Ann Lab Med.  2025 Jan;45(1):62-69. 10.3343/alm.2024.0072.

Development and Validation of a Novel Isotope Dilution-Ultraperformance Liquid ChromatographyTandem Mass Spectrometry Method for Serum C-Peptide

• Cho SE
• Han J
• You J
• Lee JH
• Yi A
• Lee SG
• Lee EH

• KMID: 2563856
• DOI: http://doi.org/10.3343/alm.2024.0072

Abstract

Background
Mass spectrometry (MS) methods exhibit higher accuracy and comparability in measuring serum C-peptide concentrations than immunoassays. We developed and validated a novel isotope dilution-ultraperformance liquid chromatography-tandem MS (IDUPLC-MS/MS) assay to measure serum C-peptide concentrations.
Methods
Sample pretreatment involved solid-phase extraction, ion-exchange solid-phase extraction, and derivatization with 6-aminoquinolyl-N-hydroxysuccinimidylcarbamate (Cayman Chemical, Ann Arbor, Michigan, USA). We used an ExionLC UPLC system (Sciex, Framingham, MA, USA) and a Sciex Triple Quad 6500 + MS/MS system (Sciex) for electrospray ionization in positive-ion mode with multiple charge states of [M+3H]3+ and multiple reaction monitoring transitions. The total run time was 50 mins, and the flow rate was 0.20 mL/min. We evaluated the precision, trueness, linearity, lower limit of quantitation (LLOQ), carryover, and matrix effects. Method comparison with electrochemiluminescence immunoassay (ECLIA) was performed in 138 clinical specimens.
Results
The intra- and inter-run precision coefficients of variation were < 5% and the bias values for trueness were < 4%, which were all acceptable. The verified linear interval was 0.050–15 ng/mL, and the LLOQ was 0.050 ng/mL. No significant carryover or matrix effects were observed. The correlation between this ID-UPLC-MS/MS method and ECLIA was good (R = 0.995, slope = 1.564); however, the ECLIA showed a positive bias (51.8%).
Conclusions
The developed ID-UPLC-MS/MS assay shows acceptable performance in measuring serum C-peptide concentrations. This will be useful in situations requiring accurate measurement of serum C-peptide in clinical laboratories.

Keyword

Tandem mass spectrometry; C-peptide; Performance

Figure

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  Fig. 1 Typical tandem mass spectrometry chromatograms of C-peptide in a patient serum sample (2.860 ng/mL). (A) Full chromatograms after 50 minutes of runtime. (B) Magnification of the major peaks in panel A.

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  Fig. 2 Effect of ion suppression. No ion suppression was observed in blank samples (A, Mass Spect Gold Human Serum; B, 2% BSA in 5% acetic acid), (C) normal sample, (D) hemolytic sample, (E) icteric sample, or (F) lipemic sample. Abbreviation: BSA, bovine serum albumin.

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  Fig. 3 Method comparison of ID-UPLC-MS/MS and the Cobas 8000 ECLIA. (A) Passing–Bablok regression scatter plot. (B) Percent bias plot. The correlation was good (R=0.995, slope=1.564) in 138 random individuals with C-peptide concentrations of 0.55–12.54 ng/mL, but positive percent bias was observed for all concentrations. Abbreviations: ID-UPLC-MS/MS, isotope dilution-ultraperformance liquid chromatography-tandem mass spectrometry; ECLIA, electrochemiluminescence immunoassay.

Reference

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