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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]

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. [ABSTRACT FROM AUTHOR]

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 R2 = 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. [ABSTRACT FROM AUTHOR]

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 C18-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. [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]