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

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 5alpha-dihydrotestosterone (DHT)-17beta-glucuronide, DHT-17beta-sulfate, and tibolone-17beta-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 740attomole 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, 16alpha-hydroxy-E2, estrone (E1), 2-methoxy-E1, 4-methoxy-EI, and 16alpha-hydroxy-E1 from 5pg/mL to 2000pg/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.

Fig. 1

LC-MS analysis of the reduction of DHTG catalyzed by human AKR1C isoforms. The ion chromatogram (m/z 450–500) of a mixture of authentic standards of DHTG and 3α-Diol-17-G; B through F, corresponding ion chromatograms of reaction samples containing no enzyme (B) and AKR1C1–4 (C–F). Samples were prepared as described in the experimental section. Reproduced with permission from the American Society of Biochemists and Molecular Biologists.

Fig. 2

LC-MS analysis of the reduction DHTS catalyzed by human AKR1C isoforms. The ion chromatogram (m/z 350–400) of a mixture of authentic standards of DHTS and 3β-Diol-17-S; B through F, corresponding ion chromatograms of reaction samples containing no enzyme (B) and AKR1C1–4 (C–F). Samples were prepared as described in the experimental section. Reproduced with permission from the American Society of Biochemists and Molecular Biologists.

Fig. 3

LC-MS analysis of the reduction of TibS catalyzed by human AKR1C isoforms. A, The ion chromatogram (m/z 200–400) of a mixture of authentic standards of TibS, 3α-OH-TibS, and 3β-OH-TibS; B through F, corresponding ion chromatograms of reaction samples containing no enzyme (B) and AKR1C1–4 (C–F). Samples were prepared as described in the experimental section. Reproduced with permission from the American Society of Biochemists and Molecular Biologists.

Fig. 4

Proposed mechanism for EC-APCI/MS. Reproduced with permission from Analytical chemistry

Fig. 5

Estrone derivatives that provide increased LC-MS sensitivity compared with underivatized estrone.

Fig. 6

Stable isotope dilution LC-ECAPCI/MRM/MS analysis of E2 and metabolites.

Fig. 7

Stable isotope dilution LC-ECAPCI/MRM/MS analysis of E1 and metabolites.

Fig. 8

Standard curves for E1 and E2 in the range of 5 pg/mL to 2000 pg/mL. Linear regression lies were obtained with r² values of 0.998 or better.