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.

Keywords: Androgens; LC-MS/MS; Method validation; Picolinic acid; Prostate cancer.

Fig. 1.

Metabolism of DHT depicting the formation of α-diol, and β-diol by the action of enzymes coded by the genes AKR1C1, and AKR1C2.

Fig. 2.

Structures of internal standards. (a) DHT-d₃; (b) α-diol-d₃; (c) β-diol-d₃.

Fig. 3.

Derivatization of analytes with picolinic acid. (a) reaction with DHT; (b) reaction with α-diol; (c) reaction with β-diol.

Fig. 4.

Binding of serum protein to stable isotope labeled analytes, i.e., DHT-d₃; α-diol-d₃; β-diol-d₃. Reaction condition is described in the Section 2.3.3. The concentration of stable isotope labeled analyte is 4.00 ng/mL.

Fig. 5.

Positive-ESI-mass spectra and product ion spectra of picolinyl derivatives of (a) DHT; (b) α-diol; (c) β-diol at an individual concentration of 500 ng/mL. The condition is described in Section 2.3.

Fig. 6.

Representative mass chromatograms of DHT and DHT-d₃ (a) in double blank pooled sera (with neither DHT nor DHT-d₃); (b) in blank pooled sera (with DHT-d₃ only, 4.00 ng/mL); (c) in pooled sera at LLOQ (0.0500 ng/mL; DHT-d₃, 4.00 ng/mL); (d) in pooled sera at 50.0 ng/mL DHT, and 4.00 ng/mL DHT-d₃; (e) in mouse serum sample # 2 at 0.0700 ng/mL DHT, and 4.00 ng/mL DHT-d₃; (f) in mouse serum sample # 3 at 0.0600 ng/mL DHT, and 4.00 ng/mL DHT-d₃.

Fig. 7.

Representative mass chromatograms of α-diol, and α-diol-d₃ (a) in double blank pooled sera (with neither α-diol nor α-diol-d₃); (b) in blank pooled sera (with α-diol-d₃ only, 4.00 ng/mL); (c) in pooled sera at LLOQ (0.0500 ng/mL; α-diol-d₃, 4.00 ng/mL); (d) in pooled sera at 50.0 ng/mL α-diol, and 4.00 ng/mL α-diol-d₃; (e) in mouse serum sample # 2 at 0.130 ng/mL α-diol, and 4.00 ng/mL α-diol-d₃; (f) in mouse serum sample # 3 at 0.0900 ng/mL α-diol, and 4.00 ng/mL α-diol-d₃.

Fig. 8.

Representative mass chromatograms of β-diol, and β-diol-d₃ (a) in double blank pooled sera (with neither β-diol nor β-diol-d₃); (b) in blank pooled sera (with β-diol-d₃ only, 4.00 ng/mL); (c) in pooled sera at LLOQ (0.0500 ng/mL; β-diol-d₃, 4.00 ng/mL); (d) in pooled sera at 50.0 ng/mL β-diol, and 4.00 ng/mL β-diol-d₃; (e) in mouse serum sample # 2 at 0.100 ng/mL β-diol, and 4.00 ng/mL β-diol-d₃; (f) in mouse serum sample # 3 at 0.110 ng/mL β-diol, and 4.00 ng/mL β-diol-d₃.