MSACL 2020 US Abstract

Dilute, Digest and Detect: Quantification of C-reactive Protein in Human Plasma by LC-MS/MS

Meng Wang (1), Junyan Shi (1), Mari L. DeMarco (1,2)
(1) Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada (2) Department of Pathology and Laboratory Medicine, St Paul’s Hospital, Providence Health Care, Vancouver, BC, Canada

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Meng Wang (Presenter) University of British Columbia

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Background: C-reactive protein (CRP) is a marker of inflammation, infection, and tissue damage and is used for risk assessment and to monitor treatment efficacy for a wide variety of diseases. Due to its broad utility, there is a high demand for measurement of CRP in routine care and in clinical research. However, discrepant CRP test results as measured by different clinical immunoassays have been reported. Moreover, the currently published LC-MS/MS methods contain costly and time-consuming sample preparation steps including affinity purification, immunodepletion or size exclusion purification. Therefore, an optimized method with metrological traceability is needed.

Objective: The primary objectives of this study were to develop a selective and quantitative LC-MS/MS method for the measurement of CRP in human plasma and serum, traceable to the newly available standard reference material.

Methods: For sample preparation, EDTA plasma samples were heat denatured, digested with trypsin and analyzed by high performance LC-MS/MS. Quantification was performed by comparison to a five-point external calibration curve, with calibrators assigned to National Institute of Standards and Technology standard reference material (SRM) 2924. The method was validated following Clinical and Laboratory Standards Institute guidelines, and a method comparison was performed against a commonly used clinical immunoturbidimetric assay.

Results: A streamlined sample preparation workflow was developed without the use of reduction alkylation steps or chemical denaturants, combined with a rapid proteolytic digestion: 10 μL of EDTA plasma was subjected to heat denaturation followed by a brief 20-minute digestion and analysis by LC-MS/MS. The lower limit of the measuring interval was 1 mg/L. The method was linear from 1 to 400 mg/L (R2=0.9948), displayed 6.0% imprecision near the medical decision limit of 10 mg/L, showed no interference with hemolysis, icterus or lipemia, and was highly correlated (R2=0.92) with a clinical immunoturbidometric method.

Conclusions: CRP in plasma can be quantified by LC-MS/MS via a simple ‘dilute, digest, and detect’ approach. This method required fewer sample preparative steps, less processing time, and less bias compared to proposed candidate reference method. Relative to immunometric clinical methods, this new LC-MS/MS requires less sample volume and has a wider analytical range.

Keywords: C-reactive protein, biomarker, quantitation, multiple reaction monitoring, liquid chromatography, tandem mass spectrometry, immunoassay.