i n a n d 20 vL o f i n t e r n a l  3 ng  w i t h i n a n d b e t w e e n d a y s was  2.11  serum  was d e t e r m i n e d by  were  spiked  with  digoxin  in  e x t r a c t e d and a n a l y z e d u s i n g t h e  assay.  The  r a t i o s were t h e n  peak  height  and  calculated.  Specificity  2.12.1 The  Steroids  steroid  samples prepared  assayed u s i n g the post-column  2.12.2 The by t h e presence  Co-Administered  i n section  2.4.5.4 were  f l u o r o g e n i c HPLC a s s a y .  Drugs  R , S - m e x i l e t i n e sample  ( s e c t i o n 2.4.5.5) was  HPLC-PC method t o e s t a b l i s h a s s a y s p e c i f i c i t y of t h i s  drug.  assayed i n the  77 2.13  Quality  Blank  Control  serum  the d i g o x i n  Procedure  samples  i n ethanol  mL)  mL  using  HPLC-PC f l u o r o g e n i c  was t h e n  were  solutions  mixing, 3 the  samples  (4  After  e x t r a c t e d and a n a l y z e d  assay.  the FPIA  13 uL o f  ( s e c t i o n 2.4.5.1).  were r e m o v e d ,  analysed with  spiked with  The r e m a i n i n g  for digoxin  at  sample  the  ACU  Laboratory.  2.14  Analysis  of D i g i t a l i z e d  Serum s a m p l e s Acute Care  Unit Hospital  received digoxin PC a n d  f r o m 25  Patient  Serum  U.B.C. H e a l t h (Vancouver,  Samples  Sciences  B.C.)  Centre  patients  who  t h e r a p e u t i c a l l y were a s s a y e d by b o t h HPLC-  FPIA methods.  The  results  f r o m t h e two  analytical  m e t h o d s were t h e n c o m p a r e d .  2.15  Analysis  of  Serum f r o m  Where H i g h DLIS L e v e l s  2.15.1  have been  Groups  Reported  Hypertension  Serum  samples  p a t i e n t s were Care U n i t  Undigitalized Patient  from  5  undigitalized  hypertensive  o b t a i n e d (U.B.C. H e a l t h S c i e n c e s C e n t r e A c u t e  H o s p i t a l , Vancouver,  B.C.)  a n d e v a l u a t e d by b o t h  t h e HPLC-PC a n d F P I A m e t h o d s .  2.15.2 Serum p a t i e n t s who  Renal  Failure  samples  from  20  were on d i a l y s i s  undigitalized  renal  failure  were o b t a i n e d f r o m t h e W i l l o w  78 D i a l y s i s Unit  (Vancouver G e n e r a l H o s p i t a l , Vancouver,  a n d a s s a y e d by b o t h t h e HPLC-PC a n d F P I A  2.15.3  failure  and  two  patient  Centre Acute  for digoxin.  Hepatic F a i l u r e  Serum s a m p l e s patients  B.C.)  from  samples  undigitalized from  were o b t a i n e d  Care U n i t  Paul's Hospital  8  one  hepatic  failure  digitalized  hepatic  f r o m U.B.C.  Hospital  Health Sciences  (Vancouver,  B.C.) a n d  St  ( V a n c o u v e r , B.C.) a n d h a v e been e v a l u a t e d by  b o t h HPLC-PC a n d F P I A  methods.  2.15.4  U m b i l i c a l Cord B l o o d Samples  A total  o f 17 m i x e d  c o r d b l o o d samples  were o b t a i n e d from C h i l d r e n ' s H o s p i t a l w e r e a s s a y e d by t h e HPLC-PC a n d F P I A  from  11 p a t i e n t s  ( V a n c o u v e r , B.C.) a n d  methods.  79 3.  3.1  RESULTS AND  Radioimmunoassay  DISCUSSIONS  of D i g o x i n  and  Its Metabolites  Samples of d i g o x i n and i t s m e t a b o l i t e s reactivity  values  molar b a s i s , Table I I . be low  using  the  magnitudes, c a l c u l a t e d  radioimmunoassay k i t s  Cross-reactivity  approximately to  of v a r y i n g  concentration  f o r the  range.  dihydrodigoxin, at  other  However,  these  concentrations  five  kits  results  using  the  therapeutic  reported  (cross-reactivity  Other  investigators  have found  c r o s s - r e a c t i v i t y with low  values  Kramer  (Malini  that t h i s  digoxin antibodies  1982)  t o as  1976, 1978; Oge 1 9 7 8 ) .  here f o r d i h y d r o d i g o x i n previously  was  h i g h as  extent of  than  1.8%).  metabolite ranging  that  has a  from  30% (Gault  The c r o s s - r e a c t i v i t y greater  than  very 1979;  observed indicated  i n the l i t e r a t u r e .  Dihydrodigoxigenin, f r a c t i o n of  i f present,  the metabolites  samples from 1968).  patients with Considerable  c r o s s - r e a c t i v i t y ) was assayed using  dihydrodigoxigenin  was  a  minor  However,  large  h a v e been o b s e r v e d i n u r i n e  increased digoxin  requirements  interference (approximately  observed  the Digi-Tab  represents  of d i g o x i n .  q u a n t i t i e s of d i h y d r o d i g o x i g e n i n  (Luchi  greater  this  indicate that  to  t h i s manufacturer  in  was f o u n d t o  range f o r d i g o x i n , i n t e r f e r e s by  a  R I A k i t b u t was t o o  within a  on  a s shown  of dihydrodigoxin  46% f o r t h e D i g i - T a b  be d e t e r m i n e d  exhibited cross-  when  kit.  t o o low  this  metabolite  The c r o s s - r e a c t i v i t y  t o be  estimated  with  22% was of the  80 other  k i t s a t these  concentrations.  a cross-reactivity d i g o x i n RIA here f o r  of l e s s  methods  (Gault  than  For d i h y d r o d i g o x i g e n i n ,  12% h a s been r e p o r t e d  1982).  d i h y d r o d i g o x i g e n i n was  The  with  i n t e r f e r e n c e found  g r e a t e r than  that  reported  in the l i t e r a t u r e .  Table I I . P e r c e n t C r o s s - R e a c t i v i t y on a M o l a r B a s i s f o r D i g o x i n M e t a b o l i t e s W i t h C o m m e r c i a l D i g o x i n RIA Kits.  Digoxin  RIA K i t s U s e d C D E 100 100 100  A 100  B 100  60  250  95  103  74  95  133  152  95  116  113  95  208  159  89  102  103  104  Digoxigenin  F 100  Digoxigenin Monodigitoxoside Digoxigenin Bisdigitoxoside Dihydrodigoxin  46  Dihydrodigoxigenin RIA K i t s U s e d : A B C D E F  For  digoxigenin,  digitoxoside, 60%  to  250%).  range i n RIA  (B)  smaller  22 D i g i - T a b RIA B i o - R I A 1-125 D i g o x i n A m e r l e x D i g o x i n RIA K i t D i g o x i n S o l i d P h a s e RIA ( B e c t o n GammaCoat D i g o x i n RIA D i g o x i n 1-125 Imusay  the  range  mono-  average c r o s s - r e a c t i v i t y These m e t a b o l i t e s  cross-reactivity kits.  digoxigenin  The o t h e r  was  exhibited  f o r the Digi-Tab four  RIA  Dickinson)  RIA  bis-  120%  (range  the  greatest  (A) and B i o -  procedures  a n d an a v e r a g e c r o s s - r e a c t i v i t y  and  showed  o f 99%  a  (range  81 74%  to  116%)  for these three  r e s u l t s agree  with previous  these metabolites kits  (Valdes The  of  reported  the  between the  kits  for  a p p e a r s t o be  necessary  the  an  intact  aglycone  was  1976;  digoxin  digoxin.  for high  (Butler  lack  of  used i n commercial Variability  in  kits  and  of  an  The  presence  the  digoxin  antibody  cross-reactivity.  However,  s u g a r s does not  greatly affect  polyclonal antibodies  1978),  extensively  are  conjugate  therefore,  l a c k of  to  specificity,  further complicated have  the  1977)  and  same  most  the  the  formed  with  an  digoxin  digoxigenin  there  Iisalo  of the  1977).  may RIA  be  is  a  initiated  due  digoxin  large  (Aronson  Inappropriate  used.  i n t e r p r e t a t i o n of  s i n c e the m e t a b o l i t e s  p o t e n c y as  i n digoxin metabolism  therapy  specificity  the  b e t w e e n t h e RIA  interact with  Digoxin  this  not  Iisalo  variability  RIA  observed.  r e s u l t s are  Gault  of  cross-react  Along with  d i g o x i n do  digoxin  emphasize  immunogenic d i g o x i n - p r o t e i n  antisera w i l l  1980;  here  observed  digitoxose  against  RIA  was  r i n g to  cross-reactivity.  moiety as  iodine-125  digoxin analysis.  metabolites  lactone  removal of  c r o s s - r e a c t i v i t y of  polyclonal antibodies  cross-reactivity  intact  These  1984).  i o d i n e - 1 2 5 RIA the  r e p o r t s on  with commercial  results  specificity  digitoxose metabolites.  1980;  of  (Aronson  interpatient Clark  1974;  changes i n p a t i e n t to  the  lack  of  82 3.2  Fluorescence  Metabolites  The  Polarization  FPIA  r e q u i r e s serum  suggested t o r e s u l t metabolites  to  Sonobe  acid  proteins to  (Skogen  each m e t a b o l i t e  1978).  although  no  digoxin from  values  with  present  is as  with  great  1.3 n m o l / L  t h e FPIA  or  greater  these  of from  assay.  t h e FPIA  than those  (91%)  The  a  cross-  for digoxigenin, bisdigitoxoside  o b s e r v e d f o r t h e s i x RIA the reported  cross-  was c o n s i d e r a b l y g r e a t e r RIA  (22%).  The  method w i t h t h e d i g o x i n than  samples.  i s therefore  and d i g o x i g e n i n  the Digi-Tab  t h e FPIA  The r e s u l t s  in  For dihydrodigoxigenin,  f o r t h e FPIA  of  samples.  metabolites  reported with  or greater  evaluated.  reactivity  i n patient  from i t s  r a n g e d f r o m 0.7 t o 4.8 n m o l / L  digoxin  problem  found  n o t be d i f f e r e n t i a t e d  was  digoxigenin monodigitoxoside  that  Therefore  were a s s a y e d by t h e F P I A .  shown i n T a b l e I I I  reactivity  of d i g o x i n and i t s  containing approximately  the FPIA  kits  been  i tis  Serum s a m p l e s  great  has  1980; S t e r n s o n  w h i c h may be p r e s e n t  significant  precipitated  (Gault  metabolites  are as  Digoxin  d i g o x i g e n i n and/or d i h y d r o d i g o x i g e n i n  that digoxin w i l l  Interference  be  1987) w h i c h  i n the transformation  anticipated  reactivity  of  i n Serum  with 5-sulfosalicylic  1977;  Immunoassay  that observed  than  cross-  metabolites  with  t h e RIA  procedure. The levels  FPIA  assay  indicated  i n the presence  of DLIS  increased  apparent  digoxin  ( B i a n c h i 1986; F r y e 1987;  83  Table  III.  E v a l u a t i o n of D i g o x i n M e t a b o l i t e s  Using  Metabolite  less  f o r Digoxin  FPIA Assay Results (nmol/L)  Actual Concentration (nmol/L)  B l a n k Serum Digoxigenin Digoxigenin Monodigitoxoside Digoxigenin Bisdigitoxoside Dihydrodigoxin Dihydrodigoxigenin  Kanan  the FPIA  Analysis  Cross-Reactivity R e p o r t e d by F P I A Manufacturer (%)  none 1.41  none 1.8  >100  1.48  4.8  >100  1.17 1.27  3.6 0.8  >100  1 .70  0.7  91  1987; S k o g e n 1987; S o l d i n 1986b; W e i n e r interference  the RIA  method  in patient  i n Serum  1 9 8 7 ) , however  f r o m D L I S was r e p o r t e d w i t h t h e F P I A  (Yatscoff  samples, the  1984).  than  For e v a l u a t i o n of d i g o x i n  F P I A may  t h e r e f o r e be b e t t e r  than  t h e R I A when D L I S i s p r e s e n t . The c l i n i c a l both  the  RIA  s i g n i f i c a n c e of and  FPIA  h i g h c r o s s - r e a c t i v i t y of  methods  with  digoxigenin  b i s d i g i t o x o s i d e and d i g o x i g e n i n m o n o d i g i t o x o s i d e s i n c e these  metabolites  retain  Digoxigenin,  dihydrodigoxin  substantially  lower  reactivity significant.  with  some p o t e n c y and  potency than the  RIA  Considering  in d i g o x i n metabolism  and  may be l o s t (Keys  dihydrodigoxigenin digoxin FPIA  so  high  methods  is  1980). have crossmore  the large i n t e r p a t i e n t v a r i a b i l i t y  (Aronson  1980) a n d t h a t f r o m 57 t o 6 0 %  84 of d i g o x i n  may be  dihydrodigoxin patient with the  (Clark  samples  low  metabolic  1974; L u c h i  potency and  potencies  as  containing high  is c l i n i c a l l y  l e v e l s of these  high c r o s s - r e a c t i v i t y are very  interference with  significant.  interference  from  Glycosides  Ultraviolet  With the  shown  Since respect  o f d i g o x i n due may  lead  to  therapy.  D e r i v a t i z a t i o n of Cardiac  w i t h HPLC-EC A n a l y s i s  3.3.1  Fujii  low w i t h  metabolites  Pre-Column 3 , 5 - D i n i t r o b e n z o y l  metabolites  exists.  Over-estimation  i t s  mainly  t h e R I A a n d F P I A methods  i n a p p r o p r i a t e changes i n p a t i e n t d i g o x i n  3.3  product,  1968), t h e p o s s i b i l i t y of  of these metabolites  to digoxin, t h e i r  to  excreted  (1983), in  Detection  3,5-DNB d e r i v a t i z a t i o n  p r o c e d u r e r e p o r t e d by  the structure of d e r i v a t i z e d digoxin  Figure  dihydrodigoxin,  8.  Trace  amounts  of  digoxigenin, dihydrodigoxigenin,  m o n o d i g i t o x o s i d e and d i g i t o x i g e n i n and  digoxigenin  formed i s digoxin,  digoxigenin  b i s d i g i t o x o s i d e as w e l l as  g i t o x i n were d e r i v a t i z e d .  The r e t e n t i o n  t i m e s o f 3,5-DNB d i g o x i n a n d i t s d e r i v a t i z e d m e t a b o l i t e s a r e shown i n by the  Table IV.  t h i s procedure derivatized  The c h r o m a t o g r a m o f d i g o x i n d e r i v a t i z e d  ( F i g u r e 9) i n d i c a t e s t h a t s m a l l a m o u n t s o f metabolites  digoxigenin monodigitoxoside digoxigenin  bisdigitoxoside  [digoxigenin  (5.96  minutes),  (9.82 m i n u t e s )  and  possibly  (17.77 m i n u t e s ) ]  are  present.  85  Table  IV.  HPLC R e t e n t i o n T i m e s o f 3,5-DNB D e r i v a t i v e s  Glycoside  Retention  time  (minutes) Digoxin  37.23  Digoxigenin  5.64  Dihydrodigoxigenin  5.63  Digoxigenin Monodigitoxoside  9.63  Digoxigenin  Bisdigitoxoside  16.90  Dihydrodigoxin 35.81 Chromatographic c o n d i t i o n s : C o l u m n : S p h e r i s o r b ODS I I 3Mm HPLC ( 4 . 6 mm x 15 c m ) ; f l o w r a t e : 1.0 mL/min.; m o b i l e p h a s e : a c e t o n i t r i l e / w a t e r (20/7); u l t r a v i o l e t detection wavelength: 254 nm.  Figure  8.  S t r u c t u r e o f 3,5-DNB D i g o x i n  86  1  i  1  r-  0  20  4 0  TIME (MIN)  Figure  9.  C h r o m a t o g r a m o f 3,5-DNB D i g o x i n  Derivatized  in Milligram  Quantities  Chromatographic c o n d i t i o n s : Column: S p h e r i s o r b ODS I I 3/xm HPLC ( 4 . 6 mm x 15 c m ) ; f l o w r a t e : 1.0 mL/min.; m o b i l e p h a s e : a c e t o n i t r i l e / w a t e r (20/7); u l t r a v i o l e t detection wavelength: 254 nm; r a n g e : 0.005; i n j e c t i o n v o l u m e : 10 uL; c h a r t s p e e d : 0.2 cm/min. Peak i d e n t i t y : 1, 3,5-DNB d i g o x i n .  87 Comparison t o  the  sample c o n f i r m s  chromatogram  that  these  obtained  peaks  are  products or contaminants of the d i g o x i n Digoxin  was  d e r i v a t i z e d on  dinitrobenzoyl chloride purified  by point  (Fujii  1983)),  (Fujii  phase and  (observed 1983)),  metabolites, (section  203 °C;  HPLC r e t e n t i o n  (20/7) m o b i l e digoxin  as d e s c r i b e d  (observed =  3,5-DNB  the  to  blank  breakdown  used. scale  with  i n section  3,5-  2.3.1 a n d  from methanol. expected =  The  203-205  °C  time with a c e t o n i t r i l e / w a t e r  ultraviolet detection  = 37.23  and t h e  due  a large  double r e c r y s t a l l i z a t i o n  melting  from  minutes; expected  order  f o r 3,5-DNB = 36 m i n u t e s  o f e l u t i o n o f 3,5-DNB  gitoxin  and  3,5-DNB  digoxin  digitoxigenin  2.3.2) c o n f i r m e d t h e i d e n t i t y o f t h e r e c r y s t a l l i z e d  3,5-DNB d i g o x i n .  3.3.2  Detection  Preliminary system  (85/15)  electrode  of  With a Single Glassy  Carbon  Electrode  r e s u l t s w i t h an a c e t o n i t r i l e / a c e t a t e b u f f e r and r e d u c t i o n  t h e 5012  using  the single glassy  screened w a l l j e t c e l l  carbon  at potentials  f r o m 0 t o -0.85 V i n d i c a t e d t h a t a p o t e n t i a l o f -0.80 V w i t h 0.1  M  acetate  Decreasing the height. M  n o t recommended  Manual). the  resolution  W i t h 0.1 limit of  gave  the  buffer concentration  Concentrations  are  4.6),  buffer  greatest  l e a d t o a l o s s i n peak  significantly f o r use  with  greater this  M sodium a c e t a t e / a c e t i c of  detection  3,5-DNB d i g o x i n  sensitivity.  0.1  d e t e c t o r (ESA a c i d b u f f e r (pH  f o r 3,5-DNB f r o m 3,5-DNB  than  digoxin  and  dihydrodigoxin  88 for  v a r i o u s m o b i l e p h a s e s i s shown i n T a b l e V.  The l i m i t o f  d e t e c t i o n was f o u n d t o be 2.2 ng o f 3,5-DNB d i g o x i n digoxin) using sensitivity,  the r e c r y s t a l l i z e d  better  sample.  r e s o l u t i o n between  3,5-DNB d i h y d r o d i g o x i n  was o b t a i n e d  With  Figure  10  shows  a  similar  3,5-DNB d i g o x i n a n d  w i t h a m o b i l e phase o f  methanol/ethanol/acetonitrile/isopropanol/buffer 22).  ( 0 . 9 8 ng  chromatogram  d e r i v a t i v e s of d i g o x i n and i t s m e t a b o l i t e s  of  (40/3/60/2/ the  3,5-DNB  using this  mobile  phase.  T a b l e V.  R e s o l u t i o n o f 3,5-DNB D i g o x i n  Dihydrodigoxin  Using  HPLC-EC w i t h V a r i o u s  a n d 3,5-DNB Mobile  M o b i l e Phase C o m p o s i t i o n Polarity Resolution MeOH EtOH ACN I PA B u f f e r o f 3,5-DNB Digoxin and 3,5-DNB Dihydrodigoxin 40 40 40 38 40 40 40 40 40 40 MeOH EtOH ACN I PA Buffer  3 3 3 3 3 3 3 3 3 3  40 40 40 40 40 40 50 60 60 60  3 3 3 3 2.75 3 3 3 2 1  15 17 18 18 18 18.4 20 22 22 22  6.08 6.16 6.19 6. 194 6.20 6.21 6.229 6.258 6.276 6.295  none none 0.86 0.80 0.70 0.67 0.60 0.50 0.75 0.70  HPLC m e t h a n o l absolute ethanol HPLC a c e t o n i t r i l e HPLC i s o p r o p a n o l 0.1 M s o d i u m a c e t a t e / a c e t i c a c i d b u f f e r  Phases L i m i t of Detection (3,5-DNB digoxin)  8.8 2.8 2.8 2.2  (pH 4.6)  Chromatographic c o n d i t i o n s : C o l u m n : S p h e r i s o r b ODS I I 3 urn HPLC ( 4 . 6 mm x 15 c m ) ; f l o w rate: 1.0 mL/min.; guard electrode: 5020 g u a r d cell electrode a t -0.85 V p r e injector; detection: 5012 s c r e e n e d wall jet c e l l recording g l a s s y c a r b o n e l e c t r o d e a t -0.80 V.  89  1  0  Figure Their  10.  5 10 TIME (MIN)  Chromatogram o f D i g o x i n  3,5-DNB D e r i v a t i v e s u s i n g  15  and i t s M e t a b o l i t e s  Electrochemical  as  Detection  Chromatographic c o n d i t i o n s : C o l u m n : S p h e r i s o r b ODS I I 3 p HPLC ( 4 . 6 mm x 15 cm); f l o w r a t e : 1.0 mL/min.; m o b i l e p h a s e : m e t h a n o l / e t h a n o l / a c e t o n i t r i l e / i s o p r o p a n o l / a c e t a t e b u f f e r (pH 4.6, 0.1 M) ( 4 0 / 3 / 6 0 / 2 / 2 2 ) ; g u a r d e l e c t r o d e : 5020 g u a r d c e l l e l e c t r o d e a t -0.85 V p r e - i n j e c t o r ; d e t e c t i o n : 5012 s c r e e n e d w a l l j e t c e l l r e c o r d i n g g l a s s y c a r b o n e l e c t r o d e a t -0.80 V. Peak identity: 1, 3,5-DNB digoxigenin; 2, 3,5-DNB digoxigenin monodigitoxoside; 3, 3,5-DNB digoxigenin bisdigitoxoside; 4, 3,5-DNB dihydrodigoxin; 5, 3,5-DNB digoxin.  90 3.3.3 To  Dual Electrode improve  electrochemical g l a s s y carbon  the  Detection stability  detection,  a 5010  and  flow c e l l ,  e l e c t r o d e s , was u s e d  the reduced  electrode  (+0.80 V ) .  Recording sensitivity (0.394  from  of  ng  p r o d u c t s were  the oxidative  0.883 ng  digoxin)  the  oxidative  baseline noise equilibrium  seen i n  2.3.2)  indicated residue  phase:  of  is  40/3/60/2/22).  alone  decreased  the  t h e r e d u c t i v e mode, a n d l o w e r e d t h e  of  milligram  quantities  HPLC a n a l y s i s  no  3,5-DNB  a l l glassware  followed  of  digoxin  described  digoxin  was  (section present.  with dimethylchlorosilane i n  by d e r i v a t i z a t i o n a n d a n a l y s i s o f 1 t o 80  d i g o x i n was  a significant  are  derivatized  the  recrystallized  comparison of  above  HPLC a n a l y s i s o f t h e  o f d i g o x i n by t h e same p r o c e d u r e  showed 3,5-DNB There  methanol/ethanol/  trace l e v e l d e r i v a t i z a t i o n of d i g o x i n  Silanization  ng s a m p l e s  3,5-DNB d i g o x i n  buffer  d i g o x i n was f o r m e d .  indicated  toluene,  o x i d i z e d a t t h e second  electrode  f o l l o w e d by  3,5-DNB  from  2.3.3)  (-0.80 V)  time f o r the system.  Derivatization (section  The  e l e c t r o d e g a v e a maximum  acetonitrile/isopropanol/acetate Recording  electrode  of r e c r y s t a l l i z e d (mobile  of  w h i c h h a s two  i n t h e r e d o x mode.  3,5-DNB d i g o x i n was r e d u c e d a t t h e f i r s t and then  sensitivity  formed  in  ( s e c t i o n 2.3.3)  the larger  loss in sensitivity  when ng  samples. samples  a s c o m p a r e d t o a n a l y s i s o f ng q u a n t i t i e s o f derivative (section  Figures  11  a n d 12.  2.3.1) a s  Figure  shown by  12 a l s o shows t h e  91 h i g h l e v e l s o f d e r i v a t i z e d m e t a b o l i t e s f o r m e d when g l a s s w a r e was  used  7.61 m i n u t e s  (3,5-DNB  a n d 3,5-DNB  5.51 m i n u t e s ) .  The  d e r i v a t i z a t i o n was (Fujii  1983).  ng  quantities  of  than p r e v i o u s l y  to the digoxin  significant.  since the  was n o t s u f f i c i e n t In  acidic  (Mousty  1986).  reduced 4- a n d  and  are  this  1986).  formed p r i o r aromatic  of  to the  reduced  In a l k a l i n e  n i t r o group (Lund  1983).  t h e HPLC-EC  serum d i g o x i n  assay  assay.  aromatic n i t r o groups a r e  in  two  successive  2-electron transfers  steps  respectively  r e d u c e d t o t h e amine  I n n e u t r a l o r b a s i c media i n t e r m e d i a t e s a r e  simultaneously reduced 1983).  was  The i n t e r m e d i a t e h y d r o x y l a m i n e f o r m e d by t h e  3,5-dinitro  individually  derivatized  1 9 8 3 ) , b u t t h e y i e l d was  4 - e l e c t r o n t r a n s f e r c a n t h e n be f u r t h e r (Mousty  digoxin  unsilanized glass,  sensitivity  aqueous s o l u t i o n s  corresponding to  indicated  Also, derivatization  for a quantitative  electrochemically  at  metabolite formation during  complete as expected ( F u j i i  not determined  monodigitoxoside  d i g o x i n and t h e d e r i v a t i z e d  to bind  a d s o r p t i o n becomes not as  digoxigenin  much g r e a t e r  m e t a b o l i t e s appear when  digoxigenin bisdigitoxoside at  amount o f  3,5-DNB  silanized  hydroxylamine groups, the t o amines i n one solution  (Mousty  1986).  n i t r o groups  i n 6-electron 12-electron selective  are  With either  t r a n s f e r s or  transfer  reduction  o f 3 , 5 - d i n i t r o a r o m a t i c compounds was  (Lund of  one  difficult  92  I  G  I  10  TIME (MIN) Figure  11.  Chromatogram o f 125 ng o f 3,5-DNB  Purified  by D o u b l e  Digoxin  Recrystallization  Chromatographic c o n d i t i o n s : C o l u m n : S p h e r i s o r b ODS I I 3 um HPLC ( 4 . 6 mm x 15 c m ) ; f l o w r a t e : 1.0 mL/min.; m o b i l e p h a s e : m e t h a n o l / e t h a n o l / a c e t o n i t r i l e / a c e t a t e b u f f e r (pH 4.6, 0.1 M) (40/3/50/3/20); guard e l e c t r o d e : 5020 guard c e l l e l e c t r o d e a t - 0 . 8 5 V p r e - i n j e c t o r ; d e t e c t i o n : 5010 a n a l y t i c a l cell with electrode 1 a t -0.80 V a n d e l e c t r o d e 2 a t + 0.80 V (recording electrode 2). Peak identity: 1 , 3,5-DNB digoxin.  93  0  10  TIME (MIN)  Figure  12.  C h r o m a t o g r a m o f 80 ng o f D i g o x i n  Derivatized  in Milligram  Chromatographic c o n d i t i o n s : Same identity: 1, 3,5-DNB d i g o x i n .  Quantities  as f o r F i g u r e  11.  Peak  94 W i t h t h e HPLC-EC s y s t e m d e v e l o p e d f o r 3,5-DNB d i g o x i n , reduction occurred acetate  buffer  i n the mixture  (pH 4.6)  of  organic  u s e d f o r t h e HPLC  Under t h e s e  conditions, i t i santicipated  groups  reduced  are  reduction of  simultaneously  one o r  system occurred  both n i t r o  was n o t  hydroxylamine formation  produced as between  1  a n d 5%  fraction  of  detector of t h e  flow  apply  i s said to g l a s s y carbon  Whether  this  HPLC-EC  possibility  a  constant  reduction, could  Typically,  i s electrolyzed.  electrolyzed  As t h e  approaches  unity,  the  The h i g h  surface  area  be c o u l o m e t r i c . e l e c t r o d e s used  here a l l o w s  them t o  A s s u m i n g a l l t h e n i t r o g r o u p s on  p e r mole  be e s t i m a t e d  and t h e r e f o r e t h e extent of u s i n g t h e peak a r e a  obtained  from i n j e c t i o n  o f a known q u a n t i t y o f 3,5-DNB d i g o x i n .  DNB  and  resolved.  3,5-DNB  Some b r e a k d o w n  during derivatization  t h e number by  of  dihydrodigoxin of digoxin  and t h e  made i t i m p r a c t i c a l t o monitoring  to  were a v a i l a b l e f o r r e d u c t i o n , t h e number o f  electrons transferred  digoxin  of  potential  the electrodes.  analyte  function coulometrically. 3,5-DNB d i g o x i n  nitro  monitor the r e s u l t a n t currents  past  of the  sample  that both  amines.  The  phase.  was a l s o n o t e v a l u a t e d .  e l e c t r o d e and analytes  mobile  groups w i t h  determined.  Amperometric d e t e c t o r s the d e t e c t i n g  to  s o l v e n t s and  to  were  i t s metabolites  assay  for  d i g o x i n u s i n g t h i s HPLC-EC a s s a y .  of e l e c t r o n s  partially  maximum s e n s i t i v i t y  d e v e l o p an  3,5-  obtained  therapeutic Therefore,  t r a n s f e r r e d and t h e p r o d u c t  r e d u c t i o n o f 3,5-DNB d i g o x i n were n o t d e t e r m i n e d .  formed  95 U n e x a m i n e d a l t e r a t i o n s t o t h e HPLC-EC a s s a y f o r 3,5-DNB digoxin that  may h a v e  l e d t o an i n c r e a s e d  sensitivity  were  modifying  the surface of the d e t e c t i n g e l e c t r o d e , i n c r e a s i n g  the  spent  time  the aqueous  in  the a n a l y t i c a l  p o r t i o n of  chemically modified to  alter  have  evaluated. the  time  spent  insufficient  produced  develop  m a t e r i a l s were  in  the  i f the time  since  DNB d i g o x i n  aqueous p o r t i o n  resolution  problems this  was  be  Improved not  i f  was  not cell,  increased  complete  cell.  by  sensitivity due  T h i s was  i n chromatographic  to not  efficiency  i n r e s o l u t i o n b e t w e e n 3,5-  dihydrodigoxin.  and p o s s i b l y  This  chromatographic  i n the  Increasing  the  of t h e m o b i l e phase would a l l o w a d d i t i o n o f  c h e l a t i n g agents be f o r m e d .  rate.  i n a decrease  a n d 3,5-DNB  related  could  the flow  the decrease  may r e s u l t  sensitivity  t h e geometry of t h e flow  reduction  spent i n  (Lund 1983)  No m o d i f i c a t i o n s  added, so  detector  with  the flow-through glassy  flow  t h e m o b i l e phase f l o w  result  attempted  Also,  Without modifying  decreasing may  f o r enhancing  may  surface modifying  Electrodes  and s e n s i t i v i t y .  n i t r o groups.  electrode  phase.  and i n c r e a s i n g  s u r f a c e s h a v e been d e v e l o p e d  suggested  r e d u c t i o n of carbon  the mobile  both s e l e c t i v i t y  been  flow c e l l  was n o t  retention  between  a more s t a b l e b a s e l i n e  evaluated time  3,5-DNB  and  would  due t o t h e i n c r e a s e i n possible  digoxin  decrease and  in  3,5-DNB  dihydrodigoxin. Other d e r i v a t i z a t i o n methods d i g o x i n have  been r e p o r t e d  f o r adding n i t r o groups t o  ( F l a s c h 1 9 8 1 ; Nachtmann  1976a,  96 1976b).  The  digoxin  carbonyl  has  been  and  compounds ( F l a s c h  chloride  other  total  ring  dinitro  acid,  aromatic  number of n i t r o  groups  i s considerably  Derivatization  with  of  less  4-nitrobenzoyl  (Nachtmann 1976a, 1976b) w o u l d a l s o r e s u l t i n f e w e r  sensitivity  added t o  d i g o x i n than  of d e t e c t i o n  groups per  m e t h o d s were other  The  lactone  dinitrobenzoic  these reactions  3,5-DNBCl.  n i t r o groups  of n i t r o  the  with  several  1981).  d i g o x i n by  than w i t h  of  derivatized  tetranitrobiphenyl  added t o  function  not  3,5-DNBCl.  i s p r o p o r t i o n a l t o the  The  total  number  m o l e c u l e o f d e r i v a t i z e d d i g o x i n so  evaluated.  electroactive  with  these  D e r i v a t i z a t i o n of d i g o x i n  a g e n t s has  not  been  reported  use  of p o l a r o g r a p h y  with  in  the  for  the  literature. Kadish  (1975) d e s c r i b e s  electrochemical The  peak  with  electrolyte. V  volt  by  determination  reductive potential  isopropanol,  +1.0  t o -0.8 using  functional  d i g o x i n was  of b o t h d i g o x i n and for digoxin  tetrabutylammonium  Carbon e l e c t r o d e s V which the  background noise. be  the  not  can  screening This  flow  a t -2.285 V and possible.  be  was  iodide  have a  -2.285 (0.01  V  electrode  to  so r e d u c t i o n  of  tenths  decrease  detector  in  M)  working range  e x t e n d e d a few  through  digitoxin.  would  as from of  a  the not  underivatized  97 3.4  P o s t - C o l u m n F l u o r o g e n i c HPLC  3.4.1  Assay  Pressure Vessel  Improvements  to  fluorescence assay  previously  methods ( D e s t a  1987; G f e l l e r  1 9 8 6 a , 1986b) a r e b a s e d on a l t e r i n g the post-column 1985)  reagents.  reported  p r e s s u r e chamber  f o r h o l d i n g t h e c o n c e n t r a t e d HC1  reduces  the  1986b) t o  chromatographic  25 m i n u t e s ] .  pressure vessel. HC1 was tube  When  d i s p l a c e d and  that  went f r o m  vessel) to  reactor.  [from  Initially  eliminated  iron rust  oxides.  which  Also, the  allowed  the a c i d .  To  h r (Kwong  vented  from  1986a,  used f o r t h e  the vessel v i a a teflon  ( i n t h e bottom of p r e s s u r e  where i t j o i n e d t h e HPLC e f f l u e n t a n d Problems occurred  Using  w i t h c o r r o s i o n of  of contaminating the  PVC f o r t h e p r e s s u r e v e s s e l  and t h e a s s o c i a t e d problems.  c o r r o d e PVC s o a b e a k e r required.  reactor, greatly  s t e e l was  t h e s t e e l a n d t h e r e was t h e p o s s i b i l i t y HC1 w i t h  1  of operating  h e x a n e was a d d e d t o t h e v e s s e l , t h e  t h e beaker  the valve  entered the  t h e post-column time  (Reh  a n d pumping h e x a n e  i n t o t h e v e s s e l by an HPLC pump t h a t i s c a p a b l e t h e r e s i s t a n c e from  1977; Kwong  t h e method o f d e l i v e r i n g  Using a  against  HPLC-PC  H C l does n o t  o r s e p a r a t e c o n t a i n e r i n s i d e was n o t PVC p r e s s u r e  f o r more c o n v e n i e n t date, degradation  v e s s e l was l i g h t c l e a n i n g and f i l l i n g of the  PVC o r  weight with  Hypalon  T M  g a s k e t m a t e r i a l ( c h l o r s u l f o n a t e d p o l y e t h y l e n e ) by t h e h e x a n e o r H C l h a s n o t been  noted.  98 3.4.2  O p t i m i z a t i o n of Post-Column Reactor  Concentrated acid  have  been  HC1,  phosphoric  used  for derivatization  fluorescent products HPLC  assays  ( F l a s c h 1981).  reported  for  of  digoxin  acid  fluorogenic  concentrated  (Desta  to  HC1  1987; G f e l l e r  solutions  (Reh 1985)  f l u o r e s c e n c e enhancement. following  c o n c e n t r a t e d HC1  chloro  sequence of  to  the  reaction  derivative  genin,  t o the  and Although  been  the  reported,  procedures  for digoxin analysis The Jork  the  of  sequence has the  HPLC-PC a s s a y  HPLC-PC  acid  derivatization.  mobile  r e p o r t e d by  m e t h a n o l / w a t e r / d i l u t e hydrogen peroxide w i t h phosphoric a d d i t i o n of  a  assay  1987; G f e l l e r 1977;  for digoxin  used  reagent  of  post-column  (1985)  14,16-  u s e d i s unknown.  previous (Desta  14,16-  phase  and  and  with  14-  14-anhydro-16-  to  structure  the  a c i d was a d d e d a s a p o s t - c o l u m n  c o n c e n t r a t e d HC1  Kwong 1 9 8 6 a ) .  of  part of the r e a c t i o n  exact  in  h y d r o l y s i s of  corresponding  f o r m e d by t h e HPLC-PC a s s a y  Dehydroascorbic along with  for digoxin  formation  dehalogenation  dianhydrogenin.  dianhydrogenin  reactions  was r e p o r t e d ( F l a s c h 1 9 8 1 ) :  glycoside  anhydrogenin,  Reh  use  Kwong 1986a) o r h y d r o g e n p e r o x i d e  The  the  and t r i c h l o r o a c e t i c  Post-column  for digoxin  d e r i v a t i z a t i o n and d e h y d r o a s c o r b i c 1977;  acid  Conditions  c o n c e n t r a t e d HC1  This allowed f o r separation of d i g o x i n  digoxigenin monodigitoxoside,  digoxigenin  d i g o x i g e n i n and  (Reh  desirable  to  for digoxin  lanatoside C resolve  digoxin  1985).  from  from  bisdigitoxoside, Since  i t is  dihydrodigoxin,  the  99  aqueous p o r t i o n partially  of a  resolve  mobile  phase  d i g o x i n f r o m d i h y d r o d i g o x i n (Kwong  was r e p l a c e d by t h e c o m b i n a t i o n s with phosphoric VI.  previously reported  of d i l u t e hydrogen  a c i d and d e h y d r o a s c o r b i c  Maximum  dehydroascorbic  peak a c i d was  height  was  acid  to  1986a)  peroxide  shown i n T a b l e  obtained  when  45  mL  used.  Table VI. E f f e c t of V a r y i n g the Aqueous P o r t i o n o f t h e M o b i l e P h a s e on Peak H e i g h t U s i n g HPLC-PC F l u o r e s c e n c e A s s a y  Aqueous P o r t i o n Of M o b i l e P h a s e  Peak  30 25 45 35 35  water/15 Hydrogen p e r o x i d e / p h o s p h o r i c water/20 Hydrogen p e r o x i d e / p h o s p h o r i c Dehydroascorbic acid Dehydroascorbic acid/10 water Dehydroascorbic a c i d /10 H y d r o g e n p e r o x i d e / p h o s p h o r i c 25 D e h y d r o a s c o r b i c a c i d /20 H y d r o g e n p e r o x i d e / p h o s p h o r i c  Height (cm)  acid acid  1.1 1.9 4.9 4.4  acid  3.5  acid  3.9  Chromatographic conditions: C o l u m n : S p h e r i s o r b ODS I I 3 Mm HPLC ( 4 . 6 mm x 15 c m ) ; m o b i l e p h a s e : m e t h a n o l / e t h a n o l / i s o propanol/aqueous (52/3/1/45); HPLC f l o w r a t e : 0.5 mL/min.; Hexane f l o w r a t e : 0.5 mL/min.; p o s t - c o l u m n r e a c t o r : 10 m i n 79 °C w a t e r b a t h ; d e t e c t i o n : f l u o r o m e t e r e q u i p p e d w i t h 360 nm e x c i t a t i o n a n d 425 nm e m i s s i o n f i l t e r s .  Both nm  360 nm  (Gfeller  (Desta  1987; Kwong 1986a; Reh 1985) a n d 350  1977) e x c i t a t i o n  r a n g i n g from  415 nm ( G f e l l e r  filters  and  derivative HPLC m o b i l e  Since  the  exact  structure  f o r m e d when d e h y d r o a s c o r b i c phase and  filters  1977) t o 480 nm ( R e h 1985) were  used w i t h p r e v i o u s methods f o r p o s t - c o l u m n digoxin.  emission  d e r i v a t i z a t i o n of of  the  digoxin  a c i d was a d d e d t o t h e  c o n c e n t r a t e d H C l was i n t r o d u c e d a s a  100 post-column  reagent  wavelength  is  unknown,  the  emission  filter  was o p t i m i z e d f o r maximum f l u o r e s c e n c e .  With  the  emission  best mobile  filters  excitation  were  filter  Maximum peak  phase, 425, then  and t h e  h e i g h t was  440  evaluated  results are  found w i t h  and  460  using  a  nm  360  nm  shown i n T a b l e V I I .  the  425  nm  emission  filter. T a b l e V I I . E f f e c t o f E m i s s i o n F i l t e r s on P e a k H e i g h t of D i g o x i n U s i n g HPLC-PC F l u o r e s c e n c e A s s a y Emission F i l t e r (nm)  Peak H e i g h t (cm) ( A v e r a g e o f n=4)  425 440 460  9.5 5.7 4.2  Chromatographic c o n d i t i o n s : Same a s f o r T a b l e V w i t h m o b i l e phase: methanol/ethanol/isopropanol/dehydroascorbic acid (52/3/1/45); d e t e c t i o n : fluorometer equipped with 360 nm excitation filter.  The p e r i s t a l t i c post-column severely  reagents  limited  pumps p r e v i o u s l y (Desta  used f o r d e l i v e r y of  1987; G f e l l e r  the choice  1977; Kwong  1986a)  of f l o w r a t e and r e a c t o r s i z e .  T h e s e l i m i t s were e f f e c t i v e l y  removed by u s i n g a s e c o n d  pump a n d  hexane d i s p l a c e m e n t  of H C l from a p r e s s u r e  The  r a t e of  flow  optimized available  HCl and  together  reactor  they  influence  for derivatization  flow rates  from  0.5  reactor, gradually The  since  post-column  shorter  time  to occur.  mL/min t o reduced  spent  both  the  i n the  HPLC  vessel.  l e n g t h were the  time  C h a n g i n g t h e hexane  1.0 mL/min,  with the  peak h e i g h t  (Table  10 m VIII).  r e a c t o r w i t h t h e 1.0 mL/min  101 flow  rate  d i d not  fluorescence.  In  without reducing m reactor  mL/min  order  was  r a t i o of 4/1). t h e 20  used, d i g o x i n  eluted  compared t o  13 m i n u t e s )  serum e l u t i n g  i n the  s e p a r a t e d from  the  of  maximum  reaction  time  r a t e b e l o w 0.5 mL/min, a 20  the  20  m  reactor,  a hexane  of  0.585  flow ng  rate  noise  early part  best  of  0.5  with  a  was a l s o  When t h e 20 m r e a c t o r  i n t h e chromatogram  allowing  the  digoxin  Reduced b a s e l i n e  m reactor. later  development  increase  with  injection  observed with  well  to  With  obtained  (direct  signal/noise  the  t h e hexane flow  was u s e d .  sensitivity  allow  was  (17 m i n u t e s  t h e endogenous peaks  from  o f t h e c h r o m a t o g r a m t o be  digoxin.  T a b l e V I I I . E f f e c t o f HC1 F l o w R a t e on Peak H e i g h t o f D i g o x i n U s i n g HPLC-PC F l u o r e s c e n c e A s s a y HC1 ( H e x a n e ) Flow Rate (mL/min)  Peak H e i g h t (cm) ( A v e r a g e o f n=4)  0.44 ( 0 . 5 0 ) 0.68 ( 0 . 7 0 ) 0.99 ( 1 . 0 0 )  9.3 7.6 5.4  Chromatographic c o n d i t i o n s : Same a s f o r T a b l e V I with d e t e c t i o n : fluorometer equipped with 360 nm e x c i t a t i o n a n d 425 nm e m i s s i o n filters.  3.4.3 The  Maximum  maximum  HPLC-PC c o n d i t i o n s  Sensitivity  sensitivity given  obtained  with  the  optimized  a b o v e was 0.5 ng d i g o x i n / i n j e c t i o n  102 directly  and  described For  other  values  serum w i t h e x t r a c t i o n a s  2.9.4.  HPLC-PC d i g o x i n  a s s a y methods,  r e p o r t e d were  0.1  here i s (1985) samples  is  still  since  dihydrodigoxin better  reporting  pump f o r  described  by Reh a n d J o r k  evaluating of  patient  digoxin  from  T h i s HPLC-PC m e t h o d h a s  than  the  to digoxin  other  (Desta  HPLC-PC  equal  methods  1987; Kwong  1986a).  the problems a s s o c i a t e d with using a p e r i s t a l t i c post-column reagent  appropriate  the for  HPLC-PC  digoxin i s  The s e n s i t i v i t y be  Kwong  here observed  significantly  improved  described  by  analysis  (Gibson  1986).  The HPLC-RIA  of  most  1980; L o o  and  analysis.  desired specificity  separation  was n o t  methods  considerably greater  methods  lack the  chromatographic  developed  o f immunoassay  HPLC-PC  methods a l o n e  may  1987;  more with by  fluorometric detectors.  The s e n s i t i v i t y  for  system  r o u t i n e use. assay  procedures f o r  d e l i v e r y (Desta  HPLC-PC  assessing different  digoxin  for  resolution  obtained.  sensitivity  make  obtained  The HPLC-PC a s s a y  preferable  partial was  1986a) a n d 10 ng  than t h a t d e s c r i b e d  sensitivity  Removal of  this  1987).  less sensitive but  1986a)  (Desta  t h e maximum  ng d i g o x i n / i n j e c t i o n  1 9 8 5 ) , 0.5 ng d i g o x i n / i n j e c t i o n (Kwong  digoxin/injection  or  d i g o x i n / 3 mL  in section  sensitivity (Reh  1.5 ng  of d i g o x i n HPLC-RIA 1977, 1981;  from  than  f o r d i g o x i n and dihydrodigoxin  methods Margot  does not d e s c r i b e  that  Immunoassay  of  digoxin  1983;  a s s a y method t h a t p a r t i a l l y  from d i h y d r o d i g o x i n  HPLC-RIA  Plum  separates  the r e s o l u t i o n  103 obtained  (Morais  1981; Wagner 1 9 8 3 ) .  assay described  here i s  serum) a r e a v a i l a b l e .  E v a l u a t i o n of D i g o x i n  Using  the  r e s o l v e d from  t h e HPLC-PC  s u p e r i o r f o r d i g o x i n a n a l y s i s when  a d e q u a t e s a m p l e v o l u m e s (3 mL  3.4.4  Therefore,  f i n a l HPLC-PC  and i t s M e t a b o l i t e s  system, d i g o x i n  is  baseline  digoxigenin, digoxigenin monodigitoxoside  digoxigenin bisdigitoxoside  a s shown  i n Figure  resolution  between d i g o x i n and d i h y d r o d i g o x i n  sufficient  for  peak h e i g h t  system s e p a r a t e s  quantitation.  13.  The  (R = 0.899) i s  Since  t h e HPLC-PC  d i g o x i n from a l l of i t s m e t a b o l i t e s ,  i s no i n t e r f e r e n c e f r o m t h e s e  metabolites  and  there  i n the a n a l y s i s of  digoxin. Resolution described  for  1980; L o o the  lack  of  digoxin  HPLC-RIA  methods  1977, 1981;  DLIS  dihydrodigoxin  1985a, 1 9 8 5 b ) ,  dihydrodigoxin  may  the p o t e n t i a l  for cross-reactivity  DLIS w i t h this to  report  t h e RIA  claiming partial does  not  with these  exists,  for  the  chromatographic  digoxin.  s e p a r a t i o n of  describe system  the  (Gibson  Along  with  of  methods.  Since  dihydrodigoxin  and  HPLC-PC method d e s c r i b e d i n  The  methods w i t h one  HPLC-RIA  d i g o x i n from  resolution  (Morais  i n t e r f e r e n c e from  HPLC-RIA  i s b e t t e r t h a n t h e HPLC-RIA  specificity  not  o f t h e RIA u s e d f o r d i g o x i n i n t h e  (Valdes occur  was  for digoxin analysis  M a r g o t 1983; P l u m 1 9 8 6 ) .  of s p e c i f i c i t y  presence of  from  method  dihydrodigoxin  obtained  1981; Wagner  respect  1983).  by  their  104  1,2 3  4  6  I i 5  15  30  TIME (MIN)  Figure  13. Its  Chromatogram o f D i g o x i n  and  M e t a b o l i t e s by HPLC-PC  Chromatographic c o n d i t i o n s : C o l u m n : S p h e r i s o r b ODS I I 3 Mm HPLC ( 4 . 6 mm x 15 c m ) ; m o b i l e p h a s e : methanol/ethanol/isopropanol/dehydroascorbic acid ( 5 2 / 3 / 1 / 4 5 ) ; HPLC flow rate: 0.4 mL/min.; H e x a n e f l o w r a t e : 0.5 mL/min.; post-column r e a c t o r : 20 m i n 79 °C w a t e r b a t h ; d e t e c t i o n : f l u o r o m e t e r e q u i p p e d w i t h 360 nm e x c i t a t i o n a n d 425 nm e m i s s i o n f i l t e r s . Peak i d e n t i t y : 1, d i g o x i g e n i n ; 2, d i h y d r o d i g o x i g e n i n ; 3, digoxigenin monodigitoxoside; 4, digoxigenin b i s d i g i t o x o s i d e ; 5, d i h y d r o d i g o x i n ; 6, d i g o x i n .  105 The for Jork  post-column  digoxin  f l u o r o g e n i c d e r i v a t i z a t i o n HPLC a s s a y s  reported  (1985)  do n o t  by  describe  dihydrodigoxin.  Baseline  dihydrodigoxin  has  sensitivity  t h i s assay  of  been  h e r e was  slightly  (Kwong 1 9 8 6 a ) . sufficient 1986b)  the  long  unreliable  from  resolution  of  digoxin  from  resolution  Analysis  of  Fluorogenic  patient  3.5.1.1 Emulsion  difficult unless  the  assay  time  from  described (R =  0.91)  of t h i s a s s a y (Kwong  (40  for  1986a,  minutes)  reagents  was  make  and it  use.  Samples  Using  The  HPLC  o f Serum E x t r a c t i o n M e t h o d  Protein Precipitation  formation  solvent  is a  of  indicate or  the  problem  e x t r a c t i o n procedure  literature  digoxin  samples  post-column  Biological  Optimization  extraction  of  the  Assay  3.5.1  reported  of  but  not adequate  HPLC-PC  chromatographic  delivery  1987)  f o r d i g o x i n was  unsuitable for routine c l i n i c a l  3.5  (Desta  the s e n s i t i v i t y  evaluating  and  digoxin  than p r e v i o u s l y r e p o r t e d  Although  for  a n d Reh  of  w i t h the  less  (1977)  separation  The  obtained  al.  reported  therapeutic monitoring. dihydrodigoxin  et  Gfeller  sample i s  aqueous found  phase  with  (Kwong 1 9 8 6 a ) .  that complete  impossible  Methods  unless  heated t o  a  and  the  previously  Reports i n the  protein precipitation i s  cold  acetone  coagulate  the  is  used  or  precipitate  1 06 (Henry  1964).  acetone i s sample  A  required  plus  concentration  for  9 volumes  e m u l s i o n formed since  final  serum o r o f 70%  i s due  increasing  t o 6 mL  (Henry  (such as  1964)  zinc plus use a c i d  may  Zinc s u l f a t e 1945)  and  methods  n o t be  were  (Henry  acetone  any  1964).  used  replacing  the  Emulsion  formation  hydroxide  e x t r a c t i o n of (Henry  for  in  this  with  the  protein  1964;  difficult to  Zinc,  d i g o x i n and than  extraction  digoxin. Somogyi  the  r e d u c e d t h e emulsi<on  extraction  method  solvent  occurred resulting  was  filtration  used.  (section  formation p r e v i o u s l y observed.  precipitation  i n the f i n a l  by u s i n g 3 mL  acetone.  1964)  procedure.  extraction  when a c e t o n e  not  precipitation,  a l s o made t h e  r e c o v e r y of  and  m e t h o d s do  in  alterations  acid)  present  still  s t e p more  removal  this extraction.  i n the  evaluated  acetone  this  solvent.  ( d i c h l o r o m e t h a n e / p r o p a n - 1 - o l 98/2), lower  in  s u l f a t e w i t h sodium h y d r o x i d e (Henry  both  The  precipitation  digoxin  copper p r e c i p i t a t i o n  be u s e d  volume  a c i d and t r i c h l o r o a c e t i c  used w i t h  with barium  copper  (1  cold  r e d u c e d t h e amount o f e m u l s i o n  degrade  b a r i u m , and so  of  63%  a r e commonly u s e d f o r t h e  picric  but would  therefore could  blood  and t h e e x t r a c t i o n  Anionic precipitants proteins  acetone)  amount  between the aqueous phase  of  whole  to incomplete protein  the  e x t r a c t i o n procedure  of a t l e a s t  e x t r a c t i o n p r o c e d u r e was  Other 3.5.1.2) Protein achieved  107 3.5.1.2 Using  the  Removal of Endogenous  HPLC-PC a s s a y d e s c r i b e d  Interference i n s e c t i o n 2.8.3  a serum  e x t r a c t i o n method p r e v i o u s l y r e p o r t e d  1986b),  serum  from  one  healthy  contained  retention  t i m e of d i g o x i n a s shown i n F i g u r e s t r u c t u r e and  observed  Hamlyn  1982;  Attempts  (Balzan  1987;  allowed  (Graves 1986). maximum s a m p l e mL)  were  sample  with  adult  DLIS  from  greater  be a f r a c t i o n o f  1983a;  required here volume  of  7.0  serum  included  micropartition  systems  This  than  removed 9 0 % o f t h e  95% r e c o v e r y  Larger  Centriflo mL)  was  also  the C e n t r i f r e e  and C e n t r i f l o s y s t e m s c o m p l e t e l y  from  samples.  the  but d i d These  i n t e r f e r i n g p e a k was r e l a t i v e l y in molecular  weight).  sample p u r i f i c a t i o n  not a l l o w results large  Ultrafiltration  s y s t e m has a  system  of serum c o n t a i n i n g t h i s  compound  of d i g o x i n  s e r u m s a m p l e s (3  Ultrafiltration  interfering  never  have  1.0 mL. so t h e  who  Vinge 1988).  The C e n t r i f r e e u l t r a f i l t r a t i o n volume o f  this  1985; D i a m a n d i s 1985;  Valdes  Graves 1986).  of  subjects  1982;  Centrifree  for  i t may  1984; C l e r i c o  eliminate  ultrafiltration  DLIS and  normal  Klingmuller  to  (Christenson  in  14.  immunoreactivity  i n t e r f e r i n g peak were n o t e v a l u a t e d ,  received digoxin  female  an i n t e r f e r i n g peak e l u t i n g c l o s e t o t h e  Although the  DLIS  1986a,  undigitalized  volunteer  the  (Kwong  and  (maximum evaluated.  i n t e r f e r i n g peak by  recovery suggest  removed t h e of d i g o x i n that  the  ( g r e a t e r t h a n 25000 Da could  s i n c e d i g o x i n was n o t  n o t be u s e d f o r  recovered.  108  1,2 3  A Kj/v^  B 15  30  TIME (MIN) Figure  14.  Chromatograms o f D i g o x i n  Metabolites  i n Ethanol  Containing  and B l a n k  Interfering  and I t s Serum  Peak  Chromatographic c o n d i t i o n s : Same a s f o r F i g u r e 13. A D i g o x i n and I t s M e t a b o l i t e s i n Ethanol B B l a n k Serum C o n t a i n i n g I n t e r f e r i n g Peak Peak i d e n t i t y : 1 d i g o x i g e n i n ; 2, d i h y d r o d i g o x i g e n i n ; 3, digoxigenin monodigitoxoside; 4, digoxigenin bisdigitoxoside; 5, dihydrodigoxin; 6, digoxin; 7, i n t e r f e r i n g peak. f  109 Removal  of  the  interfering  isooctane solvent  wash was  digoxin  after  from water  s o l v e n t wash and a l o n e was  Blank  i n t e r f e r i n g peak digoxin  and  (1.5  a  ng)  digitoxigenin)  chromatogram o b t a i n e d  was  recovered  wash.  the  f o l l o w e d by resulted  serum  8%  lower  (3 mL)  washed  mL  in l i t t l e  was  precipitation  2  with  (160  ng  mL  of  extracted.  The  (see F i g u r e  15)  removed and d i g o x i n added t o the s o l v e n t with  3  isooctane/dichloromethane  o r no e m u l s i o n  the  spiked  standard  i n t e r f e r i n g peak was  (20/5)  contained  mL)  f r o m HPLC-PC a n a l y s i s  protein  a 2  (3  and  of  t h a n when i s o o c t a n e  with  (20/5)  the  Recovery  which  internal  when d i c h l o r o m e t h a n e  Serum  modifying  isooctane/dichloromethane  and  isooctane/dichloromethane  indicated that  an  serum s a m p l e  were  by  then e v a l u a t e d .  e x t r a c t i o n was  used.  peak  mL  acetone  s o l v e n t wash  formed w i t h the  extraction  solvent. The 3  mL  final  e x t r a c t i o n procedure  acetone  for  protein  isooctane/dichloromethane  (section  precipitation  2.9.4) e m p l o y e d and  a  2  mL  ( 2 0 / 5 ) s o l v e n t wash f o r r e m o v a l  of  e n d o g e n o u s compounds t h a t i n t e r f e r e w i t h d i g o x i n a n a l y s i s .  3.5.2 The  Recovery recovery  s p i k e d w i t h 0.5, the percent  and  Precision  o f d i g o x i n was 3.0  recovery  and  10.0  determined  ng o f d i g o x i n .  f o r each c o n c e n t r a t i o n .  f o r blank Table  IX  serum shows  110  A  I  1  T  0  15  30  TIME (MIN) Figure  15.  Chromatogram o f B l a n k  with  1.5 ng D i g o x i n  Chromatographic Peak  identity:  (digitoxigenin). A  Blank  B  Spiked  Serum Serum  conditions: 1,  Serum and Serum  and I n t e r n a l  Spiked  Standard  Same a s f o r F i g u r e 13.  digoxin;  2,  internal  standard  111  Table  IX.  Recovery of Digoxin  Concentration (ng d i g o x i n / 3  mL  Recovery (%)  serum)  1.5 3.0 10.0  previously  reported  procedure  (70%)  recovery  of  digoxin  The  was  h a s been  7 8 7  observed here than w i t h a and  1986b).  (99.7%)  Number of Samples  6.4 7.2 6.1  HPLC-PC a s s a y (Kwong  e x t r a c t i o n method appropriate  recovery  Serum S a m p l e s  C.V. (%)  72.0 78.2 78.8  Better digoxin  digoxin  from Spiked  similar  extraction  Considerably using  reported  a  very  (Reh  greater different  1985) b u t i s n o t  f o r routine use.  p r e c i s i o n o f t h e HPLC-PC a s s a y was d e t e r m i n e d in  ethanol  e x t r a c t e d serum  r a t h e r than repeated  sample s i n c e  the  s a m p l e was r e q u i r e d f o r a n a l y s i s . coefficient digoxin  of  in  variation  ethanol  for  was  injections  entire  f r o m one  extracted  serum  F o r t h e HPLC-PC a s s a y t h e  1.5 a n d  4.7%  using  10 ng  (n=4)  i n j e c t i o n s of  and  3.3%  (n=l0)  respectively. The  precision  d e t e r m i n e d by samples  (3  internal  repeated  mL) s p i k e d  standard  comparison of variation  extraction  with 3  ng o f  of  procedure five  was  blank  d i g o x i n and  serum  20 ML o f  (160 ng) f o l l o w e d by HPLC-PC a n a l y s i s a n d  peak  variance  (p=0.49))  the  extraction  height  f o r w i t h i n each  A n a l y s i s of 0.84  of  ratios.  The  day  shown  is  f o r peak h e i g h t  coefficient in  of  T a b l e X.  r a t i o means ( F ( 3 g ) =  and f o r e q u a l i t y of v a r i a n c e  t  (F(  31 g  )  = 0.22  112 (p=0.88)) v e r i f i e s in  that there  the between-day A greater  reported This  for  significant difference  results.  inter-assay coefficient a similar  indicates  (Kwong 1986b)  i s no  that  e x t r a c t i o n procedure  in  comparison  the e x t r a c t i o n  described here  has  a  of v a r i a t i o n  lower  to  a  was  (Kwong  1986b).  previous  method  p r o c e d u r e and spread  (8%)  HPLC-PC  of v a l u e s  assay  in relation  to  t h e mean v a l u e s .  T a b l e X.  P r e c i s i o n of D i g o x i n  Day  Assay  C.V. (%)  3.5.3  1 2 3 4  4.6 5.7 6.6 5.3  mean  5.6  Calibration  Serum s a m p l e s d i g o x i n / m L and u s i n g the  (3 mL)  internal  calibration  correlation  ratios  curve  average values  standard,  were  shown i n  from  four  coefficient  t h i s complex  an a s s a y  ( 0 . 0 2 7 3 ) was  not  1.23).  were s p i k e d w i t h f r o m 1 t o 3.3  HPLC-PC f l u o r o g e n i c  concentration  =  Curve  of  extracted  assay. then  Figure  16  0.9876 was  was  curve  and  and the  prepared  using  determinations.  The  low  for  1981)  correlation  but  and  significantly different  Calibration  analyzed  peak h e i g h t  calculated  separate  (Falkner  The  and  ng  acceptable  the  y-intercept  from z e r o  coefficients  (t-ratio as  high  113  Figure  16.  Calibration  Curve  f o r HPLC-PC D i g o x i n  Assay  114 as  0.9999  analysis  have of  been  previously  digoxin  coefficient  observed  variability  i n the  (Kwong  1986b).  height  and  with  The  here i n d i c a t e s that  by m u t u a l d e p e n d e n c e o f  3.5.4  reported  HPLC-PC  correlation  97.5%  of the  total  weight r a t i o s i s accounted  these  for  ratios.  C o m p a r i s o n o f HPLC-PC and  FPIA Methods i n S p i k e d  Serum S a m p l e s Seven b l a n k of d i g o x i n As  shown  and  per  serum s a m p l e s s p i k e d  mL  were p r e p a r e d and  in Figure  17,  F P I A m e t h o d s was  total variability mutual dependence. the  PC  (r =  procedure  actual values The 1.25  for these  s i t e s to 1985b).  that  the  than  methods  of  the  for  the  HPLC-  better c o r r e l a t i o n with  FPIA,  gave a s l o p e  no  A possible results  displaced  binding  the  0.9895).  F P I A r e s u l t s were h i g h e r the  by  individual  l e v e l s shows t h a t  a c t u a l FPIA  weak  98%  of the  versus a c t u a l values  With  HPLC-PC  i s accounted  F P I A method ( r =  the  ng  both methods.  indicates that  a comparison  b l a n k serum was  unbound or The  a s s a y e d by  serum p r i o r t o s p i k i n g .  higher  in  two  0.9979) had  d i g o x i n added.  found i n the  This  actual digoxin  of F P I A  indicating  present  Also,  than the  plot  amount o f  the  f r o m 0 t o 3.4  c o r r e l a t i o n between the  0.9897.  of  methods w i t h  the  with  is  digoxin  that  was  DLIS  binding  (Valdes  movement o f D L I S f r o m t i g h t b i n d i n g  the  explanation  from t i g h t  sights  than  of  sites  1985a, would  115  Figure  17.  C o r r e l a t i o n B e t w e e n HPLC-PC a n d F P I A M e t h o d s For  A B C  Digoxin  HPLC-PC v e r s u s F P I A M e t h o d FPIA v e r s u s A c t u a l L e v e l s HPLC-PC v e r s u s A c t u a l L e v e l s  Analysis  116  117  ACTUAL VALUE ng/mL  118  119 make i t a v a i l a b l e f o r d e t e c t i o n 1985b) a n d  therefore  may  with  cause  t h e FPIA  higher  (Valdes  1985a,  apparent  digoxin  l e v e l s t o be o b s e r v e d .  3.5.5  Specificity  3.5.5.1  Steroids  Numerous s t e r o i d s with  digoxin  Matthewson antisera  antisera  1988). was  they  synthetic  did  procedure.  Since  not  1985;  to  the  specificity  1988;  with  digoxin  of  immunoassay  i s questionable.  Samples of  steroids  interfere  No f l u o r e s c e n t  cross-react  Longerich  cross-reactivity  these antibodies  endogenous and  reported  (Diamandis  reported,  methods u s i n g  that  have been  were e v a l u a t e d  HPLC-PC  assay  p e a k s were o b s e r v e d a f t e r  direct  these s t e r o i d s  the  injection  of  evaluated  e i t h e r do n o t e l u t e f r o m t h e HPLC s y s t e m o r do n o t  produce a  fluorescent product  therefore,  would  in  with  t o ensure  methanol.  under t h e s e  not i n t e r f e r e with  The  steroids  c o n d i t i o n s and,  t h e HPLC-PC a n a l y s i s o f  digoxin.  3.5.5.2 It  is  administered the  imperative with digoxin  analytical  digoxin  the  C o - a d m i n i s t e r e d Drugs  method.  that  be e v a l u a t e d Using a  f o l l o w i n g drugs  spironolactone,  drugs  furosemide,  which  may  be  co-  for interference  with  s i m i l a r HPLC-PC a s s a y f o r  were e v a l u a t e d disopyramide,  (Kwong  1986b):  captopril,  120 d i p y r i d a m o l , q u i n i d i n e , verapamil, propafenone, and  trimethoprim-sulfamethoxazole.  Only  furosemide  spironolactone yielded  a fluorescent  conditions  chromatographically  digoxin  used  (Kwong  mexiletine,  1986b).  was  ng  3 mL  determine  previously  if No  any  r e s p o n s e and  f l u o r e s c e n c e was  produced  f o u n d on  injection  XI.  The  f r o m 25  were  used  The was  1.13  r e s u l t s which being  within  both  0.99  + 0.73.  i n the  Serum  received HPLC-PC  in their range  mean c o n c e n t r a t i o n  digoxin and  FPIA  sensitivity  to  f o r b o t h a s s a y s were using  the  + 0 . 5 6 and t h a t f o u n d w i t h t h e  FPIA  The  i s p o s s i b l y due  included  by  methods d i f f e r  the samples  m e t h o d was  range f o r  f r o m b o t h m e t h o d s a r e shown i n T a b l e  digoxin, only  HPLC-PC a s s a y  Patient  p a t i e n t s who  assayed  results  Since these  (n=l5).  of  1973).  Serum s a m p l e s  methods.  under  be t h e maximum e x p e c t e d i n  A n a l y s i s of D i g o x i n i n D i g i t a l i z e d  therapeutically  agent,  developed  o f serum f r o m p a t i e n t s w i t h i n t h e t h e r a p e u t i c (Talbot  from  Therefore,  assay  was  the  separated  evaluated.  fluorescence  and  under  antiarrhythmic  t h e HPLC-PC  of m e x i l e t i n e which would  t h i s drug  3.6  newer  assayed using  these conditions. 7.5  The  not  m e x i l e t i n e was here to  were  procainamide  obtained  FPIA a s s a y gave h i g h e r  average  t o the m e t a b o l i t e s of  digoxin  t o t a l assay v a l u e .  W h i l e t h e r e were  individual discrepancies  between t h e a n a l y s e s of t h e  sample  by t h e  i t  assess  two  procedures,  interference with  the FPIA  was  not  possible  method t h a t w o u l d  to  l e a d t o lower  121  Table  XI. for  Patient  C o m p a r i s o n o f t h e HPLC-PC and Digoxin Analysis  in Digitalized  Digoxin Concentration  ng/mL  F P I A Methods Patients  HPLC-PC/FPIA Ratio  HPLC-PC  FPIA  1 2 3 4 5 6 7 8 9 10 1 1 12 13 14 15 16 17 18 19 20 21 22 23 24 25  1.11 1 .41 0.69 0.98 ND 1.01 0.89 0.94 1 .07 0.47 0.93 2.80 ND 0.41 0.53 0.42  0.925 0.881 0.863 0.980  0.25* 0.41 0.62 0.69 ND  1.2 1 .6 0.8 1.0 0.9 1 .0 0.9 1 .1 0.9 0.6 1 .1 3.6 0.6 0.5 0.9 0.6 0.9 0.6 0.2 0.2 0.2 0.5 0.6 0.7 0.4  mean+S.D.  0.99+0.56  0.86+0.66  0.91+0  (n=15)  (n=25)  (n=l5)  *  +  0.81  0.41 ND  q u a n t i t i e s a r e below t h e a c c e p t e d  -  1.010 0.989 0.855 1 . 189 0.783 0.845 0.778 -  -  0.589  -  0.900  -  -  1 .033 0.986 —  s i g n a l -to-noise  4:1 a n d a r e n o t i n c l u d e d i n t h e mean d a t a . ND = none  levels  detected.  found  with this  method f o r  three of  the  samples.  H o w e v e r , no i n t e r f e r e n c e h a s been shown w i t h t h e m e t a b o l i t e s of d i g o x i n  or other drugs assessed  u s i n g t h e HPLC-PC  assay.  122 Interference with this  from  endogenous s u b s t a n c e s p r e v i o u s l y  a s s a y was  e l i m i n a t e d by  altering  the  observed  extraction  method. The was  range  o f HPLC-PC  f r o m 0.589 t o 1.189.  been r e p o r t e d by Loo Nelson  (1980)  patients;  et  (0.83  1.06  +  (1981)  0.12  and  PC and  RIA p r o c e d u r e s .  0.94  for  renal  for patients  + 0.30  HPLC/immunoassay r a t i o  failure  with renal  Although v a l u e s may  HPLC and  obtained here  w i t h HPLC-PC  dialysis impairment) (1.00  d i r e c t comparison of the n o t be  j u s t i f i e d due  immunoassay methods u s e d , t h e  reported r a t i o  A n a l y s i s of  and  f r o m s e p a r a t e h o s p i t a l s ) u s i n g HPLC-  the d i f f e r e n t  3.7  (0.84 + 0 . 1 3 ) , G i b s o n  and RIA a s s a y m e t h o d s and Kwong ( 1 9 8 4 )  + 0.34  previously  samples  HPLC/immunoassay r a t i o v a l u e s h a v e al.  + 0.09  u s i n g HPLC-RIA  t o FPIA v a l u e s f o r these  and FPIA  to  ratio  methods i s s i m i l a r  to  values.  Serum f r o m  Undigitalized  Patient  Groups  Where H i g h L e v e l s o f D L I S h a v e b e e n R e p o r t e d  3.7.1  Hypertensive Patients  Serum  samples  p a t i e n t s were both the  from  obtained.  HPLC-PC f i n a l  five  undigitalized  These samples  hypertensive  were e v a l u a t e d  a s s a y p r o c e d u r e and t h e F P I A and  r e s u l t s a r e shown i n T a b l e X I I .  by the  123  Table  XII.  Comparison  Samples E v a l u a t e d  of U n d i g i t a l i z e d by  t h e HPLC-PC and  Patient  Apparent Digoxin HPLC-PC  1 2 3 4 5  ND ND ND ND ND  ND = none  The  Hypertensive  Patient  F P I A Methods  Concentration FPIA  ng/mL  ND ND 0.1 ND ND  detected.  hypertensive  patients  medications:  atenolol,  t i m o l o l and  pilocarpine.  were  spirapril,  on  the  following  enalapril,  Their  blood  pressure  that  cross-react  eltroxin, ranged  from  154/104 t o 174/110. Endogenous immunoassays  have  hypertension. fraction  compounds been  Recent  thereof,  involved  in  Wardener  1982a,  reported reports  may  hypertension  a l s o be p r e s e n t  be  in  some  suggest  the  (Buckalew  with  patients  that  natriuretic 1985;  DLIS,  with or  hormone  Cloix  1982b; G r a n t h a m 1984; W i l k i n s i n normotensive subjects  digoxin  a and  1987;  De  1985) a n d  may  ( C l o i x 1987; Hamlyn  1982). The  results  significant  presented  levels  of endogenous  w i t h the FPIA assay. interference HPLC-PC i s  from  in  Since  Table  compounds  do that  not  show  interfere  t h e HPLC-PC r e s u l t s a l s o show no  e n d o g e n o u s compounds,  as u n a f f e c t e d  XII  by e n d o g e n o u s  i t appears that the compounds  in  this  124 patient  group  as  interference with w i t h RIA  the the  FPIA  apparent  by  an  digoxin  RIA  1985;  levels  number o f p a t i e n t s may  were not  but  the  Renal F a i l u r e P a t i e n t s  Serum  samples  antacids  (Robalate,  Along with  the  A m p h o j e l , and phosphate  (iron dextran),  these,  (metoprolol,  some  propranolol,  isosorbide  by  both  renal  d i g o x i n was  not  e i t h e r method.  final  detected This  A  apparent  additional  of  greater  sample  size larger  digoxin patients  this investigation.  renal  were  on  the  failure  (multivitamins Turns), in and  following with  zinc),  B a s a l j e l (aluminum  the  gastrointestinal  vitamin  D  supplements.  other  medications  sulfisoxazole, acetylsalicylic  phenytoin,  acid,  insulin,  docusate triazolam,  hydroxyzine).  failure patient  t h e HPLC-PC  Analysis  the Willow D i a l y s i s U n i t  dinitrate,  p r e d n i s o n e , c a p t o p r i l , and The  higher  patients required  indomethacin, a l l o p u r i n o l , sodium,  Z-Bec  DLIS  l e s s than  immunoassay.  undigitalized  patients  acid,  binding  Imferon  20  d i a l y s i s at  All folic  hydroxide for tract),  from  were on  medications:  by  the c o u r s e of  3.7.2  1985).  limited  however, these  a v a i l a b l e during  were o b t a i n e d .  samples.  have r e s u l t e d i n  have r e v e a l e d  the FPIA,  p a t i e n t s whc  Nanji  method may  prohibited duplicate determination  levels with  these  FPIA method i s c o n s i d e r a b l y  m e t h o d s (Greenway  these samples  in  serum s a m p l e s w e r e  a s s a y p r o c e d u r e and i n any  of  indicates that  the  evaluated  the  FPIA  and  serum samples  by  endogenous  substances  125 were not  present  e i t h e r of as  the  specific  that  assay methods.  as  represented  in s u f f i c i e n t q u a n t i t i e s to i n t e r f e r e with  the  by  these samples.  Gibson  group  (Oldfield  interference methods.  is  1985;  be  determined  further  evaluate  the  limited  may  have  of a with  but  the  failure  DLIS a t It  not  of  with  low  p r o c e d u r e due  A n a l y s i s o f t h e s e s a m p l e s by  renal  failure patient  have p r o v i d e d  of RIA  patient  possible  l e v e l s of DLIS.  has  failure  l e v e l s too  was  1984;  degree  found  renal  this  DLIS  the  as  (DLIS)  D'Arcy  than that  FPIA.  sample s i z e .  F P I A may  to to to RIA  Evaluation  serum  samples w i t h higher  samples apparent  levels.  3.7.3  Hepatic serum  were e v a l u a t e d r e s u l t s are The  the  indicated significant  the  Ten  less  1984)  t h e s e s a m p l e s w i t h an RIA  l a r g e r number  digoxin  Yatscoff  here contained  using  1983;  1983b; K r a m e r 1 9 8 5 b ) .  possible that  samples e v a l u a t e d  Craver  F P I A a s s a y methods i n r e n a l  generally  It is  i n t h i s p a t i e n t group  E n d o g e n o u s compounds  1972;  G r a v e s 1983a,  a l s o been o b s e r v e d u s i n g patients  least  m e t h o d s h a v e been o b s e r v e d w i t h  (Bourgoignie  1980;  HPLC-PC a s s a y i s a t  FPIA f o r d i g o x i n  i n t e r f e r e - w i t h RIA  patient  The  samples from by  given  hepatic  medications: cholestyramine,  Failure Patients  both  nine  HPLC-PC  hepatic and  failure  FPIA  patients  methods.  The  in Table X I I I . failure patients  were  on  the  following  furosemide,  spironolactone,  nifedipine,  oxazepam,  co-trimoxazole,  ranitidine,  126 cephalexin,  codeine  haloperidol,  procainamide,  bisacodyl, vitamin  gluconate, tylenol,  valium,  E,  lactulose,  phosphate,  K,  thiamine, folic  p r o c h l o r p e r a z i n e were  and  XIII.  mg  heparin,  As  calcium  required,  lorazepam  Patient nine daily).  alcoholic  The  was  cirrhosis.  FPIA Methods  Patient  Apparent D i g o x i n Concentration HPLC-PC FPIA  1 2 3 4 5 6 7 8  ND ND ND ND ND ND ND ND 0.62 1 .06  ng/mL  0.6 0.2 0.6 0.5 ND ND 0.2 ND 1 .2 1.3  Digitalized = none d e t e c t e d .  Liver  Function Test  (normal  range)  Serum B i l i r u b i n T o t a l (2-23 wmole/L) A l k a l i n e P h o s p h a t a s e (30-110 U/L) A s p a r t a t e A m i n o t r a n s f e r a s e (5-47 U/L) 7 - G l u t a m y l T r a n s p e p t i d a s e (5-55 U/L) (one  Patient  and the  patients  H e p a t i c F a i l u r e P a t i e n t Samples  E v a l u a t e d by t h e HPLC-PC and  ND  cefoxitin,  chloride,  pentazocine,  administered.  as h a v i n g  tincture,  atropine,  norfloxacin.  p a t i e n t (0.125  were a l l d i a g n o s e d  opium  salbutamol, potassium acid  domperidone,  gentamycin,  pancrelipase,  dimenhydrinate,  Table  camphorated  clindamycin,  vitamin  only d i g i t a l i z e d  demerol,  values  n o r m a l - 344 n o r m a l - 610 63 - 223 122 - 393 patient at 1856)  127 Using  RIA  methods, DLIS  failure patient Nanji  serum s a m p l e s  methods i n Nanji  the FPIA  hepatic  reported  As p r e v i o u s l y d e s c r i b e d , method i s  failure  i n hepatic  ( D i P i r o 1980; G r e e n w a y 1985;  1985, 1986; Yang 1 9 8 8 ) .  interference with  l e s s than  DLIS  with  RIA  p a t i e n t s a m p l e s (Greenway 1985;  1985). For  the  consistently l e v e l s as gave t h e  undigitalized patients, blank  while  h i g h as  0.6 ng/mL.  assay and  accurately estimate hepatic  w h i l e RIA  represent  of  HPLC-PC method  digoxin levels  limited  0.6  by an  sample  For  great  the  i n these  digitalized  (1985)  In  reported  a s 1.1 ng/mL were o b s e r v e d . reported  high  levels  here  may  of DLIS.  patient,  the  f o r t h e HPLC-PC a s s a y .  FPIA  The  therefore A n a l y s i s of  results  The HPLC-PC a s s a y  of d i g o x i n  i n the  Therefore,  and endogenous  undigitalized hepatic  o r endogenous  i s unaffected compounds failure  t h e HPLC-PC r e s u l t s p r o b a b l y  true digoxin concentration  i n these  were  This difference i s  t o i n t e r f e r e n c e from m e t a b o l i t e s  the m e t a b o l i t e s  samples.  patients.  R I A method was n o t p o s s i b l e due t o t h e  compounds w i t h t h e F P I A .  were p r e s e n t  w o u l d more  size.  than those  p o s s i b l y due  whatever  0.2 ng/mL o r l e s s w i t h t h e F P I A  ng/mL  significantly  samples  higher  indicates that  digoxin  w i t h t h e FPIA d i d not a f f e c t  that the  l e v e l s of  l e v e l s as  values  these  This  f a i l u r e p a t i e n t s , N a n j i a n d Greenway  apparent d i g o x i n  FPIA  t h e HPLC-PC a s s a y was  t h e FPIA i n d i c a t e d a p p a r e n t  false p o s i t i v e values  t h e HPLC-PC  the  h a s been  samples.  by  that  patient represent  128 3.7.4  U m b i l i c a l Cord Blood  A total  o f 17  11 p a t i e n t s  mixed  u m b i l i c a l c o r d b l o o d samples  were o b t a i n e d  FPIA methods.  The  T a b l e XIV.  and a s s a y e d  from  by b o t h HPLC-PC and  r e s u l t s a r e shown i n T a b l e  XIV.  Mixed U m b i l i c a l Cord B l o o d Samples  E v a l u a t e d by t h e HPLC-PC and F P I A M e t h o d s Patient  A p p a r e n t D i g o x i n C o n c e n t r a t i o n ng/mL FPIA HPLC-PC  1  ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND ND  2 3 4 5 6 7 8 9 10 1 1 ND  0.4 0.3 0.3 0.4 0.4 ND 0.2 0.2 0.20.2 0.3 0.4 0.2 ND 0.6 0.3 0.2  = none d e t e c t e d .  The  medication  profiles  t h i s p a r t of the c l i n i c a l Digoxin-like in umbilical  immunoreactive  cord blood  involved in  available.  s u b s t a n c e s h a v e been  ( B e s c h 1976; D i a m a n d i s  1985;  Pudek 1983a,  Yatscoff  1984).  Reports  1988)  the p a t i e n t s  s t u d y were n o t  1981; Ng  Koren  for  1983b; S c h e r r m a n n  i n the l i t e r a t u r e  suggest t h a t DLIS l e v e l s  1985;  1986a, (Gonzalez  found Kelly 1986b; 1987;  i n u m b i l i c a l cord blood  129 (both venous than  and a r t e r i a l  b l o o d ) were s i g n i f i c a n t l y g r e a t e r  i n m a t e r n a l venous b l o o d . For the  17 c o r d  d i g o x i n was  present at  HPLC-PC a s s a y gave FPIA The digoxin  b l o o d s a m p l e s a s s a y e d , none  l e v e l s above the s e n s i t i v i t y  ( 0 . 5 ng/mL).  has b e e n  levels  (Greenway  1985;  than  reported the  l e v e l s are  found w i t h  significant  interference  The  RIA  1985)  to in  suggest  give the  lower  presence  of  of FPIA  DLIS  and  (0.2 ng/mL  or l e s s )  result  (up t o  may  correspond  was  not  p o s s i b l e t o e v a l u a t e t h e s e s a m p l e s by b o t h t h e  and  RIA  m e t h o d s due  S i n c e no is at FPIA  i n t e r f e r e n c e was  l e a s t as  sample s i z e  DLIS  1.1 here It FPIA  available.  f o u n d w i t h t h e HPLC-PC a s s a y , i t  u n a f f e c t e d by  i n t h i s p a t i e n t group  h i g h l e v e l s w i t h t h e RIA.  limited  RIA  that  d i g o x i n FPIA v a l u e s observed  t o the  9  apparent  t h a t e v e n when low  w i t h t h e RIA may  to r e l a t i v e l y  the  sample f o r p a t i e n t  Comparison  the FPIA  low a p p a r e n t  of  ng/mL.  N a n j i 1985).  l e v e l s reported (Nanji  ng/mL).  A l l b u t one  r e s u l t s l e s s t h a n 0.5 FPIA  indicated  e n d o g e n o u s compounds  as r e p r e s e n t e d by t h e s e  as  the  samples.  130 4.  The  results  metabolites there  is  of  obtained  dihydrodigoxin previously  partially  sugars  metabolites  prior  the FPIA  (Table  to  the  with  the  I I ) show  digoxigenin  1978; V a l d e s 1 9 8 4 ) . one  dihydrodigoxigenin  Higher RIA  to  (22%)  than  ( T a b l e I I I ) was shown a n d may be  protein precipitation Skogen  to  be  1987)  which  removed  to analysis.  dihydrodigoxigenin  of  F u r t h e r m o r e , i n t e r f e r e n c e from d i g o x i n  1984;  digitoxose  greater  and  t o the  Porter  kits  demonstrated  (46%)  with  due  (Butler  was  reported.  metabolites  s i x RIA  cross-reactivity  was e x p e c t e d  cross-reactivity  CONCLUSIONS  for cross-reactivity  digoxin with  extensive  moiety as  1984;  SUMMARY AND  reported  from The  with the  step will  (Erickson cause  digoxin  the  and i t s  cross-reactivity  of  FPIA i s s i g n i f i c a n t l y  t h a n t h a t o b s e r v e d w i t h t h e RIA m e t h o d s .  Pre-column metabolites  derivatization  with  of  digoxin  3,5-dinitrobenzoyl chloride  and  (Fujii  i t s 1983)  f o l l o w e d by HPLC a n a l y s i s w i t h e l e c t r o c h e m i c a l d e t e c t i o n was demonstrated  (section  3.3).  digoxin using  HPLC w i t h  been r e p o r t e d  i n the  To d a t e ,  a n a l y s i s o f 3,5-DNB  electrochemical detection  literature.  A maximum s e n s i t i v i t y o f  0.394 ng d i g o x i n was o b s e r v e d u s i n g d u a l e l e c t r o d e in  the  digoxin Problems  r e d o x mode. and  3,5-DNB  encountered  q u a n t i t i e s of  Partial  resolution  dihydrodigoxin with  was  derivatization  digoxin resulted  has not  in a  between also of  detection 3,5-DNB reported.  small  significant  loss  (ng) in  131 sensitivity clinical  making  this  method  impractical  for  and  dehydroascorbic acid  Hexane, d e l i v e r e d displace allowing  with  samples.  A p o s t - c o l u m n f l u o r o g e n i c HPLC a s s a y u s i n g HC1  use  the  by  a  for reliable  d e r i v a t i z a t i o n was  second  concentrated and  concentrated  HPLC  HC1  pump,  from  relatively  a  developed.  was  used  pressure  pulse-free  vessel  flow  of  acid  i n t o the  post-column r e a c t o r .  acid  was  added t o  t h e a q u e o u s p o r t i o n o f t h e HPLC m o b i l e p h a s e .  The  chromatographic metabolites  column  metabolites  of d i g o x i n may  be  digoxin  assay or  1988).  conditions. here would the  steroids  This be  by  the  patient  reported  (Diamandis  1985;  a  not  allowing  for  fluorescent  indicates that  able  cross-react  Longerich by  1988;  the  HPLC-PC  e l u t e f r o m t h e HPLC s y s t e m product  under  t h e HPLC-PC a s s a y  to q u a n t i t a t e  digoxin  i n the  these  developed  presence  of  tested. of  patient  the  HPLC-PC  and  FPIA  s a m p l e s shows h i g h e r  digoxin metabolites Evaluation  i n the  t o t a l assay  t o the  h a v e been  for  levels  i n c l u s i o n of  value.  of u n d i g i t a l i z e d p a t i e n t  DLIS l e v e l s  methods  mean d i g o x i n  F P I A a s s a y w h i c h i s p o s s i b l y due  where h i g h  its  s a m p l e s where  to  S t e r o i d samples e v a l u a t e d  produce  Comparison digitalized  h a v e been  s t u d y e i t h e r do  not  13)  from  present.  antisera  in this  do  digoxin  in d i g i t a l i z e d  Numerous s t e r o i d s  Matthewson  separated  p r i o r to d e r i v a t i z a t i o n (Figure  quantitation  with  Dehydroascorbic  to  samples from  reported  shows  groups  that  the  132 HPLC-PC a s s a y FPIA.  In the  reported  to  than the the  gives  presence of  R I A (Greenway failure  apparent d i g o x i n with  difference RIA  false positive DLIS, the  give considerably  hepatic  obtained  fewer  methods,  t h e F P I A may  and  values  1985).  umbilical  ranging Taking  FPIA assay  represent  compounds.  into  consideration  values  the  f o r t h e FPIA and  obtained  here  using  l e v e l s of i n t e r f e r i n g the  e n d o g e n o u s compounds r e s p o n s i b l e f o r t h e f a l s e p o s i t i v e  FPIA  obtained  developed here giving  here.  This  assay  samples,  f r o m 0.4 t o 0.6 ng/mL were  r e l a t i v e l y high  HPLC-PC  blood  by  values  The  positive  levels  F o r a number o f  cord  s e n s i t i v i t y t o DLIS r e p o r t e d the false  h a s been  apparent d i g o x i n  1985; N a n j i  the FPIA.  in  lower  r e s u l t s than the  was  i n d i c a t e s t h a t t h e HPLC-PC  i s unaffected  by t h e  t h e f a l s e p o s i t i v e FPIA v a l u e s  able to samples.  unaffected  s p e c i f i c a l l y quantitate  endogenous  assay  compounds  a n d t h e r e f o r e w o u l d be  digoxin i n  these  patient  133  5.  REFERENCES  A b e l R.M., L u n c h i R.L., P e s k i n G.W., Conn H.L., M i l l e r /. Pharmacol. Exp. Ther. 1 50:463 ( 1 9 6 5 ) . A d e r j a n R., B u h r H., (1979). Akera  T.  Handbook  Akera  T.,  B r o d y T.M.  Exp.  Pharmacol Trends  Rev.  R o f f m a n D.S.  Am.  T.W.  A r o n s o n J.K.  Clin.  Pharmacoki  A r o n s o n J.K.  Clin.  Sci.  .  J.  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