Sensitive LC-MS/MS Assay for Total Testosterone Quantification on Unit Resolution and High-Resolution Instruments

被引:0
|
作者
Wolken, Jill K. [1 ]
Peterson, Meghan M. [1 ]
Cao, Wenjing [1 ,2 ]
Challoner, Keith [1 ]
Jin, Zhicheng [1 ,2 ]
机构
[1] Univ Wisconsin Hosp & Clin, Madison, WI 53792 USA
[2] Univ Wisconsin Madison, Dept Pathol & Lab Med, 600 Highland Ave, Madison, WI 53792 USA
关键词
testosterone; multiple reaction monitoring; high-resolution mass spectrometry; assay harmonization; parallel reaction monitoring; TANDEM MASS-SPECTROMETRY; SERUM TESTOSTERONE; VALIDATION;
D O I
10.3390/jcm13237056
中图分类号
R5 [内科学];
学科分类号
1002 ; 100201 ;
摘要
Background: Testosterone is an androgenic hormone that plays important roles in both males and females. The circulating levels of total testosterone vary from 1 to 1480 ng/dL. High-throughput immunoassays often lack accuracy in lower concentration ranges (below 100 ng/dL), particularly when used for females or children. To address this limitation, we developed a total testosterone LC-MS/MS assay on three instruments. Methods: Sample preparation began with the dilution and conditioning of 200 mu L of serum. A supported liquid extraction cartridge was used to extract the analyte from biological matrices. Chromatographic separation was achieved using a C18 column with a runtime of 5 min per sample. This assay was validated on a Triple Quad 6500 and an API 4500 instrument. Results: Method validation was carried out according to the CLSI C62-ED2 guideline and our hospital protocol. The within-day coefficient of variation (CV) was less than 10% and the between-day CV was less than 15%. The assay had a limit of quantitation of 0.5 ng/dL with an analyte measure range of 2-1200 ng/dL. A comparison using Deming regression and Bland-Altman plots showed that this assay correlated well with a reference method. The results from the API 4500 and an Orbitrap were consistent with those from the TQ 6500. Both serum-separator tubes (BD) and serum-activator tubes were found to be suitable. Conclusions: We successfully developed and validated a robust total testosterone LC-MS/MS assay for routine clinical testing. This assay was harmonized across two triple quadrupole instruments and one high-resolution mass spectrometer.
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页数:9
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