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The EZ:Faast Family of Amino Acid Analysis Kits: Application of the GC-FID Kit for Rapid Determination of Plasma Tryptophan and Other Amino Acids

  • Abdulla A.-B. Badawy
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2030)

Abstract

Plasma tryptophan (Trp) and other amino acids (AA) can be determined rapidly by gas (GC) or liquid (LC) chromatography using the Phenomenex EZ:Faast™ family of kits. Three kits are available: (1) GC-FID or -NPD, (2) GC-MS, and (3) LC-MS. The two GC kits can determine 32 AA, whereas the LC-MS can determine 5 additional AA. All three kits, however, share the same experimental procedure up to and including the preparation of derivatized AA. The method is based on solid-phase extraction (SPE), thus saving time on prior removal of plasma or other proteins and interfering substances and can be applied to other body fluids and experimental media and to supernatants of extracts of solid material. Briefly, SPE is performed using a proprietary cation-exchange mechanism. The acid solution of the internal standard ensures that the free amino acids are in an anionic form suitable for cationic binding. The alkaline nature of the elution medium ensures that the AA cations are released prior to derivatization. The latter involves production of chloroformate derivatives of both the amino and carboxylic acid groups. With experience, six plasma samples can be so processed within 12 min. The shortest analytical run is <7 min per sample using the GC-FID/NPD kit. Despite its many steps, the procedure becomes second nature and an enjoyable task. I have now used the GC-FID kit with manual injection to process >1600 plasma and other samples. Limit of detection of AA is 1 μM or less. The procedure has been validated and optimized for Trp and its main five brain uptake competitors.

Key words

Amino acids EZ:Faast™ Kits Gas chromatography Liquid chromatography Plasma Solid-phase extraction Tryptophan Amino acid analysis 

Notes

Acknowledgments

This work was funded by the Wellcome Trust (069301). I thank Chris Morgan and Jennifer Turner for technical assistance and Ben Atkins and Lindsey Starr (Phenomenex UK) for helpful discussions and useful technical information. The author held an honorary professorial appointment at Cardiff Metropolitan University (previously University of Wales Institute, Cardiff) during the period September 2006 to September 2016.

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Copyright information

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

Authors and Affiliations

  • Abdulla A.-B. Badawy
    • 1
  1. 1.School of Health SciencesCardiff Metropolitan UniversityCardiffUK

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