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

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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.

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Data availability

The dataset supporting the conclusions of this article is available and can be accessed by contacting the corresponding author.

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Funding

This study is funded by Endocrinology and Metabolism Research Institute.

Author information

Authors and Affiliations

  1. Department of mechanical engineering, University of Bristol, Bristol, UK

    Parsa Esmati

  2. Metabolomics and genomics research center, Endocrinology and metabolism molecular-cellular sciences institute, Tehran University of medical sciences, Tehran, Iran

    Parsa Esmati, Niloufar Najjar & Babak Arjmand

  3. Department of Clinical Biochemistry, School of Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Solaleh Emamgholipour & Shaghayegh Hosseinkhani

  4. Cell Therapy and Regenerative Medicine Research Center, Endocrinology and Metabolism Molecular-Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

    Babak Arjmand

  5. Metabolic Disorders Research Center, Endocrinology and Metabolism Molecular -Cellular Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

    Amin Soleimani

  6. Endocrinology and Metabolism Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Tehran, Iran

    Ardeshir Kakaii

  7. Diabetes Research Center, Endocrinology and Metabolism Clinical Sciences Institute, Tehran University of Medical Sciences, Beneath Dr. Shariati Hospital, Gomnam Highway, Tehran, Iran

    Farideh Razi

Authors
  1. Parsa Esmati
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Contributions

All authors participated and approved the paper before submission. FR planned the study, NN, AK, and ShH did the analytical examination and analyzed the result, AS contributed in optimization and supervision of systems, BA and SE participated in the study design, PE contributed in technical issues, statistical analysis and preparation of the manuscript.

Corresponding author

Correspondence to Farideh Razi.

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The study was approved by the Endocrinology and Metabolism Research Institute, Tehran University of Medical Sciences.

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Esmati, P., Najjar, N., Emamgholipour, S. et al. Mass spectrometry with derivatization method for concurrent measurement of amino acids and acylcarnitines in plasma of diabetic type 2 patients with diabetic nephropathy. J Diabetes Metab Disord 20, 591–599 (2021). https://doi.org/10.1007/s40200-021-00786-3

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