Liquid chromatography–mass spectrometry (LC–MS) of steroid hormone metabolites and its applications

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Abstract

Advances in liquid chromatography–mass spectrometry (LC–MS) can be used to measure steroid hormone metabolites in vitro and in vivo. We find that LC–electrospray ionization (ESI)-MS using a LCQ ion trap mass spectrometer in the negative ion mode can be used to monitor the product profile that results from 5α-dihydrotestosterone (DHT)-17β-glucuronide, DHT-17β-sulfate, and tibolone-17β-sulfate reduction catalyzed by human members of the aldo–keto reductase (AKR) 1C subfamily and assign kinetic constants to these reactions. We also developed a stable isotope dilution LC–electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the quantitative analysis of estrone (E1) and its metabolites as pentafluorobenzyl (PFB) derivatives in human plasma in the attomole range. The limit of detection for E1-PFB was 740 attomole on column. Separations can be performed using normal-phase LC because ionization takes place in the gas phase rather than in solution. This permits efficient separation of the regioisomeric 2- and 4-methoxy-E1. The method was validated for the simultaneous analysis of plasma E2 and its metabolites: 2-methoxy-E2, 4-methoxy-E2, 16α-hydroxy-E2, estrone (E1), 2-methoxy-E1, 4-methoxy-EI, and 16α-hydroxy-E1 from 5 pg/mL to 2000 pg/mL. Our LC–MS methods have sufficient sensitivity to detect steroid hormone levels in prostate and breast tumors and should aid their molecular diagnosis and treatment.

Introduction

Radioimmunoassay or ELISA based methods were once considered state-of-the-art methods for measuring steroid metabolites in biospecimens. These approaches now appear to be fraught with difficultly. First, they can only measure one analyte at a time, thus multiple assays are required to measure all the metabolites from a single steroid hormone. Second, steroid metabolites have highly related structures and in a biospecimen mixtures of stereoisomers, regioisomers or compounds that differ by only the substitution of a carbonyl group for an alcohol exist. It is thus not possible to control for interference in the immunoassay from both known and unknown structurally related steroids that may be present in the biological matrix. Third, these immunological approaches do not give any structural validation of the analyte. Fourth, in many instances antisera do not exist for all the steroid metabolites of interest to allow immunodetection in the first place. This is certainly true for the detection of conjugated steroids.

The reliability of radioimmunoassays for steroid hormones has also been questioned by position papers which have documented the large inter-laboratory variability that exists in measuring plasma testosterone [1], [2], [3] and the difficulty in measuring 17β-estradiol (E2) and its metabolites in plasma and urine [4], [5], [6]. In contrast, gas chromatography–mass spectrometry (GC–MS) coupled with stable isotope dilution methodology is sensitive, specific, and accurate and has been used for the quantitative analysis of steroid hormones in biological samples such as urine and plasma [7], [8]. Unfortunately this method requires extremely tedious extraction and derivatization procedures for each sample. However, when used in conjunction with electron capture negative chemical ionization and tandem MS, very low detection limits can be obtained for plasma E2 (0.063 pg/mL) [9]. A further drawback of the GC–MS methods is that, steroid conjugates cannot be analyzed directly. LC–MS can now circumvent many of these problems. We have developed negative ion LC–ESI/MS in order to analyze multiple steroid conjugates directly. We demonstrate the utility of the method by showing that it can be used to conduct product profiling of the enzymatic reduction of endogenous DHT-conjugates and conjugates derived from the hormone replacement therapeutic tibolone catalyzed by members of the aldo–keto reductase (AKR) 1C subfamily. By contrast, LC–ESI/MS of underivatized estrogens are relatively insensitive in both positive and negative ionization modes so it cannot be used to determine the concentrations of estrogens and their metabolites which are present in the low pg/mL range [10]. To circumvent this problem, we have developed stable isotope dilution LC–ECAPCI/MS methodology, which can detect estrogen PFB derivatives in the attomole range on column [10]. This has made it possible to quantify multiple estrogen metabolites with high sensitivity in the same chromatographic run. We demonstrate the utility of this approach by analyzing E1 and E2, together with their corresponding 2- and 4-methoxy and 16 α-hydroxy metabolites in plasma. These methods can be adapted to measure targeted steroid metabolomes within prostate and breast tumor biopsy samples.

Section snippets

Materials

DHT, DHT-17β-glucuronide (DHTG), DHT-17β-sulfate (DHTS), 3α-hydroxy-5α-androstane-17β-glucuronide (3α-Diol-17G), and 3β-hydroxy-5α-androstane-17β-sulfate (3β-Diol-17S) were obtained from Steraloids (Wilton, NH, USA). The latter compound was custom synthesized by Steraloids. E2, E1, 2-methoxy-E2, 4-methoxy-E2, 16α-hydroxy-E2, 2-methoxy-E1, 4-methoxy-E1, and 16α-hydroxy-E1 were obtained from Steraloids Inc. (Newport, RI). 16,16,17-[2H3]-E2, 2,4,17-[2H3]-16α-hydroxy-E2, and 2,4,16,16-[2H4]-E1 were

Metabolism of endogenous steroid conjugates by aldo–keto reductase (AKR) 1C subfamily enzymes

The four aldo–keto reductase (AKR) 1C subfamily members found in humans (AKR1C1–AKR1C4) have been shown to catalyze the NADPH dependent reduction of 3-ketosteroids to yield 3α- or 3β-hydroxysteroids with different stereochemical preferences based on steroid substrate. For example, AKR1C2 is predominately a 3α-HSD with DHT; while AKR1C1 is predominately a 3β-HSD with the same substrate [12]. By contrast the same two enzymes convert tibolone (a hormone replacement therapeutic pro-drug) only to

Discussion

Hydroxysteroid dehydrogenases (HSDs) belong to two gene superfamilies (AKRs) and the short-chain dehydrogenase/reductases [25]. Historically many substrate specificity studies have been performed with these enzymes in which the basis of the assay is to monitor the formation or consumption of NAD(P)H linked to the oxidation or reduction of a hydroxy or ketosteroid. Assay validation is usually performed in discontinuous assays which rely upon separating substrate from product by TLC and

Acknowledgements

This work was supported by the following research grants: P30ES013587 and R01CA90744 from the National Institutes of Health and a Prostate Cancer Foundation Challenge Grant (TMP), R01CA091016 (IAB), and a Pilot-Project Grant awarded with P30ES13587 (YJ).

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    Current address: Department of Bioanalytical Chemistry, Graduate School of Pharmaceutical Science, Tohoku University, Aoba-ku, Sendai 980-8578, Japan.

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    Current address: Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.

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