Measurement of serum progesterone by isotope dilution liquid chromatography tandem mass spectrometry: a candidate reference method and its application to evaluating immunoassays
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.
KeywordsProgesterone Reference method ID-LC/MS/MS Immunoassays
We appreciate the technical support provided by the manufacturers (Abbott, Beckman, Chivd, Mindray, Roche, and Siemens) during the method comparison study.
This study was supported by grants from the National High Technology Research and Development Program of China (863 Program, No. 2011AA02A102) and The Leaping Development Project of Beijing Biomedical Industry (No. Z161100001816043).
Compliance with ethical standards
The individual serum samples were collected from the leftovers of patient samples at the clinical laboratory of Beijing Hospital, and this study had been reviewed and approved by the Ethics Committee of Beijing Hospital.
Conflict of interest
The authors declare that they have no competing interests.
- 1.Burtis CA, Ashwood ER. Tietz textbook of clinical chemistry. 2nd ed; 1994. p. 1865–6.Google Scholar
- 3.Coucke W, Devleeschouwer N, Libeer JC, Schiettecatte J, Martin M, Smitz J, et al. Accuracy and reproducibility of automated estradiol-17beta and progesterone assays using native serum samples: results obtained in the Belgian external assessment scheme. Hum Reprod. 2007;22(12):3204–9.CrossRefGoogle Scholar
- 12.External quality control for Reference Laboratories. Http://dgkl-rfb.de:81/. Accessed 10 Nov 2018.
- 13.ISO/IEC Guide 98–3:2008. Uncertainty of measurement - part 3: guide to the expression of uncertainty in measurement (GUM: 1995). Geneva: ISO; 2008.Google Scholar