Determination of dichloroacetate and its metabolites in human plasma by gas chromatography–mass spectrometry1

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Abstract

Sodium dichloroacetate (DCA) is an investigational drug for the treatment of lactic acidosis, and is also a putative environmental toxicant. We developed and validated a gas chromatography–mass spectrometry (GC–MS) technique that simultaneously measures lactate, DCA and its metabolites, monochloroacetate (MCA), glyoxylate, glycolate and oxalate in human plasma. Following administration of [13C1,2]DCA to healthy volunteers, blood samples were collected at various time points and the drug and its metabolites present in plasma were derivatized to their methyl esters by reacting with 12% boron trifluoride–methanol complex. The methyl esters were extracted with methylene chloride and analyzed by GC–MS. The quantitation limits of DCA and metabolites ranged between 0.3 and 1.5 μM. The coefficients of variation of the standards within the entire calibration range were between 0.3 and 14.5%. The bias values ranged between −16.3% and 18.7%. Total recoveries from derivatization and extraction were between 46.9% and 78.5%. The coefficients (r2) of linear regression of the calibration curves were 0.9882 to 0.9996.

Introduction

Sodium dichloroacetate (DCA) is an investigational drug with a potentially broad clinical spectrum, including utility in the treatment of acquired and congenital forms of lactic acidosis [1]. DCA is also a product of water chlorination 2, 3, and is a metabolite of certain industrial chemicals 4, 5and drugs, such as chloral hydrate [6]and chloramphenicol [7]. Neurotoxic and hepatotoxic effects of DCA in animals have focused attention on its putative role as an environmental hazard to humans, despite its relatively wide therapeutic index when administered chronically or acutely to children and adults in doses of 12.5 to 50 mg/kg of body weight [1].

Considerable interest exists, therefore, in evaluating the kinetics, metabolism and toxicology of DCA in humans. Accordingly, several analytical techniques have been developed previously to quantitate DCA and its metabolites in biological fluids, with limited success. High-performance liquid chromatography (HPLC), using an ultraviolet-visible (UV–Vis) detection, is applicable only for rather high drug concentrations, because DCA has a low UV absorption [8]. Augmenting the sensitivity of HPLC with a radioactive flow detector can be achieved 9, 10, but is impractical for clinical investigations. When DCA concentrations were measured by gas chromatography (GC) using an electron capture detector (ECD), its metabolites glyoxylate, glycolate and oxalate could not be detected 11, 12, 13, 14. Given the selectivity and sensitivity of mass spectrometry (MS), GC–MS methods have been utilized to quantitate DCA, but not its metabolites 15, 16. In this study, we developed and validated a sensitive and selective GC–MS method that is applicable to the study of DCA pharmacokinetics and metabolism in humans. Plasma lactate was measured as a means of correlating the pharmacokinetics with the pharmacodynamics of DCA.

Section snippets

Materials

Chemicals used in this investigation were [13C1,2]DCA sodium salt (>99.5%, custom synthesized by Cambridge Isotope Laboratories, Inc., Andover, MA, USA), [12C]DCA sodium salt (>99.8%) and oxalic acid dimethyl ester (TCI America, Portland, OR, USA), MCA, glycolic acid, glyoxylic acid monohydrate, 85% (d,l)-lactic acid and methyl esters of DCA, MCA, oxalic acid, (R)-(+)-lactic acid, 4-chlorobutyric acid (CBA) and 12% boron trifluoride–methanol complex (Aldrich Chemical Company, Inc., Milwaukee,

Typical total ion chromatograms (TIC) and internal standard

Presented in Fig. 1 are the TIC of the authentic compounds spiked in water (panel A) and the TIC of a plasma sample of a healthy volunteer collected one hour after intravenous administration of 25 mg/kg DCA ([12C]DCA/[13C]DCA, 50%/50%) (panel B). After investigating a number of halogenated acids, CBA, which did not interfere with analysis of other compounds, was chosen as the internal standard. The concentrations of DCA, [13C]DCA, MCA, glyoxylate, oxalate, lactate and CBA were 124.17, 84.97,

Conclusions

A GC–MS method was successfully developed and validated for simultaneous determination of lactate, DCA and its metabolites MCA, glyoxylate and oxalate in human plasma. Its accuracy and precision are comparable to those other HPLC and GC assays, while its sensitivity and selectivity are superior. In addition, derivatization of samples by our technique is simpler than silylation of organic acids [28], and it can be carried out in an aqueous environment, resulting in a high percentage yield

Acknowledgements

This study was supported by NIH grants R01ES07355, P42ES07375 and RR00082.

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