Chromatographic determination of amino acids in foods

Amino acids in foods exist in a free form or bound in peptides, proteins, or nonpeptide bonded polymers. Naturally occurring L-amino acids are required for protein synthesis and are precursors for essential molecules, such as co-enzymes and nucleic acids. Nonprotein amino acids may also occur in animal tissues as metabolic intermediates or have other important functions. The development of bacterially derived food proteins, genetically modified foods, and new methods of food processing; the production of amino acids for food fortification; and the introduction of new plant food sources have meant that protein amino acids and amino acid enantiomers in foods can have both nutritional and safety implications for humans. There is, therefore, a need for the rapid and accurate determination of amino acids in foods. Determination of the total amino acid content of foods requires protein hydrolysis by various means that must take into account variations in stability of individual amino acids and resistance of different peptide bonds to the hydrolysis procedures. Modern methods for separation and quantitation of free amino acids either before or after protein hydrolysis include ion exchange chromatography, high performance liquid chromatography (LC), gas chromatography, and capillary electrophoresis. Chemical derivatization of amino acids may be required to change them into forms amenable to separation by the various chromatographic methods or to create derivatives with properties, such as fluorescence, that improve their detection. Official methods for hydrolysis and analysis of amino acids in foods for nutritional purposes have been established. LC is currently the most widely used analytical technique, although there is a need for collaborative testing of methods available. Newer developments in chromatographic methodology and detector technology have reduced sample and reagent requirements and improved identification, resolution, and sensitivity of amino acid analyses of food samples.     

Quantitative determination of amino acids in functional foods by microchip electrophoresis

Microchip electrophoresis (MCE), a first-generation micrototal analysis system, has emerged during the miniaturization phase of food analysis. Based on the micellar electrokinetic chromatography mode, a simple and fast MCE method with light emitting diode-induced fluorescence detection was developed for quantitative analysis of amino acids in three different kinds of functional foods, viz. sports beverages, jelly-form beverages, and tablet-form functional foods. In contrast to the glass microchip, we improved the separation of amino acids on a poly(methyl methacrylate) (PMMA) chip by addition of cationic starch derivatives. 4-fluoro-7-nitro-2,1,3-benzoxadiazole, which has a short labeling time for amino acids, was used as the fluorescently labeled dye. This MCE method takes less than 10 min of total analysis time including sample preparation and analysis of amino acids in functional foods on a PMMA chip. The results show that this approach has the potential to be a fast and simple method for amino acid analysis in functional foods.     

Quantitative gas-liquid chromatography of non-protein amino acids.

The quantitative gas--liquid chromatographic analysis of non-protein amino acids, in the presence of protein amino acids, is described. The amino acids were determined as their N-trifluoroacetyl n-butyl esters on an ethylene glycol adipate column. The relative molar responses of 38 amino acids are reported.     

CE methods applied to the analysis of micronutrients in foods

This article reviews the applications of CE that are relevant to the analysis of small molecules in foods. CE has been applied to a wide range of important areas of food analysis and is rapidly being established as an alternative technique to chromatographic methods including HPLC and GC within analytical food and research laboratories. In recent years the analysis of food by CE has become more frequent and important and as such a variety of compounds have been separated and quantified. Although many other analytes have been detected by CE, this review will highlight areas relating primarily to the rather broad chemical classes of free amino acids, carbohydrates, organic acids, vitamins and a variety of antioxidants. In addition, information relating to the analyte, sample matrix, mode of CE employed, scope of the methodology and the detection and derivatization of the small molecules are considered and discussed.     

A dual spectrophotometric/contactless conductivity detector for CE determination of incompletely separated amino acids

A new application is described of a dual photometric/contactless conductivity detector to CE determination of incompletely separated compounds. These compounds are differentiated when one of them provides signals in both the cells of the detector, whereas the other yields a signal in only one cell. The technique has been applied to determination of proline and tyrosine in a dietary supplement.     

CE with sequential light-emitting diode-induced fluorescence and electro-chemiluminescence detections for the determination of amino acids and alkaloids

This paper describes the determination of alkaloids and amino acids (AAs) using CE in conjunction with sequential light-emitting diode-induced fluorescence (LEDIF) and electrochemiluminescence (ECL) detections. In the CE-LEDIF-ECL system, the ECL detector was located in the outlet of the capillary, while the LEDIF detector was positioned 12 cm from the outlet. Naphthalene-2,3-dicarboxaldehyde (NDA) was used to form fluorescent AA-NDA derivatives from AAs possessing primary amino groups, while Ru(bpy)(3) (2+) was used to obtain ECL signals for analytes having secondary and tertiary amino groups. In the presence of poly(ethylene oxide), we accomplished the CE-LEDIF-ECL separation of a mixture of 12 AA-NDA derivatives, anabasine, nicotine, and proline within 11 min. This low-cost CE-LEDIF-ECL system allows the analysis of these AA-NDA derivatives and alkaloids at concentrations in the ranges of 49 nM-0.2 microM and 0.66-4.7 microM, respectively. We applied our CE-LEDIF-ECL system to the analysis of a urine sample and also to tobacco extracts. We obtained good qualitative and quantitative results when using this method with these analytes: the RSDs were below 3.0 and 2.8%, respectively. This CE-LEDIF-ECL system provides the advantages of high efficiency, speed, and sensitivity for the analysis of analytes possessing amino groups.     

Overview of chromatographic and electrophoretic methods for the determination of branched-chain amino acids

The significance of branched-chain amino acids in diseases was clearly shown over the years. This review aims to describe the available techniques for their analytical determination. The article provides examples of the use of various analytical methods. The methods are divided into two categories: derivatization and non-derivatization approaches. Separation is achieved through different chromatography or capillary electrophoresis techniques and can be combined with different detectors such as flame ionization, ultraviolet, fluorescence, and mass spectrometry. It compares the application of various derivatization reagents or detection as such for different detectors.     

Miniaturized liquid core waveguide-based fluorimetric detection cell for capillary separation methods: application in CE of amino acids

A miniaturized post-column fluorimetric detection cell for capillary separation methods based on optical fibers and liquid core waveguides (LCWs) is described. The main part of the detection cell is a fused-silica capillary coated with Teflon AF serving as an LCW. The optical fibers are used both for coupling the excitation source with the detection domain in the LCW and for the axial fluorescence collection from the LCW end. The latter fiber is connected with a compact CCD spectrometer that serves for the rejection of the scattered excitation light and for the fluorescence signal detection. The proposed design offers a compact fluorescence detector for various microcolumn separation techniques without optical elements such as filters or objectives. Moreover, its construction and optical adjustment are very simple and the whole system is highly miniaturized. The function of the detection cell is demonstrated by CE of amino acids labelled by fluorescein-based tags. Separations of different standard amino acid mixtures and plasma samples are presented. The comparison of plasma amino acid levels of individuals being in good health with those of patients with inherited metabolic disorders is also shown.     

Chromatographic methods for the determination of pesticides in foods.

Chromatography is the most important technique available to the analyst dealing with the determination of pesticide residues in food, feed and environmental samples. Numerous methods for pesticide residues in foods have been developed in the past few years, and this paper reviews some of the most important procedures. A great variety of chromatographic methods, such as solid-phase extractions, column chromatographic clean-up methods, thin-layer, gas, high-performance liquid and supercritical fluid chromatography, and their coupling with sensitive and selective detection methods are surveyed.     

Resolution of non-protein amino acids via Carica papaya lipase-catalyzed enantioselective transesterification

Carica papaya lipase-catalyzed transesterification of the 2,2,2-trifluoroethyl esters of N-benzyloxycarbonylated dl-amino acids carrying aliphatic side chains proceeded smoothly and, in almost all the cases, enantiospecifically (E = >200), affording the l-methyl esters and leaving the d-trifluoroethyl esters intact.     

Etched bare fused-silica capillaries for online preconcentration of amino acids in CE

An online preconcentration method based on electrostatic interaction between the analytes and inner surface of the capillary column was developed for the determination of zwitterionic analytes such as amino acids in CE coupled with a DAD. The amino acids possessed positive charges when they were dissolved in an acidic solvent. When they were injected into the column, they were attracted by the negatively charged inner surface of the fused-silica capillary column. An etched column was used to increase the area of the capillary's inner surface and, consequently increase the electrostatic interaction between the amino acids and the inner surface of the capillary column. It was found that when the sample was injected at 10 psi for 1 min and the pH value of the sample was 4, the amount of amino acids attracted to the inner surface of the capillary was maximum. Under these optimized experimental conditions, the detection sensitivity of CE-DAD was enhanced by 5200, 2800, and 3100 times for asparagine, tryptophan and phenylalanine, respectively, compared with normal CE separation. The method provided good reproducibility in terms of both migration time and peak height. It can be successfully used for the preconcentration zwitterion.     

A capillary electrophoresis-tandem mass spectrometry methodology for the determination of non-protein amino acids in vegetable oils as novel markers for the detection of adulterations in olive oils

A new analytical methodology based on capillary electrophoresis-mass spectrometry (CE-MS(2)) is presented in this work, enabling the identification and determination of six non-protein amino acids (ornithine, β-alanine, GABA, alloisoleucine, citrulline and pyroglutamic acid) in vegetable oils. This methodology is based on a previous derivatization with butanol and subsequent separation using acidic conditions followed by on-line coupling to an ion trap analyzer for MS(2) detection established through an electrospray-coaxial sheath flow interface. The electrophoretic and interface parameters were optimized obtaining the separation of all compounds in less than 15 min and with resolutions higher than 5. The proposed method was validated by assessing its accuracy, precision (RSD<7% for corrected peak areas), LODs and LOQs (between 0.04-0.19 ng/g and 0.06-0.31 ng/g, respectively) and linearity range (R(2)>0.99), and it was used in order to identify the selected non-protein amino acids in soybean oils, sunflower oils, corn oils and extra virgin olive oils. MS(2) experiments performed the fingerprint fragmentation of these compounds allowing to corroborate ornithine and alloisoleucine in seed oils but not in olive oils. The method was applied to identify and quantify olive oil adulterations with soybean oil detecting in a single run the amino acids in mixtures up to 2% (w/w). The results showed a high potential in using these compounds as novel markers for the detection of adulterations of extra virgin olive oils with seed oils. Thus, the developed method could be considered a simple, rapid and reliable method for the quality evaluation of extra virgin olive oil permitting its authentication.     

Immunochemical analytical methods for the determination of peanut proteins in foods

Peanut proteins can cause allergenic reactions that can result in respiratory and circulatory effects in the body sometimes leading to shock and death. The determination of peanut proteins in foods by analytical methods can reduce the risk of serious reactions in the highly sensitized individual by allowing for the detection of these proteins in a food at various stages of the manufacturing process. The method performance of 4 commercially available enzyme-linked immunosorbent assay (ELISA) kits was evaluated for the detection of peanut proteins in milk chocolate, ice cream, cookies, and breakfast cereals: ELISA-TEK Peanut Protein Assay, now known as "Bio-Kit" for peanut proteins, from ELISA Technologies Inc.; Veratox for Peanut Allergens from Neogen Corp.; RIDASCREEN Peanut Kit from R-Biopharm GmbH; and ProLisa from Canadian Food Technology Ltd. The 4 test kits were evaluated for accuracy (recovery) and precision using known concentrations of peanut or peanut proteins in the 4 food matrixes. Two different techniques, incurred and spiked, were used to prepare samples with 4 known concentrations of peanut protein. Defatted peanut flour was added in the incurred samples, and water-soluble peanut proteins were added in the spiked samples. The incurred levels were 0.0, 10, 20, and 100 microg whole peanut per g food; the spiked levels were 0.0, 5, 10, and 20 microg peanut protein per g food. Performance varied by test kit, protein concentration, and food matrix. The Veratox kit had the best accuracy or lowest percent difference between measured and incurred levels of 15.7% when averaged across all incurred levels and food matrixes. Recoveries associated with the Veratox kit varied from 93 to 115% for all food matrixes except cookies. Recoveries for all kits were about 50% for cookies. The analytical precision, as measured by the variance, increased with an increase in protein concentration. However, the coefficient of variation (CV) was stable across the 4 incurred protein levels and was 7.0% when averaged across the 4 food matrixes and analytical kits. The R-Biopharm test kit had the best precision or a CV of 4.2% when averaged across all incurred levels and food matrixes. Because measured protein values varied by test kit and food matrix, a method was developed to normalize or transform measured protein concentrations to an adjusted protein value that was equal to the known protein concentration. The normalization method adjusts measured protein values to equal the true protein value regardless of the type test kit or type food matrix.     

Chiral capillary electrophoresis-mass spectrometry of amino acids in foods

In this work, the development of a new chiral capillary electrophoresis-mass spectrometry (CE-MS) method to separate D- and L-amino acids is shown. On-line coupling between CE and MS is established through an electrospray-coaxial sheath flow interface. Enantiomer separation is achieved by using a cheap, nonvolatile, chiral selector as beta-cyclodextrin in the background electrolyte (BGE) together with a physically coated capillary that is aimed to prevent contamination of the electrospray. The capillary coating is simple and easy to obtain as it only requires flushing of the capillary with a polymer aqueous solution for 3 min. Optimization of CE parameters (pH of BGE, type and concentration of chiral selector, and capillary inner diameter) and electrospray-MS parameters (nature and flow rate of the sheath liquid, nebulizer pressure) is carried out. Two different derivatization protocols of amino acids using dansyl chloride (DNS) and fluorescein isothiocyanate (FITC) are compared in terms of MS sensitivity and chiral resolution. Under optimum CE-MS conditions it is observed that the MS sensitivity obtained for FITC- and DNS-amino acids is similar (with limit of detection (LOD) in the microM range, corresponding to amounts injected in the fmol range) while chiral resolution is better for FITC-amino acids. The optimized method is demonstrated to provide the simultaneous analysis of 15 selected amino acids (i.e., FITC-D/L-Asp, -Glu, -Ser, -Asn, -Ala, -Pro, -Arg, and FITC-gamma-aminobutyric acid (GABA) in a single chiral CE-MS run, corresponding to the main amino acids that can be found in orange. Moreover, as a result of the high resolution achieved, it is possible to detect down to 2% of D-Asp in the presence of 98% of L-Asp. The good possibilities of chiral CE-MS in food analysis are corroborated through the detection of the main amino acids in a commercial orange juice (i.e., FITC-L-Asp, -Glu, -Ser, -Asn, -Pro, -Arg, and the nonchiral FITC-GABA) as well as the determination of the fraudulent addition of synthetic amino acids (containing D- and L-forms) to a fresh orange juice.     

Determination of organic acids by CE and CEC methods

A comprehensive overview of the analysis of low-molecular-mass organic acids employing electromigration methods in the capillary format is given. This review includes papers published since 2003 and can be seen as an update of the review paper published by Galli et al. in 2003. Tables included in this review contain application papers describing the determination of organic acids from a variety of fields like the analysis of food and beverages, environmental samples, samples from clinical origin, and from natural products.     

Rapid determination of amino acids in foods by hydrophilic interaction liquid chromatography coupled to high-resolution mass spectrometry

This study describes a rapid and sensitive analytical method for the determination of amino acids in foods and drinks. The method entailed dilution or extraction of amino acids from foods using the mixture of acetonitrile and 0.1% aqueous formic acid (50:50, v/v). Chromatographic separation of underivatized amino acids was performed using a hydrophilic interaction liquid chromatography within a runtime of 6 min. Both hydrophobicity and charge of the side chain played important roles on the elution order of amino acids under the chromatographic conditions. High-resolution mass spectrometry allowed qualitative and quantitative detection of amino acids in complex food matrices. Its response was found linear over a concentration range of 0.25-10 μg/ml. The method could be successfully applied to various foods and drinks to profile individual amino acids. Mean percentage recoveries of amino acids from different matrices were 88.5% or higher with residual standard deviation of less than 5.0%.     

CE determination of small ions: methods and techniques

This paper provides an overview on the current status of capillary electrophoresis (CE) in the analysis of inorganic and charged small organic species. The various CE strategies used to improve the separation of ionic analytes are summarized. Technical developments in the design of improved detection systems are described. A brief account of their advantages and limitations is given. The potential use of these devices for miniaturized CE systems is also described. Finally, special attention is focused on the on-capillary preconcentration techniques developed in attempts to overcome the poor detectability of CE. Recent review articles are frequently cited to provide readers with a source of information about pioneering work, theoretical treatments, and specific applications.     

Profiling methods for the determination of phenolic compounds in foods and dietary supplements

Profiling methods are needed that separate and detect all the phenolic compounds in a single extract of a food material. These methods must be comprehensive, rapid, and rich in spectral information. Fourteen methods that meet, or have the potential to meet, these criteria have been selected from the recent literature for review. In general, the methods employ a single aqueous methanol extraction, separation on a reversed-phase C column, and detection by UV/vis spectroscopy and mass spectrometry. The variations in extraction, separation, and detection are discussed. An increasingly important aspect of these methods is the archiving of data to permit cross-comparison of samples and standards and retrospective analysis. This review shows that the necessary technology is available to achieve the desired analytical goals.