Abstract：
OBJECTIVE: The standardization of cystatin C (CysC) measurement has received increasing attention in recent years due to its importance in estimating glomerular filtration rate (GFR). Mass spectrometry-based assays have the potential to provide an accuracy base for CysC measurement. However, a precise, accurate and sustainable LC-MS/MS method for CysC is still lacking.
METHODS: The developed LC-MS/MS method quantified CysC by detecting signature peptide (T3) obtained from tryptic digestion. Stable isotope labeled T3 peptide (SIL-T3) was spiked to control matrix effects and errors caused by liquid handling. The protein denaturation, reduction and alkylation procedures were combined into a single step with incubation time of 1 h, and the digestion lasted for 3.5 h. In the method validation, digestion time-course, imprecision, accuracy, matrix effect, interference, limit of quantification (LOQ), carryover, linearity, and the comparability to two routine immunoassays were evaluated.
RESULTS: No significant matrix effect or interference was observed with the CysC measurement. The LOQ was 0.21 mg/L; the within-run and total imprecision were 1.33-2.05 % and 2.18-3.90 % for three serum pools (1.18-5.34 mg/L). The LC-MS/MS method was calibrated by ERM-DA471/IFCC and showed good correlation with two immunoassays traceable to ERM-DA471/IFCC. However, significant bias was observed for immunoassays against the LC-MS/MS method.
CONCLUSIONS: The developed LC-MS/MS method is robust and simpler and holds the promise to provide an accuracy base for routine immunoassays, which will promote the standardization of CysC measurement.
METHODS: The developed LC-MS/MS method quantified CysC by detecting signature peptide (T3) obtained from tryptic digestion. Stable isotope labeled T3 peptide (SIL-T3) was spiked to control matrix effects and errors caused by liquid handling. The protein denaturation, reduction and alkylation procedures were combined into a single step with incubation time of 1 h, and the digestion lasted for 3.5 h. In the method validation, digestion time-course, imprecision, accuracy, matrix effect, interference, limit of quantification (LOQ), carryover, linearity, and the comparability to two routine immunoassays were evaluated.
RESULTS: No significant matrix effect or interference was observed with the CysC measurement. The LOQ was 0.21 mg/L; the within-run and total imprecision were 1.33-2.05 % and 2.18-3.90 % for three serum pools (1.18-5.34 mg/L). The LC-MS/MS method was calibrated by ERM-DA471/IFCC and showed good correlation with two immunoassays traceable to ERM-DA471/IFCC. However, significant bias was observed for immunoassays against the LC-MS/MS method.
CONCLUSIONS: The developed LC-MS/MS method is robust and simpler and holds the promise to provide an accuracy base for routine immunoassays, which will promote the standardization of CysC measurement.
摘要：
目的：由于胱抑素C（CysC）测量在估算肾小球滤过率（GFR）中的重要性，因此近年来标准化越来越受到重视。基于质谱的测定具有为CysC测量提供准确性基础的潜力。然而,一个精确的，CysC的准确和可持续的LC-MS/MS方法仍然缺乏。
方法：开发的LC-MS/MS方法通过检测从胰蛋白酶消化获得的特征肽（T3）来定量CysC。添加稳定同位素标记的T3肽（SIL-T3）以控制基体效应和液体处理引起的错误。蛋白质变性，还原和烷基化程序结合成一个单一的步骤，孵育时间为1小时，消化持续3.5小时。在方法验证中，消化时程，不精确,准确度,基体效应，干扰,定量限(LOQ)，结转,线性度并评估了与两种常规免疫测定的可比性。
结果：在CysC测量中未观察到明显的基质效应或干扰。LOQ为0.21mg/L；三个血清池（1.18-5.34mg/L）的运行中和总不精确度分别为1.33-2.05％和2.18-3.90％。LC-MS/MS方法通过ERM-DA471/IFCC校准,并显示与可追溯到ERM-DA471/IFCC的两种免疫测定的良好相关性。然而,与LC-MS/MS方法相比，免疫测定存在显著偏差。
结论：已开发的LC-MS/MS方法稳健且简单，有望为常规免疫测定提供准确性基础，这将促进CysC测量的标准化。
方法：开发的LC-MS/MS方法通过检测从胰蛋白酶消化获得的特征肽（T3）来定量CysC。添加稳定同位素标记的T3肽（SIL-T3）以控制基体效应和液体处理引起的错误。蛋白质变性，还原和烷基化程序结合成一个单一的步骤，孵育时间为1小时，消化持续3.5小时。在方法验证中，消化时程，不精确,准确度,基体效应，干扰,定量限(LOQ)，结转,线性度并评估了与两种常规免疫测定的可比性。
结果：在CysC测量中未观察到明显的基质效应或干扰。LOQ为0.21mg/L；三个血清池（1.18-5.34mg/L）的运行中和总不精确度分别为1.33-2.05％和2.18-3.90％。LC-MS/MS方法通过ERM-DA471/IFCC校准,并显示与可追溯到ERM-DA471/IFCC的两种免疫测定的良好相关性。然而,与LC-MS/MS方法相比，免疫测定存在显著偏差。
结论：已开发的LC-MS/MS方法稳健且简单，有望为常规免疫测定提供准确性基础，这将促进CysC测量的标准化。
