Salivary testosterone measurement by liquid chromatography tandem mass spectrometry in adult males and females

Abstract

Background: Salivary testosterone (Sal-T) may be a useful surrogate of serum free testosterone. The study aims were to use a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to determine whether Sal-T concentrations accurately reflect Sal-T concentrations in both sexes and to investigate practical aspects of sample collection. Methods: Saliva and serum samples were collected in 104 male and 91 female subjects. A more sensitive LC-MS/MS assay was developed to enable Sal-T quantitation in the low concentrations found in females. Saliva (200 mL) was extracted with 1 mL of methyl-tert-butyl ether following the addition of D5-testosterone. Quantitation was performed using a Waters TQ-S mass spectrometer. Results: The assay achieved a lower limit of quantification of 5 pmol/L, sufficiently sensitive to measure testosterone in female saliva. Sal-T showed a diurnal variation but samples taken at weekly and monthly intervals showed no significant differences. Sal-T was stable at ambient temperature for up to 5 days, after freeze-thawing and 3 years frozen storage. Reference intervals for Sal-T were 93-378 pmol/L in males and 5-46 pmol/L in females. Sal-T correlated significantly with serum calculated free-T in males (r ¼ 0.71, P < 0.001) and in females (r ¼ 0.39, P < 0.001). Conclusions: These results confirm that testosterone can be reliably and accurately measured by LC-MS/MS in both adult male and female saliva samples. These results lay the foundation for further exploration of the clinical application of Sal-T as a reliable alternative to serum testosterone in the diagnosis and management of androgen disorders and assessment of androgen status in clinical research.

Salivary Testosterone during the Minipuberty of Infancy

“…LC-MS/MS is now the gold standard for the measurement of steroid hormone levels since it has improved specificity and sensitivity [11,12]. Salivary T has previously been measured by LC-MS/MS across the full adult range in males, and it correlates significantly with serum free T [13]. Results expressed as mean ± standard deviation.…”

Salivary Testosterone Levels and Health Status in Men and Women in the British General Population: Findings from the Third National Survey of Sexual Attitudes and Lifestyles (Natsal-3)

“…We have recently demonstrated that Sal-T can be reliably and accurately measured by a highly sensitive and specific liquid chromatography–tandem mass spectrometry method (16). In a validation study comparing samples from the same individuals, we found that Sal-T in adult men and women correlated more strongly with calculated serum free-T than serum total-T (17) and was also unaffected by variations in sex hormone-binding globulin (SHBG) (18). Fiers at al (19) confirmed the good correlation in both men and women between Sal-T and serum free-T measured by equilibrium dialysis but there was a significant systematic positive bias in women, which may reflect the influence of salivary protein binding on the lower female concentrations of Sal-T.…”

Applications and Challenges in Using LC–MS/MS Assays for Quantitative Doping Analysis

“…An UHPLC-MS/MS method for direct quantification of two T metabolites (3a-glucuronide-6b-hydroxyandrosterone and 3a-glucuronide-6b-hydroxyetiocholanolone) was developed and validated by Pozo's group [54] and this was accomplished with an UHPLC-MS/MS method because three interfering compounds were noted during GC-MS/MS analysis with the most intense ion transitions. For the free T or its conjugates and other EAAS after hydrolysis, LC-MS/MS methods are available for quantification [57] in urine, plasma and serum [58][59][60][61], hair [62,63] and saliva [64]. Fabregat's group [57] described a rapid, accurate and sensitive LC-MS/MS method for measuring T fractions and its metabolites released under basic conditions.…”