Elsevier

Journal of Chromatography B

Volume 855, Issue 2, 15 August 2007, Pages 290-294
Journal of Chromatography B

Short communication
Rapid quantitation of testosterone hydroxyl metabolites by ultra-performance liquid chromatography and mass spectrometry

https://doi.org/10.1016/j.jchromb.2007.05.022Get rights and content

Abstract

A rapid and sensitive ultra-performance liquid chromatography and mass spectrometry (UPLC/MS) method was developed to simultaneously quantify seven monohydroxyl testosterone metabolites (16α-, 2α-, 7α-, 6α-, 2β-, 6β-, and 16β-hydroxyl testosterones) in rat liver microsomes. The UPLC system used a short 1.7-μm particle size column coupled to a Sciex 4000 Q trap in multiple reaction monitor (MRM) mode. All hydroxyl testosterones were resolved within 2.5 min. A 4-day validation was performed to determine the linearity, repeatability, reproducibility and accuracy of the method in rat liver microsomes. This method is applicable to the measurement of the testosterone hydroxylase activity in biological matrices such as the liver microsome incubates.

Introduction

Cytochrome P450 (CYP) family metabolizes a large number of structurally diverse endogenous and exogenous compounds. When a chemical inhibits CYPs, such as CYP3A4, it would likely to cause drug–drug interaction. The induction of CYPs could also potentially impair normal biosynthesis and metabolism of endogenous molecules and thereby interfere with regular biological processes leading to toxicological consequences. CYP inhibition and induction by drug candidates are closely monitored in drug discovery and development. Formation of different hydroxyl testosterone (OHT) metabolites has been widely used as the important marker for specific cytochrome P450 enzyme activities such as the activity of CYP3A4 in human [1], [2], [3]; CYP3A12 in dog [4]; CYP2A1, CYP2B1/2, CYP2C11 and CYP3A1/2 in rat liver microsomes [5], [6], [7].

HPLC methods with UV detection were originally used to quantify OHTs [8], [9]. Several LC/MS methods have been developed to improve detection limits [10], [11]. HPLC methods with on-line column switching techniques were applied to further increase sensitivity [12], [13]. Since OHTs share the same parent and product ion patterns, it is impossible to use mass spectrometry to distinguish them without good chromatographic separation. Therefore, to positively identify and quantify one of the OHTs, it is necessary to have a LC method that can separate these OHTs.

Most HPLC methods reported so far require a complex gradient and long chromatographic run time (usually 30 min or longer) to obtain OHT separation. Recently, ultra-performance liquid chromatography (UPLC) with <2 μm particle size column was introduced for rapid and efficient compound separation [14]. UPLC coupled with mass spectrometry is increasingly being used for rapid multiple component quantitation [15] for in vitro [16] and in vivo [17] metabolite characterization. However, no reported chromatographic method is available for full separation of OHTs, especially the efficient separation of 6β-hydroxyltestosterone, a testosterone metabolite by CYP3A, from 7α-hydroxyltestosterone. No unique mass spectrometry method is available for unambiguous identification of these OHTs simultaneously. The objective of this study was to develop a sensitive, rapid and simple UPLC/MS method to separate and simultaneously quantify seven hydroxyl testosterone metabolites.

Section snippets

Chemicals and microsomal samples

Seven most commonly analyzed hydroxyl testosterones metabolites, 16α-hydroxytestosterone (16α-OHT), 2α-hydroxyltestosterone (2α-OHT), 7α-hydroxyltestosterone (7α-OHT), 6α-hydroxyltestosterone (6α-OHT), 2β-hydroxytestosterone (2β-OHT), 6β-hydroxytestosterone (6β-OHT) and 16β-hydroxytestosterone (16β-OHT) of analytical grade were obtained from Steraloids (Wilton, NH, USA). The analytical grade corticosterone was obtained from Sigma–Aldrich (Saint Louis, MO, USA). All organic solvents were of HPLC

UPLC separation and detection of hydroxyl testosterone metabolites

Since OHTs share the same mass transition fragments in mass spectrometer analysis, LC separation before MS analysis is the only way for their confirmation and quantitation in biological matrices, such as the liver microsomes. The UPLC method developed from this study was able to achieve baseline separation for the seven OHTs within 2.5 min (Fig. 1). The separation of 6β-OHT from 7α-OHT is critical for testosterone based liver microsomes assays monitoring CYP 3A activity. The 2.1 mm × 50 mm BEH C18

Conclusion

A sensitive, rapid and simple UPLC/MS/MS method was developed and partially validated to simultaneously quantify 16α-, 2α-, 7α-, 6α-, 2β-, 6β-, and 16β-OHTs in rat liver microsomal incubates. The UPLC/MS/MS method covers a dynamic range from 0.005 to 5 μg/mL with good accuracy and reproducibility. This method reduces analysis time to less than 5 min. It is suitable for quantitation of hydroxyl testosterone metabolites in liver microsomes generated from P450 inhibition and induction assays.

Acknowledgment

We thank Dr. Scott Grimm for critical comments and suggestions throughout this study.

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