Journal of Chromatography B: Biomedical Sciences and Applications
Screening of eltanolone metabolites in dog urine by anion-exchange/reversed-phase liquid chromatography and mass spectrometry
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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