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. 2021 Mar 29;20(1):591-599.
doi: 10.1007/s40200-021-00786-3. eCollection 2021 Jun.

Mass spectrometry with derivatization method for concurrent measurement of amino acids and acylcarnitines in plasma of diabetic type 2 patients with diabetic nephropathy

Parsa Esmati  1   2 , Niloufar Najjar  2 , Solaleh Emamgholipour  3 , Shaghayegh Hosseinkhani  3 , Babak Arjmand  2   4 , Amin Soleimani  5 , Ardeshir Kakaii  6 , Farideh Razi  7
Affiliations

Mass spectrometry with derivatization method for concurrent measurement of amino acids and acylcarnitines in plasma of diabetic type 2 patients with diabetic nephropathy

Parsa Esmati et al. J Diabetes Metab Disord. .

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Supplementary information: The online version contains supplementary material available at 10.1007/s40200-021-00786-3.

Keywords: Acylcarnitine; Amino acid; Carnitine; Diabetes; Nephropathy; Tandem mass spectrometry.

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Conflict of interest statement

Conflict of interestThe authors declare that they have no conflict of interest.

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