Simultaneous measurement of serum testosterone and dihydrotestosterone by liquid chromatography-tandem mass spectrometry.
BACKGROUND
Recent reports have described inherent problems with androgen immunoassays compared with mass spectrometry analyses.


METHODS
We developed a method for measuring serum testosterone (T) and 5alpha-dihydrotestosterone (DHT) simultaneously via liquid-liquid extraction followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) with positive-mode electrospray ionization.


RESULTS
The DHT and T calibrators showed a linear response from 0.069 nmol/L to 34.4 nmol/L and 69.3 nmol/L, respectively. T interference in the DHT assay and vice versa were negligible. Within- and between-run imprecision values were <5% for both analytes. Percent recoveries of T and DHT spiked into samples at concentrations spanning the calibration curve were 100%-113% and 98%-107%, respectively. The lower limit of quantification was 0.069 nmol/L for both steroids. Serum T concentrations measured by LC-MS/MS were different from those obtained by RIA, especially at lower T concentrations. Serum DHT concentrations measured by LC-MS/MS were markedly lower than those generated by RIA because of the nonselectivity of the RIA without chromatography. The reference intervals (mean +/- 2 SDs) determined for T and DHT were 9.2-33.7 nmol/L and 0.47-2.65 nmol/L, respectively, for 113 healthy adult men and 0.33-2.02 nmol/L and 0.09-0.91 nmol/L, respectively, for 133 healthy premenopausal women.


CONCLUSIONS
We have developed and validated a selective and precise method for simultaneous measurements of serum T and DHT that can be adopted for routine measurements of these androgens in health and disease in men and women.
Shiraishi
Steve
S
General Clinical Research Center, Division of Endocrinology and Metabolism, Harbor-UCLA Medical Center and Los Angeles Biomedical Research Institute, Torrance, CA 90509, USA.
Lee
Paul W N
PW
Leung
Andrew
A
Goh
Victor H H
VH
Swerdloff
Ronald S
RS
Wang
Christina
C
D000328:Adult; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D013196:Dihydrotestosterone / Q000097:blood*; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D011863:Radioimmunoassay; D012016:Reference Values; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013739:Testosterone / Q000097:blood*
D013196:Dihydrotestosterone; D013739:Testosterone