Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry

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Abstract

Strong anion-exchange (SAX) chromatography and reversed-phase liquid chromatography (RPLC) followed by different mass spectrometric techniques for the separation and identification of conjugated and unconjugated 14C-labelled eltanolone (5β-Pregnan-3α-ol-20-one) metabolites in biological fluids are presented. Conjugates of estradiol were used as model compounds for the development of a SAX based group separation of neutral steroids, glucuronides, sulfates and di-conjugated steroids. The usefulness of the technique is demonstrated by the analysis of 14C-labelled eltanolone metabolites in dog urine. The analytical SAX column used prior to RPLC improved the capacity to separate the metabolites from each other and from endogenous components, compared to a single reversed-phase system. Liquid chromatography negative ion electrospray-mass spectrometry (LC–ESI-MS) was used for the molecular mass determination of conjugated eltanolone metabolites. Unconjugated metabolites and hydrolysed conjugates were identified using gas chromatography–mass spectrometry with an electron impact ion source (GC–MS) after trimethylsilyl (TMS) derivatization. An unexpected finding in dog urine was the diglucuronide formation of eltanolone (presumably after enolisation of its carbonyl group).

Introduction

In drug metabolism studies steroids and their metabolites generate a very complex separation pattern when a single reversed-phase column is used. Separation systems consisting of coupled columns with different separation mechanisms could be useful in these kind of studies. By expanding the separation into two dimensions, sample components which are unresolved in the first dimension can often be separated in the second. The theoretical overall peak capacity is the product of the peak capacity of each of the two systems, provided that the two columns have different separation mechanisms 1, 2.

In the present study, a system has been developed where the reversed-phase liquid chromatography (RPLC) is preceded by strong anion-exchange (SAX) chromatography, a very useful technique in steroid screening as it separates different conjugates from each other. Separation techniques using solid-phase extraction with anion-exchange columns during sample preparation have previously been reported 3, 4, 5. Here, the separation efficiency has been improved by the use of an analytical SAX column prior to RPLC.

The determination of the molecular mass of the conjugated metabolites by liquid chromatography negative ion electrospray mass spectrometry (LC–ESI-MS) is important since unpredictable elution order in the SAX chromatography has been observed. LC–ESI-MS, an atmospheric pressure ionization technique, has become routine during the last decade in studies of drug metabolism, especially for thermally unstable and ionized compounds of relatively high molecular masses such as drug conjugates 6, 7.

Unconjugated and hydrolysed conjugated analytes were identified using gas chromatography electron impact mass spectrometry (GC–MS) after trimethylsilyl (TMS) derivatization. This technique is well established and widely used [8]. Because of its limited capacity to analyse thermally unstable and non-volatile components such as conjugated and highly hydroxylated steroids, LC–ESI-MS is a good complement to GC–MS.

The aim of the present study was to develop a method suitable for the separation and direct identification of conjugated and phase I metabolites of eltanolone in biological fluids. The usefulness of the method is demonstrated by an investigation of the metabolism of eltanolone in the dog.

Section snippets

Reference compounds and chemicals

The 14C-eltanolone emulsion (4 mg/ml, specific activity 331 kBq/ml) was from Pharmacia and Upjohn, Stockholm, Sweden. The eltanolone (5β-Pregnan-3α-ol-20-one) used as a reference standard was purchased from Schering AG, Berlin, Germany.

β-Glucuronidase, Type H-1, 345 200 units/g (Helix Pomatia), 5β-Pregnane-3α,20α-diol, 5β-Pregnane-3α,20β-diol, 5β-Pregnane-3α,6α-diol-20-one, 5β-Pregnane-3α,20α-diol glucuronide, 17β-estradiol-3-sulfate, 17β-estradiol-3-(β-d-glucuronide), β-estradiol-3-sulfate-17-

Separation methods and ionization techniques

The estradiol conjugates were used as reference substances since they were commercially available and in contrast to the eltanolone derivatives detectable by UV. The elution order was found to be the following: unconjugated estradiol, estradiol monoglucuronide, estradiol disulfate, estradiol monosulfate and estradiol-3-sulfate-17-glucuronide. The elution order of the monosulfate and the disulfate was unexpected since the conjugates were expected to elute in the order of their acidity and charge

Conclusion

Gradient elution used in several dimensions provided a large difference in selectivity, which allowed efficient isolation and identification of eltanolone metabolites in dog urine, with a minimum of separation optimization. The separation technique described has with individual modifications been proved useful in metabolism studies of eltanolone in rat, dog, monkey and man. The separation system is flexible, which is necessary as the degree of hydroxylation and conjugation of the metabolites

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