I. Molnár-Perl

Derivatization and chromatographic behavior of the o-phthaldialdehyde amino acid derivatives obtained with various SH-group-containing additives.

An overview is presented of HPLC methods currently in use to determine amino acids as their o-phthaldialdyde derivatives in the presence of various SH-group-containing additives. Crucial points that proved to influence the stability of the amino acid OPA derivatives have been discussed in detail: (i) the mol ratios of the OPA-SH-group-containing additive amino acid; (ii) the preparation and storage conditions of the OPA reagents; (iii) the optimum pH conditions for the interactions and elutions; (iv) the behavior of the, believed to be, less stable amino acids, such as glycine, beta-alanine, gamma-aminobutyric acid, histidine, ornithine and lysine.

New aspects of the simultaneous analysis of amino acids and amines as their o-phthaldialdehyde derivatives by high-performance liquid chromatography. Analysis of wine, beer and vinegar.

A new high-performance liquid chromatography method is described for the simultaneous quantitation of amino acids and amines for 37 compounds (20 amino acids + 17 amines), as their o-phthaldialdehyde (OPA)-3-mercaptopropionic acid derivatives, within 53 min. Based on previously documented stoichiometric and reaction mechanism studies, derivatizations have been carried out with the OPA-SH-group = 1:50 containing reagents. Reliability and reproducibility of analyses have been considerably improved. Average reproducibility data in a wide concentration range of derivatives had RSD < or = 3.4%.

Role of chromatography in the analysis of sugars, carboxylic acids and amino acids in food.

An overview is presented of chromatographic methods currently in use to determine sugars, carboxylic acids and amino acids in foods: high-performance liquid chromatography, gas chromatography and capillary electrophoresis. As a basis of selection the following approaches can be distinguished: quantitation of constituents of several food matrices, without derivatization and in the form of different derivatives, in the presence of the matrix, or subsequently to various work-up procedures.

Simultaneous determination of sugars, sugar alcohols, acids and amino acids in apricots by gas chromatography–mass spectrometry

Abstract Our GC–MS method for the simultaneous quantitation of mono- di- and trisaccharides, sugar alcohols and acids, measured as their trimethylsilyl-oxime ether/ester derivatives, prepared in presence of the fruit matrix, from one solution by one injection has been extended. The reproducible determination of apricot constituents was performed both on the basis of total ion current (TIC) and selective fragment ion (SFI) values, ensuring identification and quantitation in a wide concentration range (∼1·10 −3 to ≥40%), calculated on dry matter basis of the samples). The GC–MS procedure was utilized to prove the advantage of the direct derivatization process, in the presence of the fruit matrix, and to determine the changes in the sugar/sugar alcohol/carboxylic acid/amino acid composition of two apricot cultivars, as a function of their harvesting dates and storage conditions. Reproducibility of quantitations, characterized by their RSD values, proved to be, 3.6% (TIC) and 4.3% (SFI).

Quantitation of amino acids and amines in the same matrix by high-performance liquid chromatography, either simultaneously or separately

A literature overview is presented of chromatographic methods currently in use to determine amino acids and mines (i) simultaneously, (ii) in the presence of each other by separate methods, or (iii) amines alone subsequent to their isolation from amino acids. Separation, derivatization and chromatographic conditions are summarized. Advantages and drawbacks of all three possibilities are discussed and criticized in detail.

Behavior and characteristics of amine derivatives obtained with o-phthaldialdehyde/3-mercaptopropionic acid and with o-phthaldialdehyde/N-acetyl-L-cysteine reagents

A comprehensive evaluation of papers dealing with the HPLC quantitation of amines as o-phthaldialdehyde (OPA) derivatives has been given and discussed in details. The stability and characteristics of selected representatives of mono [methyl-, ethyl-, n-/isopropyl, n-/isobutyl-, tert.-butyl-, sec.-butyl-, isoamyl amines and ethanolamine), di- and polyamines (ethylenediamine, 1,2-propylenediamine, 1,3-propylenediamine, agmatine, tyramine, putrescine, cadaverine, histamine, spermine, spermidine, and bis(hexamethylene)triamine] have been investigated as their OPA/3-mercaptopropionic acid (MPA) and OPA/N-acetyl-L-cysteine (NAC) derivatives, from an analytical point of view, performing photodiode array and fluorescence detection, simultaneously. All amines having in their original structure the NH2-CH2-R moiety, in accord with the amino acids of the same structure, furnished more than one OPA derivative: their initially formed species transformed to further ones. On the basis of on-line HPLC-MS the transformed derivatives were proved to be the corresponding isoindoles that contain an additional OPA molecule. In order to achieve optimum analytical conditions derivatization reagents have been applied in different composition, in parallel. The OPA and the SH-group additive contents of the reagents have been varied in the mole ratios of OPA/MPA(NAC)=1:3 and OPA/MPA(NAC)=1:50. Data obtained proved that performing derivatizations by means of the OPA/MPA(NAC)=1:50 reagents resulted in two benefits: both the stability of derivatives could have been increased and the number of the transformed derivatives decreased. In case of aliphatic amines and in ethanolamine, the transformation of the initially formed derivative can be either quantitatively avoided as in the case of ethanolamine, or considerably decreased, below 1%, as in the cases of the other aliphatic monoamines investigated. As to the behavior of di- and polyamines the stability of derivatives has been considerably improved, the number of species have been decreased from four to two with the exception of spermidine. Stability values characterized both by the UV and fluorescence responses, as a function of the reaction time (from 90 s up to 6 h) have been given in details.

Advances in the evaluation of the stability and characteristics of the amino acid and amine derivatives obtained with the o-phthaldialdehyde/3-mercaptopropionic acid and o-phthaldialdehyde/N-acetyl-L-cysteine reagents: High-performance liquid chromatography-mass spectrometry study

The composition of the amino acid and amine derivatives obtained with the o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA) and with the OPA/N-acetyl-L-cysteine (NAC) reagents was investigated by on-line HPLC-electrospray ionization MS. The initially formed derivatives proved to be, as expected, the corresponding isoindoles while their transformed species contained one additional OPA molecule. Based on the MS spectra of all transformed OPA derivatives a reaction pathway is suggested. This reaction mechanism was supported both by the molecular ions of the endproducts and by the presence of several selective fragment ions that served as an explanation to the structure of the believed to be less stable OPA derivatives. It has been shown that more than one OPA derivative forms in all those cases when the compound to be derivatized does contain the NH2-CH2-R moiety. Thus, amino acids like e.g. glycine, histidine, beta-alanine, gamma-aminobutyric acid, epsilon-aminocaproic acid, ornithine, and also several aliphatic mono- and diamines provide more than one OPA derivative. Analytical consequences of this experience were utilized by altering the reagents composition. Reagents containing mole ratios of [OPA]/[MPA] or [OPA]/[NAC]=1/50 resulted in two benefits, simultaneously: (i) in a decrease of the transformation rate of the initially formed derivative, and, (ii) in an increase of the overall stability of the total of derivatives.

Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography.

A high-performance liquid chromatographic method (HPLC) with combined diode array and fluorescence detection of amino acids and amines in various cheese samples is described. The proposal is based on acidic deproteinization, derivatization and gradient optimization studies, resulting in the identification and quantification of 21 amino acids and 9 amines from a single solution, by one injection. The optimized, simple protocol consists of deproteinization (1M perchloric acid), centrifugation, filtration and the subsequent derivatization with the o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate (OPA-ET-FMOC) reagent. The method can be characterized with a linearity of wide concentration range (6.25-1000 pM/injection), a good chromatographic reproducibility (average: 2.69% RSD) and an excellent recovery (average: 100.2%; average 3.84% RSD). The developed method was successfully applied in the determination of the amino acid and amine contents of port salut cheese, blue cheese and smoked cheese samples.

Stability and characteristics of the o-phthaldialdehyde/3-mercaptopropionic acid and o-phthaldialdehyde/N-acetyl-l-cysteine reagents and their amino acid derivatives measured by high-performance liquid chromatography

Abstract The quality and quantity of impurities present in o-phthaldialdehyde (OPA)/3-mercaptopropionic aicd (MPA) and OPA/N-acetyl- l -cysteine (NAC) reagents have been measured and characterized performing fluorescence and photodiode array detection, simultaneously. The amounts of impurities determined are considerable. Consequently, they have to be deducted from the coeluting amino acid. Stability studies, carried out with 24 amino acids, including—as believed—the less stable OPA derivatives, such as glycine, γ-aminobutyric acid (GABA), β-alanine, histidine, lysine and ornithine, proved that up to 50 min their decomposition is not significant. After 6 h reaction time, the OPA/MPA amino acids manifest higher stability (≥93%) than the corresponding OPA/NAC ones (≥88%). In order to obtain quantitative interactions: (i) extended reaction time (7–28 min) is needed to achieve 100% yield for alanine, β-alanine, GABA, isoleucine, ornithine and lysine; (ii) those amino acids which furnish more than one derivative (glycine, GABA, β-alanine, histidine, lysine and ornithine) are to be quantitated on the basis of the total of their peaks; (iii) reproducibility investigations revealed that the mol ratios of the OPA reagent/amino acids should be at least 20 times larger than the total of amino acids to be determined. The probable composition of the double derivatives on the basis of their characteristic fluorescence intensities and UV absorbances were discussed: glycine, GABA, β-alanine and histidine might furnish the 1-thiosubstituted-2-alkyl- and the 1,3-dithiosubstituted-2-alkylisoindoles, while lysine and ornithine are eluting as their mono- and dimer isoindoles.

Simultaneous quantitation of mono-, di- and trisaccharides by GC-MS of their TMS ether oxime derivatives: II. In honey

SummaryThe fructose, glucose and the minor oligosaccharide content of nineteen honeys has been determined as their TMS sugar oximes, by GC-MS, from a single solution, with one injection, without any preliminary isolation. The saccharide distribution in honey of various floral origins (8 acacia-, 8 mixed flower-,1 linden-, 1 rape-, 1 sunflower- and 1 pine sample) revealed that acacia honey contains significantly higher fructose, sucrose and minor oligosaccharides than samples from other floral sources. The simultaneous quantitation of major and minor constituents offered the possibility to determine various ratios, (maltose/turanose, sucrose/turanose and maltotriose/raffinose+erlose+melezitose) that give excellent proof of authenticity.