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Determination of serum d-lactic and l-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization

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Abstract

d-Lactic and l-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica (Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor (α) of 1.32 and a resolution (R s) of 1.98. Moreover, the features of the first elution of d-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum d-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5–101.2 and 89.0–100.7%, and the intra-day and inter-day precisions were 0.3–1.2 and 0.4–4.8%, respectively. The proposed method was applied to determine d-lactic and l-lactic acids in human serum of normal subjects and diabetic patients, showing that both d-lactic and l-lactic acid concentrations were significantly increased in the serum of diabetic patients (n=31) as compared with normal subjects (n=21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine d-lactic and l-lactic acid simultaneously. Finally, serum d-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.

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Acknowledgements

The authors thank Imtakt Co., Ltd. for kind donation of Cadenza CD-C8. Thanks are also due to graduate students in our laboratory for their participation as healthy volunteers in the present study.

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Authors and Affiliations

  1. Laboratory of Bio-Analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, Tokyo, Japan

    Hisanori Hasegawa, Takeshi Fukushima & Kazuhiro Imai

  2. Department of Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan

    Takeshi Fukushima

  3. Department of Pharmaceutical Analysis, School of Pharmacy, Taipei Medical University, Taipei, Taiwan

    Jen-Ai Lee

  4. Department of Internal Medicine, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

    Kazuhisa Tsukamoto

  5. Department of Metabolic Diseases, Graduate School of Medicine, University of Tokyo, Tokyo, Japan

    Kyoji Moriya

  6. Department of Clinical Laboratory, University of Tokyo Hospital, Tokyo, Japan

    Yoshikazu Ono

  7. Center for Research and Development, Kyoritsu College of Pharmacy, Kyoritsu, Japan

    Kazuhiro Imai

Authors
  1. Hisanori Hasegawa
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  2. Takeshi Fukushima
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  3. Jen-Ai Lee
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  4. Kazuhisa Tsukamoto
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  5. Kyoji Moriya
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  6. Yoshikazu Ono
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  7. Kazuhiro Imai
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Corresponding author

Correspondence to Kazuhiro Imai.

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Hasegawa, H., Fukushima, T., Lee, JA. et al. Determination of serum d-lactic and l-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization. Anal Bioanal Chem 377, 886–891 (2003). https://doi.org/10.1007/s00216-003-2108-6

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  • DOI: https://doi.org/10.1007/s00216-003-2108-6

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