Esmati Parsa, Najjar Niloufar, Emamgholipour Solaleh, Hosseinkhani Shaghayegh, Arjmand Babak, Soleimani Amin, Kakaii Ardeshir, Razi Farideh
Department of mechanical engineering, University of Bristol, Bristol, UK.
Metabolomics and genomics research center, Endocrinology and metabolism molecular-cellular sciences institute, Tehran University of medical sciences, Tehran, Iran.
J Diabetes Metab Disord. 2021 Mar 29;20(1):591-599. doi: 10.1007/s40200-021-00786-3. eCollection 2021 Jun.
Amino acids (AAs) and acylcarnitines play a key role in metabolic disease and can be used as biomarkers of various diseases such as malignancies, type 2 diabetes (T2D), insulin resistance, and cardiovascular diseases, therefore, designing an accurate and simple laboratory method that simultaneously measure both groups of substances, could improve the process of analytes quantification. In this research, a flow injection tandem mass spectrometry (FI-MS/MS) method for simultaneous measurement of AAs and acylcarnitines in addition to results of validation is explained.
Samples were mixed with internal standards and after derivatization (with butanolic-HCL), AAs, and acylcarnitines were quantified by tandem mass spectrometry (SCIEX API 3200) Analytical performance studies were designed based on the Clinical and Laboratory Standards Institute (CLSI) guidelines including precision, accuracy, linearity, and limit of detection-quantification (LOD-LOQ) experiments. Samples from patients with T2D in different stages of kidney disease were also analyzed to ensure the clinical usage of the method.
Performance evaluation of the method demonstrated adequate results. The mean of estimated inter-assay precision (reported as a coefficient variation) for AAs and acylcarnitines were less than 8.7% and 12.3%, the estimated mean bias was below 8.8% and 10.2% respectively. LOD of analytes ranged between 0.6-10 μmol per liter (μmol/L) for AAs and 0.02-1 μmol/L for acylcarnitines. LOQ analytes showed a range of 2-25 μmol/L and 0.05-5 μmol/L for AAs and carnitine/acylcarnitines respectively. In diabetic patients sample analysis, a significant increase in acylcarnitines (C2, C4, C5DC, C6, C8, C10, C14) and citrulline with a significant decrease in valine were seen in patients with severely increased albuminuria.
FI-MS/MS method with pre-injection derivatization with butanolic-HCL can be used for concurrent measurement of AAs and carnitine/acylcarnitines in a short time and it satisfies the analytical performance requirements. This method is applied for AAs and carnitine/acylcarnitines measurement in patient with T2DM and results show some of the acylcarnitines and AAs can be involved in diabetic nephropathy development.
The online version contains supplementary material available at 10.1007/s40200-021-00786-3.
氨基酸（AAs）和酰基肉碱在代谢性疾病中起关键作用，可作为多种疾病的生物标志物，如恶性肿瘤、2型糖尿病（T2D）、胰岛素抵抗和心血管疾病。因此，设计一种准确、简单的实验室方法同时检测这两类物质，可改善分析物定量过程。本研究解释了一种用于同时测定氨基酸和酰基肉碱的流动注射串联质谱（FI-MS/MS）方法及其验证结果。
将样品与内标混合，经衍生化（用丁醇 - 盐酸）后，通过串联质谱（SCIEX API 3200）对氨基酸和酰基肉碱进行定量。基于临床和实验室标准协会（CLSI）指南设计分析性能研究，包括精密度、准确度、线性以及检测限 - 定量限（LOD - LOQ）实验。还对不同肾病阶段的T2D患者样本进行分析，以确保该方法的临床实用性。
该方法的性能评估结果良好。氨基酸和酰基肉碱的批间精密度估计平均值（以变异系数表示）分别小于8.7%和12.3%，估计平均偏差分别低于8.8%和10.2%。氨基酸分析物的检测限范围为0.6 - 10微摩尔每升（μmol/L），酰基肉碱为0.02 - 1μmol/L。定量限分析物中，氨基酸的范围为2 - 25μmol/L，肉碱/酰基肉碱为0.05 - 5μmol/L。在糖尿病患者样本分析中，白蛋白尿严重增加的患者中，酰基肉碱（C2、C4、C5DC、C6、C8、C10、C14）和瓜氨酸显著增加，缬氨酸显著减少。
采用丁醇 - 盐酸进行注射前衍生化的FI-MS/MS方法可在短时间内同时测定氨基酸和肉碱/酰基肉碱，满足分析性能要求。该方法应用于T2DM患者中氨基酸和肉碱/酰基肉碱的测定，结果表明部分酰基肉碱和氨基酸可能参与糖尿病肾病的发生发展。
在线版本包含可在10.1007/s40200 - 021 - 00786 - 3获取的补充材料。
