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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.)

Purpose: We aimed to describe the vitamin D status and its distribution in different age groups, sexes, seasons, and provinces of a large Chinese population., Methods: This study retrospectively analyzed 1,528,685 results of serum 25-hydroxyvitamin D (25(OH)D) in the central laboratory of KingMed Diagnostics. The samples were from the individuals aged 0-119 years old in 30 provinces of China. Serum 25(OH)D was measured by an accurate commercial liquid chromatography-tandem mass spectrometry (LC-MS/MS) method from January 2017 to December 2019. The subjects were stratified by age, sex, the season of blood collection, and the province of residence., Results: The median 25(OH)D concentration was 25.5 ng/mL (interquartile range (IQR) 18.7-32.7 ng/mL) in males and 20.8 ng/mL (IQR 14.4-28.2 ng/mL) in females. Overall, the median 25(OH)D concentration decreased with age in both males and females. Males had a 0.2-2.4 ng/mL higher median 25(OH)D concentration than females in different age groups. Vitamin D deficiency (25(OH)D < 15 ng/mL for the individuals under 14 years old; < 20 ng/mL for the individuals over 14 years old) was found in 21.3% of males and 43.6% of females. Significant seasonal variation of serum 25(OH)D concentrations was repeatedly observed in 3 years, with median concentration higher in summer (25.3 ng/mL (IQR 19.3-31.9 ng/mL)) and lower in winter (18.5 ng/mL (IQR 12.3-26.6 ng/mL)). Vitamin D status varied by province. The median 25(OH)D concentration was the highest in Hainan (31.0 ng/mL (IQR 24.9-39.2 ng/mL)) and the lowest in Qinghai (14.4 ng/mL (IQR 9.6-20.0 ng/mL)). 25(OH)D 2 was detected in 12.2% of the results, and no significant seasonal variation was observed., Conclusion: In China, vitamin D deficiency is prevalent in the population participating in clinical vitamin D measurement. Age and sex differences in vitamin D levels were observed in our study. Seasonal variation and provincial differences are important aspects of serum vitamin D status. 25(OH)D 2 cannot be ignored entirely in clinical measurement practice in China., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany.)

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.

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.

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]

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]