Tianjiao Zhang

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.

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.

Commutability of control materials for external quality assessment of serum apolipoprotein A-I measurement

Abstract Background: The aim of the current study was to evaluate the commutability of commercial control materials and human serum pools and to investigate the suitability of the materials for the external quality assessment (EQA) of serum apolipoprotein A-I (apo A-I) measurement. Methods: The Clinical and Laboratory Standards Institute (CLSI) EP14-A3 protocol was used for the commutability study. Apo A-I concentrations in two levels of commercial control materials used in EQA program, two fresh-frozen human serum pools (FSPs) and two frozen human serum pools prepared from residual clinical specimens (RSPs) were measured along with 50 individual samples using nine commercial assays. Measurement results of the 50 individual samples obtained with different assays were pairwise analyzed by Deming regression, and 95% prediction intervals (PIs) were calculated. The commutability of the processed materials was evaluated by comparing the measurement results of the materials with the limits of the PIs. Results: The FSP-1 was commutable for all the 36 assay pairs, and FSP-2 was commutable for 30 pairs; RSP-1 and RSP-2 showed commutability for 27/36 and 22/36 assay pairs, respectively, whereas the two EQA materials were commutable only for 4/36 and 5/36 assay pairs, respectively. Conclusions: Non-commutability of the tested EQA materials has been observed among current apo A-I assays. EQA programs need either to take into account the commutability-related biases in the interpretation of the EQA results or to use more commutable materials. Frozen human serum pools were commutable for most of the assays.

Evaluation of serum alkaline phosphatase measurement through the 4-year trueness verification program in China

Abstract Background Alkaline phosphatase (ALP) is critical for various diseases. The International Federation of Clinical Chemistry and Laboratory Medicine had recommended the new reference procedure in 2011, but many manufacturers did not trace results to the higher procedure. Since 2012, the National Center for Clinical Laboratories (NCCL) in China has organized the trueness verification program (TV) with commutable materials. The present study summarizes the 4-year TV program to give an overview of the measurement standardization for ALP results. Methods Commutable serum-based materials with different concentrations were prepared and sent to participating laboratories. The target values were assigned by the reference lab network. Results The analytical performance was evaluated according to three indexes: trueness (bias), imprecision (CV) and accuracy (total error [TE]). The number of participating laboratories increased from 115 in 2012 to 287 in 2016. The pass rates of precision for homogeneous and heterogeneous systems were all above 85% over the 4 years; however, the pass rates of bias were much lower (<50%). Among the homogeneous systems, Roche Cobas/Modular had an obvious negative bias, whereas the mean positive bias for Beckman AU was prominent. As to the heterogeneous systems, the pass rates of bias for Sichuan Maccura (57.1%–78.6%) were higher than Roche Cobas/Modular (4.4%–33.9%) and Beckman AU (35.7%–64.8%). Conclusions The PT/EQA program with commutable materials can be used to assess the trueness against target values assigned by reference procedures. For ALP, homogeneous systems did not perform better than heterogeneous systems. The bias for ALP performance was notable and was the main obstacle to its standardization in China.

Inductively Coupled Plasma Mass Spectrometry as a Reference Method to Evaluate Serum Calcium Measurement Bias and the Commutability of Processed Materials during Routine Measurements

Background: Measuring total serum calcium is important for the diagnosis of diseases. Currently, results from commercial kits for calcium measurement are variable. Generally, the performance of serum calcium measurements is monitored by external quality assessment (EQA) or proficiency testing schemes. However, the commutability of the EQA samples and calibrators is often unknown, which limits the effectiveness of EQA schemes. The aim of this study was to evaluate the bias of serum calcium measurements and the commutability of processed materials. Methods: Inductively coupled plasma mass spectrometry was applied as a comparative method, and 14 routine methods were chosen as test methods. Forty-eight serum samples from individual patients and 25 processed materials were quantified. A scatter plot was generated from patient samples, and 95% prediction intervals were calculated to evaluate the commutability of the processed materials and measurement bias at three concentration levels was used to determine the accuracy of routine assays. Results: All assays showed high precision (total coefficient of variation [CV] <2.26%) and correlation coefficients (r > 0.99). For all assays, the mean bias for the 48 patient samples ranged from −0.13 mmol/L to 0.00 mmol/L (−5.61–0.01%), and the ranges for the three concentrations were −0.10–0.04 mmol/L (−5.71–2.35%), −0.14–−0.01 mmol/L (−5.80–−0.30%), and −0.19–0.04 mmol/L (−6.24–1.22%). The EQA samples, calibrators, and animal sera exhibited matrix effects in some assays; human serum pools were commutable in all assays; certificate reference materials were commutable in most assays, and only GBW09152 exhibited a matrix effect in one assay; and aqueous reference materials exhibited matrix effects in most assays. Conclusions: Biases for most assays were within the acceptable range, although the accuracy of some assays needs improvement. Human serum pools prepared from patient samples were commutable, and the other tested materials exhibited a matrix effect.

Determination of serum calcium levels by 42Ca isotope dilution inductively coupled plasma mass spectrometry

Abstract Background: Serum calcium level is an important clinical index that reflects pathophysiological states. However, detection accuracy in laboratory tests is not ideal; as such, a high accuracy method is needed. Methods: We developed a reference method for measuring serum calcium levels by isotope dilution inductively coupled plasma mass spectrometry (ID ICP-MS), using 42Ca as the enriched isotope. Serum was digested with 69% ultrapure nitric acid and diluted to a suitable concentration. The 44Ca/42Ca ratio was detected in H2 mode; spike concentration was calibrated by reverse IDMS using standard reference material (SRM) 3109a, and sample concentration was measured by a bracketing procedure. We compared the performance of ID ICP-MS with those of three other reference methods in China using the same serum and aqueous samples. Results: The relative expanded uncertainty of the sample concentration was 0.414% (k=2). The range of repeatability (within-run imprecision), intermediate imprecision (between-run imprecision), and intra-laboratory imprecision were 0.12%–0.19%, 0.07%–0.09%, and 0.16%–0.17%, respectively, for two of the serum samples. SRM909bI, SRM909bII, SRM909c, and GBW09152 were found to be within the certified value interval, with mean relative bias values of 0.29%, −0.02%, 0.10%, and −0.19%, respectively. The range of recovery was 99.87%–100.37%. Results obtained by ID ICP-MS showed a better accuracy than and were highly correlated with those of other reference methods. Conclusions: ID ICP-MS is a simple and accurate candidate reference method for serum calcium measurement and can be used to establish and improve serum calcium reference system in China.