A Liquid Chromatography–Tandem Mass Spectrometry Method for Salivary Testosterone with Adult Male Reference Interval Determination

To the Editor:

Our laboratory has extensive experience in developing liquid chromatography–tandem mass spectrometry (LC-MS/MS)¹ methods for routine use in the clinical laboratory. We previously developed an LC-MS/MS assay for serum testosterone and androstenedione (1). In collaboration with the National Survey of Sexual Attitudes and Lifestyles (NATSAL) working group, we have now developed an LC-MS/MS assay for salivary testosterone (Sal-T). Sal-T is a filtrate of plasma containing only the free fraction. Free testosterone (FT) is considered the physiologically active form and more accurately reflects androgen exposure in target tissues than serum total testosterone. Methods to measure serum FT are unsuitable for the routine clinical laboratory, and the reliability of mathematical algorithms to derive serum FT are questionable, with no consensus. Direct measurement of Sal-T therefore offers a potentially more accurate and convenient alternative to measuring serum FT, particularly in conditions affecting sex hormone–binding globulin.

Our method used samples collected via an unstimulated passive-drool technique. Volunteers rinsed their mouth with water, refrained from eating, and did not brush their teeth 30 min before sampling. Samples were collected by drooling down a plastic straw into a collection vial; the saliva sample was then frozen at −80 °C. Samples were thawed, mixed, and centrifuged; the clear supernatant was then used for analysis.

Sample preparation involved a liquid–liquid extraction requiring 200 μL sample with D₅-testosterone internal standard and methyl-tert-butyl ether, which was then placed at −80 °C. After 1 h at this temperature, the organic layer was transferred and evaporated by heating and gentle N₂ gas flow. The residue was reconstituted with a 500 mL/L methanol mobile phase and transferred to a 96-well microtiter plate.

Liquid chromatography was performed with a Waters ACQUITY™ Ultra Performance Liquid Chromatography (UPLC) system and a C18 ACQUITY 1.8-μm HSS T₃ column (2.1 × 50 mm) maintained at 45 °C. The mass spectrometer was a Waters Quattro Premier XE™ mass spectrometer in positive ionization mode. Binary pump mixing of mobile phases A (2 mmol/L ammonium acetate and 1 mL/L formic acid in distilled water) and B (2 mmol/L ammonium acetate and 1 mL/L formic acid in methanol) produced a linear gradient that increased from 50% to 90% methanol for 1.5 min. The total run time was 3.5 min, with testosterone and D₅-testosterone coeluting with clean, discrete, and identifiable peaks at a retention time of 1.28 min, with no ion suppression evident. The signal was optimized for the testosterone and D₅-testosterone precursor ions (seen at m/z 289.2 and m/z 294.1) and the most abundant product ions (seen at m/z 108.8 and m/z 96.8 for testosterone and at m/z 99.8 for D₅-testosterone).

Intra- and interassay CVs were <15% at 4 different concentrations, and the mean measured values were within 15% of the target values. The mean recoveries from saliva samples (n = 6) at 3 concentrations were 95.6%, 100.3%, and 95.8%, respectively.

Sample-stability studies showed no appreciable loss in testosterone, a finding supporting the feasibility of patients producing saliva samples at home and sending them to appointed laboratories. A comparison of our LC-MS/MS assay with an established Sal-T RIA found that the RIA produced testosterone concentrations approximately 30% higher than those obtained with the LC-MS/MS method and had poor correlation (Fig. 1). This difference was not due to calibration issues, because a cross-validation of calibrators used in both assays showed close agreement. The Sal-T results and poor correlation obtained with the RIA can probably be attributed to the poorer specificity of the capture antibody in the RIA method, which is known to cross-react with a number of related steroids, particularly dihydrotestosterone (2). Our LC-MS/MS method was extremely specific, with no interferences from >30 similar steroids.

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Fig. 1. Passing–Bablok analysis comparing the Sal-T LC-MS/MS method with an established RIA method for Sal-T.

Regression line (solid line) and 95% CIs (dashed lines) are indicated.

We were able to derive a reference interval of 73–343 pmol/L from Sal-T measurements obtained for 103 healthy male volunteers (16–74 years of age). The lower limit of quantification for our assay was 25 pmol/L. Thus, the method would identify testosterone deficiency in males. We observed a reduction of approximately 35% in the median Sal-T values in men >50 years of age compared to men ≤30 years of age, with a negative age trend with the following linear regression equation: y = −2.1x + 287, where y is the Sal-T concentration in picomoles per liter and x is the age in years.

In conclusion, we have developed an LC-MS/MS method for measuring Sal-T in men, which could be used as a surrogate marker for biologically available testosterone. The availability of a Sal-T assay that is reliable at low concentrations will facilitate improved clinical utility and decision-making for the laboratory assessment of androgen status in men.

• © 2011 The American Association for Clinical Chemistry