Weiyan Zhou

Rapid and Precise Measurement of Serum Branched-Chain and Aromatic Amino Acids by Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry

Background Serum branched-chain and aromatic amino acids (BCAAs and AAAs) have emerged as predictors for the future development of diabetes and may aid in diabetes risk assessment. However, the current methods for the analysis of such amino acids in biological samples are time consuming. Methods An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for serum BCAAs and AAAs was developed. The serum was mixed with isotope-labeled BCAA and AAA internal standards and the amino acids were extracted with acetonitrile, followed by analysis using LC/MS/MS. The LC separation was performed on a reversed-phase C18 column, and the MS/MS detection was performed via the positive electronic spray ionization in multiple reaction monitoring mode. Results Specific analysis of the amino acids was achieved within 2 min. Intra-run and total CVs for the amino acids were less than 2% and 4%, respectively, and the analytical recoveries ranged from 99.6 to 103.6%. Conclusion A rapid and precise method for the measurement of serum BCAAs and AAAs was developed and may serve as a quick tool for screening serum BCAAs and AAAs in studies assessing diabetes risk.

A novel and precise method for simultaneous measurement of serum HDL and LDL subfractions and lipoprotein (a) cholesterol by ultracentrifugation and high-performance liquid chromatography.

BACKGROUND We developed an ultracentrifugation and high-performance liquid chromatography (HPLC) method for simultaneous measurement of cholesterol in serum high density lipoprotein (HDL) and low density lipoprotein (LDL) subfractions and lipoprotein (a) [Lp(a)]. METHODS Serum aliquots of 0.05 ml were centrifuged at background densities of 1.006, 1.044 kg/l, and in the presence of β-mercaptoethanol (ME) at background densities of 1.044, 1.063 and 1.125 kg/l for the separation of lipoprotein subfractions and Lp(a). Cholesterol levels in the ultracentrifugal bottom fractions were analyzed by HPLC. RESULTS ME effectively dissociated Lp(a) into apolipoprotein (a) and Lp(a) remnant [Lp(a-)]. Lp(a-) showed a distinctive density distribution from that of the native Lp(a). Based on these data, a method was developed to separate lipoprotein into subfractions and Lp(a) by ultracentrifugation. The separated HDL and LDL subfractions were not contaminated with Lp(a). This method is highly precise with the total CVs for the measurement of HDL2-C, HDL3-C, LDLa-C, LDLb-C and Lp(a)-C 0.85%-2.66%, 0.87%-3.21%, 0.86%-1.11%, 2.59%-6.35% and 4.42%-12.29%, respectively. CONCLUSION A new method for the separation of HDL and LDL subfractions and Lp(a) and simultaneous measurement of cholesterol by ultracentrifugation and HPLC have been established. It is precise and sensitive and can be used in research or clinical laboratories for lipoprotein profiling.

A simple and precise LC-MS/MS method for the simultaneous determination of serum 25-hydroxyvitamin D3 and D2 without interference from the C3 epimer

Serum 25-hydroxy metabolites of vitamin D3 and D2 (25-OH D3 and 25-OH D2) are significant biomarkers for vitamin D deficiency, which has drawn widespread attention as a notable public health problem; therefore, monitoring serum 25-OH D levels plays an increasingly important role in clinical tests and preventive medicine. Some current analytical methods used to measure human serum 25-OH D3 and 25-OH D2 suffer from complex operations, inaccuracies, or interference from the C3 epimer of the metabolites. In this study, a simple, precise, and accurate LC-MS/MS method has been developed to determine serum 25-OH D3 and 25-OH D2 levels, markedly eliminating interference from the C3 epimer through successful chromatographic separation performed on a pentafluorophenyl column. Detection of the analytes was carried out in multiple-reaction monitoring mode, and quantitative analysis was supported by the use of isotope-labeled 25-OH D3 and D2 as internal standards. The relative standard deviations of intra-run and total assays were less than 2.9% and 5.5%. The detection limits were as low as 0.27 and 0.31 ng ml−1 for 25-OH D3 and 25-OH D2, with average recoveries of 99.65% and 99.93%, respectively. The accuracy of the protocol was also validated by measuring the National Institute of Standards and Technology standard serum reference materials, and the results were consistent with the certified values. In addition, the utility of the method was evaluated in a vitamin D status study to assess reference intervals and associations between vitamin D and risk factors for cardiovascular diseases in healthy Chinese volunteers. The method is simple, precise, and accurate, and can be utilized to measure 25-OH D3 and 25-OH D2 levels in human serum in clinical settings.

Simultaneous quantification of cardiovascular disease related metabolic risk factors using liquid chromatography tandem mass spectrometry in human serum

Recent observations from metabonomic studies have consistently found that branched-chain amino acids (BCAAs), aromatic amino acids (AAAs), glutamine (Gln), glutamic acid (Glu), Gln/Glu ratio, carnitine, and several species of acylcarnitines and lysophosphatidylcholines (LPCs) are possible risk factors for metabolic diseases such as diabetes mellitus (DM) and cardiovascular diseases (CVD). We described here a simple and reliable method for simultaneous quantification of these metabolic risk factors by liquid chromatography tandem mass spectrometry (LC-MS/MS). Serum samples were extracted with isopropanol, and the extracted metabolites were separated by hydrophilic interaction liquid chromatography (HILIC) and detected with electrospary ionization (ESI) inpositive ion mode with multiple reaction monitor (MRM) mode. All the metabolites were effectively separated within 5.5min. Analytical recoveries were in the range of 92.8-106.9%, with an average of 100.6%. The intra- run and total imprecisions for the measurement of these metabolites were 1.2-3.8% and 1.5-7.4%, respectively. Serum concentrations of the metabolites were analyzed in 123 apparently healthy volunteers. Significant associations between the metabolites and traditional CVD risk factors were observed. The newly developed LC-MS/MS method was simple, precise, and accurate and can be used as an efficient tool in CVD research and studies.

A rapid and precise method for quantification of fatty acids in human serum cholesteryl esters by liquid chromatography and tandem mass spectrometry

We described a rapid and precise method for simultaneous quantification of eleven fatty acids in human serum cholesteryl esters (CEFAs) by liquid chromatography and tandem mass spectrometry (LC-MS/MS). After extraction of serum lipids with isopropanol, CEFAs were separated on reversed phase liquid chromatography and detected by mass spectrometry in positive ion mode with multiple reaction monitor. Individual CEFA was quantified by peak area normalization method and expressed as molar percent of total CEFAs. The run time was less than 5 min and detection limits were from 0.31 to 14.50 × 10(-5)mmol/L. Recoveries of the CEFAs ranged from 91.85% to 104.83% with a mean of 99.12%. The intra and total CVs for the measurement of CEFAs were 0.87-7.70% and 1.02-7.65%, respectively. This LC-MS/MS method required no internal standards, eliminated natural isotope interferences, and provided reproducible and reliable results for 11 major CEFAs in human serum. This method can be used in monitoring and evaluating dietary fatty acid intake. Additional studies are needed to evaluate the associations between serum CEFAs and cardiovascular disease risk factors.

Serum cholesterol measured by isotope dilution liquid chromatography tandem mass spectrometry.

Abstract Background: Accurate cholesterol measurements are essential for the prevention and management of cardiovascular diseases. Quality assurance of cholesterol measurements requires reference methods. Methods: An isotope dilution liquid chromatography mass spectrometric (ID/LC/MS) method was developed. Serum samples were sampled volumetrically using automated dilutors, treated with potassium hydroxide and equilibrated with 3,4-13C2 cholesterol. The natural cholesterol and the internal standard were extracted with hexane and oxidized to cholest-4-en-3,6-diones with chromic acid. The oxidation products were separated on reversed phase LC and detected by tandem MS. The method was calibrated using aqueous cholesterol calibrators and the calibration function was established with a polynomial regression. Results: The correlation coefficients of the calibration curves were always >0.9999. The coefficients of variation (CV) of the volumetric sampling and the LC/MS analysis averaged 0.22% and 0.50%, respectively, and the total measurement CV was 0.60%. Other sources of measurement uncertainty were minor. Results on certified reference materials agreed within 1% of the certified values. Conclusions: An ID/LC/MS method for serum cholesterol has been developed. The method is simple and accurate and may be used as a candidate reference method.

Performance of electrolyte measurements assessed by a trueness verification program

Abstract Background: In this study, we analyzed frozen sera with known commutabilities for standardization of serum electrolyte measurements in China. Methods: Fresh frozen sera were sent to 187 clinical laboratories in China for measurement of four electrolytes (sodium, potassium, calcium, and magnesium). Target values were assigned by two reference laboratories. Precision (CV), trueness (bias), and accuracy [total error (TEa)] were used to evaluate measurement performance, and the tolerance limit derived from the biological variation was used as the evaluation criterion. Results: About half of the laboratories used a homogeneous system (same manufacturer for instrument, reagent and calibrator) for calcium and magnesium measurement, and more than 80% of laboratories used a homogeneous system for sodium and potassium measurement. More laboratories met the tolerance limit of imprecision (coefficient of variation [CVa]) than the tolerance limits of trueness (biasa) and TEa. For sodium, calcium, and magnesium, the minimal performance criterion derived from biological variation was used, and the pass rates for total error were approximately equal to the bias (<50%). For potassium, the pass rates for CV and TE were more than 90%. Compared with the non homogeneous system, the homogeneous system was superior for all three quality specifications. Conclusions: The use of commutable proficiency testing/external quality assessment (PT/EQA) samples with values assigned by reference methods can monitor performance and provide reliable data for improving the performance of laboratory electrolyte measurement. The homogeneous systems were superior to the non homogeneous systems, whereas accuracy of assigned values of calibrators and assay stability remained challenges.

Quantification of hemoglobin A1c by off-line HPLC separation and liquid chromatography-tandem mass spectrometry: a modification of the IFCC reference measurement procedure.

Abstract Background: The quality of hemoglobin A1c measurement is very important in the management of diabetes. A reference system has been established by the IFCC Working Group on HbA1c Standardization. We did some modification of the IFCC Reference Measurement Procedure with MS detection which significantly decreased the analysis time and improved the precision of the analytes by off-line HPLC separation and liquid chromatography-tandem mass spectrometry (LC/MS/MS) analysis. Methods: The samples were prepared and enzymatically cleaved according to the IFCC HbA1c reference measurement procedure. Then the digest solution was injected to a reversed phase HPLC for purification and a clean fraction was collected. The fraction was analyzed by liquid chromatography-tandem mass spectrometry with an isocratic elution on a C18 column. Liner regression was used to determine the concentration of HbA1c. Results: The total analysis time which includes the off-line HPLC separation and the LC/MS/MS analysis was reduced by at least 65% compared to the existing IFCC method. The transitions of m/z 348.4→237.2 and m/z 429.4→245.2 were selected for quantification of non-glycated and glycated hexapeptide. Fifty seven hemolysate samples used in recent 3-years’ IFCC HbA1c inter-laboratory studies (2012–2014) were analyzed by the LC/MS/MS procedure for method validation. The CVS were between 0.16% and 1.87% for 4 measurements per sample in the concentration range for HbA1c between 30.4 and 145.8 mmol/mol. The relative bias of the LC/MS/MS method was varied from -3.21% to 2.47% compared to the IFCC network assigned values. Conclusions: This method is an efficient and reliable procedure for the determination of HbA1c. After thorough evaluation within the IFCC Network this modification may be implemented in the IFCC RMP for HbA1c with MS detection.

Assessment of enzyme measurement procedures in China through a trueness verification program.

BACKGROUND Since 2003, the National Center for Clinical Laboratories (NCCL) has organized a network of reference laboratories and several survey programs to improve standardization in China. METHODS We analyzed the 2015 trueness verification program to assess the status of enzyme measurement standardization. Commutable serum-based materials were prepared and sent to 10 reference laboratories to assign target values for 2 enzymes (alanine aminotransferase-pyridoxal phosphate [ALT-pp] and γ-glutamyltransferase [GGT]) using IFCC reference measurement procedures. RESULTS Analytical performance was assessed for compliance to 3 indexes: trueness (bias), imprecision (CV), and accuracy (total error). Of the 250 participating laboratories, about half (≥124) used heterogeneous systems. More laboratories met the tolerance limit of imprecision than of trueness or accuracy. Except at the lowest concentration, the CV pass rates were >90% for the 2 enzymes. The optimal performance criterion derived from biological variation yielded pass rates for total error (ALT 77%, GGT 80%) that were higher than for bias (ALT 63%, GGT 73%). CONCLUSIONS PT/EQA results for commutable samples can be used to assess trueness against reference measurement procedures. Despite global and national standardization programs, bias remains a critical limitation of current enzyme measurement procedures in China.

Serum triglyceride measurements: the commutability of reference materials and the accuracy of results

Abstract Background We aimed to evaluate the commutability of external quality assessment (EQA) materials, aqueous solutions, and commercial reference materials (calibrators and controls), and the accuracy of routine systems for serum triglyceride measurements. Methods According to the clinical and laboratory standards institute (CLSI) EP14-A3 protocol, we analyzed 43 fresh patient specimens and 32 processed materials including lyophilized samples, human serum pools, liquid reagents, swine sera and aqueous solutions by 14 routine methods (evaluated methods) and an isotope dilution liquid chromatography tandem mass spectrometry method (ID-LC/MS/MS) (comparative method). The accuracy of the routine method was evaluated by analyzing the absolute bias, relative bias, and the bias at three medical decision levels based on CLSI EP9-A3. Results Frozen serum samples and swine sera were commutable for all of the assays. The EQA/PT materials, commercial calibrators and control materials showed matrix effects differently on routine methods. The aqueous glycerol solutions were generally noncommutable for routine method. All except one routine analytical systems met the National Cholesterol Education Program (NCEP) recommended analytical performance guideline analytical quality criteria for total error. Conclusions Matrix effects and calibration biases existed in measurements of serum triglyceride. Continued efforts are needed to improve the accuracy and comparability of routine measurements.