Development and validation of an intact c-peptide liquid chromatography tandem mass spectrometry method for serum and dry blood spot samples

Tetteh, Godwin; Costello, Ben de Lacy; Varadi, Aniko; Wark, Gwen

Abstract

Introduction
C-peptide, which is produced in the pancreatic beta cells is widely accepted as a surrogate marker for insulin secretion. It is currently used in diabetes classification, management, and investigations into non-diabetic associated hypoglycaemia. Measured mostly by immunoassay methods, C-peptide assays shows significant cross reactivity with heterophilic antibodies, biotin, insulin analogues and pro-insulin. In this research, we develop and validate an LC-MS/MS intact C-peptide method that overcome these limitations. Taking advantage of smaller sampling needs and simplified sample collection and handling, and the sensitivity offered by LC-MS/MS, we also develop a new approach for using dry blood spot (DBS) as sample type. Samples can be collected whilst patients are symptomatic saving significant cost by avoiding stimulated test method. DBS can also support large-scale clinical trials in diabetes.

Methods
The sample preparation was based on a simple protein precipitation followed by solid phase extraction. Liquid chromatographic separation was by the Waters Acquity UPLC. Targeted monitoring of transitions was by the Waters XEVO Mass Spectrometer in positive electron spray ionisation mode. Following method validation, DBS samples were verified against the serum method for systematic difference and precision at each medical decision points following the CLSI guidance

Results and conclusion
The serum assay was linear showing, r2 of >0.99, with a measuring range of 3.9pmol/L to 8000pmol/L. The CVs of within and between batch imprecision was <10%. Recovery was acceptable and carryover was <1%. LoB, LoD and LoQ were 0.047pmol/L, 1.122pmol/L and 0.215pmol/L respectively. Quantitative matrix effect was <15%. Measurement of trueness and accuracy using EQA and patient samples respectively, were acceptable. DBS precision was acceptable and there was no significant systemic difference between Serum and DBS sample types.

• Presentation Conference Type: Poster
• Conference Name: Labmed UK
• Start Date: Jun 11, 2024
• End Date: Jun 12, 2024
• Deposit Date: Oct 7, 2024
• Publicly Available Date: Oct 8, 2024
• Peer Reviewed: Not Peer Reviewed
• Public URL: https://uwe-repository.worktribe.com/output/13268486