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Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography–tandem mass spectrometry

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Abstract

Herein, we describe an accurate method for protein quantification based on conventional acid hydrolysis and an isotope dilution-ultra performance liquid chromatography–tandem mass spectrometry method. The analyte protein, recombinant human erythropoietin (rhEPO), was effectively hydrolyzed by incubation with 8 mol/L hydrochloric acid at 130 °C for 48 h, in which at least 1 μmol/kg of rhEPO was treated to avoid possible degradation of released amino acids during hydrolysis. Prior to hydrolysis, sample solution was subjected to ultrafiltration to eliminate potential interfering substances. In a reversed-phase column, the analytes (phenylalanine, proline, and valine) were separated within 3 min using gradient elution comprising 20 % (v/v) acetonitrile and 10 mmol/L ammonium acetate, both containing 0.3 % (v/v) trifluoroacetic acid. The optimized hydrolysis and analytical conditions in our study were strictly validated in terms of accuracy and precision, and were suitable for the accurate quantification of rhEPO. Certified rhEPO was analyzed using a conventional biochemical assay kit as an additional working calibrant for the quantification of EPO and improved the accuracy. The optimized protocol is suitable for the accurate quantification of rhEPO and satisfactorily serves as a reference analytical procedure for the certification of rhEPO and similar proteins.

The concept of protein quantification by amino acid analysis via acid hydrolysis using isotopedilution LC-MS

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Acknowledgement

This work was fully supported by Basic Research Projects of Korea Research Institute of Standards and Science, “Development of Protein Measurement Standards,” grant 14011043.

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Authors and Affiliations

  1. Center for Bioanalysis, Department of Metrology for Quality of Life, Korea Research Institute of Standards and Science, 267 Gajeong-ro, Youseong-gu, Daejeon, 305-340, Republic of Korea

    Jung-Hyuk Yim, Hyo-Jin Yang, Sook-Kyung Kim, Sang-Ryoul Park & Ji-Seon Jeong

  2. Department of Pharmacy Graduate School, Chungbuk National University, 52 Naesudong-ro, Heungdeok-gu, Cheongju, Chungbuk, 361-763, Republic of Korea

    Jung-Hyuk Yim & Yong-Moon Lee

  3. Department of Chemistry, University of Pennsylvania, 231S.34 Street, Philadelphia, PA, 19104, USA

    Ina Yoon

  4. Department of Bio-Analytical Science, University of Science and Technology, Daejeon, Republic of Korea

    Hyo-Jin Yang

Authors
  1. Jung-Hyuk Yim
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  2. Ina Yoon
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  3. Hyo-Jin Yang
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  4. Sook-Kyung Kim
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  5. Sang-Ryoul Park
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  6. Yong-Moon Lee
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  7. Ji-Seon Jeong
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Correspondence to Yong-Moon Lee or Ji-Seon Jeong.

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Yim, JH., Yoon, I., Yang, HJ. et al. Quantification of recombinant human erythropoietin by amino acid analysis using isotope dilution liquid chromatography–tandem mass spectrometry. Anal Bioanal Chem 406, 4401–4409 (2014). https://doi.org/10.1007/s00216-014-7838-0

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  • DOI: https://doi.org/10.1007/s00216-014-7838-0

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