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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.