Candidate Reference Measurement Procedure Research Articles

Candidate reference measurement procedure for serum 17‐hydroxyprogesterone quantification via isotope dilution liquid chromatography–tandem mass spectrometry

AbstractBackgroundAccurate quantification of 17‐hydroxyprogesterone (17‐OHP) in serum is vital for clinical and research applications. However, inter‐laboratory variability in test results exists owing to the lack of a standardized reference measurement procedure (RMP). Therefore, in this study, we developed a highly accurate, cost‐effective, and user‐friendly candidate RMP (cRMP) for analyzing 17‐OHP in serum.MethodsWe quantified 17‐OHP in serum using a one‐step liquid–liquid extraction method with the addition of 17‐OHP‐13C3, followed by liquid chromatography–tandem mass spectrometry. The ability of these methods to suppress interference was evaluated by chromatographic analysis. We assessed accuracy, specificity, the lower limit of quantitation, linearity, and matrix effects by following the standards specified by the Clinical and Laboratory Standards Institute. The consistency between the developed cRMP and the chemiluminescence method was evaluated through experiments with 120 clinical samples.ResultsThe developed cRMP required only 8 min for accurate quantification of serum 17‐OHP without bias from matrix effects or interference from 19 metabolites added as potential interferents. The method exhibited favorable measurement performance, with a quantitation limit of 0.086 ng/mL, linear range of 0.1–400 ng/mL, a total imprecision of ≤2.90%, spike recovery of 100.1%–100.6%, and relative deviations from assigned target values (RfB Institution) of −2.91% to 1.10%. The cRMP demonstrated good consistency with the conventional assay (chemiluminescence method), with a correlation coefficient R of 0.96977.ConclusionA cRMP with high accuracy, cost‐effectiveness, and convenient operation was developed for quantifying 17‐OHP in serum. Its simplicity and robust performance make it an invaluable addition to the arsenal of analytical tools available for laboratories. This method can enhance the accuracy and reliability of 17‐OHP measurements across various laboratories.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine-10,11-epoxide in human serum and plasma.

A reference measurement procedure (RMP) using isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) was developed and validated with the aim of accurately measuring carbamazepine-10,11-epoxide concentrations in human serum and plasma. To establish traceability to SI units, the absolute content of the reference material was determined using quantitative nuclear magnetic resonance (qNMR) spectroscopy. As sample preparation a protein precipitation protocol followed by a high dilution step was established. Chromatographic separation from carbamazepine and potential metabolites was achieved using a C18 stationary phase. Selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement were evaluated based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the Expression of Uncertainty in Measurement. The RMP demonstrated very good selectivity and specificity, showing no evidence of a matrix effect. This enabled accurate quantification of carbamazepine-epoxide in the concentration range of 0.0400-12.0 μg/mL. The intermediate precision was found to be less than 2.1 %, and the repeatability coefficient of variation (CV) ranged from 1.2 to 1.8 % across all concentration levels. Regarding accuracy, the relative mean bias varied from 1.4 to 2.5 % for native serum levels and from 1.4 to 3.5 % for Li-heparin plasma levels. The measurement uncertainty for single measurements ranged from 1.6 to 2.1 %. In this study, we introduce a new LC-MS/MS-based candidate RMP for accurately measuring carbamazepine-10,11-epoxide in human serum and plasma. This novel method offers a traceable and dependable platform, making itsuitable for standardizing routine assays and assessing clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of phenobarbital in human serum and plasma.

Phenobarbital serves as an antiepileptic drug (AED) and finds application in the treatment of epilepsy either as monotherapy or adjunctive therapy. This drug exhibits various pharmacodynamic properties that account for its beneficial effects as well as potential side effects. Accurate measurement of its concentration is critical for optimizing AED therapy through appropriate dose adjustments. Therefore, our objective was to develop and validate a new reference measurement procedure (RMP) for the accurate quantification of phenobarbital levels in human serum and plasma. A sample preparation protocol based on protein precipitation followed by a high dilution step was established in combination with a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method using a C8 column to separate target analytes from known and unknown interferences. Assay validation and determination of measurement uncertainty were performed based on current guidelines. Selectivity and Specificity were assessed using spiked serum and plasma samples; to investigate possible matrix effects (MEs) a post-column infusion experiment and a comparison of standard line slopes was performed. Precision and accuracy were determined within a multiday precision experiment. The RMP was shown to be highly selective and specific, with no evidence of matrix interferences. It can be used to quantify phenobarbital in the range of 1.92 to 72.0 μg/mL. Intermediate precision was less than 3.2 %, and repeatability coefficient of variation (CV) ranged from 1.3 to 2.0 % across all concentration levels. The relative mean bias ranged from -3.0 to -0.7 % for native serum levels, and from -2.8 to 0.8 % for Li-heparin plasma levels. The measurement uncertainties (k=1) for single measurements and target value assignment were 1.9 to 3.3 % and 0.9 to 1.6 %, respectively. A novel LC-MS/MS-based candidate RMP for the quantification of phenobarbital in human serum and plasma is presented which can be used for the standardization of routine assays and the evaluation of clinically relevant samples.

Entropy-based explanations of serial position and learning effects in ordinal responses to word list tests

Measuring a person’s cognitive abilities, such as memory and learning, is central in many medical conditions to reliably diagnose, treat and monitor disease progression. Common tests typically include tasks of recalling sequences of blocks, digits or words. Recalling a word list is affected by so-called serial position effects (SPE), meaning that words at the beginning or end of the list are more likely to be recalled. In our earlier work, as part of including ordinal and nominal properties in metrology, compensation for ordinality in the raw test scores has been performed with psychometric Rasch measurement theory. Thereafter, SPE have been successfully explained with construct specification equations (CSE) dominated by information theoretical entropy as candidate reference measurement procedures. Here, we present how previous German results for explaining memory difficulty in the immediate recalling (IR, trial 1) task of the Rey’s Auditory Verbal Learning Test (RAVLT) can be replicated with a Swedish cohort (the Gothenburg Mild Cognitive Impairment study, n = 251). This CSE replicability for RAVLT demonstrates comparability across the two cohorts in a kind of inter-laboratory study. Moreover, RAVLT includes repeated trials and learning through practice is expected. How memory task difficulty changes over the eight trials in RAVLT is studied: SPE are not so prominent for the delayed recalling sequences and there is an overall reduction in the task difficulty CSE intercept with trial number, interpreted as an effect of learning. To conclude, the methodology and evidence provided here can be clinically used not only to measure a person’s memory ability but also his or her learning ability, as well as understanding the relationship between learning ability and other cognitive domains.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of zonisamide in human serum and plasma.

To describe and validate an isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure (RMP) for zonisamide to accurately measure serum and plasma concentrations. Quantitative nuclear magnetic resonance (qNMR) spectroscopy was employed to determine the absolute content of the reference material used in order to establish traceability to SI units. Separation of zonisamide from known or unknown interferences was performed on a C8 column. For sample preparation a protocol based on protein precipitation in combination with a high dilution step was established. Assay validation and determination of measurement uncertainty were performed based on guidelines from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of zonisamide within the range of 1.50-60.0 μg/mL. Intermediate precision was <1.4 % and repeatability CV ranged from 0.7 to 1.2 % over all concentration levels. The relative mean bias ranged from 0.0 to 0.8 % for native serum levels and from 0.2 to 2.0 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment ranged from 1.1 to 1.4 % and 0.8-1.0 %, respectively. We present a novel LC-MS/MS-based candidate RMP for zonisamide in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of carbamazepine in human serum and plasma.

An isotope dilution liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) was developed and validated to accurately measure serum and plasma concentrations of carbamazepine. Quantitative nuclear magnetic resonance (qNMR) spectroscopy was used to determine the absolute content of the reference material, ensuring its traceability to SI units. The separation of carbamazepine from potential interferences, whether known or unknown, was achieved using a C18 column. A protein precipitation protocol followed by a high dilution step was established for sample preparation. Assay validation and determination of measurement uncertainty were performed in accordance with the guidelines of the Clinical and Laboratory Standards Institute, the International Conference on Harmonization (ICH), and the Guide to the Expression of Uncertainty in Measurement (GUM). In order to demonstrate equivalence to the already existing RMP a method comparison study was performed. The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of carbamazepine within the range of 0.800-18.0 μg/mL. Intermediate precision and repeatability (n=60 measurements) was found to be <1.6 % and <1.3 % over all concentration levels and independent from the matrix. The relative mean bias ranged from-0.1 to 0.6 % for native serum and from-0.3 to-0.1 % for Li-heparin plasma levels. The measurement uncertainties for single measurements and target value assignment were found to be <1.8 % and <1.3 %, respectively. Method comparison showed a good agreement between the Joint Committee of Traceability in Laboratory Medicine (JCTLM) listed RMP and the candidate RMP resulting in a Passing-Bablok regression equation with a slope of 1.01 and an intercept of-0.01. The bias in the patient cohort was found to be 0.9 %. We present a novel LC-MS/MS-based candidate RMP for carbamazepine in human serum and plasma which provides a traceable and reliable platform for the standardization of routine assays and evaluation of clinically relevant samples.

Routes to standardisation for tau protein measurement in CSF

AbstractBackgroundCSF biomarkers are the target of several commercially available in vitro diagnostic (IVD) kits allowing an early diagnosis of Alzheimer’s disease (AD) and eventually the monitoring of treatment effects in clinical trials. Tau hyper‐phosphorylation is one of the main hallmarks of AD and its measurement provides higher accuracy to stratify patients at an early‐stage.Due to the rapid advancements in proteomics, standardisation of measurements can be achieved through the development of reference measurement procedures (RMPs) and certified reference materials (CRMs).MethodSI‐traceable protein and peptide primary calibrators were developed for t‐tau and p‐tau: mass fraction was determined by amino acid analysis and purity was assessed by LC‐MS at high resolution. CSF pools at different tau concentration were used for the development and validation of LC‐IDMS candidate RMP and their commutability was assessed.ResultAn LC‐IDMS candidate reference method was develop and validated in CSF for t‐tau using a SI‐traceable candidate protein primary calibrator. Three different CSF pools (low, medium, high) were successfully quantified with a relative expanded uncertainty below 10%. The commutability of 13 matrix‐based CRMs was assessed involving 8 immunoassays from 5 IVD providers by measuring 40 single CSF donations.According to the AT(N) classification, high levels of p‐tau(181) are highly correlated to tau pathology and commercially available immunoassays are able to target this specific epitope. We conducted a feasibility study on the development of a candidate reference method for p‐tau(181) in CSF relying on a SI‐traceable peptide primary calibrator and an antibody‐free sample preparation workflow.New assays targeting p‐tau(217) in CSF and plasma were also developed, making it necessary the organisation of standardisation initiatives. We sourced and characterised multiple phosphopeptide primary calibrators as well as a GSK3beta‐phosphorylated protein primary calibrator for the development of an LC‐MS RMP including an immunoprecipitation step in the sample preparation workflow.Conclusiontau is considered as a priority for the IFCC WG on NDD and the development of SI traceable RMPs and CRMs is essential to underpin a worldwide standardisation of results. This will help IVD providers to address the traceability requirements of the IVDR thus facilitating regulatory approvals of the kits.

B-129 Uncertainty Calculations for Reference Measurement Procedures

Abstract Background Quantitative results based on reference measurement procedures (RMP) require the calculation of the uncertainty of the result. In order to take the uncertainty of the full traceability chain into account a modularisation of the different execution steps is needed, starting from the primary reference material until the final concentration value. Different strategies have to be applied and combined to obtain the final uncertainty.We combine different ways for calculating the uncertainty of an isotope dilution -liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) based reference measurement procedure for Cortisol, and explain the differences between Type A and Type B uncertainties and best strategies to choose from them. In addition, combinations of results from subsequent steps to a final overall uncertainty are presented. Methods An ID-LC-MS/MS based reference measurement procedure requires as a starting point a primary reference material with known purity of the analyte under consideration. Nowadays this purity assessment is often done with quantitative nuclear magnetic resonance (qNMR) spectroscopy. The qNMR uncertainty calculation is a Type A approach, based on replicate sample preparations and measurements. With this primary material a first set of primary calibrators is produced, often through multiple steps of weighings, preparation of stock solutions and dilutions. For these steps balances, volumetric flasks, or pipettes are used, all of them coming with an error budget which has to enter the uncertainty budget of the primary calibrator preparation. We will show how this can be done through Type B uncertainty calculation. Based on the primary calibrators the LC/MS instrument is calibrated and measurements of individual samples are carried out. We will introduce a hierarchical variability experiment, which takes into account the most important error sources and results in a Type A uncertainty for the LC/MS measurements.Finally, the errors of the LC/MS measurements have to be combined with those of the primary calibrator preparation to obtain the overall uncertainty of an LC/MS reference measurement procedure. Results We show the approaches and calculation strategies based on the Cortisol candidate reference measurement procedure, developed within the Roche LC/MS Reference Measurement Procedure project. The purity assessment by qNMR carries an uncertainty of 0.18%. This is incorporated into the uncertainty calculations of the primary calibrators, where in addition uncertainties from weighing and dilutions are incorporated resulting in uncertainties of the primary calibrator values between 0.6% and 1.1%. Combining these uncertainties with the variabilities of the LC/MS measurement procedure results in final uncertainties between 1.6% and 2.8%. Conclusion We have developed a sound framework for uncertainty calculation of reference measurement procedures, taking into account the different procedure steps, and included this into a process for the management of the different input variables up to comprehensive reports of the calculations.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of topiramate in human serum and plasma.

Topiramate is an antiepileptic drug (AED) used for the monotherapy or adjunctive treatment of epilepsy and for the prophylaxis of migraine. It has several pharmacodynamic properties that contribute to both its clinically useful properties and observed adverse effects. Accurate measurement of its concentration is therefore essential for dose adjustment/optimisation of AED therapy. Our aim was to develop and validate a novel reference measurement procedure (RMP) for the quantification of topiramate in human serum and plasma. An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) method in combination with a protein-precipitation-based sample preparation allows for quantification of topiramate in human serum and plasma. To assure traceability to SI units, quantitative nuclear magnetic resonance (qNMR) was applied to characterize the reference material used as primary calibrator for this RMP. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Accuracy and precision was evaluated performing an extensive five day precision experiment and measurement uncertainty was evaluated according Guide to the Expression of Uncertainty in Measurement (GUM). The method enabled topiramate quantification within the range of 1.20-36.0 μg/mL without interference from structurally related compounds and no evidence of a matrix effect. Intermediate precision was≤3.2 % and repeatability was 1.4-2.5 % across all concentration levels. The relative mean bias was-0.3 to 3.5 %. Expanded measurement uncertainties for target value assignment (n=6) were found to be≤2.9 % (k=2) independent of the concentration level and the nature of the sample. In human serum and plasma, the RMP demonstrated high analytical performance for topiramate quantification and fulfilled the requirements on measurement uncertainty. Traceability to SI units was established by qNMR content determination of the topiramate, which was used for direct calibration of the RMP. This RMP is, therefore, fit for purpose for routine assay standardization and clinical sample evaluation.

Implementing metrological traceability of C-reactive protein measurements: consensus summary from the Joint Committee for Traceability in Laboratory Medicine Workshop.

The Joint Committee for Traceability in Laboratory Medicine (JCTLM) currently lists the secondary commutable certified reference material (CRM) ERM DA-474/IFCC (DA-474) "C-Reactive Protein in Human Serum" and two generic immunoassay-based method principles as the basis for implementing the metrological traceability of C-reactive protein (CRP) measurements by end-user measurement procedures used by medical laboratories. The current metrological traceability has produced well harmonized results for clinical samples among different end-user measurement procedures. New higher-order pure substance and secondary commutable CRMs have been nominated for listing by the JCTLM. However, the data supporting performance of these new candidate CRMs, including use of new mass spectrometry based candidate reference measurement procedures (RMPs), was not clear regarding the influence that introducing these new CRMs would have on the current well harmonized results achieved with the existing metrological traceability to DA-474. The clinically relevant CRP measurand in blood serum or plasma is a pentamer of identical subunits, which adds complexity to the application of higher-order CRMs and RMPs. The JCTLM convened a workshop in December 2022 to review the appropriate implementation of metrological traceability of CRP measurements. The workshop consensus was that the extent-of-equivalence data must include considerations about the impact of a new CRM when used for its intended purpose in the calibration hierarchies of existing end-user measuring systems; and that a new RMP must compare results with another existing well validated candidate RMP or with a globally available end-user measurement system.

Estimation of measurement uncertainty for the quantification of protein by ID-LC–MS/MS

The emergence of mass spectrometry (MS)-based methods to quantify proteins for clinical applications has led to the need for accurate and consistent measurements. To meet the clinical needs of MS-based protein results, it is important that the results are traceable to higher-order standards and methods and have defined uncertainty values. Therefore, we outline a comprehensive approach for the estimation of measurement uncertainty of a MS-based procedure for the quantification of a protein biomarker. Using a bottom-up approach, which is the model outlined in the “Guide to the Expression of Uncertainty of Measurement” (GUM), we evaluated the uncertainty components of a MS-based measurement procedure for a protein biomarker in a complex matrix. The cause-and-effect diagram of the procedure is used to identify each uncertainty component, and statistical equations are derived to determine the overall combined uncertainty. Evaluation of the uncertainty components not only enables the calculation of the measurement uncertainty but can also be used to determine if the procedure needs improvement. To demonstrate the use of the bottom-up approach, the overall combined uncertainty is estimated for the National Institute of Standards and Technology (NIST) candidate reference measurement procedure for albumin in human urine. The results of the uncertainty approach are applied to the determination of uncertainty for the certified value for albumin in candidate NIST Standard Reference Material® (SRM) 3666. This study provides a framework for measurement uncertainty estimation of a MS-based protein procedure by identifying the uncertainty components of the procedure to derive the overall combined uncertainty.Graphical

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure for the quantification of levetiracetam in human serum and plasma.

To develop an isotope dilution-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for levetiracetam quantification in human serum and plasma. Quantitative nuclear magnetic resonance spectroscopy (qNMR) was used to characterize the RMP material to ensure traceability to SI units. To quantify levetiracetam, an LC-MS/MS method was optimized using a C8 column for chromatographic separation following protein-precipitation-based sample preparation. Spiked matrix samples of serum and plasma were used to test selectivity and specificity. Matrix effects were determined by performing a post-column infusion experiment and comparing standard line slopes. Precision and accuracy were evaluated over 5days. Measurement uncertainty was evaluated according to the Guide to the Expression of Uncertainty in Measurement (GUM). The RMP was proven to be highly selective and specific with no evidence of a matrix effect, allowing for quantification of levetiracetam within the range of 1.53-90.0μg/mL. Intermediate precision was <2.2% and repeatability was 1.1-1.7% across all concentrations. The relative mean bias ranged from-2.5% to-0.3% across all levels and matrices within the measuring range. Diluted samples were found with a mean bias ranging from-0.1 to 2.9%. The predefined acceptance criterion for measurement uncertainty was met and determined for individual measurements independently of the concentration level and sample type to be≤4.0% (k=2). We present a novel LC-MS/MS)-based candidate RMP for levetiracetam in human serum and plasma. Its expanded measurement uncertainty of≤4.0% meets the clinical needs in levetiracetam monitoring. Utilizing qNMR to characterize levetiracetam reference materials allowed metrological traceability to SI units.

Description and validation of an equilibrium dialysis ID-LC-MS/MS candidate reference measurement procedure for free thyroxine in human serum.

Free thyroxine (FT4) in serum is routinely measured in clinical practice to diagnose and monitor thyroid disease. Due to its concentration in picomolar range and the delicate equilibrium of free and protein-bound T4, accurate measurement is challenging. As a consequence, large inter-method differences in FT4 results exists. Optimal method design and standardization of the FT4 measurement is therefore necessary. The IFCC Working Group for Standardization of Thyroid Function Tests proposed a reference system with a conventional reference measurement procedure (cRMP) for FT4 in serum. In this study, wedescribe our FT4 candidate cRMP and its validation in clinical samples. This candidate cRMP is based on equilibrium dialysis (ED) combined with determination of T4 with an isotope-dilution liquid chromatography tandem mass-spectrometry (ID-LC-MS/MS) procedure and was developed according to the endorsed conventions. Its accuracy, reliability, and comparability was investigated using human sera. It was shown that the candidate cRMP adhered to the conventions and its accuracy, precision, and robustness were adequate in serum of healthy volunteers. Our candidate cRMP measures FT4 accurately and performs well in serum matrix.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of aldosterone in human serum and plasma.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC MS/MS)-based candidate reference measurement procedure (RMP) for aldosterone quantification in human serum and plasma is presented. The material used in this RMP was characterized by quantitative nuclear magnetic resonance (qNMR) to assure traceability to SI Units. For liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis a two-dimensional heart cut LC approach, in combination with an optimal supported liquid extraction protocol, was established for the accurate analysis of aldosterone in human serum and plasma in order to minimize matrix effects and avoid the co-elution of interferences. Assay validation was performed according to current guidelines. Selectivity and specificity were assessed using spiked serum; potential matrix effects were examined by a post column infusion experiment and the comparison of standard line slopes. An extensive protocol over 5days was applied to determine precision, accuracy and trueness. Measurement uncertainty was evaluated according to the Guide to the Expression ofUncertainty in Measurement (GUM), for which three individual sample preparations were performed on at least two different days. The RMP allowed aldosterone quantification within the range of 20-1,200pg/mL without interference from structurally-related compounds and no evidence of matrix effects. Intermediate precision was≤4.7% and repeatability was 2.8-3.7% for all analyte concentrations. The bias ranged between-2.2 and 0.5% for all levels and matrices. Total measurement uncertainties for target value assignment (n=6) were found to be≤2.3%; expanded uncertainties were≤4.6% (k=2) for all levels. The RMP showed high analytical performance for aldosterone quantification in human serum and plasma. The traceability to SI units was established by qNMR content determination of aldosterone, which was utilized for direct calibration of the RMP. Thus, this candidate RMP is suitable for routine assay standardization and evaluation of clinical samples.

Development of an equilibrium dialysis ID-UPLC-MS/MS candidate reference measurement procedure for free thyroxine in human serum

BackgroundAccurate and reliable measurement of human serum free thyroxine (FT4) is critical for the diagnosis and treatment of thyroid diseases. However, concerns have been raised regarding the performance of FT4 measurements in patient care. Centers for Disease Control and Prevention Clinical Standardization Programs (CDC-CSP) address these concerns by creating a FT4 standardization program to standardize FT4 measurements. The study aims to develop a highly accurate and precise candidate Reference Measurement Procedure (cRMP), as one key component of CDC-CSP, for standardization of FT4 measurements. MethodsSerum FT4 was separated from protein-bound thyroxine with equilibrium dialysis (ED) following the recommended conditions in the Clinical and Laboratory Standards Institute C45-A guideline and the published RMP [20,21,23]. FT4 in dialysate was directly quantified with liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. Gravimetric measurements of specimens and calibrator solutions, calibrator bracketing, isotope dilution, enhanced chromatographic resolution, and T4 specific mass transitions were used to ensure the accuracy, precision, and specificity of the cRMP. ResultsThe described cRMP agreed well with the established RMP and two other cRMPs in an interlaboratory comparison study. The mean biases of each method to the overall laboratory mean were within ±2.5%. The intra-day, inter-day, and total imprecision for the cRMP were within 4.4%. The limit of detection was 0.90 pmol/L, which was sufficiently sensitive to determine FT4 for patients with hypothyroidism. The structural analogs of T4 and endogenous components in dialysate did not interfere with the measurements. ConclusionOur ED-LC-MS/MS cRMP provides high accuracy, precision, specificity, and sensitivity for FT4 measurement. The cRMP can serve as a higher-order standard for establishing measurement traceability and provide an accuracy base for the standardization of FT4 assays.

Development of a candidate reference measurement procedure by ID-LC-MS/MS for total tau protein measurement in cerebrospinal fluid (CSF).

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.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of methotrexate in human serum and plasma.

To develop an isotope dilution-liquid chromatography-tandem mass spectrometry-(ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for quantification of methotrexate in human serum and plasma. Quantitative nuclear magnetic resonance (qNMR) was used to determine absolute methotrexate content in the standard. Separation was achieved on a biphenyl reversed-phase analytical column with mobile phases based on water and acetonitrile, both containing 0.1% formic acid. Sample preparation included protein precipitation in combination with high sample dilution, and method validation according to current guidelines. The following were assessed: selectivity (using analyte-spiked samples, and relevant structural-related compounds and interferences); specificity and matrix effects (via post-column infusion and comparison of human matrix vs. neat samples); precision and accuracy (in a five-day validation analysis). RMP results were compared between two independent laboratories. Measurement uncertainty was evaluated according to current guidelines. The RMP separated methotrexate from potentially interfering compounds and enabled measurement over a calibration range of 7.200-5,700ng/mL (0.01584-12.54μmol/L), with no evidence of matrix effects. All pre-defined acceptance criteria were met; intermediate precision was≤4.3% and repeatability 1.5-2.1% for all analyte concentrations. Bias was-3.0 to 2.1% for samples within the measuring range and 0.8-4.5% for diluted samples, independent of the sample matrix. RMP results equivalence was demonstrated between two independent laboratories (Pearson correlation coefficient 0.997). Expanded measurement uncertainty of target value-assigned samples was≤3.4%. This ID-LC-MS/MS-based approach provides a candidate RMP for methotrexate quantification. Traceability of methotrexate standard and the LC-MS/MS platform were assured by qNMR assessment and extensive method validation.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of lamotrigine in human serum and plasma.

We developed an isotope dilution (ID)-liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based candidate reference measurement procedure (RMP) for lamotrigine in human serum and plasma, using quantitative nuclear magnetic resonance-characterized reference standards to ensure traceability to the International System of Units. A sample preparation protocol based on protein precipitation combined with LC-MS/MS analysis using a C18 column for chromatographic separation was established for the quantification of lamotrigine in human serum and plasma. Assay validation was performed according to current guidelines. Spiked serum and plasma samples were used to assess selectivity and specificity; a post-column infusion experiment and comparison of standard line slopes were performed to ascertain possible matrix effects. Precision and accuracy were determined in a 5days validation experiment. Measurement uncertainty was determined per the Guide to the Expression of Uncertainty in Measurement. The method allowed the quantification of lamotrigine in serum and plasma in a range of 0.600-24.0μg/mL without any observable matrix effects. The relative mean bias (n=6) ranged from 1.7 to 3.7%; intermediate precision, including variances in between-day, -calibration, and -injection, was ≤2.4%, independent of the level and matrix. Total measurement uncertainty for a single measurement was≤2.6%; expanded uncertainty was≤5.2% (coverage factor k=2). This candidate RMP based on ID-LC-MS/MS provides a traceable and reliable platform for the standardization of routine assays and the evaluation of clinical samples.

An isotope dilution-liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS)-based candidate reference measurement procedure (RMP) for the quantification of gabapentin in human serum and plasma.

To describe and validate a reference measurement procedure (RMP) for gabapentin, employing quantitative nuclear magnetic resonance (qNMR) spectroscopy to determine the absolute content of the standard materials in combination with isotope dilution-liquid chromatograph-tandem mass spectrometry (ID-LC-MS/MS) to accurately measure serum and plasma concentrations. A sample preparation protocol based on protein precipitation in combination with LC-MS/MS analysis using a C8 column for chromatographic separation was established for the quantification of gabapentin. Assay validation anddetermination of measurement uncertainty were performed according to guidance from the Clinical and Laboratory Standards Institute, the International Conference on Harmonization, and the Guide to the expression of uncertainty in measurement. ID-LC-MS/MS parameters evaluated included selectivity, specificity, matrix effects, precision and accuracy, inter-laboratory equivalence, and uncertainty of measurement. The use of qNMR provided traceability to International System (SI) units. The chromatographic assay was highly selective, allowing baseline separation of gabapentin and the gabapentin-lactam impurity, without observable matrix effects. Variability between injections, preparations, calibrations, and days (intermediate precision) was <2.3%, independent of the matrix, while the coefficient of variation for repeatability was 0.9-2.0% across all concentration levels. The relative mean bias ranged from-0.8-1.0% for serum and plasma samples. Passing-Bablok regression analysis indicated very good inter-laboratory agreement; the slope was 1.00 (95% confidence interval [CI] 0.98 to 1.03) and the intercept was-0.05 (95% CI -0.14 to 0.03). Pearson's correlation coefficient was≥0.996. Expanded measurement uncertainties for single measurements were found to be≤5.0% (k=2). This analytical protocol for gabapentin, utilizing traceable and selective qNMR and ID-LC-MS/MS techniques, allows for the standardization of routine tests and the reliable evaluation of clinical samples.

Commutability Assessment of Candidate Reference Materials for Lipoprotein(a) by Comparison of a MS-based Candidate Reference Measurement Procedure with Immunoassays.

Elevated concentrations of lipoprotein(a) [Lp(a)] are directly related to an increased risk of cardiovascular diseases, making it a relevant biomarker for clinical risk assessment. However, the lack of global standardization of current Lp(a) measurement procedures (MPs) leads to inconsistent patient care. The International Federation for Clinical Chemistry and Laboratory Medicine working group on quantitating apolipoproteins by mass spectrometry (MS) aims to develop a next-generation SI (International system of units)-traceable reference measurement system consisting of a MS-based, peptide-calibrated reference measurement procedure (RMP) and secondary serum-based reference materials (RMs) certified for their apolipoprotein(a) [apo(a)] content. To reach measurement standardization through this new measurement system, 2 essential requirements need to be fulfilled: a sufficient correlation among the MPs and appropriate commutability of future serum-based RMs. The correlation among the candidate RMP (cRMP) and immunoassay-based MPs was assessed by measuring a panel of 39 clinical samples (CS). In addition, the commutability of 14 different candidate RMs was investigated. Results of the immunoassay-based MPs and the cRMPs demonstrated good linear correlations for the CS but some significant sample-specific differences were also observed. The results of the commutability study show that RMs based on unspiked human serum pools can be commutable with CS, whereas human pools spiked with recombinant apo(a) show different behavior compared to CS. The results of this study show that unspiked human serum pools are the preferred candidate secondary RMs in the future SI-traceable Lp(a) Reference Measurement System.