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A critical analysis of L-asparaginase activity quantification methods—colorimetric methods versus high-performance liquid chromatography

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Abstract

L-asparaginase or ASNase (L-asparagine aminohydrolase, E.C.3.5.1.1) is an enzyme clinically accepted as an antitumor agent to treat acute lymphoblastic leukemia (ALL) and lymphosarcoma through the depletion of L-asparagine (L-Asn) resulting in cytotoxicity to leukemic cells. ASNase is also important in the food industry, preventing acrylamide formation in processed foods. Several quantification techniques have been developed and used for the measurement of the ASNase activity, but standard pharmaceutical quality control methods were hardly reported, and in general, no official quality control guidelines were defined. To overcome this lack of information and to demonstrate the advantages and limitations, this work properly compares the traditional colorimetric methods (Nessler; L-aspartic acid β-hydroxamate (AHA); and indooxine) and the high-performance liquid chromatography (HPLC) method. A comparison of the methods using pure ASNase shows that the colorimetric methods both overestimate (Nessler) and underestimate (AHA and indooxine) the ASNase activity when compared to the values obtained with HPLC, considered the most precise method as this method monitors both substrate consumption and product formation, allowing for overall mass-balance. Correlation and critical analysis of each method relative to the HPLC method were carried out, resulting in a demonstration that it is crucial to select a proper method for the quantification of ASNase activity, allowing bioequivalence studies and individualized monitoring of different ASNase preparations.

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Funding

This work was developed under the scope of the Young Researcher Project financed by FAPESP (São Paulo Research Foundation Brazil) through the process 2014/16424-7. The authors also acknowledge the financial support from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, processes 163292/2015-9, and 445442/2014-0) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES and CAPES-PROEX). In addition, P. S. Barber acknowledges Earlham College for financial support.

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Authors and Affiliations

  1. Department of Bioprocesses and Biotechnology, School of Pharmaceutical Sciences, São Paulo State University (UNESP), Rodovia Araraquara-Jaú/Km 01, Campos Ville, Araraquara, São Paulo, 14800-903, Brazil

    Agnes Magri, Matheus F. Soler, André M. Lopes & Jorge F. B. Pereira

  2. Biochemistry and Technology Chemistry Department, Chemistry Institute, São Paulo State University (UNESP), Araraquara, São Paulo, 14800-900, Brazil

    Eduardo M. Cilli

  3. Department of Chemistry, Earlham College, 801 National Road West, Richmond, IN, 47374, USA

    Patrick S. Barber

  4. Department of Biochemical-Pharmaceutical Technology, Pharmaceutical Biotechnology Laboratory, University of Sao Paulo (USP), São Paulo, 05508-000, Brazil

    Adalberto Pessoa Jr

Authors
  1. Agnes Magri
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  2. Matheus F. Soler
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  6. Adalberto Pessoa Jr
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  7. Jorge F. B. Pereira
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Correspondence to Jorge F. B. Pereira.

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Magri, A., Soler, M.F., Lopes, A.M. et al. A critical analysis of L-asparaginase activity quantification methods—colorimetric methods versus high-performance liquid chromatography. Anal Bioanal Chem 410, 6985–6990 (2018). https://doi.org/10.1007/s00216-018-1326-x

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  • DOI: https://doi.org/10.1007/s00216-018-1326-x

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