Ann Lab Med.  2023 Jan;43(1):19-28. 10.3343/alm.2023.43.1.19.

Clinical Usefulness of Ultraperformance Liquid Chromatography-Tandem Mass Spectrometry Method for Low Serum Testosterone Measurement

• Cho SE
• Han J
• Park JH
• Park E
• Kim GY
• Lee JH
• Yi A
• Lee SG
• Lee EH
• Yun YM

• KMID: 2551573
• DOI: http://doi.org/10.3343/alm.2023.43.1.19

Abstract

Background
Mass spectrometry methods exhibit higher accuracy and lower variability than immunoassays at low testosterone concentrations. We developed and validated an ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay for quantifying serum total testosterone.
Methods
We used an ExionLC UPLC (Sciex, Framingham, MA, USA) system and a Sciex Triple Quad 6500+ (Sciex) MS/MS system in electrospray ionization and positive ion modes with multiple reaction monitoring transitions to evaluate precision, accuracy, linearity, lower limit of quantitation (LLOQ), carryover, ion suppression, stability, and reference intervals. For method comparison, we measured serum testosterone concentrations using this method in 40 subjects whose testosterone concentrations ranged from 0.14 to 55.48 nmol/L as determined using the Architect i2000 immunoassay (Abbott Diagnostics, Abbott Park, IL, USA) and in an additional 160 sera with testosterone concentrations <1.67 nmol/L.
Results
The intra- and inter-run precision CVs were <2.81%, and the accuracy bias values were <3.85%, which were all acceptable. The verified linear interval was 0.03–180.84 nmol/L; the LLOQ was 0.03 nmol/L. No significant carryover and ion suppression were observed. The testosterone in serum was stable at 4°C, at –20°C, and after three freeze-thaw cycles. The reference intervals were successfully verified. The correlation was good at testosterone concentrations of 0.14–55.48 nmol/L; however, the Architect assay showed positive percent bias at concentrations <1.67 nmol/L.
Conclusions
The UPLC-MS/MS assay shows acceptable performance, with a lower LLOQ than the immunoassay. This method will enable the quantitation of low testosterone concentrations.

Keyword

Tandem mass spectrometry; Testosterone; Quantitation; Performance

Figure

• 

  Fig. 1 Representative MS/MS chromatograms. (A) Representative MS/MS chromatograms of testosterone in serum samples from study subjects (4.25 ng/mL [14.73 nmol/L]). (B) The calculated ion ratio of 97.6% was close to the expected value of 98.0% for the first and second MRM transitions, implying adequate identification and specificity of the assay. (C) Representative MS/MS chromatogram of testosterone in a 1/800 dilution of NIST SRM 971a, Male Serum (LLOQ: 0.008 ng/mL [0.03 nmol/L]). Abbreviations: MS/MS, tandem mass spectrometry; MRM, multiple reaction monitoring; LLOQ, lower limit of quantitation; RT, retention time.

• 

  Fig. 2 Method comparison of UPLC-MS/MS and the Architect CMIA. Passing–Bablok regression scatter plots and percent bias plots. The correlation was good (R=0.989, slope 0.995) in 40 random subjects having testosterone concentrations of 0.04–16.00 ng/mL (0.14– 55.48 nmol/L); however, positive percent bias was observed in the percent bias plot, especially at lower concentrations (A). The Architect CMIA exhibited positive percent bias in all groups with testosterone concentrations <0.48 ng/mL in the percent bias plots: 64.46% in 40 men (B), 7.04% in 40 women (C), 29.99% in 40 boys (D), 15.76% in 40 girls (E), and 20.36% in 160 total subjects (F). (G) Results from 200 subjects (40 random subjects and 160 subjects having testosterone concentrations <0.48 ng/mL [1.67 nmol/L]). Abbreviations: CMIA, chemiluminescent microparticle immunoassay; UPLC-MS/MS, ultraperformance liquid chromatography-tandem mass spectrometry.

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