A candidate reference measurement procedure for the quantification of α-synuclein in cerebrospinal fluid using an SI traceable primary calibrator and multiple reaction monitoring.

α-synuclein aggregation is an important hallmark of neurodegenerative diseases such as Parkinson's disease (PD) and Lewy body dementia. α-synuclein has been increasingly used as a diagnostic biomarker in PD and other synucleinopathies. Current clinical assays rely on antibody-based immunoassays to detect α-synuclein, which possess high sensitivity, afford high throughput and require small sample volumes. The utility of these assays, however, may be compounded by the specificity, selectivity and batch-to-batch heterogeneity of the antibody used, which can lead to deviations in the total amount of the protein measured when comparing results among different laboratories. Similarly, current mass spectrometry-based quantification methods for α-synuclein lack well-defined, value assigned calibrators to ensure comparability of measurements. Therefore, there is still an unmet need for the standardisation of clinical measurements for α-synuclein that can be achieved by the development of reference measurement procedures (RMPs) utilising calibrators traceable to the SI (International System of Units). Here, we report a candidate RMP for α-synuclein, using an SI traceable primary calibrator and an isotope dilution mass spectrometry (IDMS) approach to address this need. The gravimetrically prepared primary calibrator was traceably quantified utilising a combination of amino acid analysis (AAA) and quantitative nuclear magnetic resonance (qNMR) for value assignment. An optimised targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction strategy was devised, followed by the development of a targeted multiple reaction monitoring (MRM) method for the quantification of α-synuclein in cerebrospinal fluid (CSF). This candidate RMP was then deployed for the sensitive detection and accurate quantification of multiple proteotypic α-synuclein peptides in patient derived CSF samples. The LC-MS based results were subsequently compared to immunoassay data to assess the overall performance of our approach. The development and adoption of this candidate RMP, along with the availability of the SI traceable primary calibrator will allow for reliable quantifications of α-synuclein in CSF by an LC-MS based assay. The RMP will potentially contribute towards the standardisation of this important biomarker and may lead to future interlaboratory comparisons.

A candidate liquid chromatography mass spectrometry reference method for the quantification of the cardiac marker 1-32 B-type natriuretic peptide.

BACKGROUND: B-type natriuretic peptide (BNP) is a 32 amino acid cardiac hormone routinely measured by immunoassays to diagnose heart failure. While it is reported that immunoassay results can vary up to 45%, no attempt of standardization and/or harmonization through the development of certified reference materials (CRMs) or reference measurement procedures (RMPs) has yet been carried out. METHODS: B-type natriuretic peptide primary calibrator was quantified traceably to the International System of Units (SI) by both amino acid analysis and tryptic digestion. A method for the stabilization of BNP in plasma followed by protein precipitation, solid phase extraction (SPE) and liquid chromatography (LC) mass spectrometry (MS) was then developed and validated for the quantification of BNP at clinically relevant concentrations (15-150 fmol/g). RESULTS: The candidate reference method was applied to the quantification of BNP in a number of samples from the UK NEQAS Cardiac Markers Scheme to demonstrate its applicability to generate reference values and to preliminary evaluate the commutability of a potential CRM. The results from the reference method were consistently lower than the immunoassay results and discrepancy between the immunoassays was observed confirming previous data. CONCLUSIONS: The application of the liquid chromatography-mass spectrometry (LC-MS) method to the UK NEQAS samples and the correlation of the results with the immunoassay results shows the potential of the method to support external quality assessment schemes, to improve understanding of the bias of the assays and to establish RMPs for BNP measurements. Furthermore, the method has the potential to be multiplexed for monitoring circulating truncated forms of BNP.

Candidate Reference Measurement Procedure for the Quantification of Total Serum Cortisol with LC-MS/MS.

BACKGROUND: Accurate measurement of serum cortisol is required to diagnose and treat adrenal disorders. Although certified reference materials (CRMs) are available to standardize cortisol measurements, External Quality Assessment (EQA) schemes still demonstrate a wide dispersion of results. We present a serum cortisol candidate reference measurement procedure that, through analysis of a Joint Committee for Traceability in Laboratory Medicine-listed panel of higher-order CRMs, provides metrologically traceable results. METHOD: Isotope-labeled internal standard was added to samples before supported liquid extraction. Extracts were analyzed with LC-MS/MS in positive electrospray ionization mode. Multiple reaction monitoring was used to detect cortisol and its corresponding internal standard transitions. We measured samples in triplicate over 3 days and calculated the mean result. RESULTS: Mean intra- and interassay imprecision were 1.3% and 1.5%, respectively, for concentrations of 154, 510, and 769 nmol/L. Ionization efficiency studies and structural analog analysis proved the method to be robust against interferences. Through analysis of 34 CRMs (83-764 nmol/L), expanded measurement uncertainty was calculated to be 5% (95% CI). The mean bias between the measured and target CRM concentrations was statistically insignificant at -0.08%. CONCLUSIONS: The accuracy and low measurement uncertainty of this method qualify it as a CRM procedure. Metrological traceability has been achieved through the analysis of higher-order CRMs. This method could be used to underpin serum cortisol EQA schemes to provide samples with a traceable target value, enabling participating laboratories to determine the accuracy and measurement uncertainty of their assays.

Fully traceable absolute protein quantification of somatropin that allows independent comparison of somatropin standards.

BACKGROUND: Measurement traceability in clinical chemistry is required to standardize clinical results irrespective of the measurement procedure and laboratory. The traceability of many protein substances is maintained by reference to the first standard produced, which may no longer exist, with values assigned by consensus. Independent methods that provide traceability to the Système d'Unité International for all relevant properties of a protein standard could remove reliance on the original standard preparations. METHODS: We developed a method based on the traceable quantification of tryptic peptides released from the protein by isotope dilution mass spectrometry to compare 2 standard preparations of somatropin (recombinant human growth hormone), WHO 98/574 and Ph.Eur.CRS S0947000. Relative quantification using isotope-coded affinity tagging, isobaric tagging for relative and absolute quantification, and standard additions were also performed to validate the digestion method used and to determine whether any modifications were present. RESULTS: The total somatropin content in both materials was determined and an uncertainty estimation undertaken [WHO 2.19 +/- 0.21) mg/vial, European Pharmacopeia 2.06 +/- 0.21 mg/vial]. Each uncertainty in this paper is a fully estimated uncertainty, with 95% CI (k = 2). Isotope coded affinity tag and standard addition results fully validated the robustness of the digestion method used. In addition, iTRAQ (isobaric tagging for relative and absolute quantification analysis) identified 2 modifications, neither of which impacted the quantification. CONCLUSIONS: An independent method that does not rely on a preexisting protein standard has been developed and validated for the traceable value-assignment of total somatropin. The methods reported here address the amount of substance (mass fraction) of the standard materials but address neither biological activity nor other characteristics that may be important in assessing suitability for use as a calibrator.

Production and certification of four frozen human serum certified reference materials containing creatinine and electrolytes.

BACKGROUND: This paper describes the preparation, analysis and certification of four frozen human serum certified reference materials (CRMs) containing creatinine and the electrolytes calcium, lithium, magnesium, potassium and sodium. These materials have been prepared to give concentrations of these analytes that cover the currently accepted analytical range. METHODS: The analysis of the materials for certification purposes has been carried out using methodology traceable to primary standards, and which is acceptable as a reference method. The certification methods include liquid chromatography-mass spectrometry (LC-MS) with exact-matching isotope dilution calibration (EM-IDMS) for creatinine, inductively-coupled plasma optical emission spectroscopy (ICP-OES), ICP-MS and isotope-dilution inductively-coupled plasma mass spectroscopy (ID-ICP-MS) for the electrolytes. RESULTS: The uncertainties estimated for these certified values include a component from the characterization measurements, as well as contributions from possible inhomogeneity and long-term instability. The certified values have been corroborated by measurements obtained in a major UK External Quality Assessment scheme, which have, with the exception of the determination of creatinine at a particularly low concentration, given excellent agreement. CONCLUSIONS: The materials are intended for use by pathology laboratories and manufacturers of in vitro diagnostic (IVD) kits for validation of existing routine methodology to a traceable standard, which will promote harmonization between the different methods, instruments and IVD kits used in these laboratories.