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Background: Potassium is an important serum ion that is frequently assayed in clinical laboratories. Quality assurance requires reference methods; thus, the establishment of a candidate reference method for serum potassium measurements is important., Methods: An inductively coupled plasma mass spectrometry (ICP-MS) method was developed. Serum samples were gravimetrically spiked with an aluminum internal standard, digested with 69% ultrapure nitric acid, and diluted to the required concentration. The 39K/27Al ratios were measured by ICP-MS in hydrogen mode. The method was calibrated using 5% nitric acid matrix calibrators, and the calibration function was established using the bracketing method., Results: The correlation coefficients between the measured 39K/27Al ratios and the analyte concentration ratios were >0.9999. The coefficients of variation were 0.40%, 0.68%, and 0.22% for the three serum samples, and the analytical recovery was 99.8%. The accuracy of the measurement was also verified by measuring certified reference materials, SRM909b and SRM956b. Comparison with the ion selective electrode routine method and international inter-laboratory comparisons gave satisfied results., Conclusions: The new ICP-MS method is specific, precise, simple, and low-cost, and it may be used as a candidate reference method for standardizing serum potassium measurements.

Background: Calcium is an important serum ion which is frequently assayed in clinical laboratories. Since quality assurance requires reference methods, the establishment of a candidate reference method for serum calcium measurement is important., Method: An inductively coupled plasma mass spectrometry (ICP-MS) method was developed. Serum samples were spiked gravimetrically with aluminum internal standard, digested with 69% ultrapure nitric acid and diluted to test concentration. Then the (44)Ca/(27)Al ratios were measured by ICP-MS in hydrogen mode. The method was calibrated using 5% nitric acid matrix calibrators and the calibration function was established with bracketing method., Results: The correlation coefficients between the measured (44)Ca/(27)Al ratios and the analyte concentrations ratios were all >0.9999. The coefficients of variation of the measurements were 0.27% and 0.16% for two spiked serum. The analytical recovery was 100.24%. The accuracy of the measurement was also verified through measurement of certified reference materials. Comparison with recognized reference method and international inter-laboratory comparisons gave satisfied results., Conclusion: New ICP-MS method is specific, precise, simple, and low in cost, and may be used as a candidate reference method in the standardization of serum calcium measurement., (Copyright © 2016. Published by Elsevier B.V.)

Aim: Assaying HbA1c in patients with haemoglobin variants has long been a technical challenge, despite methodological advances that have progressively limited the problem. The purpose of this study was to evaluate the impact of the most frequent haemoglobin variants on three routine separation methods compared with the IFCC reference method., Patients: Blood samples from heterozygous patients (AS, AC, AD, AE) were analyzed using the IFCC reference method (LC-MS), and the results compared with those obtained by capillary electrophoresis (CAPILLARYS 2 Flex Piercing, Sebia) and two HPLC methods using cation-exchange (Variant II, Bio-Rad) and affinity chromatography (Ultra(2), Primus)., Results: HbA1c values obtained by the IFCC reference method were comparable to those obtained by the three tested methods whatever the haemoglobin variant. Mean relative biases did not exceed the threshold of 7% (above which differences are generally considered clinically significant), although some individual values were above this limit with Variant II in samples with HbS and for all three methods in samples with HbE., Conclusion: This comparative study of the LC-MS reference method and three field methods has demonstrated that these assays are not clinically influenced by the presence of the most common haemoglobin variants. The present results also confirm that the interpretation of HbA1c values in patients with Hb variants remains complex and depends on the assays used and should, in some cases, take into account parameters other than analytical ones (such as differences in glycation rates and half-lives of haemoglobin variants)., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

In clinical pratice, tau protein measurement generally relies on immunoassays (IAs), whose major drawback is the lack of results comparability due to differences in selectivity and/or calibration. This underlines the importance of establishing a traceability chain for total tau (t-tau) measurements. The objective of this work is to develop a higher order candidate reference measurement procedure (RMP) for the absolute quantification of t-tau in cerebrospinal fluid (CSF). To calibrate the candidate RMP and establish metrological traceability to the SI units, a primary calibrator consisting in a highly purified recombinant protein was sourced. Its purity was evaluated by liquid chromatography coupled with high resolution mass spectrometry (LC-HRMS) and the protein mass fraction in solution was certified by amino acid analysis (AAA). An isotopically-labelled homologue was obtained to develop a candidate RMP by isotope dilution mass spectrometry (IDMS) for t-tau absolute quantification in CSF. Calibration blends and quality control (QC) materials were gravimetrically prepared and subjected to the same preparation workflow as CSF samples, followed by LC-HRMS analysis in Parallel Reaction Monitoring (PRM) mode. A primary calibrator has been developed and an IDMS candidate RMP has been validated for CSF t-tau. The candidate RMP was used to certify t-tau concentration in three pools of CSF (low, medium, high). The candidate RMP will pave the road towards global standardization of CSF t-tau measurements. Together with commutable Certified Reference Materials (CRMs), it will allow evaluating and improving the accuracy and comparability of results provided by IAs. [ABSTRACT FROM AUTHOR]

Measurement of serum progesterone is important in determining ovarian function. Most progesterone measurements are performed with immunoassays and results are often variable. Standardization of the measurements requires a reliable reference method. An isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for the measurement of serum progesterone was developed. Serum samples were spiked with 13C3-progesterone, extracted with a three-step liquid-liquid extraction, and analyzed by LC/MS/MS. A bracketing calibration was used for the analysis and samples were prepared gravimetrically. The developed method showed intra-run and total relative standard deviations (RSDs) of 0.50~0.58% and 0.71~1.33%, respectively. The analytical recoveries were 99.08~101.50%. Measurement results on certified reference materials obtained with this method agreed with the certified values within the stated measurement uncertainties. The method was applied to evaluate immunoassays through split-sample comparisons. A panel of 48 fresh frozen individual samples were measured with the ID-LC/MS/MS method, and six immunoassays and results were compared. Significant calibration biases and sample-specific deviations were observed on some of the immunoassays. [ABSTRACT FROM AUTHOR]

The total haemoglobin (Hb) concentration in blood is one of the most frequently measured analytes in clinical medicine because of its significance for evaluating the health state of a human. The spectrophotometric cyanmethaemoglobin (HiCN) method is the internationally accepted conventional reference method to determine this biomarker. It is frequently used in clinical routine diagnostics but is not traceable to the International System of Units and thus does not meet highest metrological demands. A further critical issue is the toxicity of the necessary potassium cyanide. Different methods to solve these problems are reported here. They all were validated against the HiCN method in an interlaboratory comparison by measuring the total Hb concentration present in the certified reference material JCCRM 912-2M. Methods considered were the spectrophotometric alkaline haematin detergent (AHD) method as well as several isotope dilution (ID)-based approaches. The latter include inductively coupled plasma mass spectrometry (ICP-MS), species-specific (SS) ICP-MS, organic MS and Raman spectrometry. [ABSTRACT FROM AUTHOR]

Developing reliable methodologies for detecting and quantifying allergens in processed food commodities is crucial to support food business operators in allergen risk assessment and properly implementing precautionary allergen labels whenever required to safeguard the health of allergic consumers. Multiple Mass Spectrometry (MS) methods have been developed so far and applied for single and multi-allergen detection in foods, generating a heterogeneous literature on this topic, with little attention paid to the extraction and the digestion steps, crucial in delivering accurate allergen measurements. This investigation carried out within an international consortium specifically built up to convey a prototype MS based reference method, reports on the first part of the method development, namely the optimization of the sample preparation protocol for six allergens detection (cow's milk, hen's egg, soy, peanut, hazelnut, and almond) in chocolate. The latter was chosen as model complex food matrix, having a high lipid and polyphenol content. Different steps of the sample preparation protocol have been taken into consideration: (i) sampling, (ii) composition of the extraction buffer, (iii) protein purification, (iv) protein enzymatic digestion, (v) peptide purification and pre-concentration, and some experiments were carried out by two independent laboratories and two different MS platforms to provide a first assessment of the robustness of the method under development. Fifty target peptides were monitored in multiple reaction monitoring mode and validated in different laboratories to trace the six allergenic ingredients in the incurred chocolate and the best performing protocol for sample preparation was identified. This work paves the way of the forthcoming full analytical validation of a prototype reference method for MS-based allergen quantification. • Development of a sample prep protocol for allergen detection in chocolate. • Optimization of factors influencing allergen recovery by Mass spectrometry. • Validation of peptide markers tracing for milk, egg, soy, peanut, hazelnut, almond in chocolate. [ABSTRACT FROM AUTHOR]

A method based on isotope dilution–liquid chromatography/tandem mass spectrometry (ID–LC/MS/MS) has been developed as a reference method for the accurate determination of low-level metabolite residues of 4 nitrofuran antibiotics (furazolidone, furaltadone, nitrofurantoin, and nitrofurazone) in meat. A generally adopted method based on the acid-catalyzed release of protein-bound metabolites, their conversion into 2-nitrobenzaldehyde imine-type derivatives, liquid–liquid extraction, and clean-up by solid-phase extraction cartridges was followed but with modification and further optimization for accurate and non-biased measurement results to be used as a definitive reference method. One isotope-labelled analogue for each of the 4 metabolites was used as an internal standard. Validation and performance characterization of the method were conducted by analyzing gravimetrically fortified chicken meat in the range of 0.5–3 μg/kg. The method provided accurate analytical results with the relative expanded uncertainty of 3%. Thus, the method can be used for providing reliable higher-order reference values for the quality control of analytical methods used in general testing laboratories near and below the minimum required performance limit (MRPL) set at 1 μg/kg by the EU. [ABSTRACT FROM AUTHOR]

Abstract: Objectives: Our objective was to develop a reference method to measure total cholesterol in human serum, in order to assign values and assess the accuracy of field methods in French clinical laboratories. Design and methods: A reference method based on gas chromatography coupled with mass spectrometry and isotope dilution (GC–IDMS) was developed and validated. It was then used to assign reference values to five frozen serum samples from voluntary proficiency testing schemes gathering 170 French clinical laboratories. Three peer groups were defined and bias against the reference method target value was calculated. Results: Accuracy of the reference method was assessed against NIST SRM 1951b. Bias of the reference method was less than 0.5% and imprecision was less than 1.0%. Our study indicated that field methods tended to overestimate total cholesterol concentration, mean bias being +5.02%±1.02%. The most popular methods (phenolic chromogen with spectrophotometric detection, 80% of participants) exhibited the highest bias (peer group mean bias: +5.51±1.24%). Neither these methods nor those using a non-phenolic chromogen with reflectometric detection (10% of participants, peer group mean bias: +4.20±1.44%) met NCEP recommendations according to which bias should be less than 3%. Only the methods using a non phenolic chromogen with a spectrophotometric detection met these recommendations (10% of participants, peer group mean bias: +1.39±2.75%). Conclusions: As all three peer groups provided positively biased results, the consensus mean usually used to assess the trueness of routine methods is biased as well, which results in an erroneous estimation of method bias. Therefore, this study highlights the value added by reference method target values to assess trueness of field methods and monitor performance of clinical laboratories. [Copyright &y& Elsevier]

We propose isotope-dilution mass spectrometry as a candidate reference method for determination of serum cortisol. The method uses liquid chromatography-mass spectrometry (LC-MS), interfaced with electrospray ionization, and selective monitoring of the [M+H]+ ions of cortisol and isotopically labeled cortisol. The isotope-dilution-liquid chromatography-mass spectrometry (ID-LC-MS) method simplifies sample-preparation, because samples are processed by simple solvent extraction without further clean-up and derivatization. We studied the time required for complete equilibration of endogenous cortisol and labeled cortisol spiked into serum and found it to be less than 1 h. The repeatability and the reproducibility of the method were evaluated and found to be 0.55% of the measurement value. CRM 192 and 193 from the Bureau Communautaire de Reference were analyzed for verification of the method. The results obtained from the ID-LC-MS method agreed with the certified values. The relative uncertainty of measurement results for samples in the range of a few tens of micrograms per kilogram to several hundred micrograms per kilogram was evaluated and found to be 0.56%. Immunoassay carried out by three independent clinical laboratories produced results more than 15% higher than this ID-LC-MS method, suggesting the presence of bias in the immunoassay methods. [ABSTRACT FROM AUTHOR]