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The stability of calcium glucoheptonate solutions Suryanarayanan, Rajagopalan 1981

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THE STABILITY OF CALCIUM GLUCOHEPTONATE SOLUTIONS M. Pharm., Banaras Hindu Univers i ty, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) Div is ion of Pharmaceutics We accept th is thesis as conforming to the required standard c RAJAGOPALAN SURYANARAYANAN, 1981 BY RAJAGOPALAN SURYANARAYANAN IN In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department o r by h i s o r her r e p r e s e n t a t i v e s . I t i s understood t h a t c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department of The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date - i i -ABSTRACT Calcium glucoheptonate i s o f f i c i a l i n the USP XX as Calcium Gluceptate and i s described as the calcium s a l t of D-glycero-D-gulo-heptonic a c i d which i s the a epimer of glucoheptonic a c i d . I t i s f r e e l y s o l u b l e i n water. Since l a t e 1976, s o l u t i o n s of calcium gluco-heptonate have shown a tendency to p r e c i p i t a t e on storage. The problem of p r e c i p i t a t i o n can be due to one or more of the f o l l o w i n g reasons: ( i ) change from an unstable to a s t a b l e m o d i f i c a t i o n ( i i ) presence of seed c r y s t a l s ( i i i ) d i f f e r i n g proportions of a and 3 epimers i n the calcium glucoheptonate obtained from various sources Calcium glucoheptonate was found to be amorphous while the p r e c i p i -tate was c r y s t a l l i n e . Membrane f i l t r a t i o n increased the time taken f o r p r e c i p i t a t i o n to occur while a u t o c l a v i n g r e s u l t e d i n s t a b l e s o l u t i o n s . I t can be postulated that the m a j o r i t y of seed c r y s t a l s are excluded by f i l t r a t i o n which r e s u l t s i n increased s t a b i l i t y and aut o c l a v i n g destroys the seed c r y s t a l s . When calcium glucoheptonate from d i f f e r e n t sources was used f o r the preparation of s o l u t i o n s , the time f o r p r e c i p i t a t i o n v a r i e d with the commercial source (Table 1). The most s t a b l e s o l u t i o n was prepared from a s a l t described as calcium a-B glucoheptonate and the l e a s t s t a b l e was supplied as Calcium Gluceptate USP. - i i i -Table 1. S t a b i l i t y of calcium glucoheptonate solution 26.7% w/v and proportion of a epimer in various commercial samples. Source Time for p r e c i p i t a -tion (days) Proportion of a epimer (percent) Pfanstiehl a-8' stable 51.8 Givaudan 8 71 .8 Italsintex 2 72.4 Pfanstiehl USP <1 100 It therefore seemed l i k e l y that, in addition to the presence of seed c r y s t a l s , s t a b i l i t y may depend on the r e l a t i v e proportions of the a and 3 epimers. I t i s interesting that to comply with the USP spec i f i c a t i o n s , calcium glucoheptonate should be the unstable a epimer, although no procedure i s given in the monograph for the i d e n t i f i c a t i o n or assay of the a form. Hence methods have been developed to id e n t i f y and to deter-mine the proportions of the a and 6 epimers. An aqueous solution of calcium glucoheptonate was converted into a mixture of glucoheptonic acids and their corresponding y lactones by passage through a cation exchange column. The solution was freeze-dried and the acid-lactone mixture was completely converted to the y lactones using concentrated HCl. Trimethylsilyl (TMS) derivatives of the lactones were formed by reaction with trimethylsilylimidazole in pyridine. Gas chromatography on a 3% OV-225 column using a flame ionization detector gave two peaks. - i v -A c o n t r o l experiment using the TMS d e r i v a t i v e of a reference sample of the y lactone of a-D-glucoheptonic a c i d gave a s i n g l e peak having the same r e t e n t i o n time as the second peak of the sample, thereby i n d i c a t i n g that the second peak i s due to the TMS d e r i v a t i v e of t h i s y lactone. The two GC peaks gave s i m i l a r mass s p e c t r a l patterns and su b j e c t i n g the reference m a t e r i a l to the same GC-MS a n a l y s i s , confirmed that the peak 2 was the TMS d e r i v a t i v e of the y lactone of a-p-glucoheptontecacid. Since the GC peaks 1 and 2 have d i f f e r e n t r e t e n t i o n times but the same molecular ion and s i m i l a r fragmentation p a t t e r n s , the chemical s t r u c t u r e s of the two compounds must be very s i m i l a r and hence peak 1 i s a t t r i b u t e d to the TMS d e r i v a t i v e of the y lactone of 3-D-glticohe.ptontc a c i d . The r e l a t i v e proportions of the a and 6 epimers were c a l c u l a t e d using the TMS d e r i v a -t i v e of sucrose as an i n t e r n a l standard. The c o r r e l a t i o n between the s t a b i l i t y r e s u l t s and the proportions of the a and 6 epimers i n various commercial samples of calcium glucoheptonate i s shown i n Table 1. Hence i t seems that a l l the three reasons postulated e a r l i e r have some r o l e to play i n the r e c r y s t a l l i z a t i o n of calcium glucoheptonate. By the use of elemental a n a l y s i s , IR spectroscopy, DSC and GC-MS s t u d i e s , the p r e c i p i t a t e obtained from s o l u t i o n s of calcium glucoheptonate has been i d e n t i f i e d as a hydrate of calcium glucoheptonate. Attempts were made to develop s t a b l e o r a l and parenteral s o l u t i o n s c o n t a i n i n g glucoheptonate. A l l the o r a l formulations commenced p r e c i p i t a -t i o n w i t h i n s i x months. Stable parenteral formulations can be prepared by a u t o c l a v i n g the f i n a l s o l u t i o n . I f s t e r i l i z a t i o n by f i l t r a t i o n i s d e s i r e d , then the s o l u t i o n can be s t a b i l i z e d with calcium D-saccharate or calcium gluconate. - V -TABLE OF CONTENTS Pa^e ABSTRACT i i LIST OF TABLES x LIST OF FIGURES x i LIST OF SCHEMES x i i LIST OF ABBREVIATIONS x i i i ACKNOWLEDGEMENTS x i v PART A - THE STABILITY OF CALCIUM GLUCOHEPTONATE SOLUTIONS 1. INTRODUCTION 1 1.1 METHODS OF ANALYSIS OF CALCIUM GLUCOHEPTONATE 3 1.2 CHROMATOGRAPHY OF HEPTOSES AND HEPTONOLACTONES ' 3 1.2.1 Gas chromatography 3 A. Methyl d e r i v a t i v e 4 B. T r i m e t h y l s i l y l d e r i v a t i v e 8 1.2.2 Paper chromatography 9 1.3 LACTONIZATION OF ALDONIC ACID 9 1.4 MASS SPECTROMETRY OF HEPTONOLACTONES 11 1.5 PHASE TRANSITIONS 11 1.5.1 Amorphous-crystalline t r a n s i t i o n s . 12 1.5.2 Hydrate anhydrous form t r a n s i t i o n s 13 1.5.3 Polymorphic t r a n s i t i o n s 14 - v i -Page 2. EXPERIMENTAL 15 2.1 APPARATUS 15 2.2 MATERIALS 16 2.3 STABILITY STUDIES OF CALCIUM GLUCOHEPTONATE SOLUTIONS 18 2.3.1 S o l u t i o n s made from calcium glucoheptonate a f t e r heating at 120°C 18 2.3.2 Heat treatment of calcium glucoheptonate s o l u t i o n s 19 2.3.3 Membrane f i l t r a t i o n of calcium glucoheptonate s o l u t i o n s 19 2.4 CHARACTERIZATION OF CALCIUM GLUCOHEPTONATE AND THE PRECIPITATE OBTAINED FROM SOLUTIONS OF CALCIUM GLUCOHEPTONATE 19 2.4.1 Elemental a n a l y s i s 19 2.4.2 Thermal a n a l y s i s 20 2.4.3 Infrared spectra 20 A. Preparation of s o l i d samples 20 B. Preparation of s o l u t i o n s 20 2.4.4 Heat of s o l u t i o n 21 2.4.5 X-ray d i f f r a c t i o n * 21 2.4.6 E q u i l i b r i u m s o l u b i l i t y 21 2.5 DEVELOPMENT OF A GAS CHROMATOGRAPHIC METHOD FOR ESTIMATING THE PROPORTIONS OF a AND 3 EPIMERS IN CALCIUM GLUCO-HEPTONATE 21 2.5.1 S e l e c t i o n of a substance f o r p r e l i m i n a r y s t u d i e s 21 2.5.2 Preparation of methyl d e r i v a t i v e 22 A. Preparation of sodium m e t h y l s u l f i n y l m e t h i d e 22 B. D e r i v a t i v e formation 22 - v i i -Page 2.5.3 Preparation of t r i m e t h y l s i l y l d e r i v a t i v e 23 A. N - t r i m e t h y l s i l y l i m i d a z o l e 23 B. Mixture of t r i m e t h y l s i l y l i m i d a z o l e , N , 0 - b i s ( t r i m e t h y l s i l y l ) acetamide, and t r i m e t h y l c h l o r o s i l a n e 23 C. T r i m e t h y l s i l y l i m i d a z o l e i n p y r i d i n e 24 2.5.4 Preparation of the t r i m e t h y l s i l y l d e r i v a t i v e of calcium glucoheptonate 24 2.5.5 Lactone formation 27 2.5.6 Gas chromatography-mass spectrometry .30 2.5.7 S e l e c t i o n of i n t e r n a l standard 32 2.5.8 Optimization of GC c o n d i t i o n s 32 A. S e l e c t i o n of s t a t i o n a r y phase 33 B. Temperature programming 33 C. I n j e c t i o n temperature 34 D. Detector temperature 34 E. Optimization of r e a c t i o n time 34 2.6 PREPARATION OF STANDARD CURVE OF THE y-LACTONE OF a-D-GLUCOHEPTONIC ACID 36 2.7 DETERMINATION OF THE PROPORTIONS OF a AND 3 EPIMERS IN COMMERCIAL SAMPLES OF CALCIUM GLUCOHEPTONATE 36 2.8 DETERMINATION OF THE PROPORTIONS OF a AND 3 EPIMERS IN THE PRECIPITATE OBTAINED FROM COMMERCIAL SAMPLES OF CALCIUM GLUCOHEPTONATE 38 3. RESULTS AND DISCUSSION 39 3.1 STABILITY STUDIES OF CALCIUM GLUCOHEPTONATE SOLUTIONS 39 3.2 CHARACTERIZATION OF CALCIUM GLUCOHEPTONATE AND THE PRECIPITATE OBTAINED FROM SOLUTIONS OF CALCIUM GLUCOHEPTONATE 39 - v i i i -Page 3.2.1 Elemental a n a l y s i s 39 3.2.2 Thermal a n a l y s i s 39 3.2.3 IR spectra 42 A. S o l i d samples 42 B. S o l u t i o n s 47 3.2.4 Heat of s o l u t i o n 47 3.2.5 X-ray d i f f r a c t i o n s t u d i e s 47 3.2.6 E q u i l i b r i u m s o l u b i l i t y 53 A. Calcium glucoheptonate 53 B. P r e c i p i t a t e d r i e d under vacuum at room temperature to constant weight 53 C. P r e c i p i t a t e d r i e d under vacuum at 80°C f o r 46 hours 54 3.3 IDENTIFICATION OF THE PRECIPITATE 54 3.4 POSSIBLE REASONS FOR PRECIPITATION 55 3.4.1 Change from an unstable form to a s t a b l e form 55 3.4.2 Presence of seed c r y s t a l s inducing c r y s t a l l i z a t i o n 56 3.4.3 D i f f e r i n g proportions of the a and p! epimers i n the calcium glucoheptonate from various sources 57 3.4.4 Some comments about USP s p e c i f i c a t i o n s of calcium glucoheptonate 60 PART B - DEVELOPMENT OF ORAL AND PARENTERAL LIQUID DOSAGE FORMS CONTAINING CALCIUM GLUCOHEPTONATE 1. INTRODUCTION . 63 2. EXPERIMENTAL 64 2.1 MATERIALS 64 - i x -Page 2.2 DEVELOPMENT OF ORAL FORMULATIONS 65 2.2.1 Basic formula 65 2.2.2 Use of sugar 65 2.2.3 Use of s t a b i l i z i n g agent 65 2.2.4 Method of preparation of o r a l formulations 68 2.3 DEVELOPMENT OF PARENTERAL FORMULATIONS 70 2.3.1 Basic formula 70 2.3.2 Use of s t a b i l i z i n g agents 70 2.3.3 Method of preparation of parenteral formulations 70 3. RESULTS AND DISCUSSION 3.1 STABILITY STUDIES OF ORAL FORMULATIONS 71 3.2 STABILITY STUDIES OF PARENTERAL FORMULATIONS 71 3.2.1 S t e r i l i z a t i o n by au t o c l a v i n g 71 3.2.2 S t e r i l i z a t i o n by f i l t r a t i o n 71 SUMMARY 76 REFERENCES 78 - X -LIST OF TABLES Table Page I . S t a b i l i t y of calcium glucoheptonate 40 (26.7% w/v) i n aqueous s o l u t i o n II Elemental composition of calcium gluco- 41 heptonate and the p r e c i p i t a t e obtained from the s o l u t i o n of calcium glucoheptonate I I I Heats of s o l u t i o n of calcium glucoheptonate 48 samples IV X-ray studies of the p r e c i p i t a t e obtained 49 from the s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) V Determination of the proportion of the 58 a epimer i n various commercial samples of calcium glucoheptonate VI R e l a t i o n s h i p between the proportion of 59 the a epimer and the s t a b i l i t y of calcium glucoheptonate i n s o l u t i o n VII Proportion of the a epimer i n the p r e c i p i t a t e 61 obtained from s o l u t i o n s of calcium gluco-heptonate V I I I Basic formula 66 IX Modified basic formula c o n t a i n i n g sugar as 67 sweetening agent X Oral calcium syrup-formulation d e t a i l s 69 XI Oral calcium s y r u p - s t a b i l i t y s t u d i e s 72 (calcium glucoheptonate USP ( P f a n s t i e h l ) XII Oral calcium s y r u p - s t a b i l i t y s t u d i e s 73 (calcium glucoheptonate - Givaudan) X I I I Calcium i n j e c t i o n - s t a b i l i t y s tudies 74 (calcium glucoheptonate USP - P f a n s t i e h l ) - x i -LIST OF FIGURES Figure Page 1 Str u c t u r e o f calcium s a l t s of a and $-D- 2 glucoheptonic a c i d 2 S t r u c t u r e of a and B-D-glucoheptose 5 3 St r u c t u r e of y and 6-lactones of a-D- 6 glucoheptonic'acid 4 Str u c t u r e of y and 6-lactones of 3-D- 7 glucoheptonic a c i d 5 Chromatogram of the TMS d e r i v a t i v e s of the 28 y-lactones of a and 3-D-glucoheptonic a c i d 6 Chromatogram of the TMS d e r i v a t i v e of the 29 y-lactone of a-D-glucoheptonic a c i d (reference m a t e r i a l ) 7 Chromatogram of the TMS d e r i v a t i v e of the 35 Y-lactones of a and 3-D-glucoheptonic a c i d . with the TMS d e r i v a t i v e of sucrose as i n t e r n a l standard 7a Standard curve of the y-lactone of a-D- 37 glucoheptonic a c i d 8 Infrared spectra of calcium glucoheptonate 43 and the p r e c i p i t a t e (KBr d i s c ) 9 I n f r a r e d spectra of calcium glucoheptonate 45 and the p r e c i p i t a t e ( s o l u t i o n i n chloroform) - xn -LIST OF SCHEMES L a c t o n i z a t i o n of a and B-D-glucoheptonic a c i d Fragmentation scheme of the TMS d e r i v a t i v e s of the y-lactones of a-D-glucoheptonic a c i d and B-D-glucoheptonic a c i d - x i i i -LIST OF ABBREVIATIONS DSC d i f f e r e n t i a l scanning c a l o r i m e t r y GC gas chromatography GC-MS gas chromatography-mass spectrometry IR i n f r a r e d RT room temperature TMS t r i m e t h y l s i l y l USP United States Pharmacopoeia ACKNOWLEDGEMENTS I am thankful to the f o l l o w i n g people f o r t h e i r help during the course of t h i s work: Dr. A.G. M i t c h e l l , Dr. H.M. Burt, Dr. F.S. Abbott, Dr. K.M. McErlane, Dr. J.H. M c N e i l l , Mr. R. B u t t e r s , Dr. J.N.C. Whyte, Mr. R. Burton, Mr. R. Goring, P i l l a i , Marvin, S h e i l a and Andrew. The f i n a n c i a l a s s i s t a n c e provided by the Science Council of B r i t i s h Columbia and Stanley Drug Products L t d . i s g r a t e f u l l y acknowledged. Thanks to Stanley Drug Products Ltd. f o r t h e i r generous g i f t of a number of chemicals. - X V -To Amma & Babuji - xvi -PART A THE STABILITY OF CALCIUM GLUCOHEPTONATE SOLUTIONS - 1 -1. INTRODUCTION Calcium glucoheptonate i s used i n the treatment of calcium deficiency (Wade, 1977a). According to the United States Pharmacopoeia (USP ;XX,1980) i t i s anhydrous or i t contains varying amounts of water of hydration. The a epimer of calcium glucoheptonate, also known as calcium D-gTycero-D-gulo heptonate i s o f f i c i a l in the USP. However, some of the commercially available calcium glucoheptonate appears to be a mixture of the a and 3 epimers of calcium glucoheptonate.. The 3 epimer i s chemically known as calcium-D-glycero-D-ido heptonate. These two forms d i f f e r only in the i r configuration about the C-2 carbon atom (Fig. 1). Calcium glucoheptonate i s a unique product in that i t has a very high aqueous s o l u b i l i t y . Solutions of calcium glucoheptonate containing 85 percent solids have been prepared which have not c r y s t a l l i z e d on prolonged standing (Product manual, Pfanstiehl). However, since late 1976, a tendency to c r y s t a l l i z e on storage has been reported (Muller and others, 1979; Chiu and Goring, 1979; Holstein, 1980). According to Muller and others (1979) the precipitate obtained from a solution of calcium glucoheptonate i s capable of existing in two forms -Form A and Form B. The presence of seed crystals of Form A was thought to be responsible for the pr e c i p i t a t i o n . Heating calcium glucoheptonate powder to 115-120°Cwas said to destroy the Form A c r y s t a l s , but t h i s method offered no absolute guarantee of s t a b i l i t y . Heating solutions of calcium glucoheptonate to a minimum temperature of 80^ for 30 minutes, was said to destroy the Form A crystals and offer complete protection against l a t e r c r y s t a l l i z a t i o n . Chou and Goring (1979) suggest that a - 2 -H H OH H H COO -- C - C . I - c - c OH OH H OH OH CH2OH Ca rcoo 1 -OH -1 2 c -1 H H -. 1 3c -1 OH OH - \ -1 H H - 5c -1 OH H -1 \ -1 OH I 7 CH2OH Ca calcium a-D-glucoheptonate calcium 3-D-glucoheptonate calcium D-glycero-D-gulo-heptonate calcium D-glycero-D-ido-heptonate F i g . 1. Stru c t u r e of calcium s a l t s of a and g-D-glucoheptonic a c i d - 3 -change i n the manufacturing process of calcium glucoheptonate could have introduced or removed i m p u r i t i e s which i n i t i a t e the c r y s t a l l i z a t i o n process. They heated calcium glucoheptonate at 80°Cfor 30 minutes and used i t to prepare formulations but c r y s t a l l i z a t i o n s t i l l occurred on storage. According to one manufacturer ( H o l s t e i n , 1980), the manufacture.-, of calcium glucoheptonate i n the amorphous form has been impossible i n recent years. He a t t r i b u t e d the problem of c r y s t a l l i z a t i o n to the presence of seed c r y s t a l s or to something e l s e i n i t i a t i n g c r y s t a l l i z a t i o n . H o l s t e i n suggests that i f the product i s prepared hot or autoclaved a f t e r packaging, there i s no c r y s t a l l i z a t i o n problem. 1.1 METHODS OF ANALYSIS OF CALCIUM GLUCOHEPTONATE The pharmacopoeia! (USP XX, 1980) assay method f o r calcium gluco-heptonate c o n s i s t s of the complexometric e s t i m a t i o n of calcium with e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d . The percentage p u r i t y of calcium glucoheptonate i s determined on the basis of the amount of calcium present i n the m a t e r i a l . Thus, t h i s assay method i s i n s e n s i t i v e to the sugar p o r t i o n of the molecule. A conductometric method ( N i k o l i c and o t h e r s , 1973) of assay of calcium glucoheptonate has al s o been reported. 1.2 CHROMATOGRAPHY OF HEPTOSES AND HEPT.0N0LACT0NES 1.2.1 Gas Chromatography (GC) M u l l e r and others (1979) reported a GC method f o r the determination - 4 -of the proportions of the a and 6 epimers i n calcium glucoheptonate as well as i n the p r e c i p i t a t e obtained on storage of the s o l u t i o n s . T r i m e t h y l -s i l y l (TMS) d e r i v a t i v e s were prepared and the two forms separated on a 5% methylphenyl s i l i c o n e (OV-17) column, Unf o r t u n a t e l y , d e t a i l s of the experimental c o n d i t i o n s were not presented. I t was not c l e a r whether the TMS d e r i v a t i v e of calcium glucoheptonate was prepared or whether the d e r i v a t i z a t i o n took place a f t e r conversion of calcium glucoheptonate to glucoheptonic a c i d by passage through ion-exchange r e s i n . The gas chromatography of a n o n - v o l a t i l e compound l i k e calcium glucoheptonate even a f t e r d e r i v a t i z a t i o n would be u n l i k e l y . On the other hand, i f calcium glucoheptonate was converted to glucoheptonic a c i d , then the formation of the 1,4 and 1,5 lactones of both a and 3-D-glucoheptonic a c i d s i s a p o s s i b i l i t y about which the authors make no mention. There are no other published reports on the gas chromatography of calcium glucohepto-nate. However, gas chromatography of D-glycero-D-gulo-heptose as well as the lactones of a-D-glucoheptonic a c i d . and B-D-glucoheptonic a c i d have been reported ( s t r u c t u r e s i n F i g s . 2-4) (see below). A. Methyl d e r i v a t i v e Whyte (1973) studied the chromatographic m o b i l i t y of a number of permethylated a l d i t o l s and aldonates on s t a t i o n a r y phases of varying p o l a r i t i e s . Sodium glucoheptonate was a l s o one of the compounds studie d . The best separation of the aldonates was achieved on t r i f l u o r o p r o p y l -methyl s i l i c o n e (QF-1) phase. - 5 -CHO I CHO I H - C - OH OH 1 - C - H H 1 - C - OH H 1 - C - OH • OH 1 - C - H OH 1 - C - H H I - C - OH H - C - OH H | - C - OH H - C - OH | CH2OH CH2OH D-glycero-D-gulo-heptose D-glycero-D-ido-heptose F i g . 2. Structure of a and B-D-glucoheptose - 6 -| C = 0 I H - C - OH 0 I H - C - OH I I c _ H I H - C - OH I H - C - OH I CHo0H 1,4-lactone of a-D-glucoheptonic a c i d (y-lactone of a-D-glucoheptonic acid) 0 = C 1 I H - C - OH I H - C - OH 0 I OH - C - H I H - C 1 I H - C - OH I CH20H 1,5-lactone of a-D-glucoheptonic a c i d (6-lactone of a-D-glucoheptonic acid) > F i g . 3. St r u c t u r e of y and 5-lactones of g-D-glucoheptonic a c i d . - 7 -C = 0 I OH - C - H I H - C - OH I - C - H H - C - OH I H - C - OH CH 2OH 0 = C I OH I - c 1 - H H 1 - c 1 - OH OH 1 - c 1 - H H 1 - c 1 H 1 - c - OH CH20H 1,4-lactone of 3-D-glucoheptonic a c i d ( y-lactone of 8-D-glucoheptonic acid) 1,5-lactone of 3-D-glucoheptonic a c i d (6-lactone of 8-D-glucoheptonic acid) F i g . 4. S t r u c t u r e of y and 5-lactones of 3-D-glucoheptonic a c i d . - 8 -B. T r i m e t h y l s i l y l (TMS) d e r i v a t i v e The separation and e s t i m a t i o n of carbohydrates by gas chromatography of TMS d e r i v a t i v e has been described by Sweeley and others (1963). The d e r i v i t i z a t i o n was achieved by using hexamethyldisilazane and t r i m e t h y l -c h l o r o s i l a n e . Using a methylphenyl s i l i c o n e (SE-52) column, the two anomers of D-glycero-D-gulo-Heptose were separated. A mixture of twelve a l d o n i c lactones and acids i n c l u d i n g D-glycero-D-gulo-heptono-y-lactone were separated on a methyl s i l i c o n e (SE-30) open tubular glass c a p i l l a r y column by Szafranek and others (1974). They determined the c y c l i c to l i n e a r s t r u c t u r a l r a t i o s of a number of lactones and i n case of D-glycero-D-gulo-heptono-y-lactone i t was found to be 97:3. Perry and others (1969) prepared a l l s i x t e e n p o s s i b l e heptono-1,4-lactones by applying the K i l i a n i - F i s c h e r cyanohydrin synthesis to a l l e i g h t of the p o s s i b l e D-aldohexoses. Each aldohexose gave r i s e to the expected two epimeric heptonic a c i d s which a f t e r 1 a c t o n i z a t i o n and t r i m e t h y l s i l y l a t i o n , were separated by gas chromatography. The complete separation of a l l s i x t e e n heptonolactones could not be achieved on a s i n g l e phase but a neopentyl-g l y c o l sebacate p o l y e s t e r l i q u i d phase column appeared to give the most s a t i s f a c t o r y s e p aration. Petersson and others (1967a) studied the separation of various aldono-1,4-lactones i n a number of s t a t i o n a r y phases and found the most s a t i s f a c t o r y separation i n a QF-1 column. Their s t u d i e s i n d i c a t e that i n a SE-52 column, D-glycero-D-guloheptono-lactone and D-glycero-a-mannoheptonolactone have i d e n t i c a l r e t e n t i o n times. Morrison and Perry (1966) o x i d i s e d a number of aldoses to a l d o n i c acids which a f t e r conversion to t h e i r 1,4-lactones were t r i m e t h y l s i l y l a t e d and analyzed by GC. Two s t a t i o n a r y phases were i n v e s t i g a t e d , ( i ) 10% - 9 -neopentylglycol sebacate p o l y e s t e r and ( i i ) polyethylene g l y c o l (Carbowax 20 M) and both phases were found to be only p a r t i a l l y s a t i s f a c t o r y f o r separation of the l a c t o n e s . 1.2.2 Paper chromatography A number of epimeric heptonolactones have been separated by paper chromatography using a v a r i e t y of solvent systems (Kjolberg and V e i l an, 1966). The best separation of the epimeric mixture of D-glycero-D-gulb heptono-y-lactone and D-glycero-D-ido-heptono-y-lactone was achieved i n a e t h y l a c e t a t e - a c e t i c a c i d - f o r m i c acid-water (18:3:1:4) system. However, as t h i s system was a very poor s o l v e n t , the preparative chromatography was s u c c e s s f u l l y c a r r i e d out using methyl e t h y l ketone-ethanol-water (5:2.1) as eluent. 1.3 LACT0NIZATI0N OF ALDONIC ACID An a l d o n i c a c i d i n s o l u t i o n e s t a b l i s h e s e q u i l i b r i u m with i t s 1,4-and 1,5- lactones ( I s b e l l and Frush, 1963). The e q u i l i b r i u m proportions of the c o n s t i t u e n t s vary with temperature, c o n c e n t r a t i o n , s o l v e n t , and the c h a r a c t e r i s t i c s of the p a r t i c u l a r a l d o n i c a c i d . Lactone formation i s promoted by a c i d i c c a t a l y s t s and by dehydration with a s u i t a b l e s o l v e n t . 1,5-Lactones are u s u a l l y formed more r a p i d l y than 1,4-lactones. However, a mixture of a c i d and lactone can be converted to the 1,4-lactone by d i s s o l v i n g i t i n a s u i t a b l e solvent ( f o r example, g l a c i a l a c e t i c a c i d -dioxane) c o n t a i n i n g a t r a c e of h y d r o c h l o r i c a c i d and concentrating the s o l u t i o n under reduced pressure. A f t e r repeating the process several times the s o l u t i o n i s nucleated with seed c r y s t a l s of the 1,4-lactone. - 10 -Morrison and Perry (1966) o x i d i z e d a number of aldoses to a l d o n i c aci d s and then converted them to t h e i r 1,4-lactones by the f o l l o w i n g procedure. The s o l u t i o n s were concentrated to dryness by d i s t i l l a t i o n under reduced pressure below 40°C. The residues were d i s s o l v e d i n 2N HCl and the s o l u t i o n s were d i s t i l l e d under reduced pressure. At the end of the d i s t i l l a t i o n the f l a s k s were immersed f o r two minutes i n a b o i l i n g water bath w h i l s t the l a s t t races of v o l a t i l e m a t e r i a l s were removed under vacuum. Kjolberg and V e i l an (1966) report the successful formation of 1,4-lactones without using any mineral a c i d . They prepared D-gluco-heptono-y-lactone from D-glucose by heating i t i n a b o i l i n g water-bath f o r several hours with sodium cyanide. Sodium ions were removed with a c a t i o n exchange r e s i n and the s o l u t i o n was concentrated to a small volume under vacuum. The l a c t o n i z a t i o n was achieved by heating the syrup to 100°C f o r 27 hours with mechanical s t i r r i n g . Perry and Hulyalkar (1965) converted a l d o n i c acids to the corresponding 1,4-lactones by treatment with concentrated HCl and evaporation to dryness. The f i n a l product was kept f o r 5 minutes at 100°C i n vacuum. Petersson and others (1967a) s u c c e s s f u l l y used Perry and Hulyalkar's method f o r the l a c t o n i z a t i o n of a number of a l d o n i c a c i d s . Perry and others (1969) prepared heptono 1,4-lactones by concentrating the heptonic a c i d - l a c t o n e mixture i n s o l u t i o n t o near dryness, t r e a t i n g i t w i t h few drops of 2N HCl and then reconcentrating to dryness under reduced pressure, below 60°C. - n -1.4 MASS SPECTROMETRY OF HEPTONOLACTONES The TMS d e r i v a t i v e s of some t e t r a n o , pentono, hexono and heptono-lactones were subjected to mass spectrometric s t u d i e s by Petersson and others (1967b). A weak molecular ion peak (M) was recorded f o r a-D-Glucoheptonic a c i d y-lactone at m/e 568. The ion M-15 obtained when one methyl group i s s p l i t o f f , was recorded at m/e 553. The upper part of the mass spectrum a l s o i n d i c a t e d the occurrence of M-43 (m/e 525) and M-105 (m/e 463) fragments. The base peak corresponding to the ( C H ^ S i - i o n occurred at m/e 73 f o r a l l l a c t o n e s . Ions of m/e 217, 204, 189 and 147 were a l s o recorded f o r a l l the lactones i n v e s t i g a t e d . 1.5 PHASE TRANSITIONS The r e c r y s t a l l i z a t i o n of calcium glucoheptonate may be due to a phase t r a n s i t i o n . Phase t r a n s i t i o n s from an unstable or metastable s o l i d s t a t e to a s t a b l e s t a t e are u s u a l l y of the f o l l o w i n g types: ( i ) change from an amorphous to a c r y s t a l l i n e form ( i i ) change from an anhydrous to a hydrated or solvated form ( i i i ) change from a metastable polymorphic m o d i f i c a t i o n to a s t a b l e form. One of the f i r s t steps i n formulating a s o l u t i o n i s to determine the s o l u b i l i t y of the drug i n the v e h i c l e ( H a l e b l i a n and McCrone, 1969). I f the s o l u b i l i t y i s determined using a metastable form of the drug and the c oncentration of the drug i n the formula t i o n exceeds the e q u i l i b r i u m s o l u b i l i t y of the s t a b l e form, a thermodynamically unstable preparation - 12 -r e s u l t s . S o l u t i o n s t h a t are supersaturated with respect to the s t a b l e form of the drug may remain i n t h i s s t a t e f o r a long period of time. Chance n u c l e a t i o n of the s t a b l e form, however, can r e s u l t i n c r y s t a l l i z a t i o n u n t i l e q u i l i b r i u m i s reached with the s t a b l e form. C e r t a i n phase t r a n s i -t i o n s of pharmaceutical i n t e r e s t . which have been shown to r e s u l t i n r e c r y s t a l l i z a t i o n or changes i n s o l u b i l i t y and d i s s o l u t i o n rate w i l l be b r i e f l y reviewed. 1.5.1 Amorphous-crystalline t r a n s i t i o n s Mullrhs and Macek (1960) i d e n t i f i e d two forms of novobiocin, one of which was c r y s t a l l i n e and the other amorphous. When excess of s o l i d (< 10 u s i z e ) was shaken i n 0.1 M HC1 at 25°C., the amorphous form was at l e a s t 10 times more s o l u b l e than the c r y s t a l l i n e form. The d i f f e r e n c e i n s o l u b i l i t y was found to favor the absorption of the amorphous s o l i d from the g a s t r o i n t e s t i n a l t r a c t . Unless s p e c i a l precautions are taken to maintain the s o l i d i n suspension i n the amorphous s t a t e by the a d d i t i o n of m a t e r i a l s to suppress c r y s t a l l i z a t i o n , amorphous novobiocin slowly converts to a c r y s t a l l i n e form. The formulation becomes l e s s and l e s s absorbable and f i n a l l y loses i t s therapeutic e f f e c t . Florence and S a l o l e (1975) showed th a t comminution of c r y s t a l l i n e d i g o x i n r e s u l t e d i n the appearance of an amorphous phase. Using d i g o x i n from d i f f e r e n t sources they observed increases i n e q u i l i b r i u m s o l u b i l i -t i e s between 7 and 118 percent due to the conversion from the c r y s t a l l i n e to the amorphous phase. Black and Lovering (1978) attempted to determine the d i s s o l u t i o n r a t e and apparent e q u i l i b r i u m s o l u b i l i t y of d i g o x i n samples d i f f e r i n g i n degree of crystall.tnf.ty,hoping to r e l a t e these p r o p e r t i e s , but found instead t h a t r a p i d r e c r y s t a l l i z a t i o n took place. - 13 -Chiou and Kyle (1979) attempted to determine the e f f e c t of t r i t u r a t i o n on the e q u i l i b r i u m s o l u b i l i t y of d i g o x i n but i n some samples the e q u i l i b r i u m s o l u b i l i t y was not a f f e c t e d . From dynamic s o l u b i l i t y s tudies they concluded that the absence of s o l u b i l i t y enhancement was due to conversion of the higher energy amorphous form to the more s t a b l e , lower energy c r y s t a l l i n e form during the experimental period. 1.5.2 Hydrate-anhydrous form t r a n s i t i o n s H a l e b l i a n and others (1972a) studied the i n t e r c o n v e r s i o n of seven s o l i d phases of f l u p r e d n i s o l o n e which included one tert-butylamine d i s o l v a t e , two monohydrates ( a and 6 ), three anhydrous (Forms I , II and I I I ) and one amorphous phase. A l l c r y s t a l l i n e phases were converted to the a -monohydrate upon suspension i n water. When the i n v i t r o d i s s o l u t i o n rates were compared ( H a l e b l i a n and ot h e r s , 1972b), the a-monohydrate was found to have the lowest ra t e of d i s s o l u t i o n followed by the B-monohydrate. Their s t u d i e s suggest that the a-monohydrate i s thermodynamically the most s t a b l e form. Ravin and others (1970) compared the s o l u b i l i t y and d i s s o l u t i o n r a t e of an anhydrous (Form I) and a monohydrate .(Form I*) form of an experimental a n t i h y p e r t e n s i v e . The anhydrous Form I d i s s o l v e d much f a s t e r than the corresponding hydrated Form I * . Moreover, i n aqueous suspension Form I r e a d i l y forms the hydrate (Form I * ) . Moustafa and others (1974) prepared two polymorphs, two hydrates, two s o l v a t e s and an amorphous form of s u c c i n y l s u l f a t h i a z o l e . On suspension i n water, transformation to the dihydrate Form I I occurred i n a l l i nstances. In a continued study, the same workers (Moustafa and othe r s , 1975) examined the e f f e c t of various a d d i t i v e s on the rate of transformation of the - 14 -metastable anhydrous Form I to the water-stable Form II i n aqueous suspen-si o n s . I t was concluded that the f o r m u l a t i o n of p h y s i c a l l y s t a b l e suspen-sions of s u c c i n y l s u l f a t h i a z o l e would best be achieved using water-stable Form II or a l t e r n a t i v e l y , i n c l u d i n g an e f f i c i e n t transformation retardant l i k e methyl c e l l u l o s e with Form I. 1.5.3 Polymorphic t r a n s i t i o n s Aguiar and Zelmer (1969) compared the e q u i l i b r i u m s o l u b i l i t i e s of two polymorphic forms of chloramphenicol palmita'te (Forms A and B) and found that Form B was four times more s o l u b l e than Form A. The increased e q u i l i b r i u m s o l u b i l i t y of t h i s form was predominantly a t t r i b u t e d to the-higher f r e e energy content of t h i s form. Clements and P o p l i (1973) compared the maximum concentration a t t a i n e d i n s o l u t i o n (not e q u i l i b r i u m s o l u b i l i t y ) of two forms of meprobamate. The unstable form (Form I I ) was found to be twice as s o l u b l e as the s t a b l e form (Form I ) . However, a f t e r a c e r t a i n period of time the concentration of Form II began to decrease, reaching an e q u i l i b r i u m value corresponding to the s o l u b i l i t y of Form I. Matsuda and others (1980) obtained four polymorphs of phenylbutazone by a spray drying method. The s o l u b i l i t y of one of the metastable forms was found to be 1.5 times higher than that of the s t a b l e form. - 15 -2. EXPERIMENTAL 2.1 APPARATUS Autoclave, AMSCO general purpose, American s t e r i l i z e r . Atomic absorption spectrophotometer, model AA-5, Varian Techtron. Cahn e l e c t r o b a l a n c e , Gram, Ventron Coporation. Constant temperature bath, Magni W h i r l , Blue M E l e c t r i c Company. D i f f e r e n t i a l scanning c a l o r i m e t e r with e f f l u e n t gas a n a l y z e r , DSC-1B, Perkin-Elmer. Freezer (-76°C), UC 105, K e l v i n a t o r . Freeze-drying u n i t , V i r t i s company. Gas chromatograph with a flame i o n i z a t i o n d e t e c t o r , model 5830 A, Hewlett Packard and a GC t e r m i n a l , model 18850 A, Hewlett Packard. Gas chromatographic s y r i n g e , Hamilton. I n f r a r e d spectrophotometer, Unicam SP 1000, Pye Unicam. Mass spectrometer, MAT 111, coupled to a gas chromatograph, model 5700 A, Hewlett Packard and a computer, model 620/L, V a r i a n . pH meter, model 26, Radiometer. S t e r i f i l f i l t r a t i o n system, M i l l i p o r e . Vacuum pump, Vac Torr S 35, General E l e c t r i c . V i a l s ( t e f l o n - s i l i c o n e screw-capped), P i e r c e . X-ray d i f f r a c t o m e t e r , wide angle, P h i l i p s . - 16 -2.2. MATERIALS Amberlite IR-120 ion-exchange r e s i n , Mai 1inckrodt. *Ca1cium glucoheptonate, Givaudan (supplied by May and Baker). *Calcium glucoheptonate, I t a l s i n t e x . Calcium glucoheptonate USP (pure a epimer), P f a n s t i e h l . Calcium a-B-glucoheptonate (calcium glucoheptonate, a-8 m i x t u r e ) , P f a n s t i e h l . 3% cyanopropylmethyl s i l i c o n e (SILAR I O C ) on Chromosorb W(HP) 100-120 mesh Applied Science. 3% cyanopropylphenylmethyl s i l i c o n e (OV-225) on Chromosorb W(HP) 100-120 mesh, Western Chromatography. D i m e t h y l s u l f o x i d e , BDH. 95% v/v Ethyl a l c o h o l , commercial grade and r e d i s t i l l e d . a-D-Glucoheptonic a c i d y - l a c t o n e , A l d r i c h . Hydrochloric a c i d , ACS grade, A l l i e d Chemical. Methanol, ACS grade, Caledon. 3% methyl s i l i c o n e (OV-101) on Chromosorb W (HP), 100-120 mesh, Western Chromatography. 3% phenylmethyl s i l i c o n e (OV-17) on Chromosorb W (HP), 100-120 mesh, Western Chromatography. Methyl s t e a r a t e , Matheson Coleman and B e l l . Sodium hydroxide, ACS grade, F i s c h e r . Sodium h y d r i d e / o i l d i s p e r s i o n , A l d r i c h . Sucrose, a n a l y t i c a l reagent, BDH. T r i m e t h y l s i l y l i m i d a z o l e (TSIM), P i e r c e . T r i m e t h y l s i l y l i m i d a z o l e i n p y r i d i n e (TRISIL Z ) , P i e r c e . * g i f t from Stanley Drug Products. - 17 A mixture of t r i m e t h y l s i l y l i m i d a z o l e (TSIM), N , 0 - b i s ( t r i m e t h y l s i l y l ) acetamide (BSA) and t r i m e t h y l c h l o r o s i l a n e (TMCS)-TRISIL 1TBT\ P i e r c e . Water, d i s t i l l e d . - 18 -2.3 STABILITY STUDIES OF CALCIUM GLUCOHEPTONATE SOLUTIONS The commercial formulation marketed by Stanley Drug Products contained 26.7% w/v calcium glucoheptonate i n water plus a number of a d d i t i v e s . Since these a d d i t i v e s may a l t e r the p r e c i p i t a t i o n behavior, p r e l i m i n a r y studies involved the preparation of simple s o l u t i o n s c o n t a i n i n g 26.7% w/v calcium glucoheptonate i n water. Calcium glucoheptonate from the f o l l o w i n g manufacturers was used i n the s t u d i e s : ( i ) Givaudan (through May and Baker) ( i i ) I t a l s i n t e x ( i i i ) P f a n s t i e h l . The two grades of mat e r i a l supplied by P f a n s t i e h l were: (a) USP grade (pure a form) (b) a - 3 mixture 2.3.1 S o l u t i o n s made from calcium glucoheptonate a f t e r heating a t 120°C M u l l e r and others (1979) reported that s o l u t i o n s prepared a f t e r heating the calcium glucoheptonate powder at 115-120°C often r e s u l t e d i n s t a b l e s o l u t i o n s . Thus, calcium glucoheptonate was heated at 120°C f o r 12 hours, then cooled i n a d e s i c c a t o r and used to prepare s o l u t i o n s . - 19 -2.3.2 Heat treatment of calcium glucoheptonate s o l u t i o n s (a) s o l u t i o n s were prepared and then heated at 80°C i n a water bath f o r 30 minutes. (b) s o l u t i o n s were autoclaved at 121°Cfor 20 minutes. 2.3.3 Membrane f i l t r a t i o n of calcium glucoheptonate s o l u t i o n s S o l u t i o n s were f i l t e r e d through a 0.2 ym membrane f i l t e r ( M i l l i p o r e ) i n a s t e r i l i z e d S t e r i f i l F i l t r a t i o n System. The r e s u l t s of these s t u d i e s are presented i n Table I. 2.4 CHARACTERIZATION OF CALCIUM GLUCOHEPTONATE AND THE PRECIPITATE  OBTAINED FROM SOLUTIONS OF CALCIUM GLUCOHEPTONATE The calcium glucoheptonate was d r i e d under vacuum a t 60°Cfor 16 hours. The p r e c i p i t a t e obtained from s o l u t i o n s of calcium glucoheptonate was d r i e d under the f o l l o w i n g c o n d i t i o n s : (1) at room temperature under vacuum to a constant weight. This m a t e r i a l was used f o r a l l the studies i n v o l v i n g the charac-t e r i z a t i o n of the p r e c i p i t a t e . (2) f o r 2 hours at 76°C under vacuum (5 mm). This was used i n DSC s t u d i e s . (3) f o r 46 hours at 80°C under vacuum (5 mm). This' m a t e r i a l was subjected to X-ray, IR, and s o l u b i l i t y s t u d i e s . 2.4.1 Elemental a n a l y s i s The elemental composition (carbon, hydrogen and oxygen) of calcium - 20 -glucoheptonate USP ( P f a n s t i e h l ) and the p r e c i p i t a t e obtained from the s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) were determined by Canadian M i c r o a n a l y t i c a l Service L t d . , Vancouver. The calcium content was determined i n our l a b o r a t o r y by the USP assay method (USP XX, 1980) of calcium glucoheptonate. 2.4.2 Thermal a n a l y s i s A d i f f e r e n t i a l scanning c a l o r i m e t e r equipped f o r e f f l u e n t gas a n a l y s i s was used f o r performing thermal a n a l y s i s . The m a t e r i a l s were ground i n a gla s s mortar and p e s t l e and 1-5 mg samples were weighed with a Cahn e l e c t r o -balance d i r e c t l y i n t o aluminium v o l a t i l e sample pans. Scans were made at 10°/minute using closed pans and pans with a 0.1-0.2 mm pinhole. V a p o r i z a t i o n of the water of hydration from the pans with a pinhole was detected using the e f f l u e n t gas analyzer and was estimated q u a n t i t a t i v e l y by weighing the pan a f t e r the appearence of the endothermic peak. 2.4.3 I n f r a r e d (IR) spectra A. Preparation of s o l i d samples P e l l e t s were prepared a f t e r mixing 5 mg of material with 200 mg of potassium bromide.' i n a gl a s s mortar and p e s t l e . B. Preparation of s o l u t i o n s One mg of the sample was d i s s o l v e d i n 40 mL chloroform and t h i s s o l u t i o n was used f o r o b t a i n i n g the IR spe c t r a . - 21 -2.4.4 Heat of s o l u t i o n About 500 mg of the sample was a c c u r a t e l y weighed i n t o the sample c e l l of a s o l u t i o n c a l o r i m e t e r . This was d i s s o l v e d under c o n t r o l l e d c o n d i t i o n s i n 100 g of water contained i n a double walled glass v e s s e l . Throughout the r e a c t i o n , the temperatures were sensed by a thermistor and recorded on a s t r i p chart recorder. 2.4.5 X-ray d i f f r a c t i o n This was c a r r i e d out with Ni f i l t e r e d CuKa X-rays, 40 kV, 15 ma, over a range of 20 from 10° to 60° at 1° 29/min (2 second count). Approximately 300 mg of the ground sample was used. 2.4.6 E q u i l i b r i u m s o l u b i l i t y An excess of the sample was added to 50 mL of water and the mixture was kept i n a water bath with a shaking arrangement. The water-bath was maintained at 30°C. Samples were taken p e r i o d i c a l l y , f i l t e r e d and analyzed by the USP assay method of calcium glucoheptonate. The process was continued u n t i l e q u i l i b r i u m had been a t t a i n e d . 2.5 DEVELOPMENT OF A GAS CHROMATOGRAPHIC (GC) METHOD FOR ESTIMATING  THE PROPORTIONS OF THE a AND g EPIMERS IN CALCIUM GLUCOHEPTONATE 2.5.1 S e l e c t i o n of a substance f o r p r e l i m i n a r y s t u d i e s Since carbohydrates are n o n - v o l a t i l e compounds, they cannot be analyzed by GC unless v o l t a i l e d e r i v a t i v e s are f i r s t formed (Laker, 1980). Hence the f i r s t o b j e c t i v e was the preparation of a s t a b l e , v o l a t i l e - 22 -d e r i v a t i v e of calcium glucoheptonate. The formation of an incomplete d e r i v a t i v e i s a problem often encoun-tered and t h i s i s r e a d i l y i d e n t i f i e d by the appearance of m u l t i p l e peaks i n the chromatogram. For the p r e l i m i n a r y s t u d i e s i t was necessary to use a known pure s i n g l e compound which would give a s i n g l e peak i f completely d e r i v a t i z e d . Calcium glucoheptonate could not be used because i t was suspected t h a t i t might be a mixture of the a and 3 forms,The y-lactone of a-D-glucoheptonic a c i d i s commercially a v a i l a b l e and was used f o r the p r e l i m i n a r y d e r i v a t i z a t i o n r e a c t i o n s . 2.5.2 Preparation of methyl d e r i v a t i v e The method used was a m o d i f i c a t i o n of that reported by Leclereq and Desiderio (1971). A. Preparation of sodium m e t h y l s u l f i n y l m e t h i d e An amount of sodium h y d r t d e / o i l d i s p e r s i o n c o n t a i n i n g 25 mg sodium hydride was r i n s e d 3 times with anhydrous ether. 1 mL of dry dimethyl-s u l f o x i d e (DMSO) was added and the suspension heated under n i t r o g e n , u n t i l e v o l u t i o n of hydrogen ceased.The r e s u l t i n g c l e a r s o l u t i o n was stored under nitrogen i n a r e f r i g e r a t o r . B. D e r i v a t i v e formation To 200 yg of the y-.lactone of a-D-glucoheptonic a c i d , 100 uL of DMSO was added. A stream of nitrogen was passed through the tube. Then 20 uL of sodium m e t h y l s u l f i n y l m e t h i d e was added, the v i a l was set aside f o r 15 minutes and 40 uL of methyl iodi d e was added. The tube was immersed - 23 -i n an o i l bath and heated at 40°Cfor 30 minutes. The space above the l i q u i d i n the tube was flu s h e d with n i t r o g e n , closed with the screw cap, sealed with t e f l o n tape and kept i n an oven at 58°Cfor 1 hour. The r e a c t i o n was terminated by the a d d i t i o n of 1 mL of water to the contents of the tube. The methylated product was extracted by shaking with 1 mL of chloroform and removing the water l a y e r . The chloroform l a y e r was washed 2 times with 1 mL of water and the chloroform was evaporated o f f i n a stream of nit r o g e n . The residue was r e d i s s o l v e d i n 100 uL of chloroform. When 5 uL of the above s o l u t i o n was i n j e c t e d i n t o a gas chromatograph with an OV-225 column i t r e s u l t e d i n m u l t i p l e peaks i n d i c a t i n g that methyla-t i o n was incomplete. 2.5.3 Preparation of t r i m e t h y l s i l y l d e r i v a t i v e A number of s i l y l a t i n g agents commercially a v a i l a b l e from Pierce Chemical Company, U.S.A. were i n v e s t i g a t e d as p o t e n t i a l d e r i v a t i z i n g agents p r i o r to gas chromatography on a OV-225 column. A. N - T r i m e t h y l s i l y l i m i d a z o l e (TSIM) Ffve mg of sample was d i s s o l v e d i n 0.1 mL p y r i d i n e i n a 1.0 mL v i a l . Then 0.4 mL of TSIM was added and the v i a l vortexed. The v i a l was heated at 60°Cfor 30 minutes. Five uL of the above sample was i n j e c t e d i n t o the gas chromatograph. This r e s u l t e d i n m u l t i p l e peaks i n d i c a t i n g incomplete t r i m e t h y l s i l y l a t i o n . B. Mixture of t r i m e t h y l s i l y l i m i d a z o l e , N , 0 - b i s ( t r i m e t h y l s i 1 y l ) acetamide,  and t r i m e t h y l c h l o r o s i l a n e (TRISIL 'TBT') 'Five mg of sample was d e r i v a t i z e d using TRISIL 'TBT' as above and - 24 -5 uL of the r e s u l t i n g s o l u t i o n was i n j e c t e d i n t o the gas chromatograph. This a l s o r e s u l t e d i n m u l t i p l e peaks thereby i n d i c a t i n g incomplete d e r i v a t i z a t i o n . C. T r i m e t h y l s i l y l i m i d a z o l e i n p y r i d i n e (TRISIL Z) Since t h i s reagent i s formulated i n p y r i d i n e , 5.0 mg of sample was d i r e c t l y d i s s o l v e d i n 0.5 mL of TRISIL Z. This s o l u t i o n was warmed at 60°C f o r 30 minutes and then 5 uL was i n j e c t e d i n t o the gas chromatograph. This r e s u l t e d i n a s i n g l e peak, thereby i n d i c a t i n g apparently complete t r i m e t h y l s i l y a t i o n . The s o l u t i o n was i n j e c t e d i n t o the gas chromatograph co n t a i n i n g columns of varying p o l a r i t i e s namely: ( i ) 3% 0V-101 on Chromosorb W(HP) 100-120 mesh ( i i ) 3% OV-17 on Chromosorb W(HP) 100-120 mesh ( i i i ) 3% OV-225 on Chromosorb W(HP) 100-120 mesh ( i v ) 3% SILAR 10 C on Chromosorb W(HP) 100-120 mesh In each case there was only one peak thereby confirming that the t r i m e t h y l -s i l y l a t i o n was complete. 2.5.4 Preparation of the t r i m e t h y l s i l y l d e r i v a t i v e of calcium glucoheptonate To 5 mg of calcium glucoheptonate ( P f a n s t i e h l , a-3) 0.5 mL of TRISIL Z was added and vortexed and 5 uL was i n j e c t e d i n t o the gas chromatograph with an OV-225 column. This d i d not r e s u l t i n any peaks. The chromato-graphy was unsuccessful presumably due to the n o n - v o l a t i l e nature of the calcium s a l t . Hence i t was decided to pass a s o l u t i o n of calcium gluco-heptonate through a c a t i o n exchange r e s i n and exchange the calcium f o r hydrogen to form glucoheptonic a c i d . About 10 g of the c a t i o n exchange r e s i n Amberlite IR-120 was f i r s t washed with IM NaOH s o l u t i o n . I t was then - 25 -repeatedly washed with water, then with 1 Nl formic a c i d , again with water and loaded i n t o a g l a s s column. One gram of calcium glucoheptonate ( P f a n s t i e h l , a-B) was d i s s o l v e d i n deionized d i s t i l l e d water and passed (<5 ppm) through the ion exchange column. This s o l u t i o n was analyzed f o r calcium i n the atomic absorption spectrophotometer. N e g l i g i b l e l e v e l s of calcium were observed thereby confirming the e f f i c i e n c y of the ion exchange process. This s o l u t i o n was frozen by s t o r i n g a t -76°Cin a f r e e z e r . The s o l u t i o n was f r e e z e - d r i e d to a constant weight and about 5 mg of the freeze d r i e d m a t e r i a l was t r a n s f e r r e d to a 1 mL v i a l and 0.5 mL of TRISIL Z was added. The v i a l was vortexed and heated at 60°Cfor 2 hours. Then 5 uL was i n j e c t e d i n t o the gas chromatograph with an OV-225 column. This gave four peaks presumably due to a-p-glucoheptonic a c i d , B-D-glucoheptonic a c i d , and the corresponding lactones which r e s u l t from l a c t o n i z a t i o n of the acid s i n aqueous s o l u t i o n ( I s b e l l and Frush, 1963). The formation of both 1,4-lactones as wel l as 1,5-lactones are p o s s i b l e . These r e a c t i o n s are presented i n schemes IA and IB. In order to p o s i t i v e l y i d e n t i f y these four peaks i t would be necessary to have the f o l l o w i n g pure reference compounds: ( i ) a-D-glucoheptonic a c i d ( i i ) B-D-glucoheptonic a c i d ( i i i ) y-lactone of a-D-glucoheptonic a c i d ( i v ) y-lactone of B-D-glucoheptonic a c i d (v) S-lactone of a-D-glucoheptonic a c i d ( v i ) S-lactone of B-D-glucoheptonic a c i d U n f o r t u n a t e l y , o n l y the y-lactone of a-D-glucoheptonic a c i d i s commercially a v a i l a b l e . - 26 -c 1 = 0  - c 1 - OH 1 - c 1 - OH 1 — c 1 - H 1 - c 1 - OH 1 - c - OH COOH -H90 +H20 H - C I - OH H i - c 1 - OH OH 1 - c 1 - H H 1 - c 1 - OH H 1 - c - OH -H20 +H20 0 = C 1 H i - c 1 - OH H - c 1 - OH OH 1 - c 1 - H H 1 - c 1 H 1 - c - OH CH2OH CH20H CH20H 1 , 4-lactone of a-D-glucoheptonic a c i d ( f - l a c t o n e of a-D-glucoheptonic acid) a-D-glucoheptonic a c i d 1,5 1actone of a-D-glucoheptonic ac i d ( 6-lactone of a-D-glucoheptonic a c i d ) Scheme IA. L a c t o n i z a t i o n of a-D-glucoheptonic a c i d . OH H C -I C -I c -I c -I c -0 H OH H OH OH •H20 +H20 CH20H COOH OH I - C - H H 1 - C - OH I OH I - C - H I H - C - OH H | - C - OH I 1 CH2OH •H20 +H20 0 OH H OH H H C - H. I C - OH I C - H I C I C - OH CH2OH 1 , 4-lactone of 6-D-glucoheptonic a c i d 3-D-glucoheptonic a c i d 1,5-lactone of B-D-glucoheptonic a c i d Scheme IB. L a c t o n i z a t i o n of g-D-glucoheptonic a c i d . - 27 -2.5.5 Lactone formation In the presence of h y d r o c h l o r i c a c i d a l d o n i c acids are converted i n t o t h e i r corresponding y-lactones (1,4-lactones) (Perry and H u l y a l k a r , 1965). Hence the number of peaks o c c u r r i n g i n the gas chromatographic a n a l y s i s can be reduced by t r e a t i n g the eluate from the ion exchange column with h y d r o c h l o r i c a c i d i n order to convert the glucoheptonic a c i d / l a c t o n e mixture completely to the lactones. Treatment of the ac i d - l a c t o n e mixture with h y d r o c h l o r i c a c i d a l s o avoids the p o s s i b l e c o m p l i c a t i o n due to the formation of 1,5-lactones ( I s b e l l and Frush, 1963; Perry and H u l y a l k a r , 1965). A stock s o l u t i o n of the f r e e z e - d r i e d eluate was prepared i n methanol and an a c c u r a t e l y measured volume (c o n t a i n i n g about 0.25 mg of gluco-heptonic a c i d / l a c t o n e mixture) was t r a n s f e r r e d to a 1 mL v i a l . The methanol was evaporated o f f under reduced pressure, 50 uL of concentrated HCl was added and the s o l u t i o n vortexed. The HCl was evaporated o f f under reduced pressure. A f u r t h e r 50 uL of concentrated HCl was added and t h i s process repeated three more times. F i n a l l y , 100 yL of TRISIL Z was added, the s o l u t i o n heated at 60°C f o r 30 minutes, vortexed and i n j e c t e d i n t o the gas chromatograph with an OV-225 column. This r e s u l t e d i n two peaks ( F i g . 5). A c o n t r o l experiment using the TMS d e r i v a t i v e of the Y-lactone of a-D-glucoheptonic a c i d reference m a t e r i a l ( F i g . 6) gave a s i n g l e peak having the same r e t e n t i o n time as the peak 2 ( F i g . 5) of the sample, thereby suggesting t h a t t h i s second peak i s due to the TMS d e r i v a t i v e of the y-lactone of a-D-glucoheptonic a c i d . However, evidence was necessary to e s t a b l i s h that the f i r s t peak was due to the TMS d e r i v a t i v e of the Y-Tactone of 3-D-glucoheptonic a c i d . - 28 -1. B-D-glucoheptonic a c i d Y-lactone 2. a-D-glucoheptonic a c i d Y-lactone r T 1 1 0 5 10 15 M I N U T E S F i g . 5. Chromatogram of the TMS d e r i v a t i v e s of the Y-lactones of a and B-D-glucoheptonic a c i d s . Chromatographic c o n d i t i o n s : column, 3% OV-225 on Chromosorb W (1.8 m x 4 mm); i n j e c t i o n temperature, 250°C; detector temperature, 250°C; column temperature, 200°C; c a r r i e r gas (helium) flow rate 30 mL/min. F i g . 6. Chromatogram of the TMS d e r i v a t i v e of the y-lactone of a-D-glucoheptonic a c i d (reference m a t e r i a l ) . Chromatographic c o n d i t i o n s : column, 3% OV-225 on Chromosorb W (1.8 m x 4 mm); i n j e c t i o n temperature, 250°C; detector temperature, 250°C; column temperature, 200°C; c a r r i e r gas (helium) flow r a t e 30 mL/min. - 30 -2.5.6 Gas chromatography^mass spectrometry (GC-MS) The procedure f o r preparation of the sample was the same as elaborated i n s e c t i o n 2.5.5 (lactone formation). A 1.25 m, 2.5 mm i . d . glass column packed with 3% OV-225 on Chromosorb W was used f o r the GC-MS s t u d i e s . The other experimental c o n d i t i o n s were: i n j e c t i o n temp. 250°C column temp. 200°C separator temp. 250°C c a r r i e r gas flow r a t e : 30 mL/min Beam energy 70 eV When the sample was i n j e c t e d , i t r e s u l t e d i n two peaks (as i n F i g . 5 ). The two GC peaks had s i m i l a r mass s p e c t r a l p a t t e r n s . The fragmentation p a t t e r n summarized i n Scheme 2 had the f o l l o w i n g c h a r a c t e r i s t i c s : ( i ) a molecular i o n peak (M +) recorded a t m/e 568 i n agreement with that c a l c u l a t e d f o r the f u l l y t r i m e t h y l s i l y l a t e d d e r i v a t i v e . ( i i ) the ion M-15 (obtained when one methyl group i s s p l i t o f f ) at m/e 553. ( i i i ) the ion M-43 at m/e 525. ( i v ) the ion M-105 observed at m/e 463 due to the l o s s of CH 3 and TMSOH groups. The reference m a t e r i a l i . e . the y-lactone of a-D-glucoheptonic a c i d , when subjected to GC-MS a n a l y s i s had the same r e t e n t i o n time and f r a g -mentation pattern as the peak 2 i n F i g . 5. Hence the i d e n t i t y of t h i s peak, as the TMS d e r i v a t i v e of the y-lactone of a-D-glucoheptonic a c i d - 31 -Peak 1 Peak 2 I 0 H TMS 0 - C I H - C - 0 TMS I C - H a I 0 TMS H - C - 0 TMS I CH 20 TMS C = 0 H - C - 0 TMS H - C I C I H - C 0 TMS H 0 TMS H - C - 0 TMS CH 20 TMS TMS d e r i v a t i v e of y-lactone of 3-D-glucoheptonic a c i d TMS d e r i v a t i v e of y-lactone of a-D-glucoheptonic a c i d -CH, m/e 553 M (MW 568) TMS OH m/e 463 -CH2CH2CH2 m/e 525 M (MW 568) -CH, m/e 553 TMS OH m/e 463 CH2CH 2CH 2 m/e 525 Scheme 2. Fragmentation scheme of the TMS d e r i v a t i v e s of  the y-lactones of a-D-qlucoheptonic a c i d and g-D-glucoheptonic a c i d . - 32 -was confirmed. Since GC peaks 1 and 2 ( F i g . 5) have d i f f e r e n t r e t e n t i o n times but mass s p e c t r a l patterns with i d e n t i c a l m/e values at the upper region of the spectrum, the chemical s t r u c t u r e s of the two compounds must be very s i m i l a r . Since the i d e n t i t y of peak 2 was e s t a b l i s h e d , peak 1 was ascribed to the TMS d e r i v a t i v e of the y-lactone of B-D-glucoheptonic a c i d . 2.5.7 S e l e c t i o n of i n t e r n a l standard Glucose, mannose and sucrose were chosen as compounds f o r i n v e s t i g a -t i o n as p o s s i b l e i n t e r n a l standards. About 5 mg of each of these substances was taken i n a 1 mL v i a l and 0.5 mL of TRISIL Z added. The v i a l was vortexed and heated at 60°C f o r 30 minutes. Then 5 uL was i n j e c t e d i n t o the gas chromatograph with an OV-225 column. The TMS d e r i v a t i v e s of glucose and mannose had very short r e t e n t i o n times coming o f f almost with the s o l v e n t . On the other hand, the TMS d e r i v a t i v e of sucrose had a longer r e t e n t i o n time than the compounds under i n v e s t i g a t i o n i . e . the TMS d e r i v a t i v e s of the y-lactones of a and B-D-glucoheptonic a c i d . Hence sucrose was used as the i n t e r n a l standard. I t i s a v a i l a b l e i n a h i g h l y p u r i f i e d form and the preparation of stock s o l u t i o n posed no problem. 2.5.8 Optimization of GC c o n d i t i o n s The p r e l i m i n a r y experiments were c a r r i e d out on a 1.8 m g l a s s column, 4 mm i . d . packed with 3% OV-225 on Chromosorb W under the f o l l o w i n g c o n d i t i o n s : - 33 -I n j e c t i o n temp. 250°C Column temp. 200°C Detector (F.I.D.) temp. 250°C C a r r i e r gas (helium) flow r a t e : 30 mL/min A. S e l e c t i o n of s t a t i o n a r y phase The f o l l o w i n g s t a t i o n a r y phases were i n v e s t i g a t e d f o r p o s s i b l e use: ( i ) 3% OV-17 on Chromosorb W'(HP) 100-120 mesh ( i i ) 3% OV-101 on Chromosorb W(HP) 100-120 mesh ( i i i ) 3% OV-225 on Chromosorb W(HP} 100-120 mesh ( i v ) 3% SILAR 10 C on Chromosorb W 100-120 mesh Of the fou r phases t e s t e d , the most s a t i s f a c t o r y separation was achieved with the OV-225 phase. A 1.8 m, 4 mm i . d . g l a s s column packed with 3% OV-225 on Chromosorb W was used f o r subsequent GC work. B. Temperature programming When the TMS d e r i v a t i v e of sucrose was chromatographed a t an i s o -thermal column temperature of 200°, i t had a r e t e n t i o n time of about 17 minutes. Because of the r e l a t i v e l y long r e t e n t i o n time, the peak due to sucrose was q u i t e broad. The column temperature could not be increased any f u r t h e r because the r e t e n t i o n times of the TMS d e r i v a t i v e s of y-lactones of a and B-D-glucoheptonic aci d s were clo s e to each other ( F i g . 5 ) . Hence temperature programming was done. The oven temperature was kept a t 200° f o r the f i r s t 10 minutes, then i t was increased to 215° at 5°/minute. The r e t e n t i o n time of sucrose was reduced to about 14.8 - 34 -minutes and the peak shape was als o g r e a t l y improved ( F i g . 7 ). C. I n j e c t i o n temperature Conventionally the i n j e c t i o n temperature i s 50° to 100°C higher than the oven temperature ( B u r c h f i e l d and S t o r r s , 1962). The i n j e c t i o n tempera-ture was. v a r i e d from 230° to 270°C and no d i f f e r e n c e i n the response of a-D-glucoheptonic a c i d y-lactone and B-D-glucoheptonic a c i d y-lactone was observed. V a r i a t i o n i n i n j e c t i o n temperature d i d not produce any d i f f e r e n c e i n the proportions of the a and $ epimers. Hence the i n j e c t i o n temperature was set at 250°C. D. Detector temperature The d e t e c t o r temperature was a l s o set at 250°. A v a r i a t i o n i n the detector temperature of ±20° d i d not produce any d i f f e r e n c e i n the response of y-lactones of a-D-glucoheptonic a c i d and B-D-glucoheptonic a c i d . There was no d i f f e r e n c e i n the proportions of these two compounds due to a change i n d e tector temperature. E. Optimization of r e a c t i o n time I t was necessary to determine the time required f o r the apparently complete d e r i v a t i z a t i o n of y-lactones and sucrose. The y-lactones and sucrose undergo the same d e r i v a t i z a t i o n r e a c t i o n . Hence i t was f i r s t necessary to determine the time taken f o r the optimal t r i m e t h y l s i l y a t i o n of sucrose. For t h i s purpose methyl stearate was chosen as the i n t e r n a l standard because i t does not undergo the t r i m e t h y l s i l y l a t i o n r e a c t i o n . A mixture of sucrose and methylstearate were d i s s o l v e d i n TRISIL Z, and the area r a t i o of sucrose to methylstearate was determined. This mixture was then 35 1. B-D-glucoheptonic acid y-lactone 2. a-D-glucoheptonic acid Y-lactone 3. sucrose 10 15 ~20 M I N U T E S Fig. 7. Chromatogram of the TMS derivatives of the y lactones of a and g-D-glucohetponic acids with the TMS derivative of sucrose as the internal standard. Chromatographic conditions: column, 3% OV-225 on Chromosorb W (1.8 m x 4 mm); injection temperature, 250°C; detector temperature, 250°C; column temperature, 200°C (10 min.) to 215°C at 5°C/min; carrier gas flow rate 30 mL/min. - 36 -heated at 60°C f o r 15 and 30 minutes and the area r a t i o s were again determined. There was no d i f f e r e n c e i n the area r a t i o i n d i c a t i n g that the d e r i v a t i z a t i o n r e a c t i o n of sucrose was instantaneous a t room temperature. Now using sucrose as the i n t e r n a l standard, a-D-glucoheptonic a c i d y-lactone and B-D-glucoheptonic a c i d y-lactone were subjected to a s i m i l a r s e r i e s of experiments. Again i t was observed that the apparently complete t r i m e t h y l s i -l a t i o n o f the two lactones -was accompli shea* instantaneously without heating. 2.6 PREPARATION OF STANDARD CURVE OF THE y-LACTONE OF g-D-GLUCOHEPTONIC  ACID About 60 mg of the y-lactone of a-D-glucoheptonic a c i d was a c c u r a t e l y weighed and d i s s o l v e d i n s u f f i c i e n t methanol to make up the volume to 100 mL. The f o l l o w i n g amounts of the lactone (as methanolic s o l u t i o n ) was t r a n s f e r r e d to 1 mL v i a l s - 30, 60, 90, 120, 150 and 180 yg. S i x t y -yg of sucrose (as a s o l u t i o n i n p y r i d i n e ) was added to each of the v i a l s . Then 200 yL of TRISIL Z was added to each v i a l , the v i a l s vortexed and 5 yL was i n j e c t e d i n t o the GC. The standard curve was p l o t t e d ( F i g . 7a). Each po i n t was the mean value obtained with 5 or more i n j e c t i o n s . 2.7 DETERMINATION OF THE PROPORTIONS OF a AND g EPIMERS IN COMMERCIAL  SAMPLES OF CALCIUM GLUCOHEPTONATE The proportions of the a and B epimers was determined i n the f o l l o w i n g commercial samples: ( i ) calcium glucoheptonate (Givaudan) ( i i ) calcium glucoheptonate ( I t a l s i n t e x ) - 37 -WEIGHT RATIO Fig. 7a.. Standard curve of the y-lactone of a-D-glucoheptonic a c i d . - 38 -( i i i ) calcium gluceptate USP ( P f a n s t i e h l ) ( i v ) calcium a-B-glucoheptonate(Pfanstiehl) About 1 g of calcium glucoheptonate was d i s s o l v e d i n water and passed through c a t i o n (Amberlite IR-120 H) exchange r e s i n . The eluate was f r e e z e -d r i e d to constant weight and about 70 mg was a c c u r a t e l y weighed and d i s s o l v e d i n s u f f i c i e n t methanol to 100 mL. Then 150, 200 and 250 uL of t h i s methanolic s o l u t i o n were t r a n s f e r r e d to 1 mL v i a l s and the methanol was evaporated o f f under reduced pressure. Next 100 uL of concentrated HCl was added to each v i a l , the v i a l s were vortexed and the h y d r o c h l o r i c a c i d was evaporated o f f under reduced pressure. The a d d i t i o n and evaporation of HCl was repeated three more times. About 60 mg of sucrose was d i s s o l v e d i n p y r i d i n e and the volume made up to 100 mL. From t h i s s o l u t i o n , 100 uL was added to each of the v i a l s ( i n t e r n a l standard) followed by 200 uL of TRISIL Z. The v i a l s were vortexed and a f i v e yL sample was i n j e c t e d i n t o the GC. 2.8 DETERMINATION OF THE PROPORTION OF a AND p EPIMERS IN THE PRECIPITATE  OBTAINED FROM COMMERCIAL SAMPLES OF CALCIUM GLUCOHEPTONATE The procedure followed was the same as discussed i n s e c t i o n 2.7. The p r e c i p i t a t e was subjected to GC-MS studies and the procedure followed was the same as i n s e c t i o n 2.5.6. The fragmentation pattern was the same as i n Scheme 2. - 39 -3. RESULTS AND DISCUSSION 3.1 STABILITY STUDIES OF CALCIUM GLUCOHEPTONATE SOLUTIONS The r e s u l t s presented i n Table I i n d i c a t e that the P f a n s t i e h l (USP) m a t e r i a l has the lowest s t a b i l i t y i n s o l u t i o n while the P f a n s t i e h l (a - B) sample has the g r e a t e s t s t a b i l i t y i n s o l u t i o n . F i l t r a t i o n increases the time taken f o r p r e c i p i t a t i o n to occur while a u t o c l a v i n g r e s u l t s i n s t a b l e s o l u t i o n s which have not p r e c i p i t a t e d i n more than a year. 3.2 CHARACTERIZATION OF CALCIUM GLUCOHEPTONATE AND THE PRECIPITATE  OBTAINED FROM SOLUTIONS OF CALCIUM GLUCOHEPTONATE 3.2.1 Elemental a n a l y s i s The elemental composition of calcium glucoheptonate USP ( P f a n s t i e h l ) and the p r e c i p i t a t e obtained from a s o l u t i o n of calcium glucoheptonate given i n Table II i n d i c a t e that both the i n i t i a l m a terial and the p r e c i p i t a t e have nearly i d e n t i c a l elemental compositions. 3.2.2 Thermal a n a l y s i s The calcium glucoheptonate a v a i l a b l e from each source was subjected to d i f f e r e n t i a l scanning c a l o r i m e t r i c (DSC) studies as received. On heating to 180°C there were n e i t h e r exothermic nor endothermic peaks showing that the o r i g i n a l m a t e r i a l s were anhydrous and that no polymorphic t r a n s i t i o n s occurred i n the experimental temperature range. However, the p r e c i p i t a t e showed a s i n g l e endothermic peak around 110°C with a weight l o s s f o l l o w i n g - 40 -Table I. S t a b i l i t y of calcium glucoheptonate (26.7 percent w/v) i n aqueous s o l u t i o n . Treatment C o n t r o l 9 S o l i d heated 120°C x 12 hours S o l u t i o n heated 85°C x 30 min. So l u t i o n autoclaved l"? TOT v On m i n Time f o r p r e c i p i t a t i o n to occur (days) Commercial Source Givaudan I t a l s i n t e x P f a n s t i e h l USP a - 3 2 8 1 -3 3 1 ND 13 7 1 ND F i l t e r e d 0 210 240 2 F i l t e r e d 0 and autoclaved - - -- = no p r e c i p i t a t i o n ND = not done a = s o l u t i o n prepared by d i s s o l v i n g s o l i d i n water a t room temperature; f r e q u e n t l y contaminated with m i c r o b i a l growth b = causes c a r a m e l i z a t i o n c = 0.22 ym membrane f i l t e r ( M i l l i p o r e ) Studies c a r r i e d out i n June, 1980. - 41 -Table I I . Elemental composition of calcium glucoheptonate  and the p r e c i p i t a t e obtained from the s o l u t i o n  of calcium glucoheptonate. calcium glucoheptonate USP ( P f a n s t i e h l ) T h e o r e t i c a l Experimental P r e c i p i t a t e 3 Carbon 34.3 % 33.4 32.6 % Hydrogen 5.34 % 5.48 % 5.67 % Calcium 8.17 % 8.12 % 7.88 % Oxygen 52.2 % 4 0 . l b % 42.3 b % Dried under vacuum at room temperature to constant weight. The low values (compared with t h e o r e t i c a l ) are not e x p l a i n a b l e . - 42 -the endothermic peak suggesting that the p r e c i p i t a t e might be a hydrate. When the p r e c i p i t a t e was d r i e d under vacuum a t 76°Cfor 2 hours and the DSC scan repeated, the endothermic peak disappeared. Attempts were made to determine the number of molecules of water of c r y s t a l l i z a t i o n by q u a n t i t a t i n g the weight l o s s a f t e r the appearance of the endothermic peak but the weight l o s s was h i g h l y v a r i a b l e and not c o n s i s t e n t with any s p e c i f i c value f o r waters of c r y s t a l l i z a t i o n . I t i s l i k e l y t hat the p r e c i p i t a t e from aqueous s o l u t i o n c o n s i s t s of a mixture of s t o i -c h i ometric hydrates and/or i s a hydrate of v a r i a b l e composition. 3.2.3 IR spectra A. S o l i d samples • The IR spectra i n KBr d i s c s were determined f o r : ( i ) calcium glucoheptonate (Givaudan) ( i i ) calcium glucoheptonate ( I t a l s i n t e x ) ( i i i ) calcium glucoheptonate ( P f a n s t i e h l , a-8 ) ( i v ) calcium glucoheptonate USP ( P f a n s t i e h l ) (v) p r e c i p i t a t e obtained from the s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) d r i e d under vacuum at room temperature to constant weight ( v i ) p r e c i p i t a t e obtained from the s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) d r i e d under vacuum at 80°Cfor 46 hours Calcium glucoheptonate from a l l the s u p p l i e r s showed a few large and poorly defined absorption bands ( F i g . 8A). The p r e c i p i t a t e which had been d r i e d under vacuum at room temperature had a much more c l e a r l y - 43 -F i g . 8. Infrared spectra of calcium glucoheptonate and the p r e c i p i t a t e (KBr d i s c ) . A. calcium glcoheptonate USP ( P f a n s t i e h l ) B. p r e c i p i t a t e obtained from the s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) d r i e d under vacuum at room temperature to constant weight C. p r e c i p i t a t e obtained from the s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) d r i e d under vacuum at 80°C f o r 46 hours. - 44 -3800 3500 Wawnumbai 2500 2000 F i g . 8A. WWr*twifth nm 3800 3500 Wamnumber 3000 2500 20O0 F i g . 8B. Wav*numbai F i g . 8C. - 45 -F i g . 9. In f r a r e d spectra of calcium glucoheptonate and the p r e c i p i t a t e ( s o l u t i o n i n chloroform). A. calcium glucoheptonate USP ( P f a n s t i e h l ) B. p r e c i p i t a t e obtained from the s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) d r i e d under vacuum at room temperature to constant weight C. p r e c i p i t a t e obtained from the s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) d r i e d under vacuum at 80°C f o r 46 hours - 46 -Wavelength fjm Wavenumber Fig. 9A. Wavelength fjm Wavenumber Fig. 9B. Wavenumber Fig. 9C. - 47 -defined IR s p e c t r a l p a t t e r n ( F i g . 8B.). The broad absorption found near 3300 cm 1 i s due to bonded OH s t r e t c h i n g and the numerous bands i n the region 1125-1000 cm - 1 are due to the s t r e t c h i n g of the C-0 bond. A f t e r drying under vacuum at 80°C f o r 45 hours the spectrum ( F i g . 8C) l o s t i t s sharpness and appeared l i k e the spectrum of the o r i g i n a l calcium glucoheptonate i . e . F i g . 8A. B. S o l u t i o n s Each of the samples i n v e s t i g a t e d i n s e c t i o n 3.2.3 A was d i s s o l v e d i n chloroform. The IR spectra of the s o l u t i o n s obtained were i d e n t i c a l and superimposable. The spectra of the P f a n s t i e h l USP sample and the p r e c i p i t a t e ( d r i e d under d i f f e r e n t c o n d i t i o n s ) were a l s o superimposable as shown i n F i g . 9. 3.2.4 Heat of s o l u t i o n Heats of s o l u t i o n f o r calcium glucoheptonate obtained from d i f f e r e n t sources are presented i n Table I I I . 3.2.5 X-ray d i f f r a c t i o n s t u d i e s The f o l l o w i n g samples were subjected to X-ray d i f f r a c t i o n s t u d i e s . ( i ) calcium glucoheptonate (Givaudan) ( i i ) calcium glucoheptonate ( I t a l s i n t e x ) ( i i i ) calcium glucoheptonate ( P f a n s t i e h l , a-B ) ( i v ) calcium glucoheptonate USP ( P f a n s t i e h l ) (v) p r e c i p i t a t e obtained from a s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) d r i e d under vacuum at room temperature to constant weight - 48 -Table I I I . Heats of s o l u t i o n of calcium glucohepto- nate samples. Source Heat of s o l u t i o n kJ.mol ^ I t a l s i n t e x 8.28 (1. 9 8 ) a Givaudan 10.0 (2.40) P f a n s t i e h l (a,B) 16.2 (3.88) P f a n s t i e h l (USP) 22.2 (5.31) values i n parenthesis are k.cal.mol The heat of s o l u t i o n of the p r e c i p i t a t e obtained from s o l u t i o n s of calcium glucoheptonate could not be determined because of i t s low aqueous s o l u b i l i t y . - 49 -Table IV. X-ray st u d i e s of the p r e c i p i t a t e obtained from t h e . s o l u t i o n  of calcium glucoheptonate USP ( P f a n s t i e h l ) . P r e c i p i t a t e d r i e d under P r e c i p i t a t e d r i e d under vacuum vacuum at RT at 80°cfor 46 hours d ( A ) a ( I / I 0 ) R e l a t i v e I n t e n s i t y d(A) ( I / I 0 ) R e l a t i v e I n t e n s i t y 7.82 7 7.49 21 7.56 7 - - 6.67 •8 - - 6.55 10 6.16 24 6.18 2 - - 5.85 10 - - 5.49 8 5.34 51 5.36 32 4.84 15 4.91 30 4.73 48 4.74 9 4.58 7 4.59 40 4.49 23 4.50 11 4.35 56 - -4.33 >.*-ioo 4.34 > 100 - - 4.22 9 4.13 > 100 4.19 21 cont'd a d = di s t a n c e between successive i d e n t i c a l planes of atoms i n a c r y s t a l - 50 -Table IV/cont'd P r e c i p i t a t e d r i e d under vacuum at RT P r e c i p i t a t e d r i e d under vacuum at 80°Cfor 46 hours d(A) ( I / I 0 ) R e l a t i v e I n t e n s i t y d(A) ( I / I 0 ) R e l a t i v e I n t e n s i t y - 4.03 6 - - 4.01 5 3.88 72 3.88 18 3.83 > 100 3.83 32 3.63 10 3.68 4 3.53 24 3.53 3 3.40 24 - -3.33 > 100 3.33 3.26 22 10 3.22 22 3.23 8 3.14 16 3.14 7 3.07 47 3.07 7 3.01 15 3.03 7 2.94 58 2.94 9 2.89 10 - -2.85 4 - -2.80 18 - -2.77 30 - -2.69 7 2.68 8 cont'd - 51 -Table IV/cont'd P r e c i p i t a t e d r i e d under P r e c i p i t a t e d r i e d under vacuum vacuum at RT at 80°Cfor 46 hours d(A) ( I / I 0 ) d(A) ( I / I 0 ) R e l a t i v e I n t e n s i t y R e l a t i v e I n t e n s i t y 2.67 55 - -2.65 5 - -2.59 10 - -2.56 3 - -2.51 3 - -2.46 24 2.46 5 2.44 3 - -2.39 68 2.39 20 2.34 31 2.34 10 - - 2.29 17 2.28 41 2.28 3 2.21 16 2.21 9 2.16 6 2.16 5 2.14 6 2.14 5 2.13 13 - -2.10 5 2.10 5 2.09 3 2.09 5 - - 2.06 4 2.01 22 2.02 5 1.98 44 1.98 26 cont 1 d - 52 -Table IV/cont'd P r e c i p i t a t e d r i e d under vacuum at RT P r e c i p i t a t e d r i e d under vacuum at 80°Cfor 46 hours d(A) ( I / I 0 ) R e l a t i v e I n t e n s i t y d(A) (^o) R e l a t i v e I n t e n s i t y 1.92 17 1.92 5 1.90 18 1.90 4 1.88 3 -1.85 8 -1.83 5 -1.81 9 1 .80 3 1.78 6 1.78 3 1.77 5 -1.70 4 -1.67 5 -1 .62 3 -1.59 2 -1.55 5 -1.45 4 -1.40 4 -1 .36 13 - 53 -( v i ) p r e c i p i t a t e obtained from a s o l u t i o n of calcium glucoheptonate USP ( P f a n s t i e h l ) d r i e d under vacuum at 80°C f o r 46 hrs The calcium glucoheptonate was found to be amorphous, i r r e s p e c t i v e of the source of the sample which disagrees with information received from P f a n s t i e h l that production of the amorphous form has been impossible i n recent years ( H o l s t e i n , 1980). On the other hand, the p r e c i p i t a t e had a c h a r a c t e r i s t i c X-ray d i f f r a c t i o n pattern thereby i n d i c a t i n g i t s c r y s t a l l i n e nature. When the p r e c i p i t a t e was d r i e d under vacuum at 80°C f o r several hours, there was a change i n the X-ray d i f f r a c t i o n p a t t e r n . Peaks at c e r t a i n d values disappeared and new peaks appeared but there was a reduction i n the t o t a l number of peaks (Table IV). 3.2.6 E q u i l i b r i u m s o l u b i l i t y A. Calcium glucoheptonate Attempts to determine the e q u i l i b r i u m s o l u b i l i t y of calcium gluco-heptonate were unsuccessul. The material seems to be " i n f i n i t e l y " s o l u b l e i n water. The v i s c o s i t y of the s o l u t i o n increases d r a m a t i c a l l y as more and more s o l i d goes i n t o s o l u t i o n and shaking becomes i n c r e a s i n g l y l e s s e f f e c t i v e . Moreover i n h i g h l y concentrated s o l u t i o n s r e c r y s t a l l i z a -t i o n (except f o r P f a n s t i e h l ct-B mixture) occurs much sooner and there i s no time f o r attainment of e q u i l i b r i u m . B. P r e c i p i t a t e obtained from a s o l u t i o n of calcium glucoheptonate USP  ( P f a n s t i e h l ) d r i e d under vacuum a t room temperature to constant weight This m a t e r i a l was found to have a s o l u b i l i t y of 2.5% w/v i n water. - 54 -C. P r e c i p i t a t e obtained from a s o l u t i o n of calcium glucoheptonate USP  ( P f a n s t i e h l ) d r i e d under vacuum at 80°C f o r 46 hours This m a t e r i a l was found to have a s o l u b i l i t y of 10% w/v i n water. 3.3 IDENTIFICATION OF THE PRECIPITATE Elemental a n a l y s i s (Table I I ) showed that calcium glucoheptonate and the p r e c i p i t a t e ( d r i e d under vacuum at RT to constant weight) have almost i d e n t i c a l elemental compositions. The IR studies of s o l u t i o n s i n chloroform revealed that the spectra of calcium glucoheptonate and the d r i e d p r e c i p i t a t e were superimposable ( F i g . 9). F i n a l l y , the mass s p e c t r a l p a t t e r n of calcium glucoheptonate (Scheme 2, p. 31) and the d r i e d p r e c i p i -t a t e (p. 38) were i d e n t i c a l . Hence i t was concluded that the d r i e d p r e c i p i t a t e and the i n i t i a l m a terial were chemi c a l l y i d e n t i c a l . However, from the DSC st u d i e s (Section 3.2.2) i t was concluded that the p r e c i p i t a t e was l i e k l y to be a hydrated form. The number of molecules of water of c r y s t a l l i z a t i o n was v a r i a b l e so the p r e c i p i t a t e i s l i k e l y to be a mixture of hydrates or a hydrate of v a r i a b l e composition. Drying the p r e c i p i t a t e under vacuum at 76°C f o r 2 hours r e s u l t e d i n the l o s s of water. Drying the p r e c i p i t a t e under vacuum at 80°C f o r 46 hours not only r e s u l t e d i n the l o s s of water but a l s o produced changes i n the X-ray d i f f r a c t i o n p a t t e r n (Table IV). I t seems that when the hydrated form of calcium glucoheptonate l o s e s i t s water, the c r y s t a l loses i t s l a t t i c e s t r u c t u r e but again r e c r y s t a l l i z e s . This phenomenon could e x p l a i n the change i n the d i f f r a c t i o n p a t t e r n which occurs on d r y i n g . - 55 -3.4 POSSIBLE REASONS FOR PRECIPITATION The p r e c i p i t a t i o n of calcium glucoheptonate from s o l u t i o n s could be due to one or more of the f o l l o w i n g reasons: ( i ) a change from an unstable m o d i f i c a t i o n to a s t a b l e form ( i i ) the presence of seed c r y s t a l s i n the environment or i n the m a t e r i a l which induce c r y s t a l l i z a t i o n ( i i i ) d i f f e r i n g proportions of the a and 8 epimers i n the calcium glucoheptonate obtained from various sources 3.4.1 Change from an unstable form to a s t a b l e form X-ray s t u d i e s (Section 3.2.5) i n d i c a t e d that the calcium glucohepto-nate was amorphous while the p r e c i p i t a t e was c r y s t a l l i n e . On the other hand, DSC st u d i e s (Section 3.2.2) revealed that calcium glucoheptonate i s anhydrous but the p r e c i p i t a t e i s hydrated. Hence the change that i s occuring i s from an amorphous anhydrous m a t e r i a l to a hydrated c r y s t a l l i n e p r e c i p i t a t e . The p r e c i p i t a t e has been i d e n t i f i e d as calcium glucoheptonate (Section 3.3). S o l u b i l i t y s t u d i e s (Section 3.2.6 A and B) i n d i c a t e that calcium glucoheptonate i s " i n f i n i t e l y " s o l u b l e while the p r e c i p i t a t e ( d r i e d under vacuum at room temperature to constant weight) i s only 2.5% w/v s o l u b l e i n water. This dramatic change i n s o l u b i l i t y could be due to one or other or both of these reasons: ( i ) amorphous to c r y s t a l l i n e t r a n s i t i o n ( i i ) anhydrous to hydrate t r a n s i t i o n - 56 -When the p r e c i p i t a t e was rendered anhydrous by heating i t at 80 °C f o r 46 hours under vacuum, there was a change i n the X-ray d i f f r a c t i o n p a t t e r n (Table IV). This suggested a change i n the c r y s t a l l a t t i c e s t r u c t u r e . Hence based on the studies c a r r i e d out so f a r , i t i s not p o s s i b l e to i s o l a t e which of the above two f a c t o r s i s more responsible f o r t h i s d r a s t i c change i n s o l u b i l i t y . 3.4.2 Presence of seed c r y s t a l s inducing c r y s t a l l i z a t i o n The s t a b i l i t y s t udies of calcium glucoheptonate s o l u t i o n s (Table I) i n d i c a t e that membrane f i l t r a t i o n exerts a s t a b i l i z i n g a c t i o n on the s o l u t i o n . I t can be postulated that the m a j o r i t y of the seed c r y s t a l s are excluded by f i l t r a t i o n which r e s u l t s i n increased s t a b i l i t y of the s o l u t i o n . A utoclaving could destroy the seed c r y s t a l s and thereby e x p l a i n the prolonged s t a b i l i t y of autoclaved s o l u t i o n s . These r e s u l t s i n d i c a t e that although the p o s s i b i l i t y of seed c r y s t a l s inducing c r y s t a l l i z a t i o n cannot be r u l e d out, the presence of seed c r y s t a l s cannot be the only cause of s o l u t i o n i n s t a b i l i t y f o r the f o l l o w i n g reasons: ( i ) s o l u t i o n prepared using the P f a n s t i e h l (a-8) mixture are s t a b l e . I f seed c r y s t a l s were the only causative f a c t o r f o r i n s t a b i l i t y , then the s o l u t i o n s prepared using P f a n s t i e h l (a-g) should a l s o have p r e c i p i t a t e d , ( i i ) the r e s u l t s i n Table I i n d i c a t e that the time taken f o r p r e c i p i t a t i o n to occur depends on the source of calcium glucoheptonate. Moreover, there i s a wide v a r i a t i o n i n the heat of s o l u t i o n values of calcium glucoheptonate (Table I I I ) obtained from d i f f e r e n t sources. These f a c t s suggest that the various samples of calcium glucoheptonate - 57 -behave d i f f e r e n t l y due to d i f f e r e n c e s i n t h e i r chemical composition and/or p h y s i c a l p r o p e r t i e s . 3.4.3 D i f f e r i n g proportions of the a and B epimers i n the calcium  glucoheptonate obtained from various sources For t e s t i n g t h i s hypothesis i t was necessary to: ( i ) develop methods to i d e n t i f y the a and B epimers i n the commercial samples of calcium glucoheptonate ( i i ) develop methods f o r e s t i m a t i n g the proportions of the a and B epimers i n commercial samples ( i i i ) c o r r e l a t e the proportions determined with the observed s t a b i l i t y r e s u l t s The a n a l y t i c a l method f o r estimating the proportions of the a and 8 epimers has been elaborated i n Section 2.5. The r e s u l t s i n Table V i n d i c a t e that there i s a marked v a r i a t i o n i n the p r o p o r t i o n of the a and B epimers i n the samples supplied by d i f f e r e n t manufacturers. A c o r r e l a t i o n between the proportions of the a and 8 epimers and the s t a b i l i n s o l u t i o n (from Table I) i s given i n Table VI. These r e s u l t s show that there i s a r e l a t i o n s h i p between the s t a b i l i t y i n s o l u t i o n and the propor-t i o n of the a epimer. The P f a n s t i e h l USP sample which c o n s i s t e d of the a epimer ( i . e . 100% a ) p r e c i p i t a t e d from s o l u t i o n w i t h i n one day. On the other hand, the P f a n s t i e h l ( a - B) sample which contains 51.8% a form has been s t a b l e f o r more than a year. Thus the 8 form seems to s t a b i l i z e the a form i n s o l u t i o n and i t i s p o s s i b l e t h a t there i s a c r i t i c a l concen t r a t i o n of the B f ° r m necessary f o r s t a b i l i z a t i o n . The proportions of the a and 8 epimers i n the p r e c i p i t a t e obtained - 58 -Table V. Determination of the proportion-' of the OK epimer  i n various commercial samples of calcium gluco- heptonate. Source Proportion of the (percent) a epimer Mean Standard Deviation concn. a concn. concn. May & Baker 72.52 72.20 72.04 72.25 ±0.2444 I t a l s i n t e x 71.88 71.78 71.84 71.83 ±0.0503 P f a n s t i e h l (a-B) 51.31 50.30 51.27 50.96 ±0.5719 P f a n s t i e h l USP 100.0 100.0 100.0 100.0 ±0.0000 aThe proportions i n each concentration i s the mean from 3 i n j e c t i o n s . - 59 -Table VI. R e l a t i o n s h i p between the proportion of the  a epimer and the s t a b i l i t y of calcium gluco- heptonate i n s o l u t i o n . Source Proportion of g epimer (percent) Time f o r p r e c i p i t a t i o n to occur (days) P f a n s t i e h l USP I t a l s i n t e x May & Baker P f a n s t i e h l ( a - 3 ) 100 72.4 71.8 51.8 < 1 2 8 s t a b l e - 60 -from s o l u t i o n s of calcium glucoheptonate were determined (Table VII) and the r e s u l t s i n d i c a t e that there i s an increase i n the proportion of the a epimer i n the p r e c i p i t a t e when compared with the o r i g i n a l m a t e r i a l . M u l l e r and others (1979) i n d i c a t e d that the problem of p r e c i p i t a t i o n was due to the presence of seed c r y s t a l s and that i f the seed c r y s t a l s were destroyed, t h i s would r e s u l t i n a st a b l e product. Our r e s u l t s i n d i c a t e that a l l the three reasons f o r p r e c i p i t a t i o n postulated i n Section 3.4 are p a r t i a l l y r e s p o n s i b l e although i t i s not c l e a r how a change i n the propor-t i o n s of the a and 3 epimers can produce such a marked change i n the s t a b i l i t y of calcium glucoheptonate i n s o l u t i o n . I t i s postu l a t e d t h a t the 3 epimer hinders growth of the i n s o l u b l e hydrated a form. The proportions of the a and 8 epimers i n the calcium glucoheptonate samples were not a l t e r e d by a u t o c l a v i n g . This f a c t r e i n f o r c e s the p o s s i b l e r o l e of seed c r y s t a l s as a cause of s o l u t i o n i n s t a b i l i t y . 3.4.4 Some comments about USP s p e c i f i c a t i o n s of calcium glucoheptonate Only the a form of calcium glucoheptonate i s o f f i c i a l i n the USP (USP XX, 1980). The s t a b i l i t y s t u d i e s of calcium glucoheptonate s o l u t i o n s (Table I) i n d i c a t e t h a t the calcium glucoheptonate supplied by P f a n s t i e h l which complies with the USP s p e c i f i c a t i o n s commences to p r e c i p i t a t e from s o l u t i o n w i t h i n a day. I t i s apparent that i f a sample of calcium gluco-heptonate complies with USP s p e c i f i c a t i o n s ( i . e . i t c o n s i s t s only of the a form) then i t w i l l not be s t a b l e i n s o l u t i o n . Moreover, the pharmaco-poeia o f f e r s no method f o r the i d e n t i f i c a t i o n of the a form. The USP i d e n t i f i c a t i o n t e s t s t a t e s that the IR spectrum of the sample under inves-t i g a t i o n must e x h i b i t maxima only at the same wavelength as a s i m i l a r p r eparation of USP Reference Standard of calcium glucoheptonate. I t has been found that the IR spectra (KBr p e l l e t ) of calcium glucoheptonate - 61 -Table V I I . Proportion of the a epimer i n the p r e c i p i t a t e obtained from s o l u t i o n s of calcium glucoheptonate. Source Proportion of a epimer i n Proportion of a epimer i n calcium glucoheptonate p r e c i p i t a t e (percent) (percent) P f a n s t i e h l I t a l s i n t e x Givaudan 100 72.4 71.8 100 8 4 . 6 7 9 . 9 - 62 -from different sources ( i . e . containing different proportions of the a and 8 epimers) are superimposable. Thus, IR spectroscopy i s incapable of distinguishing between the a and 8 epimers of calcium glucoheptonate. The assay method for calcium glucoheptonate consists of the complexometric estimation of calcium with disodium ethylenediaminetetra-acetate. Hence this method cannot distinguish between the a and 3 epimers. The rationale behind the choice of the pure a form of calcium glucohepto-nate in the USP i s not known. I t i s apparent that a sample of calcium glucoheptonate, in order to be stable in solution must contain approxi-mately equal proportions of the a and 8 epimers (as in Pfanstiehl (a - 8 ) mixture). Hence i t i s suggested that the USP should consider an a - 8 mixture in the monograph for calcium glucoheptonate. Since the r e l a t i v e proportion of the a and 8 epimers i s c r i t i c a l for the s t a b i l i t y of calcium glucoheptonate solutions, the monograph should include a method for estimating this proportion. In our studies, based on the close structural s i m i l a r i t i e s of the a and 8 epimers we assumed that the GC response factors of the two epimers would be very close to each other. For the absolute calculation of the r e l a t i v e proportions of a and 8 epimers i t would be necessary to have pure reference standards for a-D-glucoheptonic acid y-lactone and 8-D-glucoheptonic acid y-lactone. At present only the a-D-glucoheptonic acid y-lactone i s commercially available. - 62a -PART B DEVELOPMENT OF ORAL AND PARENTERAL LIQUID DOSAGE FORMS CONTAINING CALCIUM GLUCOHEPTONATE - 63 -1. INTRODUCTION In recent y e a r s , s o l u t i o n s of calcium glucoheptonate have shown a tendency to c r y s t a l l i z e on storage (elaborated i n Part A, Section 1). The p r e c i p i t a t e formed has been described as lumps of " c o r a l " type c r y s t a l s (Chou and Goring, 1979). Stanley Drug Products L i m i t e d , a pharmaceutical o r g a n i z a t i o n i n North Vancouver faced a s i m i l a r problem (Chou and Goring, 1979) which lead to t h i s c o l l a b o r a t i v e research p r o j e c t to develop s t a b l e o r a l and parenteral s o l u t i o n s of calcium glucoheptonate. - 64 -2. EXPERIMENTAL 2.1 MATERIALS * L a c t i c a c i d , BDH *Sodium cyclamate, May & Baker Sugar, BC sugar Sodium benzoate, BDH *Cherry f r u i t f l a v o r , Givaudan *Black raspberry f l a v o r , Givaudan Calcium D-saccharate, ICN Pharmaceuticals Calcium l a c t o b i o n a t e , ICN Pharmaceuticals * G i f t from Stanley Drug Products The other m a t e r i a l s and apparatus used have been described i n PartA, Sections 2.1 and 2.2. - 65 -2.2 DEVELOPMENT OF ORAL FORMULATIONS 2.2.1 Basic formula The b a s i c formula (Table V I I I ) was developed i n the Product Develop-ment Laboratory of Stanley Drug Products L i m i t e d . The formula t i o n contains a mixture of calcium gluconate and calcium glucoheptonate. I t has been found that calcium gluconate acts as a s t a b i l i z e r f o r calcium glucohepto-nate and v i c e versa when they are used together. I t i s claimed that a double s a l t i s formed (Product manual, P f a n s t i e h l ) . This combination permits savings since calcium gluconate i s l e s s expensive than calcium glucoheptonate. For the o r a l f o r m u l a t i o n s t u d i e s , calcium glucoheptonate from two manufacturers were used: ( i ) calcium glucoheptonate USP ( P f a n s t i e h l ) ( i i ) calcium glucoheptonate (Givaudan) 2.2.2 Use of sugar The b a s i c formula (Table V I I I ) contains sodium cyclamate as a sweetening agent. In order to t e s t the p o s s i b i l i t y of using sugar as a sweetening agent the basic formula was modified (Table IX). A l l the ora l formulations were broadly d i v i d e d i n t o two categ o r i e s based on the sweetening agent used. 2.2.3 Use of s t a b i l i z i n g agent The s t a b i l i t y of s o l u t i o n s c o n t a i n i n g calcium gluconate may be increased by the a d d i t i o n of a s u i t a b l e s t a b i l i z e r such as calcium-D-- 66-Table V I I I . Basic formula Ingredient Amount calcium glucoheptonate 9 13.2 g calcium g l u c o n a t e 9 11.2 g l a c t i c a c i d 4.0 mL sodium cyclamate 1.0 g sodium benzoate 0.11 g cherry f r u i t f l a v o r 0.17 mL black raspberry f l a v o r 0.17 mL water to 100 mL The f i n a l f ormulation contains 10.79 mg/mL calcium from calcium glucoheptonate and 10.43 mg/mL calcium from calcium gluconate. - 67 -Table IX. Modified basic formula c o n t a i n i n g  sugar as the sweetening agent. Ingredient Amount calcium glucoheptonate 13.2 g calcium gluconate 11.2 g l a c t i c a c i d 4.0 mL sugar (granulated) 33.3 g sodium benzoate 0.11 g cherry f r u i t f l a v o r 0.17 mL black raspberry f l a v o r 0.17 mL water to 100 mL - 68 -saccharate and calcium l a c t o b i o n a t e (Wade, 1977b). Hence i t was decided to t e s t the e f f e c t i v e n e s s of these s t a b i l i z i n g agents on the calcium gluconate - calcium glucoheptonate mixture. When calcium-D-saccharate or calcium l a c t o b i o n a t e was added to the basic formula, a corresponding amount of calcium gluconate was removed so that the t o t a l calcium concen-t r a t i o n i n the fo r m u l a t i o n was maintained constant. Formulations were prepared i n which 0, 2, 5 and 10% of calcium gluconate was replaced with calcium saccharate or calcium l a c t o b i o n a t e (Table X). The use of disodium edetate as a s t a b i l i z e r of an i n j e c t a b l e calcium gluconate s o l u t i o n has been reported (Welch and Scoratow, 1980). The e f f e c t of t h i s agent on the mixture of calcium gluconate-calcium gluco-heptonate has a l s o been s t u d i e d . The d e t a i l s of the formulations are given i n Table X. 2.2.4 Method of preparation of oral formulations Part A: Calcium gluconate, calcium glucoheptonate and the s t a b i l i z i n g agent (calcium-D-saccharate,calcium l a c t o b i o n a t e or disodium edetate) were d i s s o l v e d i n 40 mL of b o i l i n g water. The heat was turned o f f and the s o l u t i o n allowed to c o o l . Part B: The sugar, sodium benzoate and l a c t i c a c i d were d i s s o l v e d i n 25 mL of water with s t i r r i n g . A f t e r c o o l i n g Part A to room temperature, the contents of Part B were added to Part A and mixed. Part C: The cherry j u i c e f l a v o r and black raspberry f l a v o r were added with s t i r r i n g . The volume was made up to 100 mL and the s o l u t i o n was f i l t e r e d . - 69 -Table X. Oral calcium syrup - formulation d e t a i l s Basic formula*with sodium Basic formula** with sugar cyclamate (1%-w/y) (33.3% w/v) calcium D-a , saccharate . 0 0.13 0.32 0.64 0 0.13 0.32 0.64 (percent w/v) calcium l a c t o - c bionate 0 0.40 0.98 1.96 0 0.40 0.98 1.96 (percent w/v) disodium edetate 0 0.10 0.20 0.50 0 0.10 0.20 0.50 (percent w/v) a e q u i v a l e n t to a replacement of 0,2,5, and 10 percent of calcium gluconate r e s p e c t i v e l y . b Replacement of 2 percent of calcium gluconate with a corresponding amount of calcium D-saccharate so that the t o t a l calcium concentration i n the for m u l a t i o n was maintained constant. 11.2 g of calcium gluconate i s present i n 100 mL of the formulat i o n (Table V I I I ) . 2 percent of 11.2 g i s 0.224 g. 0.224 g of calcium gluconate contains the same amount of calcium as 0.130 g of calcium D-saccharate. Hence 0.224 g of calcium gluconate was replaced with 0.130 g of calcium D-saccharate. c e q u i v a l e n t to a replacement of 0,2,5 and 10 percent of calcium gluconate r e s p e c t i v e l y . * Table V I I I Table IX - 70 -2.3 DEVELOPMENT OF PARENTERAL FORMULATIONS 2.3.1 Basic formula The USP (USP XX, 1980 ) s p e c i f i e s that calcium glucoheptonate i n j e c -t i o n must contain between 208 and 233 mg of calcium glucoheptonate per mL. Hence i t was decided to prepare formulations c o n t a i n i n g 223 mg of calcium glucoheptonate per mL ( i . e . 22.3% w/v). The formulat i o n contained no other a d d i t i v e s . Two methods of s t e r i l i z a t i o n were used. ( i ) a u t o c l a v i n g a t 121°C f o r 20 minutes ( i i ) f i l t r a t i o n through 0.22 ym membrane f i l t e r ( M i l l i p o r e ) For the parenteral f o r m u l a t i o n development s t u d i e s , only calcium glucoheptonate USP supplied by P f a n s t i e h l was used. 2.3.2 Use of s t a b i l i z i n g agents As i n o r a l f o r m u l a t i o n s , the p o s s i b l e use of calcium D-saccharate, calcium l a c t o b i o n a t e and disodium edetate as s t a b i l i z i n g agents was explored. A p a r t of the calcium glucoheptonate was replaced with calcium gluconate and the s t a b i l i t y of t h i s combination studied (Table X I I I ) . 2.3.3 Method of preparation of parenteral formulations Calcium glucoheptonate was d i s s o l v e d i n b o i l i n g water. I f the formulati o n contained a s t a b i l i z i n g agent, i t was mixed with the calcium glucoheptonate and both s o l i d s were d i s s o l v e d i n b o i l i n g water, the s o l u t i o n was allowed to cool and then s t e r i l i z e d by aut o c l a v i n g (at 121°C f o r 20 minutes) or membrane f i l t r a t i o n . - 71 -3. RESULTS AND DISCUSSION 3.1 STABILITY STUDIES OF ORAL FORMULATIONS These st u d i e s i n d i c a t e that a l l the o r a l formulations commenced p r e c i p i t a t i o n w i t h i n 6 months of preparation. However, the use of the s t a b i l i z i n g agents has decelerated the p r e c i p i t a t i o n r e a c t i o n , i n c e r t a i n f o r m u l a t i o n s . The c o n t r o l formulation c o n t a i n i n g sugar and calcium glucoheptonate USP ( P f a n s t i e h l ) (Table XI) commenced to densely p r e c i p i -tate soon a f t e r p r e p a r a t i o n . The use of calcium D-saccharate had a sub-s t a n t i a l s t a b i l i z i n g e f f e c t on t h i s f o r m u l a t i o n . Sugar seems to a c c e l e r a t e the p r e c i p i t a t i o n r e a c t i o n . Most of the formulations (not a l l ) c o n t a i n i n g sugar commenced p r e c i p i t a t i o n w i t h i n 3 months. Moreover, dense heavy p r e c i p i t a t i o n was observed only i n formulations c o n t a i n i n g sugar. Of the three s t a b i l i z i n g agents, calcium D-saccharate seemed to exert s l i g h t l y g reater s t a b i l i z i n g a c t i o n than calcium l a c t o b i o n a t e and disodium edetate. 3.2 STABILITY STUDIES OF PARENTERAL FORMULATIONS 3.2.1 S t e r i l i z a t i o n by a u t o c l a v i n g The p r e l i m i n a r y s t a b i l i t y r e s u l t s (Table X I I I ) suggest t h a t i n j e c t a b l e formulations s t e r i l i z e d by a u t o c l a v i n g do not p r e c i p i t a t e . 3.2.2 S t e r i l i z a t i o n by f i l t r a t i o n Among the various s t a b i l i z i n g agents attempted, only calcium D-saccharate was able to s t a b i l i z e formulations s t e r i l i z e d by f i l t r a t i o n . Table XI. Oral calcium s y r u p - s t a b i l i t y s t u d i e s Calcium glucoheptonate USP - P f a n s t i e h l Formulation d e t a i l s Basic formula with sugar Basic formula with sodium cyclamate Evidence of p r e c i p i 1 3 6 tation.(months) 1 3 6 Control +++ ++ 2% of calcium gluconate replaced with calcium D-saccharate ++ 5% of calcium gluconate replaced with calcium D-saccharate + ++ 10% of calcium gluconate replaced with calcium D-saccharate + - - + 2% of calcium gluconate replaced with calcium l a c t o b i o n a t e +++ ++ 5% of calcium gluconate replaced with calcium l a c t o b i o n a t e + + + ++ 10% of calcium gluconate replaced with calcium l a c t o b i o n a t e +++ + 0.1% w/v disodium edetate added +++ ++ 0.2% w/v disodium edetate added +++ ++ 0.5% w/v disodium edetate added +++ - - ++ - c l e a r (no p r e c i p i t a t e ) + small amount of very f i n e p r e c i p i t a t e at the bottom of the c o n t a i n e r ; p r e c i p i t a t i o n apparent only on shaking the container ++ moderate amount of p r e c i p i t a t e +++ dense, heavy p r e c i p i t a t e Table X I I . Oral calcium s y r u p - s t a b i l i t y s t u d i e s  Calcium glucoheptonate - Givaudan Formulation d e t a i l s Basic with formula sugar Basic formula with sodium cyclamate 1 Evidence of prec 3 6 ' i p i t a t i o n (months) 1 3 6 Control ++ + 2% of calcium gluconate replaced with calcium D-saccharate - + + - •-• + 5% of calcium gluconate replaced with calcium D-saccharate - + + - - + 10% of calcium gluconate replaced with calcium D-saccharate - + + + 2% of calcium gluconate replaced with calcium l a c t o b i o n a t e + + + + 5% of calcium gluconate replaced with calcium l a c t o b i o n a t e + + + + 10% of calcium gluconate replaced with calcium l a c t o b i o n a t e + + + + 0.1% w/v disodium edetate added + + + + 0.2% w/v disodium edetate added + + + + 0.5% w/v disodium edetate added + + + + - c l e a r (no p r e c i p i t a t e ) + small amount of very f i n e p r e c i p i t a t e at the. bottom of the c o n t a i n e r ; p r e c i p i t a t i o n apparent only on shaking the container ++ moderate amount of p r e c i p i t a t e +++ dense, heavy p r e c i p i t a t e Table XIII. Calcium i n j e c t i o n - s t a b i l i t y s t u d i e s Calcium Glucoheptonate USP - P f a n s t i e h l Formulation d e t a i l s Autoclaved F i l t e r e d Evidence, of prec 1 3 6 i p i t a t i o n (months) 1 3 6 22.3% w/v s o l u t i o n of calcium glucoheptonate - - - +++ 2.5% of calcium glucoheptonate replaced with calcium D-saccharate - - - - - -5.0% of calcium glucoheptonate replaced with calcium D-saccharate - - - - - -2.5% of calcium glucoheptonate replaced with calcium l a c t o b i o n a t e - +++ 5.0% of calcium glucoheptonate replaced with calcium l a c t o b i o n a t e - - - +++ 25.0% of calcium glucoheptonate replaced with calcium gluconate _ +++ 50.0% of calcium glucoheptonate replaced with calcium gluconate - - - -0.1% w/v disodium edetate added +++ 0.2% w/v disodium edetate added - - - +++ - c l e a r (no p r e c i p i t a t e ) +++ dense, heavy p r e c i p i t a t e - 75 -Even when as l i t t l e as 2.5 percent of calcium glucoheptonate was replaced w i t h calcium saccharate, the formulations have been s t a b l e f o r more than 6 months. When 25 percent of calcium .glucoheptonate was replaced with calcium gluconate, the formula t i o n was unstable. However, r e p l a c i n g 50 percent of calcium glucoheptonate with calcium gluconate r e s u l t e d i n a stab l e s o l u t i o n . I n i t i a l l y calcium glucoheptonate was d i s s o l v e d i n c o l d water and then calcium D-saccharate was added to i t and t h i s system warmed ge n t l y u n t i l complete d i s s o l u t i o n of calcium D-saccharate was achieved. However, i t was observed t h a t calcium D-saccharate was unable to exert any s t a b i l i z i n g a c t i o n under such c o n d i t i o n s . On the other hand, when calcium glucoheptonate and calcium D-saccharate were added to b o i l i n g water, the formul a t i o n s were found to be s t a b l e . This suggests that probably a s t a b l e complex i s formed and t h i s r e a c t i o n takes place only at high temperature. The s t a b i l i t y s t udies of the parenteral formulations are co n t i n u i n g . I t i s planned to observe the formulations f o r a t o t a l period o f two years a f t e r t h e i r p r e p a r a t i o n . - 76 -SUMMARY 1. Only the a epimer of calcium glucoheptonate i s o f f i c i a l i n the USP. Studies i n d i c a t e that the pure a epimer i s extremely unstable i n s o l u t i o n . On the other hand, a mixture c o n t a i n i n g nearly equal proportions of the ct and 6 epimers produces s o l u t i o n s having a prolonged s t a b i l i t y . 2. A gas chromatographic method has been developed f o r the i d e n t i f i c a t i o n and e s t i m a t i o n of r e l a t i v e proportions of the a and 8 epimers of calcium glucoheptonate. 3. The i n s t a b i l i t y of calcium glucoheptonate i n s o l u t i o n s can be a t t r i b u t e d t o : ( i ) change from an unstable to a s t a b l e form ( i i ) presence of seed c r y s t a l s ( i i i ) d i f f e r i n g proportions of the a and B forms i n the calcium glucoheptonate obtained from various sources. 4. By the use of elemental a n a l y s i s , X-ray d i f f r a c t i o n , IR spectroscopy, DSC and GC-MS s t u d i e s , the p r e c i p i t a t e obtained from s o l u t i o n s of calcium glucoheptonate has been i d e n t i f i e d as a c r y s t a l l i n e hydrate of calcium glucoheptonate. 5. Attempts to develop a s t a b l e , o r a l f o r m u l a t i o n of calcium gluco-heptonate have been only p a r t i a l l y s u c c e s s f u l . - 77 -6. A s t a b l e parental formulation of calcium glucoheptonate can be prepared by a u t o c l a v i n g the f i n a l s o l u t i o n . I f s t e r i l i z a t i o n by f i l t r a t i o n i s d e s i r e d , then the s o l u t i o n can be s t a b i l i z e d with calcium D-saccharate or by r e p l a c i n g 50 percent of calcium gluco-heptonate with calcium gluconate. - 78 -REFERENCES Aguiar, A.J. and Zelmer, J.E. (1969). D i s s o l u t i o n behavior of polymorphs of chloramphenicol palmitate and mefenamic a c i d . J . Pharm. S c i . 58, 983-987. Black, D.B. and Lovering, E.G. (1978). The r e c r y s t a l 1 i z a t i o n of amorphous d i g o x i n . J . Pharm. Pharmac. 310, 380. B u r c h f i e l d , H.P. and S t o r r s , E.E. (1962). Biochemical a p p l i c a t i o n s of gas chromatography. Academic, New York, p. 79. Clements, J.A. and P o p l i , S.D. (1973). The preparation and p r o p e r t i e s of c r y s t a l m o d i f i c a t i o n s of meprobamate. Can. J . Pharm. S c i . 8_, 88-92. Chiou, W.L. and Kyle, L.E. (1979). 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S c i . 60, 1485-1488'f H a l e b l i a n , J.K., Koda, R.T. and B i l e s , J.A. (1971b). Comparison of d i s s o l u t i o n rates of d i f f e r e n t c r y s t a l l i n e phases of f l u p r e d n i s o -lone by i n v i t r o and i n vivo methods. J . Pharm. S c i . 60, 1488-1491. H o l s t e i n , A.G. (1980). P f a n s t i e h l L aboratories Inc., Waukegan, I l l i n o i s , personal communication. I s b e l l , H.S. and Frush, H.L. (1963) i n W h i s t l e r , R.L. and Wolfrow, M.L. (Eds.). Methods i n carbohydrate chemistry. V o l . I I . Reactions of carbohydrates, Academic, New York, pp. 16 and 17. K j o l b e r g , 0. and V e l l a n , E. (1966). Chromatographic separation of sugars V. Separation of some epimeric heptonolactones. Acta Chem. Scand. 20, 2081-2085. - 79 -Laker, M.F. (1980). Estimation of neutral sugars and sugar a l c o h o l s i n b i o l o g i c a l f l u i d s by g a s - l i q u i d chromatography. J . Chromatogr. 184, 457-470. 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