An LCEC method for the analysis of synthetic amino acids in fruit juices

Summary A fast and simple HPLC-method for the determination of synthetic amino acids in adulterated orange juice has been developed. The amino acid enantiomers were derivatized with a chiral reagent and the derivatives separated on a 3 m particle size C₁₈ column. An electrochemical detector operating in the oxidative mode was used for detection. The potential at which the derivatives are oxidized was determined by cyclic voltammetry.By using selective (electrochemical) detection it is possible to reduce the sample clean-up to simple centrifugation and filtration steps.     

Composition of quince (Cydonia oblonga Miller) seeds: phenolics, organic acids and free amino acids

Phenolic compounds, organic acids and free amino acids of quince seeds were determined by HPLC/DAD, HPLC/UV and GC/FID, respectively. Quince seeds presented a phenolic profile composed of 3-O-caffeoylquinic, 4-O-caffeoylquinic, 5-O-caffeoylquinic and 3,5-dicaffeoylquinic acids, lucenin-2, vicenin-2, stellarin-2, isoschaftoside, schaftoside, 6-C-pentosyl-8-C-glucosyl chrysoeriol and 6-C-glucosyl-8-C-pentosyl chrysoeriol. Six identified organic acids constituted the organic acid profile of quince seeds: citric, ascorbic, malic, quinic, shikimic and fumaric acids. The free amino acid profile was composed of 21 identified free amino acids and the three most abundant were glutamic and aspartic acids and asparagine.     

Development of a capillary electrophoretic method for the analysis of amino acids containing tablets

Ketosteril is an enteral medicinal product indicated for prevention and therapy in chronic renal insufficiency in connection with a low protein diet. Tablets of Ketosteril contain five essential amino acids like: Lys, His, Thr, Trp, Tyr and another five amino acids in the form of their hydroxy and keto analogues as calcium salts, that are: alpha-ketoleucine, alpha-ketoisoleucine, alpha-ketovaline, alpha-ketophenylalanine and alpha-hydroxymethionine. The composition of Ketosteril tablets is routinely tested with three LC methods. Capillary electrophoretic method seems to be a good alternative for amino acids and their analogues determination in multicomponent pharmaceuticals because of short analysis time and the possibility to assay all components during a single run without any pretreatment. Electrophoresis was performed in 50 microm I.D. fused-silica capillaries with 65 cm distance to the detector. Capillaries were installed in Waters Quanta 4000 electrophoretic equipment with a positive power supply and on-line UV detection at 214 nm. Separations were done in a buffer containing 40 mM Tris and 160 mM boric acid titrated with NaOH to pH 10. The method developed allows the separation of all investigated analytes with an efficiency of n = 230,000 and 20 min analysis time. The method was applied for determination of all components of Ketosteril in commercial tablets.     

Multiparametric analysis of amino acids and organic acids in rat brain tissues using GC/MS

Using design of experiment (DOE) theory coupled with multivariate statistical analysis, we have developed a simple and reliable GC/MS-based analytical assay for simultaneous analysis of amino acids and organic acids in rat brain tissue samples. The process of water extraction (pH 10.0) was extensively evaluated using brain tissue samples and a set of 21 reference standards. Acceptable calibration curves were obtained over a wide concentration range, 0.2-35.0 microg/mL for standards and 15.0-2.4 mL/g (tissue) for brain tissue samples. The precision was mostly better than 10% for both the mixed standards and the brain tissue samples. The brain tissue samples exhibited good stability within 48 h with RSD generally less than 15%. Furthermore, the developed analytical method was successfully applied in distinguishing the subtle variation among different parts of the brain tissues, such as cerebral cortex, hippocampus, and thalamus.     

Simplification of determination method for standard materials using post-column reaction GC/FID

For the simple and fast preparation of highly reliable standard materials, a post-column reaction GC/FID system was developed and evaluated on the mixture of oxygen-containing organic compounds. The oxygen-containing organic compounds mixing solution were determined with the post-column reaction GC/FID system using n-dodecane as an internal calibration standard. Required value of relative expanded uncertainty as an original source of SI-traceable standard materials was within 1% and it aimed at this value as accuracy of the quantitative analysis. The results showed good agreement between the prepared concentrations and analytical values using post-column reaction GC/FID system. These results indicated that the post-column reaction GC/FID system would be used for getting SI-traceable values.     

High-performance liquid chromatographic analysis of free amino acids in fruit juices using derivatization with 9-fluorenylmethyl-chloroformate

A simple, rapid, and reliable reversed-phase high-performance liquid chromatographic method for the analysis of 16 amino acids of main interest in commercial fruit juices (pear, orange, grapefruit, pineapple, peach, and apricot) is described. No sample cleanup is required. The pH of the fruit juices is adjusted to alkaline value (8.5) using 200 mM borate buffer, then amino acid is converted to stable derivatives using 9-fluorenylmethyl-chloroformate. The excess of derivatization reagent is removed by a hydrophobic amine, 1-amino-adamantane hydrochloride. The derivatization procedure is simple, fast, and described in detail. Amino acids are detected at 263 nm and eluted within 35 min. The calibration, precision (< or = 6.1%), and recovery (102% +/- 4%) of the method are reported. The conditions of separation are optimized; however, serine partially overlapped with aspartic acid. The amino acid profile of fruit juices is consistent with data from the literature.     

Stability of the FID sensitivity during an analysis in capillary GC

The sensitivity of an FID may change when the carrier gas flow rate changes during a chromatographic run. Sample parts which are eluted at reduced FID sensitivity produce a reduced peak area, hence are discriminated as compared to other components. Sensitivity changes were studied for hydrogen as carrier gas. For the detector tested, differences in the carrier gas flow rates of 1 ml/min shifted the FID sensitivity by 1 to 5% (depending on the fuel gas supply). Thus the stability of the sensitivity is no longer ensured as soon as the carrier gas flow rate is changed manually or by an automatic programmer during an analysis. Sensitivity drifts may also occur during temperature programmed runs with a pressure regulated carrier gas supply since the gas flow through the capillary drops with increasing temperature. Such shifts in the response became noticeable as soon as relatively high carrier gas flow rates combined with long range temperature programmes were used. The typical patterns of such discriminations are shown, closing with a discussion on the possibilities for minimizing such undesired effects.     

Statistical evaluation of an analytical GC/MS method for the determination of long chain fatty acids

In-depth evaluation of an analytical method to detect and quantify long chain fatty acids (C₈-C₁₆) at trace level concentrations (25-1000 μg/l) is presented. The method requires derivatization of the acids with methanolic boron trifluoride, separation, and detection by gas chromatography-mass spectrometry. The calibration experiments passed all the tested performance criteria such as linearity, homoscedasticity, and ruggedness. The detection limits and related quantities were computed by applying the method detection limit, and the calibration line approximation. The values obtained by applying the latter approach were more reliable and consistent with the actual statistical theory of detection decisions and yielded the following concentrations: C₈, 87.6 μg/l; C₁₀, 45.2 μg/l; C₁₁, 39.9 μg/l; C₁₂, 37.7 μg/l; C₁₄, 41.4 μg/l and C₁₆, 40.6 μg/l. Two different gas-liquid chromatographic columns were tested and similar results achieved, which shows the ruggedness of the method.     

Phenylacetylene as a new surrogate standard for the determination of BTEX by GC/FID

Benzene, toluene, ethylbenzene, xylene (short form: BTEX) and other monoaromatic compounds are environmental contaminants which are often analyzed by GC/FID. For the calculation of BTEX concentrations in water samples normally external quantification with defined BTEX solutions is sufficient. However, for accurate quantification of BTEX in complex matrices it is necessary to use internal standards, e.g. ¶1-chlorohexane. Isotopes of BTEX are usually the best alternative but they are only applicable to GC/MS, because their retention times are similar to the original BTEX. 1-Chlorohexane and phenylacetylene were compared with respect to their quality as internal standards. Good results were obtained with ¶the monoaromatic phenylacetylene as a surrogate standard. The physical properties of phenylacetylene are very similar to BTEX species and it normally does not occur in environmental samples. 1-Chlorohexane was more strongly adsorbed on the used soil than BTEX during sample preparation. This fact suggests that the single aromatic rings of BTEX and phenylacetylene are mainly responsible for the adsorption behavior.     

Phenylacetylene as a new surrogate standard for the determination of BTEX by GC/FID

Benzene, toluene, ethylbenzene, xylene (short form: BTEX) and other monoaromatic compounds are environmental contaminants which are often analyzed by GC/FID. For the calculation of BTEX concentrations in water samples normally external quantification with defined BTEX solutions is sufficient. However, for accurate quantification of BTEX in complex matrices it is necessary to use internal standards, e.g. 1-chlorohexane. Isotopes of BTEX are usually the best alternative but they are only applicable to GC/MS, because their retention times are similar to the original BTEX. 1-Chlorohexane and phenylacetylene were compared with respect to their quality as internal standards. Good results were obtained with the monoaromatic phenylacetylene as a surrogate standard. The physical properties of phenylacetylene are very similar to BTEX species and it normally does not occur in environmental samples. 1-Chlorohexane was more strongly adsorbed on the used soil than BTEX during sample preparation. This fact suggests that the single aromatic rings of BTEX and phenylacetylene are mainly responsible for the adsorption behavior.     

Improved and simplified liquid chromatography/atmospheric pressure chemical ionization mass spectrometry method for the analysis of underivatized free amino acids in various foods

An improved analytical method which offers rapid, accurate determination and identification of 22 amino acids in a variety of matrices, e.g. baby foods, juices, honey is reported. The amino acids were extracted from the matrixes using acidified water. Simultaneous determination of 22 underivatized amino acids was carried out by a liquid chromatography-mass spectrometry (LC/MS). A narrow-bore column allowed rapid screening and quantitative analysis by positive LC/atmospheric pressure chemical ionization (APCI) MS with only acidified mobile phase. Retention times of the 22 amino acids were in the range of ca. 0.9-7.5 min. Sample preparation without clean-up followed by fast chromatographic analysis allowed the analysis to be completed in <25 min.     

Kinetic method for the simultaneous chiral analysis of different amino acids in mixtures

The kinetic method has been extended to enantiomeric excess (ee) determinations on amino acids present in mixtures. Singly charged trimeric clusters [Cu(II)(ref*)(2)(A(m)) - H](+) are readily generated by electrospraying solutions containing Cu(II), a chiral reference ligand (ref*), and the amino acids (analytes A(m), m = 1-3). A trimeric cluster ion for each amino acid is individually mass-selected and then collisionally activated to cause dissociation by competitive loss of either the reference ligand or the analyte. For each analyte in the mixture, as shown from separate experiments, the logarithm of the ratio of the fragment abundances for the complex containing one enantiomer of this analyte expressed relative to that for the fragments of the corresponding complex containing the other enantiomer is linearly related to the enantiomeric composition of the amino acid. Formation and dissociation of each trimeric complex ion are shown to occur independently of the presence of other analytes. Chiral selectivity appears to be an intrinsic property and the chiral selectivity R(chiral(m)) measured from the mixture of analytes is equal to R(chiral) measured for the pure analyte. The sensitive nature of the methodology and the linear relationship between the logarithm of the fragment ion abundance ratio and the optical purity, characteristic of the kinetic method, allow the determination of chiral impurities of less than 2% ee in individual compounds present in mixtures by simply recording the ratios of fragment ion abundances in a tandem mass spectrum.     

Optimization of the Extraction and Analysis of Natural Androgen Steroids and Their Metabolites in Urine by GC/MS and GC/FID

A multiresidue method was developed for screening, quantification, and confirmation of nine natural androgen steroids and their metabolites in urine. Steroids were first extracted from urine by solid phase extraction, enzymatically deglucuronated, re-extracted using a liquid/liquid extraction for purification, and finally acetylated for GC/MS and GC/FID analysis. Each step of sample preparation, as well as analysis, was optimized: solid phase extraction, liquid/ liquid extraction, and derivatization reaction … Therefore, a rugged sample preparation procedure was developed leading to extracts of sufficient purity (recoveries >66% and few matrix compounds). The whole methodology allowed reliable detection and quantification of the nine steroids at low concentration levels. Linearity and repeatability were established and were found to be satisfactory (R² > 0.996, RSD < 11%). Finally, the method was applied to quantify compounds of interest in real samples collected from healthy volunteers and patients treated with 4-androstenedione or dehydroepiandrosterone.     

A new reversed-phase liquid chromatographic/tandem mass spectrometric method for analysis of underivatised amino acids: evaluation for the diagnosis and the management of inherited disorders of amino acid metabolism

Seventy-six compounds of biological interest for the diagnosis of inherited disorders of amino acids (AA) metabolism have previously been demonstrated to be detectable in positive mode electrospray ionisation tandem mass spectrometry (ESI-MS/MS), after separation by ion-pairing reversed-phase liquid chromatography (RPLC). The separation method used tridecafluoroheptanoic acid as ion-pairing agent, and a gradient of acetonitrile for the elution of the most retained compounds. This method had previously been demonstrated to be suitable for the qualitative diagnosis of many AA disorders, and for the quantitative measurement of 16 AA in biological fluids, using their stable isotope labelled (SIL) AA as internal standard. For quantification of the other AA, an internal standard was chosen among the available SIL-AA, as close as possible to the analyte to be measured, in terms of structural analogy, and of retention time in the chromatographic system. The performances of the quantitative analysis of the other AA to be measured are reported here. They show validated results for several AA, allowing their accurate quantification, with another SIL-AA as internal standard. For some other AA, quantitative results were not accurate, allowing only semi-quantitative or qualitative determination for these parameters.     

Electrophoretic clean-up of organic acids from coffee for the GC/MS analysis

The clean-up presented here includes a free flow field step electrophoresis followed by ultrafiltration. Thus, organic acids can be separated from non-acidic and high-molecular compounds in roasted and instant coffee. The acids are identified by gas chromatography/mass spectrometry after freeze-drying and trimethylsilylation. With the method presented, 31 acids could be identified in commercial roasted coffee blends and in instant coffee, among them for the first time in coffee: 3-hydroxypropionic, 2-oxobutyric, glyceric, 2,4-dihydroxybutyric, 5-hydroxymethylfuran-2-carboxylic and 2-hydroxyglutaric acid.     

Electrophoretic clean-up of organic acids from coffee for the GC/MS analysis

The clean-up presented here includes a free flow field step electrophoresis followed by ultrafiltration. Thus, organic acids can be separated from non-acidic and high-molecular compounds in roasted and instant coffee. The acids are identified by gas chromatography/mass spectrometry after freeze-drying and trimethylsilylation. With the method presented, 31 acids could be identified in commercial roasted coffee blends and in instant coffee, among them for the first time in coffee: 3-hydroxypropionic, 2-oxobutyric, glyceric, 2,4-dihydroxybutyric, 5-hydroxymethylfuran-2-carboxylic and 2-hydroxyglutaric acid.     

Metrological effectiveness of an analytical method for volatile organic compounds standard materials using post-column reaction GC/FID system

The metrological effectiveness of an analytical method using the post-column reaction GC/FID system was evaluated. The SI-traceable certified reference material (CRM), the eight ester phthalates mixture standard solution, was used as a sample. We assigned specific value to n-eicosane and it was used as an internal standard. A known quantity of n-eicosane was added to the CRM, and the mixture was measured with the post-column reaction GC/FID system. Six phthalate esters were chromatographically separated and determined. The assigned values by our system are in good agreement with the certified values of the CRM, and the combined uncertainties of the measurements by the present system were better than those of the CRM. Our method is classified as a primary ratio method and the specific values of many organic compounds can be assigned very precisely by using a small number of reference materials. Also, the method can make it possible to avoid the purity determination of raw materials and directly to assign their specific values after the preparation of the standard mixture. Conclusively, this post-column reaction GC/FID system is very effective for the chemical metrology.