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.

Specificity and predictive value of circulating testosterone assessed by tandem mass spectrometry for the diagnosis of polycystic ovary syndrome by the National Institutes of Health 1990 criteria.

OBJECTIVE: To determine whether liquid chromatography-tandem mass spectrometry (LC-MS/MS) determination of total (TT) and free (FT) testosterone is more specific than extraction chromatography-radioimmunoassay (RIA) for distinguishing women with polycystic ovary syndrome (PCOS) from controls and whether differing cutoff values should be used depending on the setting. DESIGN: Prospective case-control cohort study. SETTING: Tertiary care center and reference laboratory. INTERVENTION(S): Blood sampling. PATIENT(S): One hundred patients with PCOS and 100 controls. MAIN OUTCOME MEASURE(S): Circulating TT measured by RIA and LC-MS/MS and FT measured by equilibrium dialysis using RIA or LC-MS/MS-generated TT values. RESULT(S): T measurement by RIA and LC-MS/MS similarly differentiated patients with PCOS from controls; detection of PCOS was higher for FT for both methods. TT values demonstrated greater overlap between patients with PCOS and controls than did FT for both RIA (80% vs. 42% overlap) and LC-MS/MS (52% vs. 67% overlap). A lower cutoff value by LC-MS/MS was better suited for the study of patients seen in the clinical (referred) setting (35 and 4.0 ng/dL for TT and FT, respectively) than in the screening of a general population (50 and 5.0 ng/dL for TT and FT, respectively). CONCLUSION(S): Extraction chromatography-RIA and LC-MS/MS measurements of T have similar performance for differentiating patients with PCOS from healthy controls; LC-MS/MS may be preferable given its relative ease of automation. Compared with FT, measurement of TT has relatively limited value for distinguishing PCOS from normal. Finally, different cutoff values should be considered depending on the clinical/investigative setting, with higher values being used in the study of biased (e.g., clinical or referred) populations.

Validation of a total testosterone assay using high-turbulence liquid chromatography tandem mass spectrometry: total and free testosterone reference ranges.

Accurate measurement of testosterone concentration is of critical importance when diagnosing and treating male hypogonadism, congenital adrenal hyperplasia, premature or delayed puberty, and androgen excess in polycystic ovary syndrome or other virilizing conditions. However, some assays have inherent limitations and biases that affect measurement of low-testosterone values. Therefore, we developed a highly specific online mass spectrometry method. Sera were extracted online using high-turbulence flow liquid chromatography coupled to analytical HPLC and atmospheric pressure chemical ionization tandem mass spectrometry (HTLC-APCI-MS/MS). Analyte ions were monitored by multiple reaction monitoring (MRM). Total analysis time was 1.15 min per sample when using the multiplexing system. Testosterone concentrations were measured directly from 150 microL of serum or plasma without derivatization or liquid-liquid extraction. The lower limit of quantification was 0.3 ng/dL, and the assay was linear up to 2000 ng/dL. The method compared very well with an established RIA: y=1.02x+1.5, r(2)=0.994. Comparison with a platform immunoassay confirmed the previously reported ICMA positive bias at low concentrations. Male and female adult and pediatric reference ranges were developed for this very sensitive and accurate high-throughput LC-MS/MS method. This method is suitable for measuring the expected low-testosterone concentrations seen in women, children, and hypogonadal males and for monitoring testosterone suppressive therapy in prostate cancer patients.