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Objective:To establish a candidate reference method for the determination of angiotensin Ⅱ in human plasma by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) and to evaluate its performance.Methods:Using [ 13C 6- 15N]-angiotensin Ⅱ as the internal standard, the plasma was accurately weighed by gravimetric method and mixed with a certain amount of internal standard. At the same time, enzyme inhibitor was added. After zinc sulfate solution protein precipitation and reversed-phase solid-phase extraction plate treatment, it was analyzed by liquid chromatography tandem mass spectrometry. The multi reaction ion monitoring mode(MRM)was selected by mass spectrometry to detect specific ion fragments of angiotensin Ⅱ and internal standard. The linearity, sensitivity, precision, recovery rate and uncertainty of the performance of the established method were evaluated according to ISO15193. Results:Angiotensin Ⅱ had good linearity in the range of 10-1 000 pg/g ( r=0.999 5), the lower limit of quantification was 7.68 pg/g, the analytical recoveries were 97.14% to 102.85%, intra-batch imprecision≤3.21%, inter-batch imprecision≤2.96%, and total imprecision≤3.67%. Conclusion:A method for the determination of plasma angiotensin Ⅱ was established by ID-LC-MS/MS. The method is accurate and reliable, and is expected to be a reference method for the determination of plasma angiotensin Ⅱ.
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Electrolytes for sodium, potassium, magnesium, and calcium are important serum ions that are frequently assayed in clinical laboratories. In this study, we assessed the trueness of routine analytical systems for four cations using an inexpensive candidate reference method aimed to promote the standardization of serum electrolyte detection. An ion chromatography (IC) method with Cesium as an internal standard was developed and evaluated. The residual clinical serum samples at Chaoyang Hospital were collected and prepared into three human serum pools of electrolytes, which were used for the trueness evaluation of five routine analytical systems. Furthermore, the agreement between routine methods and the IC method was verified using 40 individual human samples. The recovery rates of sodium, potassium, magnesium and calcium were 99.69%, 100.34%, 100.43% and 99.89%, respectively. The intra-batch standard deviation and intra-laboratory precision of NIST SRM 956c were all less than 1% for the four ions. The certified values were within the validation range, and the deviation between the results and the certified values were less than 0.5%. The three serum pools were homogeneous and stable. All routine systems aligned with the IC method for four cations and achieved the analytical quality specifications for potassium and magnesium at 3 different concentrations. The developed IC method is simple, practical, accurate, and precise, which can be used as a candidate reference method for serum electrolytes measurement. Five routine analytical systems for electrolytes measurement had the acceptable bias for potassium and magnesium and their results showed good concordance.
Subject(s)
Chromatography/methods , Electrolytes/blood , Bias , Humans , Reference StandardsABSTRACT
BACKGROUND: Owing to the increasing interest in public health research of antioxidant micronutrients and the inaccuracy of routine serum concentrations of the fat-soluble vitamins A (retinol) and E (DL-α-tocopherol) measurements, we developed a reliable, highly sensitive, robust and rapid method for the quantification of two clinically important lipophilic antioxidants in serum using a reverse-phase HPLC/DAD method. METHOD: Sample preparation and analytical conditions that would affect extraction efficiency and quantitative results of vitamins A and E were investigated and optimized. Vitamins A and E were extracted from serum via liquid-liquid extraction (LLE). After adequate sample preparation, the samples were injected directly into the HPLC system with diode-array detector (DAD). Chromatographic separation was completed in 7 minutes for vitamins A and E. With vitamin A acetate and vitamin E acetate as internal standards, the method was applied to the measurement of vitamins A and E in human serum. RESULTS: We evaluated method linearity, accuracy (recovery rate and trueness), precision, carryover, limit of quantitation and limit of detection, and measurement uncertainty. The method was evaluated for trueness using NIST Standard Reference Material SRM 968f. The serum concentration of the studied compounds had a good linear relationship in the range of 0.05 ~ 3.0 µg/mL concentration (r ï¼ 0.9998), with 0.0077 µg/mL detection limit and 0.025 µg/mL quantitative limit for vitamin A, respectively, and 1.0 ~ 60.0 µg/mL concentration (r ï¼ 0.9999), with 0.40 µg/mL detection limit and 0.50 µg/mL quantitative limit for vitamin E, respectively. The intra- and inter-assay coefficients of variation were calculated by using three concentrations (1, 2, and 3) of the studied compounds in human serum samples. Intra-assay and inter-assay precision were 1.23%-4.97% and 0.97%-3.79% for vitamin A, respectively, and 0.64%-4.07% and 0.81%-5.96% for vitamin E, respectively. The average recovery rates were 100.98% for vitamin A, and 99.21% for vitamin E, respectively. The carryover rate of vitamins A and E was below 1%. As for the evaluation of accuracy, the biases were <± 5% by comparing with NIST standard reference material SRM 968f. CONCLUSION: The method is a simple sample treatment procedure for the determination of fat-soluble vitamins A and E in human serum with high sensitivity and specificity. The proposed method could be recommended as a candidate reference method for the determination of serum concentrations of the fat-soluble vitamins A and E in human serum.
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BACKGROUND: We developed an ion chromatography (IC) method for measurement of chloride in human serum which was regarded as a simple, rapid, accurate, and sensitive technique. The method will be hopefully selected as a candidate reference method. METHOD: Serum aliquots of 0.1 mL were diluted 500 times with Milli-Q water, and chloride in serum samples was measured by IC with a gradient elution procedure using a KOH eluent generator. RESULTS: Based on the data, chloride in human serum was well detected by IC. The calibration curve for chloride was linear in the concentration range from 0 to 0.42 mmol/L with a correlation coefficient of .99995 under the optimum experimental conditions. The chloride concentration had a good linear relationship with the peak areas of chloride. This method was sensitive because of the low limit of detection (LOD) and the low limit of quantification (LOQ) 9.87 × 10-5 mmol/L and 3.27 × 10-4 mmol/L, respectively. Besides, the method was highly precise with the within-run coefficient of variations (CVs) for the measurement of low, medium, and high concentration level samples 0.32%, 0.73%, and 0.50%. As for the evaluation of accuracy, the biases were less than ±1% and 2% by comparing with National Institute of Science and Technology (NIST) standard material SRM 956d and 2013-2018 IFCC-RELA samples, respectively. Finally, the biases between IC method and the inductively coupled plasma mass spectrometry (ICP-MS) method were less than 1% which showed good agreement. CONCLUSION: Ion chromatography is a simple sample treatment procedure for the determination of chloride in human serum with high sensitivity and specificity. The proposed method could be recommended as a candidate reference method for the determination of serum chloride in human serum.
Subject(s)
Chlorides/blood , Chromatography, Ion Exchange/methods , Humans , Limit of Detection , Linear Models , Mass Spectrometry , Reproducibility of ResultsABSTRACT
BACKGROUND: To improve the accuracy of the routine methods in laboratory medicine, ion chromatography with a simple sample treatment procedure, which can completely remove the proteins and/or organics in human serum, has been developed for the determination of serum cations. METHODS: Chromatographic conditions for the separate and simultaneous determination of K, Na, Ca, and Mg were investigated. Furthermore, various factors influencing the mineralization of human serum, such as the selection and amount of oxidant, were also examined systematically and optimized. RESULTS: The optimized experimental conditions are as follows: 1.0 mL of serum specimen digested with 2 mL nitric acid (120°C) followed by 2 mL hydrogen peroxide (80°C). The specimens were then redissolved and determined by ion chromatography under the optimum eluent concentration of 32 mmol/L methanesulfonic acids. The measurement accuracy and precision are less than 1.0% for all the analytes by analyzing NIST certified reference materials, IFCC-RELA specimens and serum specimens. The results were also comparable with the reference values obtained by the inductively coupled plasma mass spectrometry (ICP-MS), which were found to be in good agreement. CONCLUSIONS: Ion chromatography with a simple sample treatment procedure for the determination of cations in human serum with high sensitivity and specificity was developed. The proposed method could be recommended as a candidate reference method for the determination of serum cations.
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Objective To develop a candidate reference method for the determination of serum creatinine and to evaluate the ac-curacy of conventional detection systems though method comparison to achieve traceability .Methods The candidate reference method was established according to the sarcosine oxidase and the accuracy and reliability of the method was verified through par-ticipation in international reference laboratories EQA activities (IFCC-RELA) .20 fresh single human serum samples with different concentration and calibrator were simultaneously measured by using conventional detection system and candidate reference method . Results The calibration curve for serum creatinine was linear in the concentration range from 50-2 000 μmol/L with a correlation coefficient of 0 .999 9 under the optimum experimental conditions (the linear equation was Y=0 .000 884 2X-0 .000 325 3) and the imprecision was less than 1 .0% .The proposed method has been applied to the determination of RELA samples with satisfactory re-sults .The measured results with conventional detection systems were consistent with candidate reference method ,and the slope of the regression equation was 1 .005 6 .Conclusion The candidate reference method of serum creatinine is successfully established and which can be used for traceability and standardization .It may provide an effective way for conventional detection system traceable to the reference method or reference material .
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Objective To establish a candidate reference method for the determination of serum lithium based on ion chromatog-raphy and evaluate its analytical performance.Methods A simple sample treatment procedure,which can be remove the proteins and/or organics in human serum,has been developed for the determination of serum lithium.Method precision was evaluated with different concentration of fresh human serum and EQA sample RELA-A/B.Method accuracy was investigated with the recovery ex-periments in fresh human serum and RELA-A/B sample.Results The linear equation was Y =0.817 1X -0.001 3 with a correla-tion coefficient of 0.999 95 under the optimum experimental conditions,the detection limit (3S/N)for lithium was 6 μg/L and the imprecision was less than 1.0%.The results of the recovery experiments indicated that the recoveries were reasonable for the deter-mination of serum lithium,in a range of 99%-101%.Conclusion The candidate reference method of lithium was successfully es-tablished and which can be used for traceability and standardization.It may provide an effective way for routine testing of lithium traceable to the reference method/reference material.