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

Abstract

Amino acids are the building blocks of proteins and the analysis of their (13)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(13)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 3-, 5-25 mM). The total run time was 70 min. The average δ(13)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 (13)C amino acid analysis in ecology, archaeology, forensic science, nutrition and protein metabolism.

Online wet oxidation/isotope ratio mass spectrometry method for determination of stable carbon isotope ratios of water‐soluble organic carbon in particulate matter

“…The Bland–Altman plot showed a bias of −0.61‰ for five reagents (Figure B). This bias was slightly larger than in previous studies . The difference was largest for succinic acid (−1.51‰).…”

“…All the organic compounds are oxidized to CO 2 by thermally activated peroxodisulfate, as shown in Equation for ethanol: The generated CO 2 is separated from the liquid by means of a membrane and is transported by a carrier gas to the IRMS inlet. Recently, this LC/IRMS method has been used to determine δ 13 C values for various compounds, including sugars, amino acids, alcohols, pesticides, and black carbon . However, LC/IRMS has not been widely used to determine δ 13 C values for environmental field samples.…”

Position‐specific 13C/12C analysis of amino acid carboxyl groups – automated flow‐injection analysis based on reaction with ninhydrin

“…A number of previous studies have described the stable carbon isotopic analysis of individual amino acids, typically derived by hydrolysis of proteins, from various biological sources . This compound‐specific isotope analysis (CSIA) is achieved either by gas chromatography of derivatised amino acids coupled with isotope ratio mass spectrometry (IRMS) or by high‐pressure liquid chromatography (HPLC) of underivatised amino acids coupled to IRMS using chemical oxidation to convert the carbon present in organic compounds into CO 2 (LC/CO/IRMS) .…”

Isotope Ratio Mass Spectrometry Technique to Follow Plant Metabolism

“…This allows the direct analysis of d 13 C in sugars and other soluble organics. Abaye, Morrison, & Preston, 2011;Dunn, Honch, & Evershed, 2011;Hettmann & Gleixner, 2012;Krummen et al, 2004), but the comparison of GC-C-IRMS and LC-C-IRMS results on amino acids showed a relatively good agreement of d 13 C values (Dunn et al, 2011), indicating that LC-C-IRMS is a very promising technique. However, three major difficulties remain.…”