An SI traceable primary calibrator was used for the development of a reference measurement procedure for α-synuclein. A targeted proteomics workflow allowed for the SI traceable quantification of α-synuclein in cerebrospinal fluid.

AbstractObjectivesα-synuclein aggregation is an indicator of neurodegenerative diseases such as Parkinson’s disease (PD) and recent advances have suggested that this protein could serve as a potential biomarker. It has been indicated that soluble and oligomeric α-synuclein in biological fluids could have diagnostic applications for PD. Clinical laboratories currently rely on antibody-based assays to detect α-synuclein. These assays have limited specificity, low sensitivity and poor inter-lab reproducibility, which prevents the validation of α-synuclein as a biomarkers. This study aims to fill the unmet need for the standardisation of clinical measurements for α-synuclein.MethodsWe report the first candidate reference method for α-synuclein, using an SI traceable primary calibrator for α-synuclein and isotope dilution mass spectrometry. The primary calibrator was traceably quantified utilising a combination of amino acid analysis and nuclear magnetic resonance. A targeted sample clean-up procedure involving a non-denaturing Lys-C digestion and solid-phase extraction allowed for the sensitive detection of multiple proteotypic α-synuclein peptides in cerebrospinal fluid (CSF) samples.ResultsThe candidate reference method procedure showed linearity across three orders of magnitude, covering the physiological levels of α-synuclein in CSF (LOQ = 0.1 ng/g). The method was used to quantify a cohort of CSF samples and the measurements were correlated with immunoassay-based quantifications.ConclusionsThe SI traceable quantification of α-synuclein in complex biological matrices means that the role of this protein can be further elucidated in synucleinopathies. This candidate reference method would lead to the harmonisation of α-synuclein measurements, which may allow for development of high throughput clinical tests.

Determination of the purity of a substance traceable to the International System of Units (SI) is important for the production of reference materials affording traceability in quantitative measurements. Post-column isotope dilution using liquid chromatography-chemical oxidation-isotope ratio mass spectrometry (ID-LC-CO-IRMS) has previously been suggested as a means to determine the purity of organic compounds; however, the lack of an uncertainty budget has prevented assessment of the utility this approach until now. In this work, the previously published ID-LC-CO-IRMS methods have not only been improved by direct gravimetric determination of the mass flow of 13C-labelled spike but also a comprehensive uncertainty budget has been established. This enabled direct comparison of the well-characterised ID-LC-CO-IRMS method to quantitative nuclear magnetic resonance spectroscopy (qNMR) for purity determination using valine as the model compound. The ID-LC-CO-IRMS and qNMR methods provided results that were in agreement within the associated measurement uncertainty for the purity of a sample of valine of (97.1 ± 4.7)% and (99.64 ± 0.20)%, respectively (expanded uncertainties, k = 2). The magnitude of the measurement uncertainty for ID-LC-CO-IRMS determination of valine purity precludes the use of this method for determination of purity by direct analysis of the main component in the majority of situations; however, a mass balance approach is expected to result in significantly improved measurement uncertainty.

The comparison required the assignment of the mass fraction of folic acid present as the main component in the comparison sample. Performance in the comparison is representative of a laboratory's measurement capability for the purity assignment of organic compounds of medium structural complexity [molecular weight range 300–500] and high polarity (pKOW < −2). Methods used by the eighteen participating NMIs or DIs were based on a mass balance (summation of impurities) or qNMR approach, or the combination of data obtained using both methods. The qNMR results tended to give slightly lower values for the content of folic acid, albeit with larger associated uncertainties, compared with the results obtained by mass balance procedures. Possible reasons for this divergence are discussed in the report, without reaching a definitive conclusion as to their origin. The comparison demonstrates that for a structurally complex polar organic compound containing a high water content and presenting a number of additional analytical challenges, the assignment of the mass fraction content property value of the main component can reasonably be achieved with an associated relative standard uncertainty in the assigned value of 0.5% Main text To reach the main text of this paper, click on Final Report. Note that this text is that which appears in Appendix B of the BIPM key comparison database kcdb.bipm.org/. The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).