Abstract
A method for selective and sensitive quantification of amino acids is described. The combination of established derivatization procedures of secondary and primary amino groups with 9-fluorenylmethoxycarbonyl chloride (Fmoc-Cl) and subsequent detection of derivatized amino acids by LC-ESI-MS/MS using multiple reaction monitoring provides high selectivity. The attachment of an apolar moiety enables purification of derivatized amino acids from matrix by a single solid-phase extraction step, which increases sensitivity by reduced ion suppression during LC-ESI-MS/MS detection. Additionally, chromatography of all amino acids can be performed on reversed-phase HPLC columns using eluents without additives, which are known to cause significant decreases in signal to noise ratios. The method has been routinely applied for amino acid profiling of low amounts of liquids and tissues of various origins with a sample throughput of about 50–100 samples a day. In addition to a detailed description of the method, some representative examples are presented.
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Acknowledgments
We thank Pascal Rudewig for excellent technical assistance. We are also grateful for the financial assistance provided by HEC-Pakistan and DAAD and for support by core funding to the Leibniz Institute of Plant Biochemistry from the state of Saxony-Anhalt and the Federal Republic of Germany.
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Ziegler, J., Hussain, H., Neubert, R.H.H., Abel, S. (2019). Sensitive and Selective Amino Acid Profiling of Minute Tissue Amounts by HPLC/Electrospray Negative Tandem Mass Spectrometry Using 9-Fluorenylmethoxycarbonyl (Fmoc-Cl) Derivatization. In: Alterman, M. (eds) Amino Acid Analysis. Methods in Molecular Biology, vol 2030. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9639-1_27
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DOI: https://doi.org/10.1007/978-1-4939-9639-1_27
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