Simultaneous determination of dihydrotestosterone and its metabolites in mouse sera by LC-MS/MS with chemical derivatization

Abstract

Androgens play a vital role in prostate cancer development, and their elimination and blockade are essential in the disease management. DHT is the key ligand for androgen receptor (AR) in the prostate. It is locally synthesized from testosterone. In the prostate, DHT is predominantly metabolized to α-diol and β-diol. Recent studies indicate that impaired DHT catabolism is associated with prostate cancer, signifying the necessity of a sensitive quantitative method for the determination of DHT and its metabolites. In this work, an LC-MS/MS method for the simultaneous quantification of DHT and its metabolites was developed and validated. Steroid-free sera were prepared and used for the preparation of sera calibrators and quality controls (QCs). DHT and its metabolites along with their respective stable heavy isotope labeled analytes representing internal standards were first extracted with methyl tertiary-butyl ether (MTBE) and derivatized with picolinic acid (PA). The derivatized analytes were then extracted again with MTBE, dried under nitrogen and reconstituted in the mobile phase (80% methanol and 0.2% formic acid in water). Baseline chromatographic separation of the derivatized analytes was achieved isocratically on XTerra C18 column (2.1 × 100 mm) using the mobile phase at a flow rate of 0.25 mL/min. Quantitation was performed using multiple-reaction-monitoring mode with positive electrospray ionization. The method has calibration ranges from 0.0500 ng/mL to 50.0 ng/mL for DHT and its two metabolites with acceptable assay precision, accuracy, recovery, and matrix factor. It was applied to the determination of DHT and its metabolites in an animal study.

Establishment of a novel prognostic prediction model through bioinformatics analysis for prostate cancer based on ferroptosis-related genes and its application in immune cell infiltration

“…In prostate cells, AKR1C2 acts as a 3-ketosteroid reductase to reduce dihydrotestosterone (DHT) levels and block the activation of androgen receptor (26). Selective deletion of AKR1C2 in PCa may promote clonal expansion of tumor cells by reducing DHT metabolism and enhancing androgen-dependent cell proliferation (27). SRC is a proto-oncogenic non-receptor tyrosine kinase that plays a role in tumor cell proliferation, survival, and invasion.…”

Development of a Liquid Chromatography/Mass Spectrometry-Based Inhibition Assay for the Screening of Steroid 5-α Reductase in Human and Fish Cell Lines

“…Similar to the acylation reaction of fluorinated anhydrides and the hydroxyl group of the seventeenth carbon position in the steroid reaction, derivatization using PA showed a higher sensitivity in the detection of 17-OH steroids, such as corticosteroids, in ESI-LC/MS [ 17 ]. Recently, LC-MS based quantification methods for androgens such as DHT that utilize various sample sources were developed, and 5AR inhibition studies were conducted [ 16 , 18 , 19 , 20 , 21 , 22 ]. The method in the present study requires an additional derivatization step compared to the direct measurement.…”

“…However, compared to these reports, the LLOQ of DHT (14.5 pg/mL) in the present study showed higher sensitivity than hydroxylamine hydrochloride derivatization [ 18 ] or direct measurement [ 19 , 23 ] by using LLE after PA derivatization. Also, methods using solid-phase extraction have been developed for the detection of steroids, but these methods are not efficient in time and cost -effective compared with liquid-liquid extraction [ 16 , 24 , 25 ]. The present study also used 2 times the liquid-liquid extraction step using MTBE after and before derivatization, this process increased the recovery of target compounds from 69 to 74% to 89–108% [ 16 ].…”

“…Also, methods using solid-phase extraction have been developed for the detection of steroids, but these methods are not efficient in time and cost -effective compared with liquid-liquid extraction [ 16 , 24 , 25 ]. The present study also used 2 times the liquid-liquid extraction step using MTBE after and before derivatization, this process increased the recovery of target compounds from 69 to 74% to 89–108% [ 16 ]. The lower limit of quantification of other studies using spectrophotometric method for DHT were from 0.2–10 nM [ 12 , 22 , 26 ], and other studies using radioactive substrates were range of 25 to 250 ng.…”

“…The liquid chromatography-tandem mass spectrometry (LC-MS/MS) method can be used for high-throughput screening (HTS) techniques, and combinational chemistry during drug discovery and development has led to a tremendous increase in the number of compounds to be evaluated for potential 5AR inhibition [ 14 , 15 ]. Recently, sensitive chemical derivatization methods for DHT detection in LC-MS/MS were developed [ 16 ].…”