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A novel, structure-related derivatization principle has been developed in order to quantify cathinone-type synthetic drugs (CTSDs), focusing on the most common pentedrone (PENT), including also 4-fluoromethcathinone (4-FMC), methcathinone (MCTN), 4-methylethcathinone (4-MEC), 3,4-dimethylmethcathinone (3,4-DMMC), and 4-ethylmethcathinone (4-EMC). Firstly, oximated and, secondly, trimethylsilylated CTSD derivatives were characterized by mass fragmentation patterns using GC/MS that led to the development of a harmonized, quantitative, two-steps derivatization methodology. The two-step process involved i) oximation with hydroxylamine hydrochloride; and ii) trimethylsilylation with N-methyl-N-(trimethylsilyl)-trifluoroacetamide (MSTFA). Next, the oximated-trimethylsilylated species were characterized by retention and mass fragmentation properties. Due to α-cleavage decomposition at their C1 C2 bonds without exception, CTSDs uniformely exhibited a structure-related fragmentation pattern. The practical utility of this newly recognized mechanism was validated in urine samples employing an extraction-free and time-, work-, cost- and solvent-effective protocol in accordance with Green Chemistry. The centrifuged urines (10–40 μL) were evaporated to dryness, followed by derivatization. The analytical performance of the methodology was characterized by repeatability (RSD%, varying between 1.43% and 5.44%), limit of quantitation (LOQ, 15–24 μg/mL), linearity (R2, 0.9976–0.9998) and recovery (97–99%) values. The new principle was tested on drug users’ urine: one specimen provided 56.8 μg/mL PENT (3.8 RSD%). Simple trimethylsilylation of CTSDs confirmed their special fragmentation patterns, not yet described. The instantenous combination of the primarily formed, characteristic fragments with the mass m/z 44, led to the special equilibrium of products: confirming that direct trimethylsilylation of CTSDs is not suitable for their quantitation.

A literature criticism is given on methods using currently gas chromatography mass spectrometry (GC/MS) to determine cathine (CAT), cathinone (CTN) and norephedrine (NE), jointly khatamines. In this study, khatamines' oximation, trimethylsilylation and mass fragmentation properties-applying N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), its trimethyliodosilane (TMIS) catalyst containing version (MSTFA(TMIS)), N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and hexamethyldisilazane (HMDS)-was highlighted, at first. Derivatization, mass fragmentation and quantitation related, optimized model investigations have been carried out as a function of the reaction times and conditions. Special emphasis was put (i) on the stability of the primarily formed (CAT-2TMS, NE-2TMS, CTN-TMS(TMS-oximes)1,2), then transformed, fully derived (CAT-3TMS, NE-3MTS, CTN-2TMS(TMS-oximes)1,2) species, and, (ii) on the proportionally formed stable products, suitable to selective quantitation of all three natural amines, simultaneously. Results, as novelty to the field confirmed that (i) TMIS catalyzed trimethylsilyation triggers to form fully derivatized species unfortunately, in part only; while, (ii) khatamines' simultaneous quantitation needs to be carried out in a two steps derivatization process consisting of oximation (1st step, hydroxylamine in pyridine) and trimethylsilylation (2nd step, MSTFA), to the CAT-2TMS, NE-2TMS, CTN-TMS(TMS-oximes)1,2. These species were characterized with their retention, mass fragmentation and analytical performance properties, in model solutions and in the presence of plant tissues, as well: R(2), limit of quantitation (LOQ) data, expressed in pg/1μL injection basis, proved to be 62.5pg (CAT), 20pg (NE) and 62.5pg (CTN), respectively. The practical utility of proposal was enormously enhanced by the novel, direct sample preparation method. In this process, the freshly harvested, freeze-dried, then pulverized leaves of Catha edulis FORKS were directly derivatized, in the presence of the matrix. Reproducibility (in average 2.07 RSD% varying between 0.15 and 5.5 RSD%), linearity (0.9990-0.9994) and recovery (95.7-99.1%) values of the new sample preparation protocol was confirmed by the standard addition method for CAT, NE and CTN equally. From plant leaf, 0.061w/w% CAT and 0.014w/w% NE contents were obtained. In this tissue CTN was not found. Very likely attributable to the unfavorable climate for the plant: grown in Hungary of temperate zone and naturalized in the tropical Africa.

Quantification, stability and unique spectroscopic properties of indolinyl-caged glutamates (ICGs), with the o-phthalaldehyde-3-mercaptopropionic acid (OPA–MPA) reagent, were described, at first. As new principle to the field, reactivity and stoichiometry of variously substituted OPA–MPA derivatized ICGs, such as 4-methoxy-7-nitroindolinyl-(MNI-Glu), 4-methoxy-5,7-dinitroindolinyl-(DNI-Glu), 2-dimethylamino-propoxy and dimethylamino-isobutoxy alternatives (2DMA-1PO-DNI-Glu, 1DMA-2P-DNI-Glu and 3DMA-1iBU-DNI-Glu), was demonstrated. Derivatives’ stability was determined using high performance liquid chromatography (HPLC) applying simultaneous photodiode array (DAD) and fluorescence (Fl) detections, while their structural identity was confirmed by HPLC–time of flight mass spectrometry (HPLC–TOF-MS). The SH-additive of the reagents was also varied. ICGs react unequivocally, with one OPA-SH-group molecule, in the molar ratios of ([OPA-SH-additive]/[ICG] = 1/1, resulting in species with the characteristic isoindole spectral property (EEx/EEm = 337/454 nm; λmax = 337 nm). ICGs’ isoindole derivatives, due to their sandwich structure, are manifesting the π–π-stacking phenomenon: they fail to show fluorescence. ICGs’ stability decreased in the order of MNI-Glu, 2DMA-1PO/1DMA-2PO, 3DMA-1iBU and DNI-Glu, correspondingly, resulting in increasing order of free glutamic acid (GA), as their decomposition product. GA and ICGs were determined as their OPA/MPA derivatives while uncaged species (MNI, DNI and its substituted alternatives) in their initial forms. The practical utility of the method was confirmed analyzing ICGs and their decomposition products, simultaneously. Quantifications’ reliability and reproducibility were characterized with the relative standard deviation percentages of responses (RSDs%): for GA 0.41–12 RSD% for ICGs 0.057–7.0 RSD% were obtained. Stability properties of variously substituted, recently introduced ICGs, prepared in laboratories of Institute of Experimental Medicine, were defined.

This paper, as a novelty to this field, presents the deproteinization and derivatization of plasma's free amino acids (PFAAs), simultaneously, in a single step, with the acetonitrile (ACN) containing 9-fluorenylmethyloxycarbonyl chloride (FMOC) reagent. Deproteinization and derivatization, were studied with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection, simultaneously. Model investigations have been carried out as a function of the FMOC concentration, reaction time and reaction conditions: with standard solutions, with human plasma samples in its initial condition and fortified with standard amino acids (excluding tryptophan because it co-elutes with the hydrolyzed FMOC). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 3.0 mM FMOC concentration, at pH 9; derivatization time - 20 min), and characterized with the relative standard deviation percentages of their responses (

A literature overview is given of HPLC methods currently in use to determine amino acids as their 9-fluorenylmethyloxycarbonyl (FMOC) derivatives. On the basis of the detailed literature overview an exhaustive derivatization study was performed with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection simultaneously, in order to clear up the controversial points of FMOC derivatization. Model investigations have been carried out as a function of the reaction time and reaction conditions, such as the molar concentration of the reagent, the molar ratios of the reactants, the pH and the solvent composition of the reaction medium. Special emphasis was put (i) on the evaluation of the blank values of the reagents, as a function of the composition and that of the pH of the reaction medium, (ii) on the unambiguous quantitation of all amino acids, including the less reactive aspartic and glutamic acids, as well as on the formation and transformation of histidine and tyrosine, existing partly, as single (N-FMOC-histidine, N-FMOC-tyrosine), partly as double labeled species (N,NH-FMOC-histidine, N,O-FMOC-tyrosine). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 0.5mM FMOC concentration corresponding to the molar ratios of [FMOC]/[amino acids](T)=5.5/1 (note: the superscript 'T' means the total of amino acids), with acetonitrile containing reagents, at pH 9, derivatization time=20 min), and characterized with the relative standard deviation percentages of their responses (

This paper reports a multiresidue analysis procedure which permits the identification and quantification of sixty-three water-soluble pollutants. Subsequent to their solid-phase extraction (SPE) enrichment, analyses of species have been carried out from one solution, by a single injection, as their trimethylsilyl-oxime ether/ester derivatives, by gas chromatography-mass spectrometry, within 31min. Based on our optimized extraction, derivatization and mass fragmentation studies separation have been performed in the total ion current mode, identification and quantification of compounds have been carried out on the basis of their selective fragment ions. Including various pharmaceuticals, benzoic acid, its substituted species, different aromatic carboxylic acids, cholic acids, unsaturated and saturated fatty acids, aliphatic dicarboxylic acids, as well as synthetic pollutants of various origins (2,4-di-tert-butylphenol, different phthalates). Standard compounds were added to 500 mL effluent wastewater samples, at three concentrations (1-5 microg/L, 5-10 microg/L and 10-20 microg/L). Recoveries, using the Waters Oasis cartridges performing extractions at pH 2, pH 4 and pH 7 proved to be the optimum at pH 4 (average recoveries (94.5%), except for cholesterol (10%), paracetamol (18%) and 2,5-dihydroxybenzoic acid (25%). Carbamazepine could be recovered at pH 7, only. Responses, obtained with derivatized standards proved to be linear in the range of 4-80 microg/L levels. Limit of quantitation values varied between 0.92 ng/L (4-hydroxyphenylacetic acid) and 600 ng/L (dehydrocholic acid) concentrations. One of the most important messages of this work is the confirmation of the origin of blank values. It was shown that contaminants, mainly 2,4-di-tert-butylphenol, different phthalates and fatty acids, are sourced both from the reagents and mainly from the SPE procedure, independent on the cartridge applied. Reproducibilities, characterized with the relative standard deviations (RSDs) of measurements, varied between 0.71% and 10%, with an average of 4.38% RSD. The practical utility of the method was shown by the identification and quantification of the pollutant contents of Hungarian influent and effluent wastewaters (for six consecutive months and that of the Danube River for 2 months).

This paper presents a derivatization, mass fragmentation study relating to the most common six cholic acids, such as cholic, lithocholic, chenodeoxycholic, ursodeoxycholic, 3-hydroxy,7-ketocholanic and dehydrocholic acids, identified and quantified as pollutants in the aquatic environment at the first time. Derivatizations have been performed with the two-step process (1: oximation, 2: silylation) varying the time and temperature of both reactions. Optimum responses have been obtained after 30 min oximation with hydroxylamine.HCl and 90 min silylation with hexamethyldisilazane and trifluoroacetic acid at 70 degrees C. Fragmentation patterns of the trimethylsilyl (oxime) ether/ester derivatives of all six cholic acids provided the theoretically expected, fully derivatized compounds. Reproducibility/linearity of derivatives calculated on the basis of the corresponding selective fragment ions, characterized by the relative standard deviation percentages of measurements, proved to beor =4.9 (RSD%). The practical utility of the method was shown by the identification and quantification of cholic acids as pollutants in the aquatic environment. Subsequently to a solid phase extraction study varying the pH of extractions (pH 2, pH 4 and pH 7), applying the OASIS cartridges, it has been confirmed that the recoveries for all six cholic acids are acceptable, varying between 77% and 104%, and are independent on the pH. The total cholic acid content of a Hungarian wastewater plants' influent wastewater varied between 184 microg/L and 356 microg/L, while the Danube rivers' cholic acid content was 4.1 microg/L, only.

The main aims of this work were (a) to present the characteristics and stability of the o-phthalaldehyde (OPA)-ethanethiol (ET) derivatives of 22 amino acids, including the believed-to-be less stable OPA derivatives providing glycine, gamma-aminobutyric acid, beta-alanine, histidine, ornithine, lysine and the C(1)-C(5) aliphatic amines; (b) to compare the stability properties of the most common amino acids and amines as OPA-ET-fluorenylmethyl chloroformate (FMOC) derivatives to the corresponding ones obtained from OPA reagents containing various (SH)-additives; (c) to show the molar responses of alanine and lysine depending on the OPA reagent's composition; as well as (d) to prove the practical utility of these basic researches, by the simultaneous HPLC separation of 22 amino acids and 15 amines as their OPA-ET-FMOC derivatives. Investigations have been carried out by varying the composition of the reagents, the molar ratios of reactants and the reaction time, applying diode array and fluorescence detections simultaneously. Average reproducibility of quantitations, characterized with the relative standard deviations (RSDs) based on the fluorescence intensities of derivatives, in the order of listing, proved to be 1.2-5.9% for amino acids and 1.1-8.7% for amines. The practical utility of the method is demonstrated by the analysis of the amino acid and amine contents of mouse tissues, with an average reproducibility of 3.5%.

The fragmentation patterns and quantitation possibilities of three anthocyanidins (pelargonidin, cyanidin, malvidin), one flavonol (quercetin), two flavones (apigenin, luteolin) and two flavanones (naringenin, hesperetin) have been investigated as trimethylsilyl and as trimethylsilyl (oxime) derivatives by gas chromatography-mass spectrometry. Results proved that anthocyanidins and flavanones form trimethylsilyl (oximes), while flavonol and flavones provide simple trimethylsilyl derivatives. In all cases, characteristic fragments of high masses are formed proper for quantitation purposes. Hydrolysis conditions for naringin, hesperidin and rutin have been optimized, resulting in the quantitative release of naringenin, hesperetin and quercetin together with their corresponding saccharides. These basic studies made possible the identification and quantification of the flavonoid, carboxylic-/amino acid and sugar constituents of citrus fruit juices and albedos, without any extraction/enrichment procedure. In total 33 compounds have been determined in hydrolyzed samples, such as 2 flavonoids (naringenin and hesperetin), 6 phenolic acids (trimethoxybenzoic, 4-hydroxybenzoic, vanillic, quinic, chlorogenic and rosmarinic acids), 3 aliphatic carboxylic acids (levulinic, malic, citric acids), phosphoric acid, 4 amino acids (aspartic, glutamic acids, alanine, proline), 9 monosaccharides (xylose, arabinose, rhamnose, fucose, fructose, galactose, glucose, galacturonic acid, sedoheptulose), inositol, sugarphosphate, 5 disaccharides and tocopherol. Measurements were carried out as the trimethylsilyl (oxime) ether/ester derivatives of constituents, in the concentration range of 2 x 10(-3) to 49.9%. Identification level of samples varied between 26.4 and 77.5%, expressed in dry matter content of juices and albedos.

The extraction of ornithine, lysine, putrescine, cadaverine, 1,7-diaminoheptane, spermidine and spermine from biological tissues was optimized for HPLC quantitation as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate (OPA/ET/FMOC) derivatives. In applying perchloric acid deproteinization two approaches have been followed: (i) deproteinization with subsequent neutralization by potassium hydroxide and lyophilization, and (ii) deproteinization without neutralization and lyophilization. Neutralization and lyophilization resulted in the loss of free biogenic amines. HPLC analysis of ornithine (Orn), lysine (Lys), putrescine (Put), cadaverine (Cad), 1,7-diaminoheptane (Dah), spermidine (Spd) and spermine (Spm) content of biological tissues as their OPA/ET/FMOC derivatives was performed in the supernatant of perchloric acid-deproteinized samples (model solutions and tissues) with an average reproducibility of ≤2.6% relative standard deviation (RSD), including recovery of sample treatment and chromatography.

The possibilities in the identification and quantitation of the constituents of Rubia tinctorum L.'s root, called also madder root, was described and compared by gas chromatography (GC)-MS, high-performance liquid chromatography (HPLC)-UV/photodiode array detection (DAD) and HPLC-MS: chromatographic analyses were carried out in parallel, from the same samples/extracts/hydrolyzates. Anthraquinone glycosides, anthraquinones, carboxylic acids and sugars were determined directly in the presence of the matrix and in its extracts without and subsequently to hydrolyses. Hydrolyses were performed as a function of time, with hydrochloric and trifluoroacetic acids, as well as enzymatically. Data revealed that as hydrolyzing agent trifluoroacetic acid is to be preferred. Madder root's anthraquinones (pseudopurpurin/purpurin, alizarin, lucidin, munjistin, nordamnacanthal) were identified on the basis of their absorption spectra (HPLC-DAD) and fragmentation patterns by GC-MS and HPLC-MS, equally. Reproducibility of anthraquinone's quantitation, by HPLC-DAD and GC-MS, in the concentration ranges of 4 x 10(-5) to 3 x 10(-2)g/g dried sample, provided an average reproducibility of 4.2% (varying between 0.9 and 9.4% relative standard deviation (RSD percentages)). Carboxylic acids (malic, citric, quinic, rosmarinic acids) and saccharides (xylose, ribose, fructose, glucose, sucrose, primverose) were quantified as their trimethylsilyl (oxime) ether/ester derivatives by GC-MS, in the concentration ranges of 10(-5)g to 10(-2)g/g dried sample, with an average reproducibility of 4.7% RSD.

The stability and characteristics of the ornithine (Orn), lysine (Lys), putrescine (Put), cadaverine (Cad), 1,7-diaminoheptane (Diah), spermidine (Spd) and spermine (Spm) derivatives obtained with the o-phthalaldehyde (OPA)-ethanethiol (ET)-fluorenylmethyl chloroformate (FMOC) reagent has been investigated. The stoichiometry of the introduced, two-step derivatization process has been followed by photodiode array (DAD) and fluorescence (FL) detections, simultaneously, while the composition of derivatives was confirmed by on-line HPLC-electrospray ionization (ESI) MS measurements. Depending on the composition of the OPA reagents, in addition to the secondary amino group-containing Spd and Spm, under common aqueous conditions also Orn and Lys do react with FMOC resulting in derivatives of various compositions. Applying the OPA-ET reagent of increasing methanol (Met) content (38-80%, v/v) the formation of the FMOC group containing Orn and Lys derivatives could be considerably decreased. Optimum elution condition (18 min, including equilibration) was developed for the simultaneous quantitation of Orn, Lys, Put, Cad, Diah, Spd and Spm, in the presence of the rest of protein amino acids. The practical utility of the method was demonstrated by the analysis of mouse tissues. Average reproducibility of quantitations, characterized with the relative standard deviation percentages of fluorescence intensities and UV responses, in order of listing, proved to be 2.1% and 2.1%, respectively.

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 literature overview is presented of chromatographic methods currently in use to determine amino acids and amines (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.

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.

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.

As is well known, the knowledge of the qualitative and quantitative distribution of sugars and acids, present in various biological (urine fermentation liquor) and several natural matrices (fruits, vegetables, drug- and industrial plants, mushrooms, honeys) proved to be of primary importance from several points of view. In accordance with the chronological order of the development of the chromatographic methods, first, the possibilities of gas chromatography, thereafter, those of high-performance liquid chromatography have been shown. The advantages/disadvantages of these two main chromatographic methods, relating to this special topic will be presented in details.

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).

The separation and identification possibilities of 27 PTC-amino acids (with particular attention to those present in apples in free forms), are reported on seven RP columns such as, Nucleosil, 3 and 5 microns: 150(+20 guard) x 4.0 mm; Gromsil 3 microns; 150(+10 guard) x 4.0 mm; Hypersil 5 microns: 130(+20 guard) x 4.0 mm, 150(+20 guard) x 4.0 mm and 200(+20 guard) x 4.0 mm, as well as, Hypersil 3 microns: 150(+20 guard) x 4.0 mm: a UV range photodiode array (PDA) detection was employed. Optimization studies carried out in model solutions, as a function of the temperature (30-55 degrees C) and flow-rate (0.8-2.5 ml/min) of eluents proved that optimum resolutions are associated 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 of 30-50 degrees C. Twenty-seven amino acids, characteristic in apples in free forms, have been separated and determined on all seven columns, performing the same gradient program, (the main component asparagine, present in overwhelming excess, and the minor constituents glutamine, beta-alanine, gamma-aminobutyric acid, homoserine, homoarginine and 1-aminocyclopropane-1-carboxylic acid). Optimum conditions, at 2.1 ml/min, at 50 degrees C, with 40 min run time, including equilibration, have been obtained with the Hypersil, 150(+20 guard) x 4 mm column, performing elutions. Responses of the corresponding amino acids proved to be independent of the column used; reproducibility in the concentration range of 15-1500 pmol was < 4.0% R.S.D. (relative standard deviation). Detailed study of the PDA spectra revealed that in addition to the identification/peak purity possibilities further characteristics can be obtained taking advantage of the difference in maximum values and of those of their special ratio values, respectively. The utility of the protocol was shown in the quantitation of the free amino acid content of three apple varieties.

Derivatization conditions of amino acids with the o -phthaldialdehyde (OPA)/3-mercaptopropionic acid (MPA) and with the OPA/N-acetyl- l -cysteine (NAC) reagents have been exhaustively reviewed and investigated. The existence and extent of the self fluorescence of both reagents have been demonstrated and measured. The stability of the OPA/MPA and OPA/NAC isoindoles have been determined and compared to each other, from the same working solution, at pH=9.4 and at pH=7.2, by UV and fluorescence photometry. Data obtained proved that only slight differences can be expected applying the OPA/MPA or the OPA/NAC reagents in terms of the corresponding molar absorptivity, isoindole's stability and concentration linearity values. The relative molar fluorescence intensities of the OPA/MPA derivatives proved to be higher by relatively 10–55%, in comparison to the corresponding OPA/NAC ones. Stabilized background fluorescence can be obtained with both reagents prepared at least 90 min before use and stored in a refrigerator. Reproducibility values determined in the linear concentration ranges for both derivatives were much better applying UV photometry in comparison to the fluorescence intensity measurements.

Some of the general problems known in the analysis of tryptophan (both in its free form, alone or together with its metabolites, as well as in hydrolyzates of peptides or proteins, alone or together with all other amino acids) are described. This review includes an exhaustive literature overview using the author's experience with the preparation of derivatives and with various conditions arising from the analytical procedure itself. The special requirements of various tryptophan containing matrices (biological tissues or fluids, food and feed stuffs, etc.) are also taken into account. For the sake of completeness, in addition to the most common HPLC techniques, GC and spectrophotometry (in selected cases very important procedures), are also discussed.

An approach presented recently for the high-performance liquid chromatographic determination of dl- and l-cystines and cysteines as phenylthiocarbamyl (PTC) derivatives has been completed (i) with the determination of d-cystine/cysteine, (ii) by a study relating to the spectral characteristics of the PTC cystine/cysteine derivatives performed with diode-array detection and (iii) by the fragment analysis of the PTC-cyst(e)ine derivatives, applying the extremely soft conditions of the liquid chromatography-atmosphere pressure ionization mass spectrometry (LC-API-MS). In accordance with earlier experiences it has been repeatedly demonstrated that cystines and cysteines can be determined on the basis of two or four derivatives, with the same retention times and molar responses, without any special pretreatment, furnishing a simple possibility for the determination of the total cystine/cyteine in protein hydrolysates. The UV spectra of all four PTC derivatives provided by diode-array-detected purity parameters (PuP) showed that the corresponding PTC derivatives of dl-l- and d-cystine and -cysteine are identical and homogeneous, and do not show any characteristic differences. On the basis of the fragmentation patterns of seventeen PTC-amino acids and those of six PTC-cyst(e)ines, obtained under the very soft conditions of the LC-API-MS procedure, it can be stated that two characteristic PTC derivatives are formed: (i) the main product (“a” and “b” diastereoisomers, >80% of the total), shown to be the fragment of m/z = 255, a monomeric cysteine derivative, and (ii) the other characteristic constituent (“c” and “d” stereoisomers

A GC-MS method is reported for establishing the reproducibility of the determination of widely different amounts of sugars and acids as their trimethylsilyl derivatives simultaneously, from one solution with one injection. Optimum conditions were achieved on a 30-m DB-5 column. The determination of the components was based on their TIC and on selected ion monitoring. Data furnished by a Varian Saturn II GC-MS system equipped with a Varian Model 8200 AutoSampler showed that 4–20 ng of the minor constituents, in the presence of 50–250 ng of the main components, could be determined with a relative standard deviation of 10.6% or less. The utility of the procedure was demonstrated by the analysis of the composition of six different apple varieties, gathered at three different time of ripeness, in two consecutive years (1991, 1992), and stored for various periods of time. The separated carboxylic acids and sugars were phosphoric, succinic, pyruvic, 5-hydroxy-N-valeric and malic acid, butanal, 3-methyl-2-hydroxy-2-butenoic acid, 1,2-hydroxycyclohexene, pimelic acid, 2-deoxy- d -erythrose, tartaric acid, xylitol, arabinose, caffeic acid, d -ribose, citric acid, rhamnose, quinic acid, d -erythro-tetrafuranose, talose, 2-ketogluconic acid, mannitol, sorbitol, fructose, galactose, glucose, fructose (open form), glucaric and galacturonic acid, lactose, meso-inositol, gluconic, linoleic, glucuronic, stearic and arachidic acid, sucrose, turanose, maltose, chlorogenic acid, β-sitosterol, raffinose and maltotriose.

An exhaustive study was made to determine the amino acid composition of lysozyme in hydroysates performed in a CEM microwave oven under various conditions [0.621–0.827 MPa (90–120 p.s.i.g.) for 5, 10, 20 and 40 min], determined by HPLC as their phenylthiocarbamyl derivatives. The results showed that the CEM microwave hydrolysis system works reproducibly up to 0.689 MPa (100 p.s.i.g.) pressure only. Uniformly optimum parameters for all components present in hydrolysates were not found. The recoveries of the components at 0.621 MPa (90 p.s.i.g.) (after 20 and 40 min) and at 0.689 MPa (100 p.s.i.g.) (after 10 20 and 40 min) varied from 63 to 111%. The reproducibility of the measurements was

Some of the general problems commonly encountered in the analysis of phenylthiocarbamyl amino acids are described. This review includes experiences associated with the preparation and storage of derivatives prior to analysis and those originating from the analytical procedure itself. The issue of the quantification of the phenylthiocarbamyl derivatives of tryptophan and cyst(e)ine, together with all other amino acids from the same hydrolysate, is also discussed. The possible reproducibility of the measurements, as a function of the conditions applied is considered.

Tryptamine [3-(2-aminoethyl)indole] as an additive to 6 M HCl largely prevented the decomposition of tryptophan in proteins, and also in gas-phase hydrolysis, using a Pico-Tag Work Station, at 145°C for 4 h. This procedure proved to be excellent for amino acid analysis using conventional high-performance liquid chromatography with derivatization with phenyl isothiocyanate. The recovery of tryptophan from model solutions and from proteins was 80–98%. The results proved to be as good as any other obtained by the analysis of special alkaline or organic acid hydrolysates, most of which are suitable exclusively for the determination of tryptophan. The most important advantage of tryptamine was that it did not affect the quantitative recovery of other amino acids of proteins, as shown by the composition of lysozyme hydrolysates obtained in the absence and presence of various amounts of tryptamine. The reproducibility of the measurements was 3.7% (relative standard deviation) or less.

The optimum conditions for the cation-exchange clean-up of amino acids, present in protein hydrolysates, prior to their gas chromatographic determination were investigated. The results of exchaustive study monitoring the amounts of amino acids as N,O(S)-trifluoroacetyl isobutyl esters, revealed that the recovery of amino acids from the column was affected appreciably by either the particle size or the divinylbenzene content of the resins. Quantitative recovery and reproducible determination of amino acids require (i) a 50-fold excess of resin (calculated as equivalent capacity relative to the amino acids present) and (ii) sufficient amounts of eluates: the volumes both of distilled water and of 7 M ammonia solution must be about six times the volume of the wet resin applied. Under optimum conditions the recoveries of alanine, glycine, threonine, serine, valine, leucine (isoleucine), proline, hydroxyproline, methionine, aspartic acid, phenylalanine, ornithine, glutamic acid, tyrosine, lysine, arginine and cystine, were quantitative, both without and after hydrolysis, and that of tryptophan was ca . 85%.

The simultaneous determination of organic acids (succinic, malic, pimelic, tartaric, shikimic, citric, quinic, cafeeic and chlorogenic acids), sorbitol and sugars (arabinose, rhamnose, fructose, galactose, glucose, sucrose, maltose and raffinose) as trimethylsilyl (TMS)-oxime and TMS derivatives in a single solution has been developed. The optimum conditions for the simultaneous derivatization and the rapid quantitative evaluation of the nineteen compounds by capillary gas—liquid chromatography are reported in this paper. Reproducibility data reveal that ⪖0.02 μg of acids in the presence of ⪕12 μg of sugars can be determined with a relative standard error (R.S.E.) of ⪕11.2%, whereas the m ain constituents are determined with an R.S.E. of ⪕3.6%.

Relative retention times and detector responses of the trimethylsilyl ether and oxime derivatives of the components of electrochemically reduced glucose syrups and commercial sorbitol samples are reported. d -Glucose, 2-deoxy- d -glucose, d -glucitol and 2-deoxy- d -glucitol each gave a single main peak on Dexsil 300 stationary phase a second peak of d -glucose was about one quarter the height of the main peak. Amounts of 0.13–13 μg of all four components in the presence of each other, even in ratios of 1:100, have been measured with relative standard deviations of 2.5–5.3%. The amount of 2-deoxy- d -glucitol in commercial sorbitols varied from >0.1 to 0.8% (w/w).

A simple and rapid gas—liquid chromatographic method for the simultaneous determination of aliphatic mono-, di- and tricarboxylic acids, cyclic acids (shikimic + quinic), aromatic acids and sugars, all as trimethylsilyl derivatives in a single solution, was developed. Optimum conditions for the simultaneous derivatization and for the quantitative evaluation of organic acids and sugars were established. Reproductibility data showed that 0.25–1 μg of the characteristic acids of apples in the presence of a 700–2500-fold excess of sugars can be determined with a relative standard deviation (R.S.D.) of 10.7% or less. The main components (malic acid, fructose, glucose and sucrose) were determined with an R.S.D. of 4.0% or less.

Relative retention times and detector responses of trimethylsilyl and trifluoroacetyl derivatives of the components of maltitol (maltotriitol) syrups on Dexsil GC 300 liquid phase are reported. Glucose, sorbitol, maltose, maltitol, maltotriose and maltotriitol gave a single main peak, whereas as trifluoroacetylated derivatives two or three separated peaks could be observed, which probably showed products of incomplete trifluoroacetylation. A method for rapid separation and quantitative estimations obtained by gas-liquid chromatography for the constituents of maltitol (maltotriitol) syrups are given.

An improved ion-exchange chromatographic method for rapid separation and quantitative estimation of deoxyfructosyllysines and their hydrolysis products (pyridosine and furosine) is described. The proposed elution procedure has good reproducibility; the optimum hydrolysis conditions for the highest molar ratio of pyridosine and furosine are given. The furosine/pyridosine molar ratios obtained by ⌈dhydrolysis” of model fructosyllysine as well as of natural protein matrices (soya beans, tomato powder, barley and malts) are in good agreement and without exception > 1. The smallest amount of pyridosine determined was 0.009% (w/w). The relationship between the compounds pyridosine, furosine, lysine and carbohydrate units present in the parent moiety is discussed.

The esterification of homologous series of fatty acids, aliphatic and aromatic di- and polycarboxylic acids, with aliphatic alcohols in the presence of water and a mineral acid as catalyst, is described. The efficiency of derivatization as well as the detector responses, referred to the organic carbon content of the derivatized products, obtained with two alcohols are compared to each other to establish the optimum conditions for various series of acids. The molar ratios of water/alcohol which yielded quantitative esterifications are given, as is the maximum water content of the esterifying mixture permitted in order for esterification to proceed to completion. The optimum conditions for a reasonable ester yield in the presence of anhydrous sodium sulphate are also presented.