Sample Multiplexing: Increased Throughput for Quantification of Total Testosterone in Serum by Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: For high-volume assays, optimizing throughput reduces test cost and turn-around time. One approach for liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays is sample multiplexing, wherein the analyte of interest is derivatized in different specimens with reagents of different molecular weight (differential mass tagging). Specimens can then be combined and simultaneously analyzed within a single injection to improve throughput. Here we developed and validated a quantitative, sample-multiplexed LC-MS/MS assay for serum total testosterone (TT) based on this approach. METHODS: For the sample-multiplexed assay, calibrators, controls, and patient specimens were first extracted separately. After mass tagging with either methoxyamine or hydroxylamine, they were combined and injected into the LC-MS/MS system. To evaluate assay performance, we determined limit of quantification (LOQ), linearity, recovery, and imprecision. A method-comparison study was also performed, comparing the new assay with the standard LC-MS/MS assay in 1574 patient specimens. RESULTS: The method was linear from 2.5 to 2000 ng/dL, with accuracies from 93% to 104% for both derivatives. An LOQ of 1.0 ng/dL was achieved. Intra-assay and total CVs across 4 quality control concentrations were less than 10%. The assay demonstrated good agreement (Deming regression, 1.03x + 6.07) with the standard LC-MS/MS assay for the patient specimens tested (TT, 3 to 4862 ng/dL). CONCLUSION: Sample multiplexing by differential mass tagging of TT increases LC-MS/MS throughput 2-fold without compromising analytical accuracy and sensitivity.

Measurement of Cortisol and Testosterone in Athletes: Accuracy of Liquid Chromatography-Tandem Mass Spectrometry Assays for Cortisol and Testosterone Measurement in Whole-Blood Microspecimens.

Fragala, MS, Goldman, SM, Goldman, MM, Bi, C, Colletti, JD, Arent, SM, Walker, AJ, and Clarke, NJ. Measurement of cortisol and testosterone in athletes: Accuracy of LC-MS/MS assays for cortisol and testosterone measurement in whole-blood microspecimens. J Strength Cond Res 32(9): 2425-2434, 2018-Biomarker monitoring provides insight into athletes' training tolerance but is limited by the need for office-based specimen collection. To facilitate self-collection during training, we developed liquid chromatography-tandem mass spectrometry-based tests that measure circulating total cortisol and testosterone using a finger stick volumetric absorptive microsampler. Here, we describe the analytical validation of these tests. Forty-six Division I athletes (18-22 years, 30 women, 16 men) provided a 20-µL finger stick microspecimen and a 5-ml venous blood specimen from the forearm; the venous blood sample was analyzed using both normal volume serum analysis and analysis of dried whole blood (from the microsampler). Liquid chromatography-tandem mass spectrometry on standard serum specimens obtained by venipuncture yielded total cortisol levels of 26.2 ± 11.6 µg·dl (women and men), and total testosterone levels of 37 ± 17 ng·dl in women and 564 ± 171 ng·dl in men. Analytical measurement ranges of the microspecimen assay were 0.3-440 µg·dl (CV <9%) for cortisol and 15 to 20,000 ng·dl (CV <9%) for testosterone. Deming regression and Pearson correlation indicated good test accuracy for the microspecimen tests compared with venipuncture tests for cortisol (y = 0.98x + 1.34, 95% CI of slope = 0.83-1.14; r = 0.92, p < 0.0001) and testosterone (y = 1.06x - 0.01, 95% CI of slope = 0.99-1.14; r = 0.99, p < 0.0001). Similarly, high agreement was observed between finger stick and venous microspecimens for cortisol (y = 1.00x + 0.65, 95% CI of slope = 0.9-1.11; r = 0.96, p < 0.001) and testosterone (y = 0.97x + 2.75, 95% CI of slope = 0.9-1.03; r = 0.99, p < 0.001). These findings suggest the viability of finger stick collection whole-blood microspecimens for assessment of total cortisol and testosterone in athletes.