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To solve long-term lack of traceability of commercial calibrator kits and standardize clinical routine assays, we developed a human serum matrix-based unconjugated estriol (uE3) reference material (RM) with five concentration gradients. The RMs of uE3 were certified by the National Institute of Metrology (NIM) with the codes of GBW (E) 091048, GBW (E) 091049, GBW (E) 091050, GBW (E) 091051, and GBW (E) 091052. The RMs were determined by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method which was developed in our group and recommended by the Joint Committee on Traceability on Laboratory Medicine (JCTLM). GBW09224 is intended for use as a primary reference material to enable the SI-traceable measurement of uE3. This study describes the development process of these certified RMs. The candidate material was prepared by collecting from the remaining serum samples after routine clinical testing. Satisfactory homogeneity and stability were shown in these RMs. They are also commutable between the reference method and the three routine clinical immunoassay systems. To improve the accuracy of value assignment, a collaborative study in nine reference laboratories was conducted which was performed according to ISO/WD 15725-1 and all of the reference laboratories have been confirmed by China National Accreditation Service for Conformity Assessment (CNAS). The raw results were statistically analyzed and processed, coupled with uncertainty evaluation, to obtain the certified value: GBW (E) 091048 is 22.1 ± 1.3 nmol/L, GBW (E) 091049 is 33.6 ± 1.6 nmol/L, GBW (E) 091050 is 10.4 ± 0.8 nmol/L, GBW (E) 091051 is 15.5 ± 1.0 nmol/L, GBW (E) 091052 is 47.0 ± 2.0 nmol/L. The preparation process of human serum matrix-based reference material and the lack of these type of secondary (commutable) reference material of unconjugated estriol lead to the interruption of its traceability chain, which is a problem to be solved in its standardization as mentioned in the metrological traceability in ISO 17511, 2020., (© 2022. Springer-Verlag GmbH Germany, part of Springer Nature.)

A candidate reference measurement procedure (RMP) for measurement of unconjugated estriol in human serum has been developed and validated. The proposed method is highly reliable and uses isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) and requires no derivatization. An appropriate amount of serum was accurately weighed and spiked with an isotopically labeled internal standard. Unconjugated estriol and its internal standard were extracted from serum matrix using liquid-liquid extraction prior to reversed-phase LC-MS/MS. Calibrator bracketing was used to give higher specificity and accuracy for assigning serum level. The accuracy of the candidate RMP was validated by split-sample comparison to established RMPs. The lowest limit of detection (LLoD) and lowest limit of quantification (LLoQ) for developed RMP was estimated to be 0.14 nmol/L and 0.35 nmol/L, respectively. Both intra- and inter-assay imprecisions were ≤2.19% at 1.39, 17.34 and 69.35 nmol/L, respectively. Recoveries were 98.54% to 100.34% and linear response ranged from 0.35 to 173.38 nmol/L. No interference was observed. Biases were 5.6% and 2.8% against the targets of RELA2015A (3.87 nmol/L) and RELA2015B (40.62 nmol/L), respectively. Moreover, the candidate RMP was successfully applied to measure level of unconjugated estriol in serum samples of pregnant women (n = 3) and compared with two immunoassays in clinical laboratory. Our developed method is simple, accurate, and can be used as a candidate RMP to determine total unconjugated estriol level in human serum. Further improvement of certain immunoassays in accuracy and precision is needed. Graphical abstract Selected ion chromatograms by LC-MS/MS using a C18 column for uE 3 from a serum sample.

Estrogen measurements are important in the assessment of female reproductive function and have expanding roles in other fields. A simple, accurate, highly sensitive and specific isotope-dilution liquid chromatography-tandem mass spectrometry method was developed and evaluated to simultaneously measure three endogenous estrogens in serum: estrone (E 1 ), 17β-estradiol (E 2 ), and estriol (E 3 ). Chromatographic separation was achieved on a C 18 column before electrospray ionization triple-quadrupole mass spectrometry in multiple reaction monitoring mode. The sample preparation in this assay requires no derivatization and extraction by liquid-liquid extraction. After optimization of the extraction conditions, the final extraction efficiency of E 1 , E 2 , and E 3 was 83.8%, 78.9%, and 77.3% respectively. The metabolites and structural analogs that have the same molecular masses as the estrogens were separated under the optimized liquid chromatography conditions. Method validation showed satisfactory linearity over the concentration range of 20-10000 pg mL -1 for all three estrogens (r 2  > 0.997). The limits of quantification were 5, 10, and 10 pg mL -1 for E 1 , E 2 , and E 3 respectively, and their recoveries ranged from 94.7% to 103.5%. The accuracy of the proposed method was further evaluated with use of certified reference materials BCR-576, BCR-577, and BCR-578 for E 2 and 2014 International Federation of Clinical Chemistry and Laboratory Medicine External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine samples for E 3 , whose certified values were determined by reference methods. Great agreement was observed between the measured values and the certified values. Satisfactory precision (coefficients of variation less than 7.44%) was also obtained for the three estrogens. Moreover, the proposed method was successfully applied to measure the three estrogens in serum samples of pregnant women in the second trimester and to assess the accuracy of chemiluminescent immunoassays in clinical laboratories by determination of E 2 and unconjugated E 3 in serum samples. Graphical Abstract Schematic representation of the simultaneous quantitation of three major endogenous estrogens in human serum by ID-LC-MS/MS.