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 (ID-UPLC-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.

中文翻译：

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血清 C 肽的新型同位素稀释-超高效液相色谱-串联质谱法的开发和验证。


背景 质谱 (MS) 方法在测量血清 C 肽浓度方面表现出比免疫测定更高的准确性和可比性。我们开发并验证了一种新型同位素稀释-超高效液相色谱-串联 MS (ID-UPLC-MS/MS) 测定法来测量血清 C 肽浓度。方法 样品预处理包括固相萃取、离子交换固相萃取和 6-氨基喹啉基-N-羟基琥珀酰亚胺基氨基甲酸酯（Cayman Chemical，安娜堡，密歇根州，美国）衍生化。我们使用 ExionLC UPLC 系统（Sciex，Framingham，MA，美国）和 Sciex Triple Quad 6500+ MS/MS 系统 (Sciex) 在正离子模式下进行电喷雾电离，具有 [M+3H]3+ 和 [M+3H]3+ 的多种电荷态多重反应监测转变。总运行时间为 50 分钟，流速为 0.20 mL/min。我们评估了精密度、真实度、线性、定量下限 (LLOQ)、残留和基质效应。对 138 份临床标本进行了与电化学发光免疫分析 (ECLIA) 的方法比较。结果批内和批间精密度变异系数<5%，正确性偏差值<4%，均可接受。验证的线性区间为 0.050-15 ng/mL，LLOQ 为 0.050 ng/mL。没有观察到显着的残留或基质效应。该ID-UPLC-MS/MS方法与ECLIA相关性良好(R=0.995，斜率=1.564)；然而，ECLIA 显示出正偏差（51.8%）。结论 开发的 ID-UPLC-MS/MS 测定法在测量血清 C 肽浓度方面显示出可接受的性能。这在临床实验室需要精确测量血清 C 肽的情况下非常有用。