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Negative ion chemical ionization GCMS analysis of valproic acid and its metabolites Kassahun, Kelem


Valproic acid (VPA) is a major anticonvulsant drug widely used in the treatment of absence seizures. VPA is extensively metabolized in humans. Several VPA metabolites possess anticonvulsant activity and other metabolites are implicated in rare but fatal cases of hepatotoxicity. A highly sensitive and more specific analytical method was required to analyze the large number of VPA metabolites, some of which are present at trace levels. The objective of this study was to develop such a method and to make a preliminary application of the method to the determination of trace VPA levels and to search for new VPA metabolites. The suitability of analyzing halogenated derivatives of VPA and its metabolites by negative ion chemical ionization (NICI) GCMS was evaluated for the desired sensitivity and specificity. An assay was thus developed for VPA in serum and saliva based on NICI-GCMS of the pentafluorobenzyl (PFB) derivative. The NICI spectrum of the PFB ester of VPA was dominated by a single fragment ion, the m/z 143 ([M-181]⁻) ion. When the m/z 143 ion was monitored the lower limit of detection was 2 ng/mL of VPA in serum or saliva. Using [²H₆]-VPA as the internal standard, the intra- and inter-assay variations were less than 10 % at serum VPA concentrations of 10 to 800 ng/mL. Linearity was observed over the concentration range of 10 ng/mL to 25 µg/mL. The NICI assay was employed to quantitate VPA in serum (total and free) and saliva in five healthy volunteers who took part in a drug interaction study between VPA and carbamazepine (CBZ). A total of 63 paired saliva and serum samples were analyzed by NICI-GCMS; 33 before the administration of CBZ and 30 after CBZ. The % decrease in the average VPA concentration after CBZ was 27.91 ± 3.48, 36.85 ± 13.64, and 48.13 ± 7.70, for serum total, serum free and saliva VPA, respectively. There was a significant reduction (p<0.025) in the average VPA concentration in all three biological fluids. The average saliva to serum free VPA ratio was 18.92% ± 6.25 before CBZ and 16.37% ± 2.82 following CBZ. The average saliva to serum total VPA ratio was 2.43% ± 0.86 before CBZ and 1.67% ± 0.50 following CBZ, indicating that the saliva to serum total VPA ratio was concentration dependent. A strong correlation was found between saliva and both serum free (r = 0.9035 ± 0.0784) and serum total VPA (0.9058 ± 0.0450) (after CBZ). The free fraction of VPA did not increase after CBZ administration suggesting that the decrease in VPA concentration after CBZ was not related to changes in the free fraction of VPA. PFB derivative formation of VPA metabolites was facile and resulted in uniform derivatization of all metabolites studied. In the NICI mass spectra most of the ion current was carried by the [M-181]⁻ fragment ion, the only exception being that of 3-keto VPA. The base peak in the NICI spectrum of PFB derivatized 3-keto VPA was [M-181-C0₂]⁻. Isolated metabolites were identified with the help of twin ions (deuterated and undeuterated) in the mass spectra and by comparison of mass spectra and retention times with synthetic reference compounds. Urine or serum metabolites were analyzed in one chromatographic run and SIM chromatograms obtained. Serum and urine controls showed no interfering peaks and the analytical method appears suitable for a sensitive assay of VPA metabolites. The NICI method employing PFB derivatives was sensitive enough to detect VPA metabolites in saliva. Seven metabolites were detected. The ratio of Z to E isomers of 2-ene VPA was much greater in saliva than in serum (3.82 vs. 0.458), suggesting differences in the transport or plasma protein binding properties of these two isomers. A new VPA metabolite, assigned the structure 4¹-keto-2-ene VPA was detected in urine. The mass spectrum and retention time of this new metabolite matched that of one compound which was present in a synthetic mixture containing 4¹-keto-2-ene VPA. Another new metabolite which appears to be 2-(2¹-propenyl)-glutaric acid was also detected in urine. The synthesis of 4¹-keto-2-ene VPA was attempted using two different synthetic methods. The first method which involved the dehydrogenation of the 0-TMS dialkyl ketene acetal of ethyl 2-propyl-4-oxopentanoate apparently resulted in the formation of the positional isomer, 4-keto-2-ene VPA. The second synthetic route was based on the dehydration of 4-carboethoxy-2-ethylenethioketal-5-hydroxyheptane and produced 4¹-keto-2-ene VPA. However, it was not possible to isolate sufficient product for NMR characterization.

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