Evaluation of low-density lipoprotein cholesterol equations by cross-platform assessment of accuracy-based EQA data against SI-traceable reference value.

OBJECTIVES: Low-density lipoprotein cholesterol (LDLC) is the primary cholesterol target for the diagnosis and treatment of cardiovascular disease (CVD). Although beta-quantitation (BQ) is the gold standard to determine LDLC levels accurately, many clinical laboratories apply the Friedewald equation to calculate LDLC. As LDLC is an important risk factor for CVD, we evaluated the accuracy of Friedewald and alternative equations (Martin/Hopkins and Sampson) for LDLC. METHODS: We calculated LDLC based on three equations (Friedewald, Martin/Hopkins and Sampson) using the total cholesterol (TC), triglycerides (TG), and high-density lipoprotein cholesterol (HDLC) in commutable serum samples measured by clinical laboratories participating in the Health Sciences Authority (HSA) external quality assessment (EQA) programme over a 5 years period (number of datasets, n=345). LDLC calculated from the equations were comparatively evaluated against the reference values, determined from BQ-isotope dilution mass spectrometry (IDMS) with traceability to the International System of Units (SI). RESULTS: Among the three equations, Martin/Hopkins equation derived LDLC had the best linearity against direct measured (y=1.141x - 14.403; R2=0.8626) and traceable LDLC (y=1.1692x - 22.137; R2=0.9638). Martin/Hopkins equation (R2=0.9638) had the strongest R2 in association with traceable LDLC compared with the Friedewald (R2=0.9262) and Sampson (R2=0.9447) equation. The discordance with traceable LDLC was the lowest in Martin/Hopkins (median=-0.725%, IQR=6.914%) as compared to Friedewald (median=-4.094%, IQR=10.305%) and Sampson equation (median=-1.389%, IQR=9.972%). Martin/Hopkins was found to result in the lowest number of misclassifications, whereas Friedewald had the most numbers of misclassification. Samples with high TG, low HDLC and high LDLC had no misclassification by Martin/Hopkins equation, but Friedewald equation resulted in ∼50% misclassification in these samples. CONCLUSIONS: The Martin/Hopkins equation was found to achieve better agreement with the LDLC reference values as compared to Friedewald and Sampson equations, especially in samples with high TG and low HDLC. Martin/Hopkins derived LDLC also enabled a more accurate classification of LDLC levels.

Determination of purity values of amino acid reference materials by mass balance method: an approach to the quantification of related structure impurities.

A systematic procedure for the determination of purity values of amino acid reference materials was developed by use of mass balance method where four categories of impurities (related structure impurities (RSIs), water, organic solvent residue (OSR), and non-volatile residue (NVR)) were quantified separately. The amount of RSIs was determined using a combination of three quantification methods. To ensure metrological traceability in the determination of RSIs, at least one such impurity in each candidate amino acid reference material was quantified using liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS). Other RSIs were determined using external calibration liquid chromatography-tandem mass spectrometry (LC-MS/MS) or o-phthaldialdehyde (OPA) derivatization, followed by liquid chromatography-ultraviolet (LC-UV) measurement. As the UV absorption of most RSIs came basically from the same chromophore after OPA derivatization, a relative peak area approach was used in the LC-UV method to quantify the amount of RSIs by comparing their peak areas with that of a reference RSI. The reference RSI was pre-selected and the amount determined by LC-IDMS/MS separately. The absence of D-amino acids was confirmed using Marfey's reagent derivatization, followed by LC-UV analysis. The amounts of water, OSR, and NVR were measured using Karl Fischer coulometry, gas chromatography-mass spectrometry (GC-MS) and thermogravimetry, respectively. By using this procedure, four amino acid (L-valine, L-leucine, L-isoleucine, and L-phenylalanine) certified reference materials (CRMs) were developed from the candidate materials. The homogeneity and stability of the CRMs were demonstrated by use of LC-IDMS/MS or OPA-LC-UV method, following the principles in ISO 17034 and ISO Guide 35.Graphical abstract.

Enhancing the accuracy of measurement of small molecule organic biomarkers.

Over two decades, the Organic Analysis Working Group (OAWG) of the Consultative Committee for Amount of Substance: Metrology in Chemistry and Biology (CCQM) has organized a number of comparisons for clinically relevant small molecule organic biomarkers. The aim of the OAWG community is to be part of the coordinated international movement towards accuracy and comparability of clinical measurements that will, in turn, minimize the wastage of repeat testing and unnecessary therapy to create a sustainable healthcare industry. International and regional directives/requirements on metrological traceability of calibrators and control materials are in place. Metrology institutes worldwide maintain infrastructure for the practical realization of metrological traceability and demonstrate the equivalence of their measurement capabilities through participation in key comparisons organized under the auspices of the CCQM. These institutes provide certified reference materials, as well as other dedicated value-assignment services benefiting the in-vitro diagnostic (IVD) industry, reference (calibration) laboratories and the clinical chemistry laboratories. The roles of these services in supporting national, regional, and international activities to ensure the metrological traceability of clinical chemistry measurements are described. Graphical abstract.

Commutable whole blood reference materials for hemoglobin A1c validated on multiple clinical analyzers.

Background The measurement of hemoglobin A1c (HbA1c) is important for diagnosing diabetes mellitus as well as assessing glycemic control in diabetic patients. Commutable whole blood certified reference materials (CRMs) are needed in the measurement of HbA1c for method validation and/or as quality controls. Methods We developed three levels of hemolyzed whole blood CRMs for HbA1c. The certified values were determined using liquid chromatography-isotope dilution tandem mass spectrometry method (LC-IDMS/MS) where two "signature" hexapeptides of HbA1c and hemoglobin A0 (HbA0) were used as the calibration standards. The concentrations of the hexapeptide solutions were determined by amino acid analysis by the LC-IDMS/MS method using amino acid CRMs as the calibration standards. The commutability study was conducted by measuring 25 patient specimens and the whole blood CRMs by both LC-IDMS/MS method and various routine methods using six different clinical analyzers. Results The certified values were determined to be 35.1±2.0, 50.3±1.9 and 65.8±2.6 mmol/mol, respectively. These CRMs showed good commutability on five of the six clinical analyzers but showed poor commutability on one of the clinical analyzers that used similar method as two other analyzers where good commutability was observed. Conclusions With certified target values based on metrological traceability and good commutability on most of the clinical analyzers, the developed whole blood CRMs can be used for method validation or as quality control materials in the measurement of HbA1c. The commutability study results also underscored the need of commutability testing of clinical CRMs using various clinical analyzers.

Achieving comparability with IFCC reference method for the measurement of hemoglobin A1c by use of an improved isotope-dilution mass spectrometry method.

The development of reference measurement methods for hemoglobin A1c (HbA1c) is important for quality assurance in diabetes management. The IFCC reference method using purified proteins as calibration standards is the recommended accuracy-based reference method for the standardization of HbA1c measurement. We developed a highly precise and accurate liquid chromatography-isotope-dilution tandem mass spectrometry (LC-IDMS/MS) procedure, which can serve as an alternative accuracy-based method for HbA1c measurement. In this method, enzymatic proteolysis was applied to sample preparation, followed by LC-IDMS/MS measurement of hemoglobin A0 (HbA0) and HbA1c, using two "signature" hexapeptides for calibration. The concentrations of the signature hexapeptide calibration solutions were, in turn, determined using a hydrolysis method with HCl, followed by LC-IDMS/MS measurement using amino acid solutions as calibration standards. These solutions were gravimetrically prepared from pure amino acid certified reference materials (CRMs). The developed LC-IDMS/MS method was used in participation in an IFCC ring trial for reference laboratories (RELA 2013 and 2014) for HbA1c, where our results were compared with those using the IFCC reference method. The deviations were found to be 0.4-1.7 mmol mol(-1) [or 0.04-0.16% in National Glygohemoglobin Standardization Program (NGSP) units], revealing good comparability with the IFCC reference method. The relative expanded uncertainty of the LC-IDMS/MS was in the range of 2.6% to 2.8% (1.6% to 2.2% after converting to NGSP units). With excellent method precision, good comparability with the IFCC reference method, and a small measurement uncertainty, the developed LC-IDMS/MS method may be used as an alternative accuracy-based reference method for HbA1c measurement.

Determination of total thyroxine in human serum by hollow fiber liquid-phase microextraction and liquid chromatography-tandem mass spectrometry.

Determination of total thyroxine in human serum using hollow fiber liquid-phase microextraction (HF-LPME) has been accomplished for the first time. HF-LPME serves as an inexpensive sample pretreatment and the cleanup method that is nearly solvent-free. Thyroxine was extracted through a water immiscible organic solvent immobilized in the wall pores of a polypropylene hollow fiber into 20µl of an aqueous acceptor phase inside the lumen of the hollow fiber. This technique produced extracts that had comparable cleanness with those obtained using solid-phase extraction (SPE). Serum samples with endogenous thyroxine were spiked with isotopically-labeled thyroxine and analyzed by liquid chromatography-tandem mass spectrometry after HF-LPME extraction. Extraction parameters including the organic phase, acid/base concentration of acceptor phase, stirring speed and extraction time were optimized. The calibration range was found to be linear over 1-1000ng/g with the limit of detection (LOD) of 0.3 ng/g. For quantification of total thyroxine in human serum, 6 subsamples were prepared and the results indicated very good precision with a relative standard deviation of <1.3%. The difference from the SPE method was less than 1.2%, with independent t-test showing insignificant bias. Two reference materials of human serum were analyzed, and our obtained values were compared with the reference values. The results showed very good precision with RSD around 0.2% and the deviation from the reference values were -3.1% and -2.1%. The newly developed method is precise, accurate, inexpensive, and environmentally friendly.

An improved reference measurement procedure for triglycerides and total glycerides in human serum by isotope dilution gas chromatography-mass spectrometry.

BACKGROUND: Triglycerides are widely tested in clinical laboratories using enzymatic methods for lipid profiling. As enzymatic methods can be affected by interferences from biological samples, this together with the non-specific nature of triglycerides measurement makes it necessary to verify the accuracy of the test results with a reference measurement procedure. Several such measurement procedures had been published. These procedures generally involved lengthy and laborious sample preparation steps. In this paper, an improved reference measurement procedure for triglycerides and total glycerides was reported which simplifies the sample preparation steps and greatly shortens the time taken. METHODS: The procedure was based on isotope dilution gas chromatography-mass spectrometry (IDGC-MS)with tripalmitin as the calibration standard. Serum samples were first spiked with isotope-labeled tripalmitin. For the measurement of triglycerides, the serum samples were subjected to lipid extraction followed by separation of triglycerides from diglycerides and monoglycerides. Triglycerides were then hydrolyzed to glycerol, derivatized and injected into the GC­MS for quantification. For the measurement of total glycerides, the serum samples were hydrolyzed directly and derivatized before injection into the GC-MS for quantification. RESULTS: All measurement results showed good precision with CV <1%. A certified reference material (CRM) of lipids in frozen human serum was used to verify the accuracy of the measurement. The obtained values for both triglycerides and total glycerides were well within the certified ranges of the CRM, with deviation <0.4% from the certified values. The relative expanded uncertainties were also comparable with the uncertainties associated with the certified values of the CRM. The validated procedure was used in an External Quality Assessment (EQA) Program organized by our laboratory to establish the assigned values for triglycerides and total glycerides.

High accuracy analysis of glucose in human serum by isotope dilution liquid chromatography-tandem mass spectrometry.

BACKGROUND: An isotope dilution mass spectrometry (IDMS) technique has been developed for high accuracy analysis of glucose in human serum. Currently, all the IDMS methods for glucose analysis are based on gas chromatography-mass spectrometry (GC-MS). In this study, isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS) was investigated. METHODS: NIST SRM 965b glucose in frozen human serum was analyzed by linear regression IDMS based on both LC-MS/MS and GC-MS. Serum samples were spiked with isotope labeled glucose and deproteinized by acetonitrile. For LC-MS/MS measurement, the supernate was injected directly after filtration and dilution. For GC-MS measurement, the supernate was evaporated to dryness and went through a two-step derivatization before injection. RESULTS: All measurements had good precision with CVs of <1%. Results from GC-MS agreed very well with results from LC-MS/MS, with a difference of <0.7%. The final reporting values in this study, based solely on LC-MS/MS, were within the certified ranges. The relative expanded uncertainties were within the range of 1.37% to 1.69% for the 4 levels of glucose, which were comparable with uncertainties from the certificate. CONCLUSIONS: The IDMS method based on LC-MS/MS is precise and accurate. It does not require lengthy derivatization steps and thus, greatly simplifies the sample preparation procedure.