Nantong University, Nantong 226001, China; Department of Laboratory Medicine, Affiliated Hospital of Nantong University, Nantong 226001, China.
Department of Laboratory Medicine, The Second Affiliated Hospital of Nantong University, Nantong 226001, China.
J Chromatogr B Analyt Technol Biomed Life Sci. 2022 Nov 15;1211:123487. doi: 10.1016/j.jchromb.2022.123487. Epub 2022 Oct 3.
Beta 2-microglobulin (B2M) is a commonly used detection index in clinical laboratories. Currently, it is used as a sensitive indicator for the early detection of kidney disease. Immunoassay is the most commonly used method for B2M detection in clinical practice. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) has changed the face of laboratory testing by providing high-throughput analysis with better specificity than detection methods using only antibodies. An isotope-dilution LC-MS/MS (ID-LC-MS/MS) method was developed for serum B2M quantification. After B2M denaturation, reduction and alkylation of cysteine residues, trypsin was added for digestion of B2M. Then, it was purified by a solid-phase extraction column, and the sample was injected into a high-performance LC-MS/MS for measurement. A signature peptide (VNHVTLSQPK) was selected as a surrogate for B2M. A stable isotope-labeled peptide (VNHVT[CN]LSQP) was used as the internal standard to quantify B2M based on the calibration curve method. The linear range of serum B2M calibration curve was from 0.25 to 40 mg/L. The limit of quantification was 0.25 mg/L, and limit of detection was 0.06 mg/L. At different concentrations of serum B2M, the precision (coefficients of variation, CV%) ranged from 1.47% to 3.97%, and accuracy (relative error, RE%) was within -3.15% and 6.80% of nominal values. The applicability and reliability of the method were verified by measuring B2M in the ERM-DA470k/IFCC and serum samples. The validated LC-MS/MS method was successfully applied to a clinical study involving quantification of serum B2M in patients with acute renal insufficiency and healthy individuals. Deming analysis showed that the ID-LC-MS/MS and immunoassay were in good agreement. The LC-MS/MS method we developed is sensitive and reliable for the absolute quantification of serum B2M.
β2-微球蛋白（B2M）是临床实验室常用的检测指标之一。目前，它被用作检测肾脏疾病早期的敏感指标。免疫测定法是临床实践中最常用于检测 B2M 的方法。液相色谱-串联质谱法（LC-MS/MS）通过提供比仅使用抗体的检测方法更高的特异性的高通量分析，改变了实验室检测的面貌。建立了一种用于血清 B2M 定量的同位素稀释 LC-MS/MS（ID-LC-MS/MS）方法。B2M 变性、半胱氨酸残基还原和烷基化后，加入胰蛋白酶进行 B2M 消化。然后，通过固相萃取柱进行纯化，将样品注入高效 LC-MS/MS 进行测量。选择特征肽（VNHVTLSQPK）作为 B2M 的替代物。将稳定同位素标记的肽（VNHVT[CN]LSQP）用作内标，根据校准曲线法定量 B2M。血清 B2M 校准曲线的线性范围为 0.25 至 40mg/L。定量下限为 0.25mg/L，检测限为 0.06mg/L。在不同浓度的血清 B2M 下，精密度（变异系数，CV%）在 1.47%至 3.97%之间，准确度（相对误差，RE%）在名义值的-3.15%至 6.80%范围内。通过测量 ERM-DA470k/IFCC 和血清样品中的 B2M 验证了该方法的适用性和可靠性。该验证的 LC-MS/MS 方法成功应用于一项涉及急性肾功能不全患者和健康个体血清 B2M 定量的临床研究。Deming 分析表明，ID-LC-MS/MS 和免疫测定法具有良好的一致性。我们开发的 LC-MS/MS 方法对血清 B2M 的绝对定量具有灵敏性和可靠性。
