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Measurement of serum progesterone by isotope dilution liquid chromatography tandem mass spectrometry: a candidate reference method and its application to evaluating immunoassays
- Wang, Yufei, Zhang, Tianjiao, Zhao, Haijian, Zhou, Weiyan, Zeng, Jie, Zhang, Jiangtao, Zhang, Chuanbao, Chen, Wenxiang
- Analytical and bioanalytical chemistry 2019 v.411 no.11 pp. 2363-2371
- animal ovaries, blood serum, immunoassays, isotope dilution technique, liquid chromatography, liquid-liquid extraction, progesterone, reference standards, standard deviation, tandem mass spectrometry, uncertainty
- Measurement of serum progesterone is important in determining ovarian function. Most progesterone measurements are performed with immunoassays and results are often variable. Standardization of the measurements requires a reliable reference method. An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for the measurement of serum progesterone was developed. Serum samples were spiked with ¹³C₃-progesterone, extracted with a three-step liquid-liquid extraction, and analyzed by LC/MS/MS. A bracketing calibration was used for the analysis and samples were prepared gravimetrically. The developed method showed intra-run and total relative standard deviations (RSDs) of 0.50~0.58% and 0.71~1.33%, respectively. The analytical recoveries were 99.08~101.50%. Measurement results on certified reference materials obtained with this method agreed with the certified values within the stated measurement uncertainties. The method was applied to evaluate immunoassays through split-sample comparisons. A panel of 48 fresh frozen individual samples were measured with the ID-LC/MS/MS method, and six immunoassays and results were compared. Significant calibration biases and sample-specific deviations were observed on some of the immunoassays.