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.

Commutability assessment of human urine certified reference materials for albumin and creatinine on multiple clinical analyzers using different statistical models.

Urine albumin concentration and albumin-creatinine ratio are important for the screening of early-stage kidney damage. Commutable urine certified reference materials (CRMs) for albumin and creatinine are necessary for standardization of urine albumin and accurate measurement of albumin-urine ratio. Two urine CRMs for albumin and creatinine with certified values determined using higher-order reference measurement procedures were evaluated for their commutability on five brands/models of clinical analyzers where different reagent kits were used, including Roche Cobas c702, Roche Cobas c311, Siemens Atellica CH, Beckman Coulter AU5800, and Abbott Architect c16000. The commutability study was conducted by measuring at least 26 authentic patient urine samples and the human urine CRMs using both reference measurement procedures and the routine methods. Both the linear regression model suggested by the Clinical and Laboratory Standard Institute (CLSI) guidelines and log-transformed model recommended by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) Commutability Working Group were used to evaluate the commutability of the human urine CRMs. The commutability of the human urine CRMs was found to be generally satisfactory on all five clinical analyzers for both albumin and creatinine, suggesting that they are suitable to be used routinely by clinical laboratories as quality control or for method validation of urine albumin and creatinine measurements.

Improving the accuracy of chloride measurements through participation in regular external quality assessment programme.

BACKGROUND: A chloride test is an integral part of a basic metabolic panel that is essential for the assessment of a patient's acid-base and electrolyte status. While many methods are available commercially for the routine measurement of chloride, there is a need to address the accuracy and variability among the measurement results, especially with the prevalence of patients seeking treatment across different healthcare providers for alternative opinions. METHOD: A method based on sector field inductively coupled plasma isotope dilution mass spectrometry (SF-ICP-IDMS) was developed for the measurement of chloride in human serum. The SF-ICP-IDMS method was then used to assign the target values in the Health Sciences Authority (HSA) External Quality Assessment (EQA) Programme to evaluate the results of chloride test from participating clinical laboratories. RESULTS: The accuracy of the measurements was evaluated by comparing the results with the certified values of Electrolytes in Frozen Human Serum Certified Reference Materials (SRM 956c and SRM 956d) from the National Institute of Standards and Technology (NIST) at different chloride concentration levels. Over a five-year period from 2014-2018, the number of clinical laboratories which participated in the EQA Programme increased from 23 to 33. Comparison of robust means from the laboratories' results with our assigned target values revealed a reduction in relative deviation over time. The relationship between the deviation of each brand of clinical analysers and the chloride levels was established, where a larger deviation was uncovered at low chloride concentration. The SF-ICP-IDMS method was further demonstrated to be comparable with methods used by other metrology institutes in an international comparison organised by HSA under the auspice of the Consultative Committee for Amount of Substance - Metrology in Chemistry and Biology (CCQM). CONCLUSION: The use of metrologically traceable assigned target values enabled the study of method biasness from a small pool of dataset in each of the four brands of clinical analysers in HSA EQA Programme. This work underscores the need to improve the accuracy of chloride measurements by regular participation in an accuracy-based EQA Programme.

Measurement of urine albumin by liquid chromatography-isotope dilution tandem mass spectrometry and its application to value assignment of external quality assessment samples and certification of reference materials.

OBJECTIVES: Urine albumin is measured in clinical laboratories by immunoturbidimetry. However, large biases are observed among the different routine methods. To standardize the measurement of urine albumin, a reference measurement procedure (RMP) and urine albumin certified reference materials (CRMs) are needed. METHODS: A candidate RMP for urine albumin based on liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) using human serum albumin as calibration standard was developed. Isotope-labeled human albumin was used as internal standard. Urine samples were digested using trypsin and eight resulting "signature" peptides of albumin were quantified by LC-IDMS/MS. The candidate RMP was employed in value assignment of external quality assessment (EQA) samples and certification of urine albumin reference materials. The commutability of the developed CRMs was assessed against patient samples. RESULTS: The candidate RMP (recovery 101.5-103.2% and CV 1.2-3.3% at about 7-40 mg/L) met optimal performance goal. The lower limit of quantification was 0.03 mg/L as determined by signal-to-noise method. The EQA results from clinical laboratories using different immunoturbidimetric methods were generally comparable with assigned target values determined by the candidate RMP, with albumin concentrations ranging from 5 to 226 mg/L. Urine albumin reference materials (two levels) certified using the candidate RMP showed good commutability in a preliminary study. CONCLUSIONS: With optimal method precision and trueness, as well as comparability with routine methods, the developed RMP may be used for value assignment of EQA samples or certification of reference materials, which are important pillars in urine albumin method standardization.

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.

Simple and accurate candidate reference measurement procedure for total testosterone in human serum by one-step liquid-liquid extraction coupled with isotope dilution mass spectrometry.

Testosterone in human serum is commonly tested in clinical laboratories using immunoassay methods as well as liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. To standardize and ensure the accuracy of the measurement results, reference procedures with higher metrological order are required. A simple measurement procedure based on one-step liquid-liquid extraction (LLE) and liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) was developed for total testosterone in human serum. The procedure involved serum spiked with 13C3-testosterone, equilibration for 2 h, and extraction with an organic solvent. Testosterone certified reference material (CRM) was used as the calibration standard to ensure the traceability to the International System of Units (SI). Testosterone in serum CRMs from the National Institute for Standards and Technology (NIST) and LGC were used to validate the accuracy of the newly developed method. The deviations of the obtained values from the NIST and LGC certified values ranged from -0.55% to 0.45%. Similarly, the coefficient of variations (CVs) of the replicate measurements were in the range of 0.55% and 0.78%, respectively. The relative expanded uncertainties were comparable with those of the certified materials. The newly developed LC-IDMS/MS procedure demonstrated adequate trueness and precision, and was simple to perform. The method can be used for value assignment of testosterone in external quality assessment (EQA) materials as well as certification of CRMs in the future. 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.

Optimisation of extraction methods and quantification of benzo[a]pyrene and benz[a]anthracene in yerba maté tea by isotope dilution mass spectrometry.

A gas chromatography-isotope dilution mass spectrometry (GC-IDMS) technique was developed for the quantification of two heavy polyaromatic hydrocarbons (PAHs), benz[a]anthracene and benzo[a]pyrene, in yerba maté tea (maté). The optimisation of two extraction methods, namely liquid-liquid extraction and accelerated solvent extraction, was carried out. Both optimised methods were validated using a certified reference material of fine dust and the results were within the expanded uncertainties at 95% confidence level. Recoveries of 99.2-106.7% with RSD of measurements of 1.1-2.3% were achieved for benz[a]anthracene. Recoveries of 95.7-101.9% with RSD of measurements of 0.4-1.4% were achieved for benzo[a]pyrene. The validated methods were applied for the extraction of benz[a]anthracene and benzo[a]pyrene in maté powder from NIST. A metrological approach was undertaken to ensure the traceability of measurement results. The uncertainties associated with the results were rigorously evaluated and also reported herein. Graphical abstract Quantification of benz[a]anthracene and benzo[a]pyrene using IDMS.

Simple and accurate measurement of carbamazepine in surface water by use of porous membrane-protected micro-solid-phase extraction coupled with isotope dilution mass spectrometry.

To achieve fast and accurate analysis of carbamazepine in surface water, we developed a novel porous membrane-protected micro-solid-phase extraction (µ-SPE) method, followed by liquid chromatography-isotope dilution tandem mass spectrometry (LC-IDMS/MS) analysis. The µ-SPE device (∼0.8 × 1 cm) was fabricated by heat-sealing edges of a polypropylene membrane sheet to devise a bag enclosing the sorbent. The analytes (both carbamazepine and isotope-labelled carbamazepine) were first extracted by µ-SPE device in the sample (10 mL) via agitation, then desorbed in an organic solvent (1 mL) via ultrasonication. Several parameters such as organic solvent for pre-conditioning of µ-SPE device, amount of sorbent, adsorption time, and desorption solvent and time were investigated to optimize the µ-SPE efficiency. The optimized method has limits of detection and quantitation estimated to be 0.5 ng L(-1) and 1.6 ng L(-1), respectively. Surface water samples spiked with different amounts of carbamazepine (close to 20, 500, and 1600 ng L(-1), respectively) were analysed for the validation of method precision and accuracy. Good precision was obtained as demonstrated by relative standard deviations of 0.7% for the samples with concentrations of 500 and 1600 ng kg(-1), and 5.8% for the sample with concentration of 20 ng kg(-1). Good accuracy was also demonstrated by the relative recoveries in the range of 96.7%-103.5% for all samples with uncertainties of 1.1%-5.4%. Owing to the same chemical properties of carbamazepine and isotope-labelled carbamazepine, the isotope ratio in the µ-SPE procedure was accurately controlled. The use of µ-SPE coupled with IDMS analysis significantly facilitated the fast and accurate measurement of carbamazepine in surface water.

Determination of cyanuric acid using exact matching isotope dilution mass spectrometry.

A high-accuracy double isotope dilution mass spectrometric method using an exact matching approach with GC coupled to a mass spectrometer for the analysis of cyanuric acid in fortified milk powder was developed. Various parameters for sample clean-up such as the type of SPE cartridge, GC column and type of derivatizing agent used were investigated. The method was found to be linear in the concentration range of 0.03 to 2 mg/kg of cyanuric acid in milk powder. LOD and LOQ were found to be 0.9 and 3 µg/kg, respectively. Recoveries in the range of 95.7 to 102.2% were obtained for the in-house fortified milk powder samples, with RSD of measurements in the range of 0.2 to 3.0%. A metrological approach was undertaken to examine all possible biases that contributed to the combined measurement uncertainty of the method. This high-accuracy method can serve as a reference method for techniques commonly applied in routine testing laboratories.

Determination of melamine in milk powder using gas chromatography-high-resolution isotope dilution mass spectrometry.

A GC-high-resolution isotope dilution MS (IDMS) method for the quantification of melamine in milk powder is described. The developed technique is compared to the LC-IDMS/MS technique, typically used for the determination of melamine in various matrices. The accuracy of the GC-high-resolution IDMS method was demonstrated when a small degree of equivalence was obtained in a regional comparative study involving the determination of melamine in milk powder.

Bridged triphenylamine-based dendrimers: tuning enhanced two-photon absorption performance with locked molecular planarity.

Triphenylamine derivatives bridged by methylene units afford a near planar molecular platform in a series of one dimer and two oligomers exhibiting increased structural rigidity compared to that of their parent triphenylamines. This series of dendrimers show significantly enhanced two-photon absorptions that are up to 3-fold that of triphenylamines with similar molecular size and structure.

Infinite three-dimensional polymeric metalloporphyrin network via six-coordinate Zn(II) and two axial oxygen donors.

[structure: see text]. An X-ray crystallographic study of zinc(II) 5,15-di-(2-methoxymethylphenyl)-porphyrin indicates that it forms a coordination polymer through ligation of the ether oxygen atoms on the porphyrin peripheries to the metal centers of two identical adjacent porphyrins. This gives a novel extensively linked, three-dimensional polymeric structure in which the zinc(II) metal forms a six-coordination center. The uniquely structured network has cavities between 4.81 and 9.27 angstroms, which makes it resemble molecular sieve materials.