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Rapid and Sensitive Determination of Branched-Chain Amino Acids in Human Plasma by Capillary Electrophoresis with Contactless Conductivity Detection for Physiological Studies

  • Petr TůmaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1972)

Abstract

Capillary electrophoresis (CE) with contactless conductivity detection (C4D) represents a strong tool for determining amino acids in clinical samples. This chapter provides detailed instructions for CE/C4D determination of the branched-chain amino acids (BCAAs) valine, isoleucine, and leucine in human plasma, which can be readily employed in physiological studies. Baseline separation of all the BCAAs is achieved on a short separation length equal to 18 cm in optimized background electrolyte consisting of 3.2 M acetic acid dissolved in 20% v/v methanol with addition of 1.0% v/v INST-coating solution. The analysis time does not exceed 3 min and the limit of detection is 0.4 μM for all BCAAs. The pretreatment of human plasma is very simple and is based on fourfold plasma dilution by acetonitrile and subsequent filtration. Only 50 μL of plasma is used for the analysis. The high sensitivity of the CE/C4D method is achieved by injecting a large volume of sample, combined with application of negative pressure to flush the acetonitrile zone out of the capillary.

Key words

Branched-chain amino acids Capillary electrophoresis Contactless conductivity detection Human plasma Large-volume sample stacking Pressure-assisted analysis Rapid determination 

Notes

Acknowledgements

This work was supported by the Grant Agency of the Czech Republic, Grant No. 18-04902S.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Hygiene, Third Faculty of MedicineCharles UniversityPragueCzech Republic

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