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

Abstract

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.

Keywords: 5α-Dihydrotestosterone; Androstenedione; Derivatization; Liquid chromatography mass spectrometry; Testosterone.

Fig. 1

(A) Synthetic reaction of HMP from FMP-TS; 2-fluoro-1-methylpyridinium p-toluene sulfonate (FMP-TS) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HMP) (B) Exact mass of HMP was confirmed by FTICR-MS analysis, HMP elemental formula of C₆H₁₀N₃ and the theoretical mass (i) m/z 124.0872 were aligned with accurate mass (ii) m/z 124.0869. Counts per second (cps).

Fig. 2

Formation of steroid-HMP derivatives, showing examples of derivatization of DHT (representative of derivatives forming on the A ring) and DHEA (derivatization on the D Ring) and putative fragmentation with product ions scans and confirmation using accurate mass data; difference (Δ), 5α-dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA) and 2-hydrazino-1-methylpyridine (HMP). Products ion scan of further analytes and internal standards can be found in Supplementary Fig. S1. The two product ions selected for analysis were derived from the derivatization reagent.

Fig. 3

Putative fragmentation of HTP derivatives; testosterone (T), androstenedione (A4), 5α-dihydrotestosterone (DHT), 5α-dihydroandrostanedione (DHA), epitestosterone (EpiT), dehydroepiandrosterone (DHEA), 2,3,4-¹³C_3-testosterone (¹³C₃-T), 2,3,4-¹³C₃-androstenedione (¹³C₃-A4), 2,3,4-¹³C₃-5α-dihydrotestosterone (¹³C₃-DHT) and 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP).

Fig. 4

Total ion chromatograms of quantifier mass transitions of HMP and HTP derivatives; (A) HMP (DHT-HMP m/z 396→108; T-HMP, DHA-HMP and EpiT-HMP m/z 394→108; A4-HMP m/z 392→108; DHEA-HMP m/z 394→109) and (B) HTP (DHT-HTP m/z 451→288; T-HTP and EpiT-HTP m/z 449→257; A4-HTP m/z 447→269; DHA-HTP m/z 449→286; DHEA-HTP m/z 449→286), testosterone (T), androstenedione (A4), 5α-dihydrotestosterone (DHT), 5α-dihydroandrostanedione (DHA), epitestosterone (EpiT), dehydroepiandrosterone (DHEA), 2-hydrazino-1-methylpyridine (HMP), 2-hydrazino-4-(trifluoromethyl)-pyrimidine (HTP) and counts per second (cps).

Fig. 5

Retention times of quantifier mass transitions of HMP derivatives; (i) DHT-HMP (4.9 min), (ii) T-HMP (4.6 min) and (iii) A4-HMP (5.1 min). Testosterone (T), androstenedione (A4), 5α-dihydrotestosterone (DHT), 2-hydrazino-1-methylpyridine (HMP) and counts per second (cps).

Fig. 6

Mass chromatograms of quantifier mass transitions of DHT-HMP (m/z 396→108) and internal standard, ¹³C₃-DHT-HMP (m/z 399→108) derivatives in extracts of plasma from Males (A and B) and post-menopausal females (C and D) respectively. 5α-dihydrotestosterone (DHT) was detected at levels of 30 pg and 14 pg/sample ((1.0 nmol/L and 241 pmo/L) in males and post-menopausal female plasma samples respectively. 2,3,4-¹³C₃-5α-dihydrotestosterone (¹³C₃-DHT) was added as 100pg. 2-Hydrazino-1-methylpyridine (HMP) and counts per second (cps).