Validation of a testosterone and dihydrotestosterone liquid chromatography tandem mass spectrometry assay: Interference and comparison with established methods

Abstract

Testosterone (T) and its metabolite dihydrotestosterone (DHT) are androgens with different biologic profiles. T and DHT measurements are required for assessment of patients with ambiguous genitalia, hirsutism, during 5 alpha reductase treatment of prostate disorders, and new androgen formulations. Our laboratory has developed and validated a method to simultaneously measure serum T and DHT with liquid chromatography tandem mass spectrometry (LC-MS/MS) for use in a clinical chemistry laboratory. Analysis of sera from blood collected in tubes containing clot activator gave results of T that were fourfold higher than blood collected in plain tubes. Changing the ion pair selected for monitoring eliminated this interference by clot activators. Blood collected in fluoride-coated tubes gave serum T and DHT levels that were 20 and 15% lower, respectively than levels measured in blood collected in plain tubes (no additives). Addition of T enanthate to blood collected in plain tubes caused a dose related increase serum T levels due to the action of non-specific esterases in the red cells. This esterase activity could be avoided by using fluoride tubes for blood collection. Serum DHT levels were consistently lower when measured by LC-MS/MS versus radioimmunoassay. The differences were concentration dependent and the variance for the difference was large when serum DHT concentration was low. Celite chromatograph prior to radioimmunoassay reduced the differences between the two methods, thus confirming that higher levels of DHT obtained by immunoassays were probably due to interfering substances which were partially removed by Celite chromatography.

Figures

Fig. 1

Selected ion chromatographs by LC-MS/MS for serum T at a concentration of 10 nmol/L (289ng/dL) from a sample where the blood was collected in a tube with a clot activator (Fig. 1 A) or without any additive (Fig. 1B). The ion pair monitored in the left panels was 289.3/97.1 and in the right panel was 289.2/109.0.

Fig. 1

Selected ion chromatographs by LC-MS/MS for serum T at a concentration of 10 nmol/L (289ng/dL) from a sample where the blood was collected in a tube with a clot activator (Fig. 1 A) or without any additive (Fig. 1B). The ion pair monitored in the left panels was 289.3/97.1 and in the right panel was 289.2/109.0.

Fig. 2

Comparison of serum DHT measured by LC-MS/MS versus RIA after potassium permanganate treatment. Fig. 2A. Deming’s regression; Fig. 2B. Bland and Altman plot of the differences in serum DHT versus the average measured by LC-MS/MS and RIA; Bland and Altman plot of the percent differences versus the average serum DHT concentration measured by LC-MS/MS and RIA. (◆Untreated subjects, o subjects after oral T treatment, and ■ subjects after transdermal T or DHT treatment)

Fig. 2

Comparison of serum DHT measured by LC-MS/MS versus RIA after potassium permanganate treatment. Fig. 2A. Deming’s regression; Fig. 2B. Bland and Altman plot of the differences in serum DHT versus the average measured by LC-MS/MS and RIA; Bland and Altman plot of the percent differences versus the average serum DHT concentration measured by LC-MS/MS and RIA. (◆Untreated subjects, o subjects after oral T treatment, and ■ subjects after transdermal T or DHT treatment)

Fig. 2

Comparison of serum DHT measured by LC-MS/MS versus RIA after potassium permanganate treatment. Fig. 2A. Deming’s regression; Fig. 2B. Bland and Altman plot of the differences in serum DHT versus the average measured by LC-MS/MS and RIA; Bland and Altman plot of the percent differences versus the average serum DHT concentration measured by LC-MS/MS and RIA. (◆Untreated subjects, o subjects after oral T treatment, and ■ subjects after transdermal T or DHT treatment)

Fig. 3

Comparison of serum DHT measured by LC-MS/MS versus RIA after potassium permanganate treatment followed by Celite chromatography. Fig. 3A. Deming’s regression; Fig. 3B. Bland and Altman plot of the differences in serum DHT versus the average measured by LC-MS/MS and RIA; Bland and Altman plot of the percent differences versus the average serum DHT levels measured by LC-MS/MS and RIA after Celite Chromatography. (◆Untreated subjects, o subjects after oral T treatment, and ■ subjects after transdermal T or DHT treatment)

Fig. 3

Comparison of serum DHT measured by LC-MS/MS versus RIA after potassium permanganate treatment followed by Celite chromatography. Fig. 3A. Deming’s regression; Fig. 3B. Bland and Altman plot of the differences in serum DHT versus the average measured by LC-MS/MS and RIA; Bland and Altman plot of the percent differences versus the average serum DHT levels measured by LC-MS/MS and RIA after Celite Chromatography. (◆Untreated subjects, o subjects after oral T treatment, and ■ subjects after transdermal T or DHT treatment)

Fig. 3

Comparison of serum DHT measured by LC-MS/MS versus RIA after potassium permanganate treatment followed by Celite chromatography. Fig. 3A. Deming’s regression; Fig. 3B. Bland and Altman plot of the differences in serum DHT versus the average measured by LC-MS/MS and RIA; Bland and Altman plot of the percent differences versus the average serum DHT levels measured by LC-MS/MS and RIA after Celite Chromatography. (◆Untreated subjects, o subjects after oral T treatment, and ■ subjects after transdermal T or DHT treatment)