Strong anion exchange liquid chromatographic separation of protein amino acids for natural 13C-abundance determination by isotope ratio mass spectrometry

Abaye, D., Morrison, D. and Preston, T. (2011) Strong anion exchange liquid chromatographic separation of protein amino acids for natural 13C-abundance determination by isotope ratio mass spectrometry. Rapid Communications in Mass Spectrometry, 25(3), pp. 429-435. (doi:10.1002/rcm.4844)

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Abstract

Amino acids are the building blocks of proteins and the analysis of their <sup>13</sup>C abundances is greatly simplified by the use of liquid chromatography (LC) systems coupled with isotope ratio mass spectrometry (IRMS) compared with gas chromatography (GC)-based methods. To date, various cation exchange chromatography columns have been employed for amino acid separation. Here, we report strong anion exchange chromatography (SAX) coupled to IRMS with a Liquiface interface for amino acid δ<sup>13</sup>C determination. Mixtures of underivatised amino acids (0.1–0.5 mM) and hydrolysates of representative proteins (prawns and bovine serum albumin) were resolved by LC/IRMS using a SAX column and inorganic eluents. Background inorganic carbon content was minimised through careful preparation of alkaline reagents and use of a pre-injector on-line carbonate removal device. SAX chromatography completely resolved 11 of the 16 expected protein amino acids following acid hydrolysis in underivatised form. Basic and neutral amino acids were resolved with 35 mM NaOH in isocratic mode. Elution of the aromatic and acidic amino acids required a higher hydroxide concentration (180 mM) and a counterion (NO<sub><b style="text-decoration:overline">3</b></sub>., 5–25 mM). The total run time was 70 min. The average δ<sup>13</sup>C precision of baseline-resolved peaks was 0.75‰ (range 0.04 to 1.06‰). SAX is a viable alternative to cation chromatography, especially where analysis of basic amino acids is important. The technology shows promise for 13C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Preston, Professor Thomas and Morrison, Dr Douglas and Abaye, Dr Daniel
Authors: Abaye, D., Morrison, D., and Preston, T.
Subjects:G Geography. Anthropology. Recreation > GE Environmental Sciences
College/School:College of Science and Engineering > Scottish Universities Environmental Research Centre
Journal Name:Rapid Communications in Mass Spectrometry
Publisher:John Wiley & Sons Ltd.
ISSN:0951-4198
ISSN (Online):1097-0231
Published Online:07 January 2011

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