Sensitive and specific liquid chromatographic–tandem mass spectrometric assay for dihydroergotamine and its major metabolite in human plasma

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Abstract

A sensitive and specific procedure for the simultaneous determination of dihydroergotamine (DHE) and its 8′-hydroxylated metabolite (8′-OH-DHE) in human plasma was developed and validated. The analytes were extracted from plasma samples by liquid–liquid extraction, separated through a Zorbax C18 column (50×2.1 mm I.D.) and detected by tandem mass spectrometry with an electrospray ionization interface. Caroverine was used as the internal standard. The method has a lower limit of quantitation (LOQ) of 10.0 and 11.0 pg/ml for DHE and 8′-OH-DHE, respectively. The intra- and inter-run precision was measured to be below 9.1% for both DHE and 8′-OH-DHE. The inter-run accuracy was within 4% for the analytes. The overall extraction recoveries of DHE and 8′-OH-DHE were determined to be about 58 and 52% on average, respectively. The chromatographic run time was approximately 2.5 min. More than 120 samples could be assayed daily with this method, including sample preparation, data acquisition and processing. The method developed was successfully used to investigate plasma concentrations of DHE and 8′-OH-DHE in a pharmacokinetic study of volunteers who received DHE orally.

Introduction

The ergot alkaloids are a family of chemical entities that have many pharmacologic effects. Their diversity results from their interaction with multiple receptors, their variable receptor affinity and intrinsic activity, and their variable organ-specific receptor access. Ergotamine was one of the first ergot alkaloids to be isolated. Dihydroergotamine (DHE, Fig. 1) is synthesized by reducing an unsaturated bond in ergotamine. This modification results in a changed pharmacologic profile. DHE exhibits greater α-adrenergic antagonist activity and much less potent arterial vasoconstriction and emetic potential, which is used to prevent or treat vascular headaches and orthostatic hypotension in clinical treatments [1]. After oral administration, DHE undergoes extensive first-pass metabolism, resulting in very low plasma concentrations from pg/ml to low ng/ml. Several metabolites have been identified in humans and 8′-hydroxydihydroergotamine (8′-OH-DHE, Fig. 1) is expected to be the most important one [2], [3]. Plasma concentrations of the metabolite after oral administration of DHE were shown to exceed those of the parent compound by several fold and its pharmacodynamic activity could be demonstrated in receptor binding studies [3].

Analytical techniques with sufficient sensitivity and specificity are needed to measure the low concentrations of DHE and its active metabolite in biological fluids. Preliminary pharmacokinetic parameters of DHE were obtained by radiolabelled drug and radioimmunoassay (RIA) methods, which were very sensitive, but lacked selectivity [4], [5], [6]. Meanwhile, some high-performance liquid chromatography (HPLC) methods with fluorescence detection were described with the limit of quantitation (LOQ) ranging from 0.1 to 0.6 ng/ml using 2.0 or 3.0 ml plasma, which did not offer a sensitivity necessary for the study of DHE pharmacokinetics following oral administration to volunteers [7], [8], [9].

The purpose of this study is to develop a highly sensitive and specific liquid chromatographic–tandem mass spectrometric (LC–MS–MS) method for simultaneous determination of DHE and its major metabolite 8′-OH-DHE in plasma suitable for use in human pharmacokinetic investigations after oral administration of DHE.

Section snippets

Chemicals and reagents

DHE mesylate was obtained from the United States Pharmacopoeia (Rockville, MD, USA). 8′-OH-DHE (95.7% by HPLC) was supplied by Dr. Willmar Schwabe Pharmaceuticals (Karlsruhe, Germany), which was prepared from rat liver microsomes. 1H- and 13C-nuclear magnetic resonance (NMR) spectra indicated that 8′-OH-DHE was a mixture composed of two stereoisomers 8′α- and 8′β-OH-DHE in a ratio of 78:22. Caroverine (internal standard, I.S.) was obtained from Phafag (Schaanwald, Germany). Acetonitrile and

Mass spectrometry

DHE, 8′-OH-DHE and the I.S. caroverine were at first characterized by MS and MS–MS by flow injection analysis to ascertain their precursor ions and to select product ions for use in SRM, respectively.

Fig. 2 shows the full-scan Q1 mass spectra of DHE and 8′-OH-DHE in the positive-ion mode. Both formed protonated molecular ions [M+H]+ as major ion peaks. Small amounts of solvent clustered ions [M+H+CH3CN]+ were also found. The product ion spectra of [M+H]+ showed several fragment ions at m/z 253,

Conclusion

An LC–MS–MS method was developed and validated for the simultaneous determination of DHE and 8′-OH-DHE in human plasma. The method is very sensitive, selective and reliable with an LOQ of 10.0 pg/ml for DHE and 11.0 pg/ml for 8′-OH-DHE. It was proved superior in sensitivity and selectivity than the reported HPLC assay with fluorescence detection, or the RIA methods. The method was used successfully to evaluate the pharmacokinetics of DHE and 8′-OH-DHE after an oral administration. More than 120

Acknowledgements

This paper was supported in part by the grant 39930180 of the National Natural Science Foundation of China. The authors would like to thank Farmasan Arzneimittel GmbH, Germany, for the support of the clinical study.

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