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A novel, multifunctional group mediated one-step derivatization (MGOD) method has been developed employing the couples of hexamethyldisilazane (HMDS) and perfluorocarboxylic acid (PFCA). 1 MGOD relies on the unique reactivity of HMDS and PFCA to provide simultaneous trialkylsilylation and acylation in a single step. While primary aliphatic amines and diamines are generally nonreactive (certainly secondary and tertiary amines are nonreactive either), MGOD allows for trialkylsilylation of amino acids (AAs), amino alcohols, amino sugars, 2,6-diaminoheptanedioic acid, di- and tripeptides, and in turn moderates the basicity of their free amino groups and facilitates subsequent acylation. The identification, quantification and stoichiometry of MGOD were characterized by selective mass fragmentations and monitoring by GC–MS. Reaction conditions were optimized and analytical performance characteristics were established with respect to a reproducibility of 5.6 RSD%, a linearity of R2 = 0.996 and limit of quantitation values (LOQ) of 41–75 pg/μL. Derivatives were prepared without preliminary extraction steps, avoiding the use of organic solvents. The practical utility of MGOD was evidenced by derivatization of amino acids in a branched- chain amino acid (BCAA), nutritional supplement (recovery data of standard addition to BCAA varied between 92 and 110%.), in 100, 200 and 300 µL volumes of human urine demonstrating the proportionality of the method, and in peptide hydrolysates.

A simple, effective and environmentally friendly method was developed to prepare the precious lignan arctigenin, in as high as 88.1% and 85.2% purity, from the fruits of Serratula tinctoria and Arctium lappa. In the course of this study, arctiin, the glycoside of arctigenin already known in A. lappa, was determined as the main compound in S. tinctoria fruit for the first time. In both samples, hydrophilic arctiin was converted into its hydrophobic aglycone arctigenin by endogenous enzymatic hydrolysis, allowing the preparation of high-purity arctigenin extract (HPAE) by an optimized three-step process. Firstly, fruits were purified with diethyl ether to remove all of their hydrophobic constituents. Thereafter, the arctiin remaining in the fruits was converted into arctigenin in aqueous medium by enzyme treatment. Finally, formed arctigenin was extracted with diethyl ether to prepare HPAE. Thus, 11.3 mg and 10.1 mg HPAE containing 9.96 mg and 8.61 mg arctigenin was isolated from 200.0 mg S. tinctoria and A. lappa fruits respectively, using only 16 mL ether and 0.5 mL water. The applicability of the method to a large-scale HPAE isolation was also confirmed. Arctigenin and its structurally related compound trachelogenin were proved to significantly inhibit the microtubular system of SW480 adenocarcinoma cells.

The molecular constituents of Cirsium brachycephalum fruits were identified, quantified and isolated for the first time. The lignan glycoside tracheloside was the main compound, which was transformed quantitatively into its aglycone trachelogenin by endogenous enzymatic treatment of the fruit. Following this transformation by high performance liquid chromatography (HPLC) hyphenated with UV and mass spectrometry (MS) detections on a quantitative basis, the enzyme-hydrolyzed fruit was found to be the richest raw material containing trachelogenin (17.2mg/g) reported to date. Thus, the enzyme-hydrolyzed fruit was used to isolate trachelogenin using preparative HPLC in order to (1) unambiguously confirm its identity by gas chromatography-MS, nuclear magnetic resonance spectroscopy and optical rotation, and (2) investigate its in vitro antiproliferative activities against the SW480 colon adenocarcinoma cell line. Trachelogenin significantly affected the phosphorylation of key proteins such as β-Catenin, c-Myc and GSK3 in the β-Catenin signaling pathway in a concentration-dependent manner. These changes account for the antiproliferative effects of trachelogenin.

Small scale laboratory experiment series were performed to study the suitability of a cyclodextrin-based sorbent (s-cyclodextrin bead polymer, BCDP) for modelling the removal of micropollutants from drinking water and purified waste water using simulated inflow test solutions containing target analytes (ibuprofen, naproxen, ketoprofen, bisphenol-A, diclofenac, β-estradiol, ethinylestradiol, estriol, cholesterol at 2–6 μg/L level). This work was focused on the preliminary evaluation of BCDP as a sorbent in two different model systems (filtration and fluidization) applied for risk reduction of emerging micropollutants. For comparison different filter systems combined with various sorbents (commercial filter and activated carbon) were applied and evaluated in the filtration experiment series. The spiked test solution (inflow) and the treated outflows were characterized by an integrated methodology including chemical analytical methods gas chromatography–tandem mass spectrometry (GC–MS/MS) and various environmental toxicity tests to determine the efficiency and selectivity of the applied sorbents. Under experimental conditions the cyclodextrin-based filters used for purification of drinking water in most cases were able to absorb more than 90% of the bisphenol-A and of the estrogenic compounds. Both the analytical chemistry and toxicity results showed efficient elimination of these pollutants. Especially the toxicity of the filtrate decreased considerably. Laboratory experiment modelling post-purification of waste water was also performed applying fluidization technology by s-cyclodextrin bead polymer. The BCDP removed efficiently from the spiked test solution most of the micropollutants, especially the bisphenol-A (94%) and the hormones (87–99%) The results confirmed that the BCDP-containing sorbents provide a good solution to water quality problems and they are able to decrease the load and risk posed by micropollutants to the water systems.

Dibenzylbutyrolactone-type lignan glycosides (tracheloside and carthamoside), their aglycones (trachelogenin and carthamogenin) and feruloyl-serotonin isomers were determined in the fruits of Leuzea carthamoides by using LC-UV, LC-MS/MS and GC-MS techniques. The composition of the embryo and wall parts of the fruits was analysed before and after their hydrolysis. As a result of these studies, fruit part-specific accumulation of lignan glycosides and feruloyl-serotonins were confirmed, demonstrating that the embryo contains a high amount of lignan glycosides (tracheloside 32.9 mg/g, carthamoside 45.3 mg/g), while the wall part of the fruit accumulates feruloyl-serotonins (63.0 mg/g). Enzymatic hydrolysis of the embryo resulted in the quantitative transformation of lignan glycosides into their corresponding aglycones, allowing selective isolation of trachelogenin and carthamogenin. These aglycones were subjected to an antiproliferative study against the SW480 colon adenocarcinoma cell line. In this test, moderate activity of carthamogenin and a significant effect of trachelogenin were demonstrated in a concentration range of 22-185 μM.

Irregular tissue outgrowths (called galls) in the inflorescences of three European Fraxinus species ( F. angustifolia , F. excelsior and F. ornus ), were analyzed for their phytochemical composition for the first time. The main goal of this study was to demonstrate the significance of Fraxinus galls as the new and abundant sources of drug candidate metabolites. The comparable quantitative results of nuclear magnetic resonance (NMR) spectroscopy and high performance liquid chromatography (HPLC)-mass spectrometry (MS) analyses confirmed extraordinarily high amounts of the valuable phenylethanoid glycoside (PhEG) acteoside (in the galls of F. angustifolia 86.7 mg/g – 139.9 mg/g and F. excelsior 71.7 mg/g – 161.2 mg/g) and that of the coumarin glucoside cichoriin (in the galls of F. ornus 143.0 mg/g – 232.0 mg/g). Optimized, consecutive treatments of acteoside, in distilled water (100 °C, 300 min) and acidic media (2 M trifluoroacetic acid, 100 °C, 15 min), resulted in an equilibrium of acteoside and isoacteoside as well as that of desrhamnosyl acteoside and desrhamnosyl isoacteoside, following isomerization and derhamnosylation, respectively. Three related PhEGs could thus also be isolated after optimized treatments, in addition to acteoside, in the highest yield reported so far. Their structures were identified by NMR spectroscopy and high-resolution Orbitrap-MS/MS techniques, also confirming the distinction of the regioisomers desrhamnosyl acteoside and desrhamnosyl isoacteoside by MS/MS. Molecular modeling was used to study the mechanism of isomerization. Isolated PhEGs and cichoriin showed no cytostatic activity in non-human primate Vero E6 cells and no hemolyis of human erythrocytes. Our results highlight the significance of Fraxinus galls in the production of PhEGs and cichoriin as easily available, high-yield harvestable, new raw materials for these pharmacologically important metabolites.

An overview is presented on the advancement of the two most frequently used derivatization protocols applying the o-phthalaldehyde (OPA)-thiol and the fluorenylmethyloxycarbonyl (FMOC) chloride reagents, prior to the high performance liquid chromatographic analysis of amino acids. This review pays special attention (i) to the blank value, to the composition, to the stability and to the life time of the reagents, (ii) to the optimum pH conditions for the interactions and derivatives' elutions, (iii) to the characteristics and behavior of those amino acids which might provide more than one derivative correlating with the molar ratios of the reagent to the reactants, and (iv) on the practical applicability of the optimized protocols.

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 (

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 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.

In this paper we identify/determine the composition of the extracts of Dracocephalum moldavica L. and D. ruyschiana L. with special emphasis on their flavonoids, aliphatic, aromatic carboxylic acids and sugars. The plant materials were extracted using methanol-acetone 1:1 (v/v) acidified with HCl (0.05%). Composition of extract's most red fractions was identified by HPLC, while the constituents of the entire extract were identified and quantified as their trimethylsilyl-(oxime) ether/ester derivatives by GC-MS. On the basis of HPLC analyses - (performed on Phenomenex Luna 5 µm C18 column, 250× 4.6 mm I.D.; eluent: acetonitrile - trifluoroacetic acid (0.1 %); isocratic/gradient conditions) - delphinidin, cyanidin and pelargonidin (in traces) were identified by spectral analysis and on the basis of authentic standard's addition. GC-MS analyses were carried out immediately with extracts, as well as subsequently to extract's hydrolysis (trifluoroacetic acid: 2M, 2h, 4h). Constituents were identified and quantified as their trimethylsilyl-(oxime) ether/ester derivatives, from a single run, on the basis of their total (TIC) and selective fragment ion (SIM) responses. Calculations were related to the dry matter content of extracts. As main constituents monosaccharides, sugar alcohols di- and trisaccharides, aliphatic (phosphoric, succinic, levulinic, malic, tartaric, fatty acids) and aromatic (quinic, chlorogenic, caffeic, ferulic, 3,4-dihydroxyphenyllactic, rosmarinic) carboxylic acids and their corresponding esters; apigenin, luteolin flavon aglycons and tocopherol, in total 33 constituents were quantitated partly by their TIC, partly by their SIM responses. Identification/quantification proved to be in total of 35–69% (expressed in the dry matter content of extracts).

Our gas chromatography-mass spectrometry method--developed for the simultaneous quantitation of mono-, di-, and trisaccharides, sugar alcohols, caboxylic and amino acids, measured as their trimethylsilyl-(oxime) ether/ester derivatives, from one solution by a single injection, prepared in the presence of the fruit matrix--has been extended/utilized for special purposes. The compositions of (i) freshly harvested and stored sour cherries (Prunus cerasus), (ii) apples obtained from organic and integrated productions (Malus domestica), and (iii) green and ripe bers (Zizyphus mauritiana L.) were compared. On the basis of earlier, basic researches (derivatization, quantitation, and fragmentation studies of authentic compounds), we demonstrate the reproducible quantitation of the main and minor constituents in a wide concentration range (approximately 1 x 10(-)(3) to >/=40%, in total up to < or =98%, calculated on dry matter basis of the fruit matrices). Reproducibility of quantitations, calculated on the basis of their total ion current values, provided an average reproducibility of 3.3 (sour cherries), 6.2 (apple), and 4.3 (ber) RSD %, respectively.

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%.

A literature overview is given of HPLC methods currently in use to determine amino acids (AAs) and amines (As) as theiro-phthaldialdyde (OPA)/2-mercaptoethanol (MCE) derivatives. This is followed by an exhaustive derivatization study performed using the classical proteinogen AAs, C1–C4 aliphatic As, several polyamines (PAs) and the OPA/MCE reagent. Model studies have been carried out as a function of reaction time and reagent composition. The primary importance of reagent composition (mole ratios of OPA to SH-group additive) is repeatedly proven. Stabilities of the OPA/MCE derivatives have been compared with corresponding OPA/3-mercaptopropionic acid (MPA) and/or OPA/N-acetyl-L-cysteine (NAC) compounds, obtained under the same conditions. Crucial factors proven to influence the stability of OPA derivatives of AAs and As are discussed in detail: (i) preparation and storage conditions of the OPA reagents, (ii) special behavior of the currently believed to be particularly unstable, AAs and As associated with their original molecular structure, e.g. glycine, β-alanine, γ-amino butyric acid (GABA), histidine, ornithine and lysine, several As, as well as (iii) the mole ratios of the OPA/SH-additive/AA and/or A.

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.

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.

Fragmentation patterns of the essential amino acids (AAs) as their silyl derivatives have been obtained with the aid of ion trap detection (ITD). Three derivatizing reagents, hexamethyldisilazane+trifluoroacetic acid (HMDS+TFAA),bis-(trimethylsilyl)trifluoroacetamide (BSTFA) andN-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide (MTBSTFA) were used. Simple and multiple derivatives obtained with each reagent have been investigated, with regard to their sensitivity and selectivity.

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.

This paper describes the fragmentation patterns and the GC-MS quantitation possibilities of the trimethylsilyl derivatives of thirty-one aromatic carboxylic acids, using ion trap detection (ITD). Sixteen aralkyl carboxylic acids, including those containing a saturated aliphatic side chain {phenylacetic, 2-phenylbutyric, phenylglycolic (mandelic acid), β-phenyllactic, 3-hydroxyphenylacetic, β-phenylpyruvic and 3-(4-hydroxyphenyl)-propionic acids} and those with an unsaturated aliphatic side chain {cinnamic, 2-hydroxycinnamic (o-coumaric), 4-methoxycinnamic, 3-hydroxycinnamic (m-coumaric), 4-hydroxycinnamic (p-coumaric), 4-hydroxy-3-methoxycinnamic (ferulic acid), 3,4-dihydroxycinnamic (caffeic), and 4-dihydroxy-3,5-dimethoxycinnamic (sinapic) acids}, as well as, the fifteen hydroxy(methoxy) benzoic acids {benzoic, 2-hydroxybenzoic (salicylic), 3-hydroxybenzoic, 4-hydroxybenzoic, 3,5-dimethoxybenzoic, 3,4-dimethoxybenzoic (veratric), 2,6-dihydroxybenzoic (γ-resorcylic), 3-methoxy-4-hydroxybenzoic (vanillic), 2,5-dihydroxybenzoic (gentisic), 2,4-dihydroxybenzoic (β-resorcylic), 3,4-dihydroxybenzoic (protocatechuic), 3,5-dihydroxybenzoic (α-resorcylic), 2,4,5-trimethoxybenzoic (asaronic), 3,5-dimethoxy-4-hydroxybenzoic (syringic) and 3,4,5-trihydroxybenzoic (gallic) acids}, provided distinct fragmentation characteristics that were very useful for their identification and simultaneously quantitation. Based on 1–20 ng amounts of acids, very informative ions of high mass with considerable intensities ([M+TMS]+, [M+1]+), $$([M]^{_ \cdot ^ + } )$$ , ([M−CH3]+) were obtained. In the case of the cinnamic acid derivatives, several odd electron fragments are formed by the loss of CO, HCHO and/or Si(CH3)4 molecules. In the case of benzoic acids the molecular ion $$([M]^{_ \cdot ^ + } )$$ proved to be abundant in three, the [M−CH3]+ ion in nine cases out of fifteen. The special MacLafferty rearrangement product ([C6H5Si(CH3)2]+) was obtained in different yields. In addition to the TIC values, at least three, and in most cases four, selective fragment ions could be utilized for quantitation. The reproducibility of the data in the concentration range of 1–20 ng acids proved to be between 1.2 and 13.0% (R.S.D.).

A GC-MS procedure is described for the simultaneous quantitatation of the minor and major constituents of honeys, as their trimethylsilyl derivatives, from one solution, by one injection. Selected minor components (aliphatic and aromatic carboxylic acids, members of various homologous series, together with o-phosphoric acid, proline and hydroxymethylfurfurol), have been determined on the basis of their characteristic fragment ions, in the presence of extremely high excess of honeysaccharides. Selective fragmentation of these minor compounds in the ion trap detector provided possibilities for distinguishing them. The method permitted the simultaneous quantitation of o-phosphoric, malic, shikimic, citric/isocitric, quinic, margaric, oleic and stearic acids, hydroxymethylfurfurol and proline with the extremely high sugar contents of honeys (fructose, glucose, galacturonic acid, inositol, sucrose, trehalose, turanose, maltose, gentiobiose, isomaltose, raffinose, erlose, melezitose, maltotriose, panose, isomaltotriose) and allowed the fast evaluation of sugar and acid constituents of fifteen honeys from various floral and geological origin. Results revealed that (i) the minor components varied in the concentration range of 0.0001 to 0.43%, and, (ii) together with the saccharides of honeys made up the total of identified and determined constituents from 87.8% to 98.5%. Quantitative evaluation of the minor constituents was performed on the basis of their selective fragment ion values with an average reproducibility of 6.7% (RSD).

The simultaneous quantitation of acids and sugars as their trimethyl silyl (TMS) derivatives has been extended in order to identify and quantitate the simple acid and sugar constituents in the hydrolyzates of various immunostimulant, water-soluble polysaccharides obtained from various Basidiomycetes, such as Armillariella mellea, Auricularia auricula-judae, Coriolus versicolor, Flammulina velutipes, Fomes fomentarius, Ganoderma applanatum, Ganoderma lucidum, Pleurotus ostreatus, Schizophyllum commune, Trametes hirsuta. Optimum hydrolysis conditions, performed with 2 M trifluoroacetic acid (TFAA) for five hrs, proved the presence of several sugars and acids with maximum recovery. (i) the total sugar/sugar alcohol content of polysaccharides varied between 20- and 65% and consisted of arabitol (0.01–10.2%), arabinose (0.09–1.3%), ribose (0.2–1.8%), fucose (0.3–1.2%), mannitol (0.01–5.3%), sorbitol (0.01–0.05%), galactiol (0.04%), fructose (0.08–0.8%), galactose (0.9–29%), glucose (10–53%), uronic acids (0.14–3.7%), sucrose (0.03–2%), trehalose (0.2–1%), cellobiose (0.01–0.6%), maltose (0.2–1.9%), other disaccharides (0.2–8%). (ii) The total of acids varied from 1.5 to 30% including o-phosphoric (1.3–19%), malic (0.08–4.7%), citric (0.08–4.7%), isocitric; (3%) and C16−C18 fatty acids (1–6%).

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.

The 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.

This paper describes the separation and quantitation of mono- di- and trisaccharides in model solutions. Optimized conditions for identification and determination of common sugar constituents in several natural matrices by GC-MS, from one solution by one injection, are given. Applying a 52 min temperature program to a 30 m capillary column and taking advantage of the stable ratios of the syn- and anti oximes eluting either alone or together with one or two other anomers: provides the basis of calculation for co-eluting compounds. Saccharides (5–200 ng), such as fructose, glucose, sucrose, trehalose, cellobiose, maltose, turanose, gentiobiose, palatinose, melibiose, isomaltose, erlose, raffinose, melezitose, maltotriose, panose and isomaltotriose, in the presence of each other in various ratios, have been measured as silyloxime compounds with a reproducibility of ≤5 R.S.D. %. The possibility of measuring all these saccharides as their TMS methoximes is also presented.

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

The behavior of the flavonoid diglycosides, relevant constituents of parsley ( Petroselinum crispum ) fruit (PFr) and leaf (PLe) samples was characterized upon their enzymatic hydrolyses applying complementary liquid chromatography-ultraviolet (LC-UV) and gas chromatography mass selective (GC–MS) detections. Analyses were performed in quantitative manner, from the same extracts as a function of hydrolysis times. Both in fruit and leaf tissue extracts, in intact and in enzyme hydrolyzed ones, apigenin, chrysoeriol, their glycosides, sugars, sugar alcohols, carboxylic acids and phytosterols, in total 17 constituents were identified and quantified. Based primarily on the selective mass fragmentation properties of the trimethylsilyl (oxime) ether/ester derivatives of constituents, we confirmed several novelties to the field. (i) It was shown for the first time that in parsley tissues different types of glycosidase enzyme are active. In PFr samples, both the stepwise and disaccharide specific endogenous mechanisms were certified, quantifying simultaneously the continuous release of apigenin, chrysoeriol, 2- O -apiosyl-apiose, apiose and glucose. (ii) 2- O -Apiosyl-glucose was demonstrated as disaccharide due to its formation under derivatization conditions from parsley glycosides. (iii) Both in PFr and in PLe samples even the invertase enzyme activity was attainable: sucrose decomposition in both tissues was going on with the same intensity. Three different types of enzymatic glycosidase processes were followed with their specific hydrolysis products by means of HPLC-UV and GC–MS, simultaneously.

The organic chemistry of the 1,4-cyclohexanedione (CHD)-bromate-sulfuric acid oscillatory system has been revealed by following the reaction of 1,4-CHD with bromate using a GC/MS technique. We could identify 1,4-dihydroxybenzene as an intermediate, 1,4-benzoquinone as the main oxidation, and mono- and dibromocyclohexanedione as the main bromination products. Acid bromate does not cleave the alicyclic ring.

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

A GC-MS method has been developed for the analysis of the sugar and carboxylic acid content of citrus fruits, including the quantitation of isocitric acid in the presence of a large excess of citric acid. Optimum conditions have been determined for the analysis (from one solution, by one injection, on a 30 m DB-5 column) of the TMS oxime and/or TMS derivatives of sugars and acids obtained from various lemon, grapefruit, and orange samples. Quantitation was based on the monitoring of the total ion current and of selective fragments. The utility of the method has been demonstrated by the determination of isocitric acid in the presence of 10- to 200-fold excess of citric acid.

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.

Classical liquid phase hydrolysis of proteins with hydrochloric acid in the presence of tryptamine [3-(2-aminoethyl)indole] has shown that tryptophan can be protected from destruction. An exhaustive study has been made to establish the optimum conditions for protein hydrolysis, in the gas phase using the Pico-TagTM Work Station, as a function of time and temperature. The amino acid content, including tryptophan and cyst(e)ine, of standard proteins such as lysozyme, human albumin and α-chymotrypsin were determined as phenylthiocarbamyl (PTC) derivatives. On 5 μm Hypersil columns (15×4.6 mm) the quantitation of twenty PTC amino acids requires 22 minutes. The reproducibility of the measurements was 5.8% (relative standard deviation) or less.

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.

A new approach is described, and a novel explanation presented, for the high performance liquid chromatographic analysis of cystine and cysteine as their phenylthiocarbamyl derivatives. PTC cystine and cysteine have been eluted with the same retention times and molar responses, most probably due to electrophilic attack of phenylisothiocyanate on cystine resulting in the scission of the disulfide bond yielding two moles of cysteine. Further, total PTC cystine and cysteine have been measured both in model solutions and in standard protein hydrolyzates (lysozyme, bovine albumin, ribonuclease) with the same linearity as the other ineteen amino acids. The reproducibility of the measurements, at the 250–750 pmole level, proved to be 4.1% (Relative Standard Deviation %) or less.

The selective reaction of lysine, δ-hydroxylysine, ornithine and α,e-diaminopimelic acid with furfuraldehyde, in anhydrous media, was investigated at 80, 85, 90 and 95°C by spectrophometry, in the presence of various additional materials, such as potassium pyrosulphite, ascorbic acid and lead( II ) acetate. Second-order rate constants and molar absorptivities were determined. Optimum conditions were established for the selective determination of lysine in the presence of intact proteins or in protein hydrolysates in the presence of a wide range of other amino acids including ornithine. Under optimum conditions, in the concentration range 2 × 10 −8 −3 × 10 −7 M, molar absorptivities and reproducibility data were measured with a standard error of 0.9–5.0%, depending on the procedure applied and on the amount of lysine present.

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%.

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.

Amino acids interact with carbohydrates to form Maillard browning products. Such reactions reduce the nutritional value of foods containing amino acids and carbohydrates and may lead to the formation of compounds that are mutagenic and clastogenic or chromosome-damaging. A need therefore exists to inhibit these heat-induced interactions. To demonstrate whether SH-containing sulfur amino acids minimize nonenzymatic browning, beta-alanine, N alpha-acetyl-L-lysine, glycylglycine, and a mixture of amino acids were each heated with glucose in the absence and presence of the following potential inhibitors: N-acetyl-L-cysteine, L-cysteine, reduced glutathione, sodium bisulfite, and urea. Inhibition was measured as a function of temperature, time of heating, and concentration of reactants. The extent of browning was estimated by absorbance measurements at 420 nm. Inhibition was independent of the amino group containing reactant. The minimum concentrations for optimum inhibition, in moles of inhibitor per mole of D-glucose, were as follows: sodium bisulfite, 0.02; L-cysteine, 0.05; N-acetyl-L-cysteine, 0.2; reduced glutathione, 0.2; urea, 8. An "index of prevention" (IP) was used to calculate the inhibition at the optimum mole ratio range, where IP = 100 - [molar absorptivity value (MAV) of the amine compound + glucose + inhibitor] X 100/(MAV of the amine compound + glucose). The calculated values were about 90% in all cases. Possible mechanisms of browning prevention are discussed.

Dye-binding stoichiometry and selectivity of the reactions of several standard proteins and protein matrices with the phthalein dye cresol red (CR) are presented. Spectrophotometric measurements show that the interactions are selective; amino acids, peptides, and albumin-type proteins do not react at all. The optimum analytical conditions of the reactive proteins—human gamma globulin (HGG), casein (C), milk protein (MP), soya bean (SBM), meat (MM), and fish meals (FM)—with CR are given. Dye-binding values obtained with soya bean meals are complemented by the sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns (SDS-PAGE) of the same samples, in order to show a qualitative picture of the molecular weight distribution of three untreated samples when comparing them to their respective denatured ones.

Summary 1. Based on stoichiometric and mass spectrometric studies of the OPA derivatives of AAs the nightmare of their instability has been solved. 2. It was confirmed, for the first time, that the common characteristic of the believed to be less stable OPA derivatives is associated with their initial molecular structure: they do contain the -NH 2 -CH 2 -R moiety and all of them furnish more than one OPA derivative. 3. In the light of the common structure of the multiple OPA derivatives providing compounds a plausible reaction pathway has been described. According to the proposed mechanism hydrogen atoms of the -CH 2 - group, neighbor to the primary amino group, play the key role in the reaction with an additional molecule of OPA: resulting in consecutive OPA-derivatives transformed from the initially obtained species. 4. As analytical consequences, the author of this review, based on recent experience, takes the responsibility to state: OPA derivatives of AAs are very much more stable than they are generally believed to be, considering that some of them are providing more than one reaction product (For the time being the successively formed products have not been taken into account). Consequently, in the HPLC analysis of AAs the following requirements are to be followed: (i) the molar ratios of the OPA/SH-group additive/AA should be close to 20/60/1, (ii) the reagents, saved in the refrigerator at ≤ 4 °C, should be at least 90 min and maximum 9 days old, (iii) the reaction time between the OPA reagent and the AAs should be at least 7 min, (iv) the OPA derivatives should not be acidified before loading them onto the chromatographic column, (v) the HPLC elution procedure should be proper for the separation and quantitation of all components formed, including the more than one OPA derivative providing AAs, (vi) last but not least, in order to be able to get reliable AA contents, in particular when working in the low pM level, the blank value of the reagent should be carried out at least every day and the impurity values are to be substracted from the coeluting AA derivative.

Summary An overview is presented of chromatographic methods currently in use to determine AAs and As (i) simultaneously in a single run, (ii) in the presence of each others by separate methods, or (iii) amines alone subsequently to their isolation from AAs. Separation, derivatization and chromatographic conditions are summarized. The advantages and drawbacks of all three possibilities were characterized on the basis of recovery, reproducibility values, time and cost phenomena of methods, (i) From the point of view of the analytical chemist the simultaneous quantitation of AAs and As seems to be the best choice: however to find optimum conditions, with the time not being considered, for AAs and As simultaneously occurring in a matrix, is an unambiguous challenge. In order to solve the problem quickly it helps to have practice in the derivatization and chromatographic conditions of the two groups (AAs, As), separately. (ii) In cases when knowledge of the A content of the sample only is needed, the best choice could be the fast chromatographic elution of AAs without separation: prior to the slow, well-resolved separation of As, such as putrescine, cadaverine, spermine and spermidine, etc. Certainly, As of low molecular weight, inserted into the AA derivatives and are eluting together with them. (iii) On the basis of the unfortunately low recoveries of As, (determined subsequently to their isolation/extraction from AAs), this possibility can be regarded as the worst solution of the task.

Summary Some of the general problems encountered in the analysis of amino acids derivatized with various isothiocyanates, determined both as phenylthiocarbamoyl (PTC) and as phenylthiohydantoin (PTH) derivatives, are presented. Based on our own experience and the literature associated with sample preparation, stability, optimum chromatographic and quantitation conditions are discussed. Twenty-seven PTC-AAs have been separated on seven columns, with the same gradient program, applying various temperatures and eluent flow rates, within 40 min, including equilibration time. Response values and spectral characteristics of the 27 AAs proved to be independent of the chromatographic conditions performed. Particular attention was paid to the issue of sensitive amino acids under hydrolysis (cystine/cysteine, methionine, tryptophan), and derivatization (cystine/cysteine) conditions as well. The special behavior of the PTC-cystine/cysteine has been examined by LC/API/MS measurements showing that, irrespective of the initial compound to be derivatized, the same stereoisomer pairs are formed at m/z = 255, PTC-cysteines (∼80 % of the total) and their oxidized version at m/z = 287 (∼20 % of the total). Whether there is an advantage of the microwave irradiation procedure is still not decided: however the hydrolysis time of lysozyme has been shortened considerably, but optimum hydrolysis conditions were not uniformly found for all essential amino acids.

A GC-MS method - developed for the simultaneous quantitation of mono-, di- and trisaccharides, sugar alcohols and acids, as their trimethylsilyl (oxime) ether/ester derivatives prepared in the presence of the fruit matrix has been extended. The reproducible determination of derivatized sour cherry constituents was performed both on the basis of total ion current (TIC) and selective ion values; ensuring their identification and quantitation over a wide concentration range (∼1 × 10−2 to ≥ 41%, 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/flavonoid hydrolysis products of three sour cherry cultivars, as a function of their harvesting dates and storage conditions. Reproducibility of quantitations in the case of the main constituents, calculated on the basis of their TIC values provided an average reproducibility of 2.3 and 3.6 RSD%, for components present in concentrations of ≥1% and ≤1%, respectively.

The simultaneous quantitation of organic acids (glycolic, lactic, oxalic, sorbic, benzoic, succinic, malic, pimelic, tartaric, citric+isocitric, quinic, ascorbic, palmitic, caffeic, linoleic, stearic, arachidic, behenic, and chlorogenic acids), mannitol, sorbitol and sugars (arabinose, xylose, rhamnose, fructose, galactose, mannose, glucose, sucrose, maltose, isomaltose, raffinose and maltotriose) are reported. Optimum conditions for simultaneous silylation and quantitation of the 33 possible components of orange, lemon, grape, banana, tomato, apple and pear, by capillary gas chromatography are detailed. The transformation of the organic acid and sugar composition of pears during storage is also described.

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.

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