Derivatization with 2-hydrazino-1-methylpyridine enhances sensitivity of analysis of 5α-dihydrotestosterone in human plasma by liquid chromatography tandem mass spectrometry

• Published in: Journal of Chromatography A Vol. 1640; p. 461933
• Main Authors: Faqehi, Abdullah MM, Denham, Scott G, Naredo, Gregorio, Cobice, Diego F, Khan, Shazia, Simpson, Joanna P, Sabil, Ghazali, Upreti, Rita, Gibb, Fraser, Homer, Natalie ZM, Andrew, Ruth
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 15.03.2021; Elsevier
• Subjects: 5α-Dihydrotestosterone; Adult; Androgens - blood; Androstenedione; Androstenedione - blood; Biological Assay; Calibration; Chromatography, Liquid; Derivatization; Dihydrotestosterone - blood; Female; Humans; Hydrazines - chemical synthesis; Hydrazines - chemistry; Liquid chromatography mass spectrometry; Male; Pyridines - chemical synthesis; Pyridines - chemistry; Reference Standards; Reproducibility of Results; Tandem Mass Spectrometry - methods; Testosterone; Testosterone - blood; Testosterone; Liquid chromatography mass spectrometry; Androstenedione; 5α-Dihydrotestosterone; Derivatization
• ISSN: 0021-9673; 1873-3778
• DOI: 10.1016/j.chroma.2021.461933

•Quantitative analysis of low abundance androgens in human plasma.•Quantitation of androgens over physiological range in men and post-menopausal women.•Use of hydrazine derivatives improves analytical sensitivity. Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is the gold-standard approach for androgen analysis in biological fluids, superseding immunoassays in selectivity, particularly at low concentrations. While LC-MS/MS is established for analysis of testosterone and androstenedione, 5α-dihydrotestosterone (DHT) presents greater analytical challenges. DHT circulates at low nanomolar concentrations in men and lower in women, ionizing inefficiently and suffering from isobaric interference from other androgens. Even using current LC-MS/MS technology, large plasma volumes (>0.5 mL) are required for detection, undesirable clinically and unsuitable for animals. This study investigated derivatization approaches using hydrazine-based reagents to enhance ionization efficiency and sensitivity of analysis of DHT by LC-MS/MS. Derivatization of DHT using 2-hydrazino-1-methylpyridine (HMP) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP) were compared. A method was validated using an UHPLC interfaced by electrospray with a triple quadruple mass spectrometer , analyzing human plasma (male and post-menopausal women) following solid-phase extraction. HMP derivatives were selected for validation affording greater sensitivity than those formed with HTP. HMP derivatives were detected by selected reaction monitoring (DHT-HMP m/z 396→108; testosterone-HMP m/z 394→108; androstenedione-HMP m/z 392→108). Chromatographic separation of androgen derivatives was optimized, carefully separating isobaric interferents and acceptable outputs for precision and trueness achieved following injection of 0.4 pg on column (approximately 34 pmol/L). HMP derivatives of all androgens tested could be detected in low plasma volumes: male (100 µL) and post-menopausal female (200 µL), and derivatives were stable over 30 days at -20°C. In conclusion, HMP derivatization, in conjunction with LC-MS/MS, is suitable for quantitative analysis of DHT, testosterone and androstenedione in low plasma volumes, offering advantages in sensitivity over current methodologies.