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The irregular behavior of histidine in its reaction with the o-phthaldialdehyde (OPA) reagents has been studied. Histidine provides more than one OPA derivative. Similarly to all those primary amino group-containing compounds that do have in their initial structure the -CH2-NH2 moiety. The ratio of histidine's initially formed and transformed OPA derivatives depends on the temperature: very likely due to the fact that elevated temperature favors the intra-molecular rearrangement of histidine resulting in the formation of the -CH2-NH2 moiety-containing tautomer(s). The higher the temperature the higher the amount of the transformed species. The composition of the initially and transformed OPA derivatives of histidine were identified on the basis of their on-line HPLC-electrospray ionization (ESI) MS spectra and computations. The initially formed species has been identified as the classical isoindole, while the transformed one contains an additional OPA molecule.

The stability and characteristics of the C6C8 n-aliphatic and phenylethylamines have been investigated as their o-phthaldialdehyde (OPA)/3-mercaptopropionic acid, OPA/N-acetyl- l -cysteine, OPA/2-mercaptoethanol and OPA/ethanethiol derivatives. Stoichiometric studies have been followed by photodiode array and fluorescence detection, simultaneously, while the composition of derivatives was confirmed by on line HPLC–electrospray ionization (ESI)-MS measurements. All four amines having in their original structure the NH2–CH2– moiety in accordance with the C1C4 aliphatic, mono and diamines and amino acids of the same structure—furnished more than one OPA derivative: their initially formed isoindoles transform to further ones. Depending on the composition of the OPA reagents and on the pH of derivatizations different type of transformed species have been identified, in various proportions. Applying the OPA/SH additive reagent in the molar ratio of 1/3, favors the formation of one additional OPA molecule-containing isoindole, while using the OPA/SH additive (1/50) reagent resulted in the formation of one additional SH additive-containing species, identified and measured at the first time by HPLC. Transformation rate and stability of derivatives proved to be associated with the composition of the OPA reagent, with the type of the SH additive, with the pH of derivatizations, and, in selected cases also with the chain length of the amine. Results of stoichiometric and mechanism studies have been utilized to define optimum analytical conditions.

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%.

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 reagent's 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.

The behavior of o-phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA) and OPA/N-acetyl-L-cysteine (NAC) derivatives of amino acids (AAs) have been studied from analytical view point, based on their UV and fluorescence responses, simultaneously. The particular aim was to clarify the background of the believed instability of AAs, that give more than one OPA-derivative, such as glycine, γ-amino butyric acid (GABA), β-alanine, histidine, ornithine and lysine. To confirm the stoichiometric correlation and to improve reproducibility of interaction the mode ratios of OPA to the SH-group-containing additive, the concentration of reactants, pH of injected analyte, temperature of interaction and reactivity of substituted AAs toward the OPA/MPA reagent have been investigated. Varying the mole ratios of [OPA] to [MPA] and [OPA] to [NAC], it is shown that the higher the concentration of SH-group-containing additive the lower the transformation rate of the initially formed OPA-derivative to those forthcoming. Performing all reactions with the same mole ratios, at higher concentrations resulted in increased amounts of the succesively-formed OPA-derivatives. Decreasing the pH of OPA-AAs from 9.3 to 7.2 before injecting them onto the column, lead to considerable loss of response. Increasing the temperature of reactions from 4°C to 30°C resulted in increased decomposition rate of derivatives. The stability, and hence reproducibility of reactions can be improved (i) by applying the the same mole ratios of OPA and SH-group-containing additive, (ii) by injecting the analyte of derivatives at the derivatization pH, and, (iii) performing reactions with refrigerated stock solutions and maintained before injection possible at 4°C.

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 reagent's 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.

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.

The separation/identification of 25 amino acids as their o-phthaldialdehyde-3-mercaptopropionic acid (OPA/MPA) and o-phthaldialdehyde-N-acetyl-L-cysteine (OPA/NAC) derivatives have been optimized [paying particular attention to those amino acids which elute with more than one derivative (glycine, histidine, gamma-aminobutyric acid, beta-alanine, ornithine, lysine) and that are expected to be present in apples in their free form]. Optimum separation conditions are reported on six reversed-phase columns: Nucleosil 3 and 5 microm, 150(+20 guard)x4.0 mm; Gromsil 3 microm, 150(+10 guard)x4.0 mm; Hypersil 5 microm, 150(+20 guard)x4.0 mm and 200(+20 guard)x4.0 mm; and Hypersil 3 microm, 150(+20 guard)x4.0 mm. Elutions were followed, simultaneously, with photodiode array and fluorescence detectors connected in line. Optimization studies carried out in model solutions as a function of temperature (30-55 degrees C) and eluent flow-rate (0.8-2.5 mL/min) demonstrated that optimum resolutions are obtained with the highest flow-rate applicable (remaining on the safe side with a column pressure of << 3500 p.s.i.; 1 p.s.i.=6894.76 Pa) in the temperature range 30-50 degrees C. Twenty-five amino acids, eluting in 31 separate, characteristic derivatives, were determined on all six columns (the main component, asparagine, present in overwhelming excess, together with the minor constituents glutamine, beta-alanine, gamma-aminobutyric acid, homoserine, and homoarginine). Optimum conditions in the case of both derivatives were obtained on the same type of column (Hypersil, 5 microm), as follows: for the OPA/MPA amino acids with programmed flow-rate [1.3-2.3 ml/min; column, 200(+20 guard)x4 mm], at 50 degrees C, while, for the OPA/NAC amino acids at 2.1 ml/min flow rate, at 30 degrees C [column, 150(+20 guard)x4 mm], with 40 and 37 min run times, including equilibration. Responses of the corresponding amino acids proved to be independent of the column used; reproducibility in the concentration range 6-12,000 pmol, related to the injected amount of amino acids, was