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Quantification of homocysteine and cysteine by derivatization with pyridoxal 5'-phosphate and hydrophilic interaction liquid chromatography

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Abstract

A simple and rapid assay using pyridoxal 5'-phosphate (PLP) as a derivatizing reagent was developed for the simultaneous determination of homocysteine (Hcy) and cysteine (Cys) in human plasma. Derivatization with PLP affords UV-absorbing tetrahydrothiazine and thiazolidine derivatives of Hcy and Cys, respectively. Separation of these derivatives was achieved in 5 min using a hydrophilic interaction liquid chromatography, followed by UV detection at 330 nm. Linearity in detector response was observed over the range of 0.25–20 μM for Hcy and 10–300 μM for Cys. The limit of quantification (LOQ) values for Hcy and Cys were 0.25 and 2.5 μM, respectively. The method was successfully applied to plasma samples donated by apparently healthy volunteers.

A flow chart diagram of analytical procedure for total plasma Hcy and Cys determination

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Abbreviations

Cys:

Cysteine

Hcy:

Homocysteine

TCEP:

Tris(2-carboxyethyl)phosphine hydrochloride

PLP:

Pyridoxal 5'-phosphate

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Acknowledgments

This work was supported in part by grants from the National Science Center (2012/07/B/ST5/00765, 2012/07/B/NZ7/01178, 2013/09/D/ST5/03909, 2013/09/B/NZ5/02794, 2013/11/B/NZ1/00091), the American Heart Association (0855919D, 12GRNT9420014), the University of Łódź, and the Marshal of Łódzkie Voivodeship (3/SMN/2015).

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Authors and Affiliations

  1. Faculty of Chemistry, Department of Environmental Chemistry, University of Łódź, 163 Pomorska Str., 90-236, Łódź, Poland

    Rafał Głowacki, Justyna Stachniuk & Kamila Borowczyk

  2. Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers New Jersey Medical School, Newark, NJ, 07103, USA

    Hieronim Jakubowski

  3. Department of Biochemistry and Biotechnology, University of Life Sciences, 60-637, Poznań, Poland

    Hieronim Jakubowski

  4. Institute of Bioorganic Chemistry, 61-704, Poznań, Poland

    Hieronim Jakubowski

Authors
  1. Rafał Głowacki
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  2. Justyna Stachniuk
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  3. Kamila Borowczyk
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  4. Hieronim Jakubowski
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Corresponding author

Correspondence to Rafał Głowacki.

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The University of Łódź Ethical Committee approved the study and all participants provided written informed consent.

Conflict of interest

The authors declare that they have no competing interests.

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Głowacki, R., Stachniuk, J., Borowczyk, K. et al. Quantification of homocysteine and cysteine by derivatization with pyridoxal 5'-phosphate and hydrophilic interaction liquid chromatography. Anal Bioanal Chem 408, 1935–1941 (2016). https://doi.org/10.1007/s00216-016-9308-3

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  • DOI: https://doi.org/10.1007/s00216-016-9308-3

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