Derivatization with 2-Hydrazino-1-methylpyridine Enhances Sensitivity of Analysis of 5α-Dihydrotestosterone in Human Plasma by Liquid Chromatography Tandem Mass Spectrometry

Abdullah Faqehi, Scott Denham, Gregorio Naredo, Diego Cobice, Shazia Khan, Ghazali Sabil, Joanna Simpson, Rita Upreti, Fraser Gibb, Natalie Homer, Ruth Andrew

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7 Citations (Scopus)
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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.

Original languageEnglish
Article number461933
Early online date23 Jan 2021
Publication statusPublished (in print/issue) - 15 Mar 2021

Bibliographical note

Funding Information:
The authors acknowledge the financial support of NHS Research Scotland (NRS) through the Mass Spectrometry Core of the Edinburgh Clinical Research Facility and SIRCAMS, School of Chemistry for technical expertise. AF was funded through a scholarship from the Royal Embassy of Saudi Arabia. We are also grateful to the British Heart Foundation ( RE/08/001 ), the Chief Scientist Office ( CZB_4_642 ) and the Wellcome Trust ( 202794/Z/16/Z , 080397/Z/06/Z , 108468/Z/15/Z ) for funding.

Publisher Copyright:
© 2021


  • 5α-Dihydrotestosterone
  • Androstenedione
  • Derivatization
  • Liquid chromatography mass spectrometry
  • Testosterone


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