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A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood

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Abstract

The presence of reduced aminothiols, including homocysteine (Hcy), cysteine (Cys), cysteinyl-glycine (CG), and glutathione (GSH), is significantly increased in the pathological state. However, there have been no reports on the relationship between reduced aminothiols (Hcy, Cys, CG, and GSH) and different genders, ages, and drug combinations in human blood. The accurate quantification of these reduced thiols in biological fluids is important for monitoring some special pathological conditions of humans. However, the published methods typically not only require cumbersome and technically challenging processing procedures to ensure reliable measurements, but are also laborious and time-consuming, which may disturb the initial physiological balance and lead to inaccurate results. We developed a hollow fiber centrifugal ultrafiltration (HFCF-UF) method for sample preparation coupled with a high-performance liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) method and used it to determine four reduced aminothiols (Hcy, Cys, CG, and GSH) in human blood for the first time. A total of 96 clinical patients were enrolled in our study. The influence of different genders, ages, and drug combinations on the levels of four reduced thiols in human blood was also discussed by SPSS 24.0. The sample preparation was simplified to a single 5 min centrifugation step in a sealed system that did not disturb the physiological environment. The validation parameters for the methodological results were excellent. The procedure was successfully applied to monitoring the concentrations of four reduced aminothiols (Hcy, Cys, CG, and GSH) in 96 clinical blood samples. There were no significant differences in Hcy, Cys, CG, or GSH for the different genders, ages, or combinations with methotrexate or vancomycin (P > 0.05). However, there was a significant increase in Hcy concentration in patients treated with valproic acid who were diagnosed with epilepsy (p=0.0007). It is advisable to measure reduced Hcy level in patients taking valproic acid. The developed HFCF-UF method was simple and accurate. It can be easily applied in clinical research to evaluate oxidative stress in further study.

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Funding

The authors received the financial support provided by the Wu Jieping Medical Foundation (No. 320.6750.2020-04-11) and the Science and Technology Foundation of Hebei Province of China (Project No. 19277728D). The research was also supported by the National Natural Science Foundation of China (82002281) and the Natural Science Foundation of Hebei Province (H2021206299).

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Author notes

  1. Wei-Chong Dong and Jia-Liang Guo contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, Shijiazhuang, 050017, Hebei Province, China

    Wei-Chong Dong, Meng-Qiang Zhao, Yi-Xuan Cui, Jing-Ying Feng, Chen-Xiao Zhang & Ye Jiang

  2. Department of Pharmacy, The Second Hospital of Hebei Medical University, Shijiazhuang, 050051, Hebei Province, China

    Wei-Chong Dong, Xi-Kun Wu & Zhi-Qing Zhang

  3. Department of Orthopaedics, The Third Hospital of Hebei Medical University, Shijiazhuang, 050000, Hebei Province, China

    Jia-Liang Guo

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  1. Wei-Chong Dong
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Correspondence to Ye Jiang or Zhi-Qing Zhang.

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The study protocol was approved by the Ethics Committee of Hebei Medical University. All the blood samples in our present work were obtained from the Second Hospital of Hebei Medical University.

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Dong, WC., Guo, JL., Zhao, MQ. et al. A simple and accurate HFCF-UF method for the analysis of homocysteine, cysteine, cysteinyl-glycine, and glutathione in human blood. Anal Bioanal Chem 413, 6225–6237 (2021). https://doi.org/10.1007/s00216-021-03578-z

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