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.     

Analytical approaches to determination of total choline in foods and dietary supplements

Choline is a quaternary amine that is synthesized in the body or consumed through the diet. Choline is critical for cell membrane structure and function and in synthesis of the neurotransmitter acetylcholine. Although the human body produces this micronutrient, dietary supplementation of choline is necessary for good health. The major challenge in the analysis of choline in foods and dietary supplements is in the extraction and/or hydrolysis approach. In many products, choline is present as choline esters, which can be quantitated individually or treated with acid, base, or enzymes in order to release choline ions for analysis. A critical review of approaches based on extraction and quantitation of each choline ester as well as hydrolysis-based methods for determination of total choline in foods and dietary supplements is presented.     

CE methods for the determination of non-protein amino acids in foods

In addition to the 20 amino acids universally distributed as protein constituents in living organisms, there are other amino acids of non-protein origin that can be found in foods. The determination of these non-protein amino acids is interesting since they can be indicative of the quality and safety of foods. This work presents for the first time an updated and comprehensive review devoted to show the possibilities of capillary electrophoresis for the determination of non-protein amino acids in food samples. The results reported have been classified according to the chemical structure of the non-protein amino acid studied. Separation conditions as well as detection systems used have been detailed since most of these amino acidic compounds do not possess chromophore groups detectable by conventional UV-Vis detection, being in this case necessary a previous derivatization step. Finally, the application of microchip electrophoresis to the determination of non-protein amino acids in foodstuffs is also included in this review.     

Lipophilicity determination of some fully protected peptides and amino acids

Summary The lipophilicity of 21 fully protected peptides and amino acids was determined by reversed-phase thin-layer chromatography. The R_M values decreased linearly with growing methanol concentration of the eluent. The sequence and conformation of molecular substructures did not significantly influence the lipophilicity. The presence of salt and ammonia in the eluent had a negligible impact on retention; the effect of the pH value was also low. In the presence of 1M and 2M acetic acid the retention of each compound considerably decreased. Acetic acid also changed markedly the selectivity. Our data suggest that the acetic acid has a preponderant role in changing the retention and selectivity of fully protected peptides and amino acids in reversedphase thin-layer chromatography.     

Status of methodology for the determination of fat-soluble vitamins in foods, dietary supplements, and vitamin premixes

Fat-soluble vitamins (FSVs) include vitamin A, carotenoids, vitamins D, E, and K. New legislation is being introduced in many countries to reinforce regulatory compliance of declared concentrations of vitamins and other micronutrients in food products and dietary supplements. The levels of FSVs are likely to be more closely scrutinized due to their potential health risks associated with overdosing, in particular of vitamin D. However, a proviso of stricter regulatory compliance is that analytical methods must be fit-for-purpose, providing adequate accuracy and precision. Official methods have been published by organizations such as AOAC INTERNATIONAL, European Committee for Standardization, International Dairy Federation, U.S. Pharmacopeia, and International Organization for Standardization. The methods available for foods, dietary supplements, and vitamin premixes are evaluated in this review. In general, these methods show adequate precision for regulatory compliance; however, the field of application has not often been evaluated for a sufficiently large range of food matrixes. Gaps have been noted in the range of published official procedures, particularly for carotenoids and vitamin premixes. The potential of some recent developments in sample preparation and chromatographic techniques were evaluated to provide improved procedures for FSV analysis the future.     

Quantitative NMR spectroscopy for accurate purity determination of amino acids,and uncertainty evaluation for different signals

Purity evaluation of amino acids using nuclear magnetic resonance spectroscopy is reported. Three amino acids (aspartic acid, valine, and arginine) and certified reference materials (CRMs), such as acidic, neutral, and basic amino acids, as well as a low pure sample of valine were used as the analytes. DCl solution, D₂O, and NaOD solution were used as the preparation solvents. The quantitative values were obtained from all observed signals and compared with the certified values of the CRMs. When an amino acid was dissolved in water, a strong HOD signal due to proton exchange was observed. When the signal adjoining the HOD signal was considered in the evaluation, the accurate quantitative value could not be obtained. Therefore, under optimized conditions, the analyte signals separated from the HOD signal were chosen for purity determination of amino acids. As a result, the quantitative values were in agreement with the certified values of CRMs. An expanded uncertainty was estimated to be approximately 0.002 kg kg^?1. We also discuss the effect of impurities on purity determination based on all signals and conclude that agreement of quantitative values determined from different signals in a molecule is a good indication of the accuracy of the results.     

Rapid evaluation of molecular electrostatic potential maps for amino acids,peptides, and proteins by empirical functions

A simple method for evaluating the molecular electrostatic potential (MEP) map without self-consistent field molecular orbital (SCF-MO) calculation is extended, and the parameters for amino acids, peptides, and proteins are determined. In this method, the electrostatic potentials due to electrons in the valence shells are calculated by a set of simple empirical functions at various origins, and those due to the core electrons and nuclei by point charge approximation. For application of the method to amino acids, peptides, and proteins, the functions for the σ and π bonds and lone-pair electrons involved in these species were determined, and the MEP maps calculated by the empirical functions were compared with those calculated by an ab initio method. It is shown that the method reproduces correctly the shape of ab initio MEP map even for the repulsive MEP region. The method is shown to be very useful for rapid evaluation of reliable MEPs for large biological molecules. © 1996 by John Wiley & Sons, Inc.     

非蛋白氨基酸对生物活性多肽的修饰及其构效关系的研究进展

简述了非蛋白氨基酸的结构特征和生理活性。介绍了某些天然存在的含非蛋白氨基酸的生物活性肽及非蛋白氨基酸残基在相应活性肽中的重要作用。着重总结了非蛋白氨基酸对生物活性肽进行修饰的方式和构效关系研究的进展。     

Molecular recognition properties of acyclic cucurbiturils toward amino acids,peptides, and a protein

The binding of 1 and 2 toward 19 amino acid amides by ¹H NMR and ITC is reported. Hosts 1 and 2 bind to aromatic or hydrophobic residues by cavity inclusion leaving the cationic residues at the C=O portals. K_a values range from 10² to >10⁶ M^?1 with H-Phe-NH₂, H-Trp-NH₂, and H-Tyr-NH₂ displaying sub-micromolar K_d values. Hosts 1 and 2 bind tightly to dicationic H-Lys-NH₂ and H-Arg-NH₂ which are poor guests for CB[7]. Comparison of the affinity of 1 and 2 toward the amino acid amide, N-acetyl-amino-acid amide, and amino acid forms of Phe revealed that the removal of the NH₃⁺ to O=C and SO₃^? electrostatic interactions costs 3.8 kcal/mol whereas the introduction of an unfavourable CO₂^? to O=C and SO₃^? electrostatic interactions costs 2.1 kcal/mol. Hosts 1 and 2 bind to insulin with low micromolar affinity. Acyclic CB[n] display high affinity toward a wider range of N-terminal amino acids residues than CB[n] which suggests a broad range of applications.     

Preparation of trimethylsilyl derivatives of amino acids and peptides for peptide synthesis

Russian Chemical Bulletin -     

Simultaneous determination of sibutramine and N-Di-desmethylsibutramine in dietary supplements for weight control by HPLC-ESI-MS

A high-performance liquid chromatographic method, coupled with UV detection and electrospray ionization mass spectrometry (HPLC-UV-ESI-MS), is developed for the simultaneous determination of the illegal additives sibutramine and its metabolite N-di-desmethylsibutramine in dietary supplements for weight control. The separation is achieved on a Spherisorb C8 reversed-phase column, employing acetonitrile and an aqueous 0.2% formic acid solution containing 20mM ammonium acetate as mobile phases in a gradient mode. UV detection is used for quantitation at a wavelength of 223 nm. Identification of target compounds is completed by ESI-MS using selected ion recording at m/z 280 for sibutramine and m/z 252 for N-di-desmethylsibutramine. Calibration curves are linear over the range of 0.025-1.0 mg/mL for sibutramine and N-di-desmethylsibutramine. Correlation coefficients are better than 0.9990. The intra- and inter-day precision and accuracy for sibutramine and N-di-desmethylsibutramine are acceptable. The method is successfully applied to the analysis of natural dietary supplement samples.     

Primary constituents of blue cohosh: quantification in dietary supplements and potential for toxicity

Dietary supplements containing dried roots or extracts of the roots and/or rhizomes of blue cohosh (Caulophyllum thalictroides) are widely available. This botanical has a long history of use by Native Americans and its use continues to the present day. The primary constituents of blue cohosh are its alkaloids and saponins. The structures of the alkaloids magnoflorine, baptifoline, anagyrine, and N-methylcytisine have been known for many years. The last 10 years have seen a great increase in isolation and identification of the large number of saponins present in blue cohosh. Important developments in nuclear magnetic resonance techniques have contributed substantially to the increase in elucidation of the structures of the complex saponins. Several authors have described quantitative methods for both the alkaloids and saponins in blue cohosh. Such methods have made it possible to quantify these constituents in dietary supplements containing this botanical ingredient. Concentrations of both alkaloids and saponins vary substantially in dietary supplements of blue cohosh. The nicotinic alkaloid, N-methylcytisine, a potent toxicant, has been found in all dietary supplements of blue cohosh analyzed. The teratogenic alkaloid anagyrine has been found in some but not all dietary supplements.     

Electrochemical strategies for the label-free detection of amino acids, peptides and proteins

Electrochemical methods for the detection of amino acids, peptides, and proteins in a variety of media are reviewed. Label-free strategies in which the detection is based on the inherent electrochemical properties of the analyte are discussed. Various processes such as direct or mediated (in solution or immobilised) redox processes and interfacial ion transfers have been employed for the electrochemical detection and determination of the target analytes. The various methods covered encompass voltammetry at uncoated and modified electrodes and at immiscible liquid-liquid interfaces, potentiometry at polymer membrane electrodes and electrochemical impedance spectroscopy. The determination of the target analytes in complex biological matrices is discussed. The various approaches highlighted here illustrate the rich capabilities of electrochemical methods as simple, low-cost, sensitive tools for the determination of these important biological analytes at trace and ultra-trace levels.     

Automated determination of neuroactive acidic sulphur-containing amino acids and gamma-glutamyl peptides using liquid chromatography with fluorescence and electrochemical detection

A column liquid chromatographic method is presented for the determination of trace levels of acidic sulphur-containing amino acids and gamma-glutamyl di- and tripeptides in microdialysates sampled from rat brain in vivo. Automated precolumn derivatization was performed with o-phthaldialdehyde-beta-mercaptoethanol. The derivatives were separated by reversed-phase liquid chromatography with electrochemical and fluorescence detection. The mean relative standard deviation (n = 10) was 1.03 and 4.59% for retention times and peak heights, respectively. The mean correlation coefficient of linearity (r) was 0.9982 in the range 4.5-450 pmol (n = 15), and the lowest detectable amount was 200 fmol for the homocysteinesulphinic acid derivative, (k' = 5.4, at a signal-to-noise ratio of 3). A microcolumn electrochemical detection method, developed for volume-limited samples, produced a fifteen-fold increase in mass sensitivity. Neurochemical applications using microdialysis in vivo are presented.     

In vitro determination of the release kinetics of peptides and free amino acids during the digestion of food proteins

The kinetics of peptide release during in vitro digestion of 4 protein sources (casein, cod protein, soy protein, and gluten) were investigated. Samples were sequentially hydrolyzed with pepsin (30 min) and pancreatin (2, 4, or 6 h) in a dialysis cell with continuous removal of digestion products. Nondialyzed digests were fractionated by ion-exchange chromatography and ultrafiltration. Animal proteins were digested at a greater rate than plant proteins. Target amino acids of specific enzymes appeared more rapidly in the dialyzed fractions when compared to other amino acids. Throughout the hydrolysis, nondialyzed digests contained a higher proportion of peptide mixtures with basic-neutral properties. Except for gluten, peptide fractions with molecular weights that exceeded 10 kDa (basic-neutral, BN > 10) were rapidly hydrolyzed during the first 2 h of pancreatin digestion. The kinetics of release and the composition of peptide fractions were different when the protein sources were compared. The analysis of amino acids revealed that threonine and proline proportions were relatively high in BN > 10 and in peptide fractions with molecular weight between 10-1 kDa (BN 10-1), while tyrosine, phenylalanine, lysine, and arginine predominated in the low molecular weight (<1 kDa) fractions. More resistant peptides were generally rich in proline and glutamic acid. The role of in vitro digestion assays in dietary protein quality evaluation is discussed.     

Linked scans of peptides and protein digests: amino acid sequence determination of components of complex mixtures

Two-sector linked-scan analysis of an unpurified proteolytic digest of a pyruvate decarboxylase enzyme (60,000 Da) has allowed the discovery and assignment of an amino-terminal post-translational modification and processing event. A difference in amino acid sequence from that predicted by a recently published nucleotide sequence has also been found. These results illustrate both the use and considerable potential of linked-scan methods for the analysis of complex biopolymer mixtures.