Development and validation of an LC-MS/MS intact C-peptide method and a protocol for testing dry blood spot samples for the diagnosis and management of Diabetes Mellitus

Tetteh, Godwin

Abstract

C-peptide, which is produced in the pancreatic β-cells, is widely accepted as a surrogate marker for insulin secretion and estimation assist in the diagnosis and management of Diabetes Mellitus. Currently, C-peptide is predominantly measured by immunoassay methods, which have a number of potential limitations such as interferences from biotin, proinsulin, and heterophilic antibodies. We have developed and validated an LC-MS/MS intact C-peptide method for serum and plasma samples that overcomes these analytical limitations. We have also developed a new approach for using dry blood spot (DBS) as an alternative sample type.

Assay calibrants and in-house internal quality control were prepared using Cerilliant C-peptide certified reference material spiked into charcoal stripped bovine serum. The internal standard used was (Tyr⁰)-C-Peptide. Sample preparation was based on simple protein precipitation followed by solid phase extraction using the Oasis HLB µElution plate. LC separation was done by the Waters Acquity UPLC system.

The method was first validated for serum and plasma samples by the evaluation of accuracy, precision, recovery, carry over, sensitivity and interferences. DBS was verified against the serum method for systematic differences by method comparison studies and an evaluation of imprecision at medical decision points following CLSI guidance EP35.

The assay calibration curve for serum and plasma samples was linear, showing R2 of >0.99, with a measurement range of 3.9 pmol/L to 8000 pmol/L. The within batch and between batch imprecision was <10%. Sample recovery was between 101.9% and 108.4% and carryover was <1%. There was no cross-reactivity with proinsulin or insulin, concentrations assessed was up to 5000 pmol/L and 100,000 pmol/L respectively. The Limit of Blank, Limit of Detection and Limit of Quantification were 0.0474 pmol/L, 0.08295 pmol/L, and 0.227 pmol/L respectively. There was no significant matrix effects. Measurement trueness using EQA samples, Z-scores were all within +/-2. Patient sample comparison R2 was >0.99. DBS imprecision were all below the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) biological variation allowable limits of 8.3%. The systematic difference of DBS was -15.7%, -17.3% and -9.6% at 0 – 300pmol/L, 300-700pmol/L, and 700-600pmo/L clinical decision target ranges respectively.

This intact C-peptide LCMS-MS assay is sensitive with a low serum and plasma sample volume of 50 µl. DBS sampling and analytical workflow is convenient, and can support large scale clinical trials.

• Thesis Type: Thesis
• Deposit Date: Oct 6, 2024
• Publicly Available Date: Feb 10, 2025
• Public URL: https://uwe-repository.worktribe.com/output/13268481
• Award Date: Feb 10, 2025