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Neuroblastoma (NB), an embryonic tumor of the autonomous nervous system, poses a significant threat to the health and lives of children. Accurate measurement of vanillylmandelic acid (VMA) and homovanillic acid (HVA) in human urine is crucial for screening and diagnosis of NB. Although various laboratories have developed liquid chromatography tandem mass spectrometry (LC-MS/MS) method to detect VMA and HVA, the comparability between the results obtained from different laboratories and methods was poor. The absence of reference method for VMA and HVA hinders the standardization of their measurements. Therefore, a candidate reference measurement procedure (cRMP) based on isotope dilution LC-MS/MS (ID-LC-MS/MS) for the detection of VMA and HVA in human urine was established. Urine samples were spiked with VMA-d3 and HVA-d5 as internal standards and extracted using a protein precipitation method. The cRMP exhibited desirable precision with the total imprecision below 5 %. The accuracy of this cRMP was demonstrated by the high analytical recovery (98.64 % - 102.22 % and 98.41 % - 100.97 % for VMA and HVA, respectively), and comparability between different reference systems. The limit of detection for HVA and VMA were 15.625 ng/mL and 3.906 ng/mL, respectively; the quantification limits were 62.5 ng/mL and 7.813 ng/mL, respectively, which can meet the clinical detection requirements. The linear range was from 78.125 ng/mL to 20 μg/mL. Specificity evaluations showed no corresponding interference from structurally similar analogs. In conclusion, we have established a cRMP based on ID-LC-MS/MS for the measurement of VMA and HVA in urine samples, demonstrating well-defined method performance including accuracy, precision, and specificity. This newly established cRMP is suitable for routine assay standardization and evaluation of clinical samples. Furthermore, this method has the potential to significantly enhance the diagnostic accuracy for neuroblastoma., Competing Interests: Declaration of Competing Interest All authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A candidate reference measurement procedure (RMP) for serum theophylline via isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. With a single-step precipitation pretreatment and a 6-min gradient elution, the method achieved baseline separation of theophylline and its analogs on a C18-packed column. A bracketing calibration method was used to ensure repeatable signal intensity and high measurement precision. The intra-assay and inter-assay imprecisions were 1.06%, 0.84%, 0.72% and 0.47%, 0.41%, 0.25% at concentrations of 4.22 µg/mL (23.40 µmol/L), 8.45 µg/mL (46.90 µmol/L), and 15.21 µg/mL (84.43 µmol/L), respectively. Recoveries ranged from 99.35 to 102.34%. The limit of detection (LoD) was 2 ng/mL, and the lowest limit of quantification (LLoQ) was 5 ng/mL. The linearity range extended from 0.47 to 60 µg/mL (2.61-333.04 µmol/L). No ion suppression and carry-over (< 0.68%) were observed. The relative bias for this candidate RMP that participated in 2023 External Quality Control for Reference Laboratories (RELA) conducted by the International Federation of Clinical Chemistry (IFCC) was within a range of 0.17 to 0.93%. Furthermore, two clinical immunoassay systems were compared with this candidate RMP, demonstrating good correlations. The results of the Trueness Verification Plan indicate significant differences among routine systems, highlighting the need for standardization efforts. The developed candidate RMP for serum theophylline serves as a precise reference baseline for standardizing clinical systems and assigning values to reference materials., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Advanced glycation end products (AGEs), derived from the non-enzymatic glycation reaction, are defined as glycotoxins in various diseases including aging, diabetes and kidney injury. Exploring AGEs as potential biomarkers for these diseases holds paramount significance. Nevertheless, the high chemical structural similarity and great heterogeneity among AGEs present a formidable challenge when it comes to the comprehensive, simultaneous, and accurate detection of multiple AGEs in biological samples. In this study, an UPLC/MS/MS method for simultaneous quantification of 20 free AGEs in human serum was firstly established and applied to quantification of clinical samples from individuals with kidney injury. Simple sample preparation method through protein precipitation without derivatization was used. Method performances including imprecision, accuracy, sensitivity, linearity, and carryover were systematically validated. Intra- and inter- imprecision of 20 free AGEs were 1.93-5.94 % and 2.30-8.55 %, respectively. The method accuracy was confirmed with good recoveries ranging from 96.40 % to 103.25 %. The LOD and LOQ were 0.1-3.13 ng/mL and 0.5-6.25 ng/mL, respectively. Additionally, the 20 free AGEs displayed excellent linearity (R 2 >0.9974) across a wide linear range (1.56-400 ng/mL). Finally, through simultaneous quantitation of 20 Free AGEs in 100 participants including kidney injury patient and healthy controls, we identified six free AGEs, including N 6 -carboxyethyl-L-arginine (CEA), N 6 -carboxymethyl-L-lysine (CML), methylglyoxal-derived hydroimidazolones (MG-H), N 6 -formyl-lysine, N 6 -carboxymethyl-L-arginine (CMA), and glyoxal-derived hydroimidazolone (G-H), could well distinguish kidney injury patients and healthy individuals. Among them, the levels of four free AGEs including CML, CEA, MG-H, and G-H strongly correlate with traditionally clinical markers of kidney disease. The high area under the curve (AUC) values (AUC=0.965) in receiver operating characteristic (ROC) curve indicated that these four free AGEs can be served as combined diagnostic biomarkers for the diagnosis of kidney disease., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

We developed and evaluated two-level, namely 2017011 and 2017012, serum-based reference materials (RMs) for 17 beta-estradiol (17 β-E2) by the reference method of isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) from the remaining serum samples after routine clinical tests, to help improve clinical routine testing and provide the traceability of results. This paper describes the development process of these RMs. The National Metrology Institute of Japan (NMIJ) certified reference material (CRM) 6004-a was used as the primary RM for the measurement of 17 β-E2. These serum-based RMs showed satisfactory homogeneity and stability. They also assessed the commutability between the reference method and the three routine clinical immunoassay systems. Besides, a collaborative study was carried out in five reference laboratories, all of which had been accredited by the China National Accreditation Service for Conformity Assessment (CNAS) in accordance with ISO/WD 15725-1. Statistical analysis of raw results and uncertainty assessment obtained certified values: 2017011 was 445.2 ± 39.0 pmol/L, and 2017012 was 761.9 ± 35.5 pmol/L., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

The distribution of 16 photoinitiators (PIs)-benzophenone (BP) and its five derivatives, 4-methylbenzophenone (4-MBP), methyl-2-benzoylbenzoate (M2BB), 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB), and the other 7 PIs-in packaged juice and milk was assessed using an UPLC-MS/MS with micro-QuEChERS. The validated method demonstrated robust linearity (≥0.9908), LOQs (juice: 0.006-7.26 ng/mL; milk: 0.004-7.14 ng/mL), intra- and inter-day accuracy (93.1%-110.1% for juice and 66.8%-114.6% for milk with relative standard deviations (RSDs) of ≤16.8%), and matrix effect with RSDs of ≤14.8%. BP exceeded the LOQ in 100% of 136 packaged juice (14.22 ± 7.60 ng/mL) and 51 milk (8.45 ± 3.25 ng/mL) samples, and the quantitation rates for M2BB and EHDAB in packaged juice were 100%. Notably, the 4-MBP in both juice and milk was significantly related to the packaging material. The method has been successfully applied to evaluate the occurrence and distribution of PIs in packaged juice and milk., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Cyclocarya paliurus is an edible and medicinal plant exhibiting significant hypoglycemic effect. However, its active components are still unclear and need further elucidation. In this research, the active components of the leaves of C. paliurus responsible for the α-glucosidase inhibitory activity were screened and identified based on a spectrum-effect relationship study in combination with ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) analysis. The 70% ethanol eluate fraction of the leaves of C. paliurus with the strongest α-glucosidase inhibitory activity was obtained after extraction and purification with macroporous resin. Their chromatographic fingerprints (15 batches) were established by UPLC analysis and 32 common peaks were specified by similarity analysis. Their IC 50 values for α-glucosidase inhibition were measured by an enzymatic reaction. Several multivariate statistical analysis methods including hierarchical cluster analysis, principal component analysis, partial least square analysis and gray relational analysis were applied to explore the spectrum-effect relationship between common peaks and IC 50 values, and the chromatographic peaks making a large contribution to efficacy were screened out. To further elucidate the active components of leaves of C. paliurus, the 70% ethanol eluate fraction was characterized by UPLC-MS/MS analysis, and 10 compounds were identified. This study provides a valuable reference for further research and development of hypoglycemic active components of C. paliurus., (© 2022 John Wiley & Sons, Ltd.)

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.)

Accurate quantitation of aldosterone is clinically important in standardized testing for primary aldosteronism. The results are often variable when performed by clinical immunoassays. To standardize and ensure the accuracy of clinical systems, reference measurement procedures (RMPs) with higher metrological order are required. A simple and reliable isotope dilution LC-IDMS/MS-based measurement procedure for human plasma aldosterone has been developed. This method involved plasma spiked with a deuterium-labelled internal standard, equilibrated for 0.5 h, and extracted by liquid-liquid extraction (LLE) without derivatization. Aldosterone and its structural analogues were baseline separated with a C18-packed UHPLC column with gradient elution within 7 min. The signal intensity variability and measurement imprecision were reduced by bracketing calibration during plasma aldosterone value assignment. The limit of detection (LoD) was 19.4 pmol/L with a signal-to-noise ratio (S/N) > 3. The lowest limit of quantification (LLoQ) was 27.7 pmol/L (S/N > 10 and CV < 10.0%). LLE was performed with 1 mL of n-hexane/ethyl acetate (3:2, v/v), and the extraction recovery was determined to be 92.15 ± 3.54%. The imprecisions were ≤ 3.18% for samples at 124.8, 867.0, and 2628.5 pmol/L. The recoveries were 98.11-101.61%. The relative bias between this candidate RMP and the established RMP was 2.76-1.89%. The linearity response ranged from 27.7 to 2774.4 pmol/L with R 2  = 0.999. The method performance met the requirements of RMPs (≤ 5% total CV and ≤ 3% bias). Furthermore, the developed method was applied to evaluate immunoassays through 41 patient sample comparisons. The calibration and measurement capability (CMC) of this method were also evaluated by measuring these samples. The candidate RMP can serve as an accurate reference baseline for routine methods and can be used for value assignment for reference materials. Selected ion chromatograms by LC-MS/MS using a C18 column for aldosterone and its structural analogues.

Background: The aim of this study is to verify the analytical performance of four homocysteine detection systems made in China and to explore the comparability of homocysteine detection systems by isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) reference method., Methods: The intra-batch precision, inter-batch precision, accuracy, and linear range of four homocysteine detection systems were evaluated. The ID-LC-MS/MS reference method was used to evaluate the comparability and accuracy of fresh frozen serum samples in four different detection systems of homocysteine. The ID-LC-MS/MS reference method is used to assign samples as calibrators to calibrate each system. The variation and deviation of fresh serum samples between different systems before and after calibration were compared., Results: The intra-batch imprecision of the four detection systems was less than 5%, and the coefficient of variation of inter-batch imprecision was less than 6.7%. The precision met the clinical requirements. Before calibration, the results measured by detection system 2 are consistent with the ID-LC-MS/MS reference method, which meets the requirements of accuracy verification. The regression equation of R² ≥ 0.975 in the regression equation of linear analysis of the four systems, the linearity of the four detection systems is good in the range of evaluation concentration, and all of them can meet the declared linear range. The absolute average bias of fresh serum measured by the four detection systems after calibration decreased from 3.76 μmol/L, 0.96 μmol/L, 1.30 μmol/L, -1.56 μmol/L to 0.31 μmol/L, 0.28 μmol/L, 0.4 μmol/L, 0.40 μmol/L, respectively. The relative average bias decreased from 22.6%, 7.50%, 11.0% and -8.50% to 1.98%, 1.78%, 2.59%, 2.34%, respectively. After calibration, the slope and intercept of the regression curve of the fresh serum measured by the four detection systems and the reference method are closer to 1 and 0 than before calibration., Conclusions: The precision, reference interval, and linear evaluation of the four detection systems are good. The ID-LC-MS/MS reference method assigning fresh frozen serum samples as calibrators can improve the accuracy and comparability of the results of different detection systems.

A candidate reference measurement procedure (RMP) based on isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated for the quantification of 17α-hydroxyprogesterone (17-OHP) in human plasma. 17-OHP spiked with a deuterium-labeled internal standard was extracted from plasma by liquid-liquid extraction with 1 mL n-hexane/ethyl acetate (3:2, v/v). Reversed-phase chromatography and positive electrospray ionization were used in the ID-LC-MS/MS. Gradient elution coupled with use of a C 18 -packed ultrahigh-performance liquid chromatography column allowed complete baseline resolution of 17-OHP from its structural analogue desoxycorticosterone in 6 min. To determine the 17-OHP level in human plasma, a bracketing calibration method was used to give higher accuracy and precision. The limit of detection and the lower limit of the measuring interval for the candidate RMP were 2.1 pg/mL (6.4 pmol/L) and 4.6 pg/mL (13.9 pmol/L), respectively. Extraction recovery was determined to be (96.08 ± 3.03)% (n = 3). Imprecision (intra-assay and interassay) was 4.03% or less at 0.83, 15.19, 64.22, and 313.46 ng/mL (2.51, 45.97, 194.34, and 948.56 nmol/L, respectively). Recoveries ranged from 98.05% to 102.24%. When comparing our RMP results with those obtained with an established RMP via International Federation of Clinical Chemistry and Laboratory Medicine external quality assessment scheme for reference laboratories in laboratory medicine (RELA) samples, we found that the biases ranged from -1.99% to 3.08% against the targets. No interference was observed, and the linear response ranged from 0.47 to 958.63 ng/mL (1.42 to 2900.90 nmol/L). Moreover, the candidate RMP was used to measure the concentration of 17-OHP in human plasma and was compared with an immunoassay using 40 plasma samples. The performance of the method meets the needs of an RMP (total coefficient of variation of 5% or less and bias of 3.08% or less). This method can be used for reference material value assignment of 17-OHP in human plasma matrix. It could also serve as an accurate reference baseline for routine methods to increase the accuracy and precision of certain clinical laboratory measurements. Graphical abstract Selected ion chromatograms obtained by liquid chromatography-tandem mass spectrometry with a C 18 column for 17α-hydroxyprogesterone (17-OHP) from a plasma sample.

Urea is an important indicator of liver and kidney disease, and very frequently determined in clinical chemistry. The reference measurement procedures (RMPs) for serum urea recognized by the Joint Committee for Traceability in Laboratory Medicine are spectrophotometry, and isotope dilution (ID)-mass spectrometry (MS) coupled with gas chromatography. This study investigated a candidate RMP (cRMP) for detecting serum urea directly via ID-liquid chromatography (LC)-MS/MS, without derivatization, which simplifies pre-processing samples. The cRMP was developed and evaluated relative to the recognized RMP, inter- and intra-laboratories. The intra-precisions were 1.35%, 1.98% and 1.47% at 4.95, 24.74 and 31.36 mmol/L, respectively; inter-precision was 2.10%, 2.60% and 2.10%. The relative bias for the measurement of standard human serum (SRM 909c) was -0.49%. The relative biases were 0.41% and 0.02% for IFCC-RELA (International Federation of Clinical Chemistry and Laboratory Medicine-External Quality Assessment Scheme [EQAS] for reference laboratories) 2015A and 2015B. The linearity response between 2.4 mmol/L and 53.7 mmol/L was R 2  = 0.9989. No carryover, ion suppression, or interference was detected. Correlation was acceptable with the reference spectrophotometry (R 2  = 0.9985, P <  0.0001). Between our laboratory and other reference laboratories, the absolute deviation range for IFCC-RELA 2016A and 2016B was from -0.63 to 1.52 mmol/L. This well-characterized cRMP for urea can provide a base of accuracy for the traceability of clinical systems., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Influenza hemagglutinin is a surface glycoprotein related to virus invasion and host immune system response. Understanding site specific glycosylation of hemagglutinin will increase our knowledge about virus evolution and can improve the design and quality of vaccines. In our study, we used glycoproteomic analysis based on multienzyme digestion followed by LC tandem MS analysis to determine the glycosylation of Influenza hemagglutinin (H1/A/California/04/2009) using the following steps: PNGaseF treatment combined with trypsin or pepsin digestion was used to determine the glycosites and glycan occupancy. Three enzymes, trypsin, AspN, and pepsin, were used separately to generate suitable glycopeptides for online LC tandem MS analysis. The glycan structure of a given glycopeptide was determined by collision-induced dissociation MS/MS fragmentation, and the peptide backbone information was provided by collision-induced dissociation (CID)-MS3 fragmentation. With this approach, 100% sequence coverage of the hemagglutinin sample was obtained. Six glycosylation sites fitting the sequon N-X-S/T were successfully confirmed, and the glycan heterogeneity as well as the ratios of glycoforms were determined at each site.

Uric acid is a precipitating factor for gout and renal disease, and regular monitoring of uric acid concentration is essential for health management. Hence, accurate measurement is indispensable for clinical diagnosis. Reference measurement procedure (RMP) is an essential element to ensure accuracy and comparability of tested results. In this study, the optimization of a candidate RMP (cRMP) adopting isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method for human serum uric acid measurement has been developed. The accuracy of this cRMP was validated by measuring certified reference materials (CRMs) and participating in the external quality assessment (EQA) scheme initiated by International Federation of Clinical Chemistry (IFCC) and National Center for Clinical Laboratory (NCCL). Biases were −0.64-1.51%, which were within the required equivalent limit. Excellent precision was obtained with intra- and inter-assay CVs of 0.42–0.73% and 0.20–0.43%, respectively. The limit of detection (LOD) and limit of quantification (LOQ) was determined to be 0.24 and 1.19 μmol/L, respectively. A good liner response was obtained ranged from 30.0 to 895 μmol/L. Compared with the previous RMP for uric acid, the much easier one-step protein precipitation method without further evaporation, the simplified isocratic elution of chromatographic separation condition, and the shorter analysis running time in only 5 min allowed simpler and faster analysis. Moreover, this optimized cRMP was also applied for value assignment for clinical serum samples covered a wide concentration range of uric acid and obtained a decent calibration and measurement capability (CMC). [Display omitted] • The optimization of a candidate RMP (cRMP) adopting ID-LC-MS/MS method for serum uric acid measurement has been developed. • The method performance was fully validated. • The method was applied to clinical samples and compared with routine methods. [ABSTRACT FROM AUTHOR]

Abstract: The liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods were used to identify the pharmacologically active n-butanol extract from Compound Indigowoad Root Granule. As a result, eighteen compounds belonging to various structural classes such as nucleosides, purines, flavonoids and amino acid were unambiguously identified. Then an in vitro neuraminidase (NA) inhibition assay was carried out to examine the inhibitory activity of the standard samples and extracts on NA. After which, ultrafiltration liquid chromatography with photodiode array detection coupled to electrospray ionization tandem mass spectrometry (ultrafiltration LC–MS/MS) was used to study NA inhibitory activity of standard flavones and n-butanol extract of Compound Indigowoad Root Granule. This method is highly selective and sensitive, and it could be used for characterization of bioactive compounds and botanical extracts. The result provides some enlightenment for the explanation of the antiviral activity of Compound Indigowoad Root Granule and some guidance for natural anti-influenza medicine development. [Copyright &y& Elsevier]

Glycocholic acid (GCA) is an important identified biomarker for various hepatobiliary diseases. However, results obtained from routine clinical measurement significantly varied due to the interference from the extremely similar structure analogues, which cause confusion to clinical diagnosis. In this study, a bracketing calibration-based isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method was developed. The established method exhibited good performance criteria through the adoption of isotope dilution, calibrator bracketing, and gravimetric measurements. The satisfactory precision (0.16%–2.01%) and good recoveries were achieved at five spiked levels (98.0–100.9%). The limit of detection (LOD) and limit of quantification (LOQ) were 0.01 ng/mL and 0.05 ng/mL, respectively. No interference, matrix effect, and carryover were observed. The sample preparation process was easier to operate, without further solvent evaporation and time-saving. The optimized method was successfully applied to quantify GCA with a wide range of concentrations in clinical human serum samples and also compared with clinical routine systems. [Display omitted] • An isotope-dilution based LC-MS/MS method for serum GCA determination was developed. • The method performance was validated. • The method was applied to clinical samples and compared with routine methods. [ABSTRACT FROM AUTHOR]

• A highly reliable, sensitive, accurate, and precise ID-LC–MS/MS-based candidate reference measurement procedure for E 2 measurement was developed and validated. • Bracketing calibration method (BCM) compared to classical calibration curve. • BCM has demonstrated to be higher accurate and precise and in good agreement with the need of RMPs. • Sample preparation requires no derivatization, and appropriate retention of E 2 and high resolution of structural analogs were achieved within a short time span. • The validated method was successfully applied to measure the E 2 level in serum samples and to assess the accuracy of three immunoassays in a clinical laboratory. A highly accurate liquid chromatography tandam mass spectrometry (LC–MS/MS) based candidate reference measurement procedure (cRMP) for the determination of human serum 17β-estradiol (E 2) was developed. A bracketing calibration method (BCM) based on internal standard (IS) concentrations for each sample used to best match the analyte concentration was compared to the classical calibration curve-based method with a constant IS concentration. Precision was analyzed according to the inter-assay reproducibility (n = 15) and intra-assay repeatability (n = 9). Accuracy was confirmed using certified reference materials (CRMs) and further validated by comparison to the established RMPs. Sixty patient serum samples were collected, and the E 2 levels were measured by the BCM isotope dilution (ID) procedure coupled with the LC–MS/MS method to evaluate three immunoassays commonly used in clinical laboratories in China. Limits of quantification (LoQ) as low as 5 pg/mL (18 pmol/L) were achieved. The isomer of E2 (17α-estradiol) was successfully separated by optimizing the LC method. The results of method validation showed that the intra- and inter-assay imprecisions were 1.84 ∼ 2.91% vs 2.08 ∼ 9.53%, while the analytical recoveries were 98.73 ∼ 100.77% vs 93.72 ∼ 102.39% for the BCM and classical calibration curve quantification method, and the BCM demonstrated a higher agreement with CRMs and established RMPs. Bland-Altman plots of the E 2 results revealed concentration-dependent biases. In conclusion, a precise, facile, and reliable ID-LC–MS/MS method is developed in this study. It is demonstrated that the use of the BCM could achieve higher precision and accuracy, which better meet the needs of RMPs. [ABSTRACT FROM AUTHOR]