Complete amino acid analysis in hydrolysates of foods and feces by liquid chromatography of precolumn phenylisothiocyanate derivatives

The amino acid analysis method using precolumn phenylisothiocyanate (PITC) derivatization and liquid chromatography was modified for accurate determination of methionine (as methionine sulfone), cysteine/cystine (as cysteic acid), and all other amino acids, except tryptophan, in hydrolyzed samples of foods and feces. A simple liquid chromatographic method (requiring no derivatization) for the determination of tryptophan in alkaline hydrolysates of foods and feces was also developed. Separation of all amino acids by liquid chromatography was completed in 12 min compared with 60-90 min by ion-exchange chromatography. Variation expressed as coefficients of variation (CV) for the determination of most amino acids in the food and feces samples was not more than 4%, which compared favorably with the reproducibility of ion-exchange methods. Data for amino acids and recoveries of amino acid nitrogen obtained by liquid chromatographic methods were also similar to those obtained by conventional ion-exchange procedures.     

Development of a new high-performance liquid chromatography-electrospray ionization time-of-flight mass spectrometry method for the determination of low molecular mass organic acids in plant tissue extracts

A liquid chromatography-electrospray ionization time-of-flight mass spectrometry method has been developed for the direct and simultaneous determination of 10 low molecular mass organic acids in different plant tissue extracts. The method does not include a derivatization step. Quantification was accomplished using (13)C-labeled malic and succinic acids as internal standards. Good limits of detection (0.05-1.27 pmol) were obtained for malic, 2-oxoglutaric, succinic, quinic, shikimic, cis-aconitic, and citric acids, whereas for oxalic, ascorbic, and fumaric acids limits of detection were 255, 25, and 11 pmol, respectively. Repeatability values for the retention time and peak area were <5%, with the exception of ascorbic acid. Analyte recovery was between 92% and 110% in most cases, with the exception of oxalic (39-108%), 2-oxoglutaric (44-69%), and ascorbic (22-86%) acids, which may require specific extraction procedures and use of the corresponding (13)C-labeled organic acid as internal standards to improve accuracy. The method was applied to three types of plant materials: sugar beet leaf extracts, tomato xylem sap, and commercial orange juice.     

Determination of nonprotein amino acids and betaines in vegetable oils by flow injection triple-quadrupole tandem mass spectrometry: a screening method for the detection of adulterations of olive oils

A novel screening method using an automated flow injection electrospray ionization tandem mass spectrometry system is proposed for the simultaneous determination of five nonprotein amino acids (β-alanine, alloisoleucine, ornithine, citrulline, pyroglutamic acid) and three betaines (glycine betaine, trigonelline, proline betaine) after derivatization with butanolic HCl. MS/MS experiments were carried out in a triple-quadrupole instrument using multiple reaction monitoring mode in <2 min. The proposed method provided high fingerprinting power to identify the presence of five of the studied compounds in different types of vegetable oils (soybean, sunflower, corn, olive) with LODs at parts per billion levels. The method was validated, and different mixtures of extra virgin olive oil with seed oils were analyzed, achieving the typification for the detection of adulterations in extra virgin olive oils up to 2% w/w. The nonprotein amino acid ornithine was confirmed as a marker for adulteration in the olive oils analyzed.     

Analysis of neurotoxin 3-N-oxalyl-L-2,3-diaminopropionic acid and its alpha-isomer in Lathyrus sativus by high-performance liquid chromatography with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatization

A method was developed for the quantitative determination of the neurotoxic nonprotein amino acid, 3-N-oxalyl-L-2,3-diaminopropionic acid (beta-ODAP), and its nontoxic alpha-isomer, 2-N-oxalyl-L-2, 3-diaminopropionic acid (alpha-ODAP), in the plant samples of Lathyrus sativus after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) by reversed-phase high-performance liquid chromatography (HPLC). Hippuric acid was used as an internal standard. A linear response was recorded in the concentration rang 0.32-32 nmol with r > 0.999. The RP HPLC detection limit for both isomers is 1.8 ng. According to different experimental needs, a ternary gradient system can be used to determine toxin and other nonprotein amino acids. The RP HPLC method and a colorimetric method were compared for measuring ODAP.     

Antiangiogenic potential of three triterpenic acids in human liver cancer cells

Three triterpenic acids, oleanolic acid, ursolic acid and maslinic acid, at 2 or 4 μmol/L were used to study their antiangiogenic potential in human liver cancer Hep3B, Huh7 and HA22T cell lines. The effects of these compounds upon the level and/or expression of hypoxia-inducible factor (HIF)-1α, basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), interleukin (IL)-8, urokinase plasminogen activator (uPA), reactive oxygen species (ROS), nitric oxide (NO) and cell invasion and migration were examined. Results showed that these triterpenic acids at 4 μmol/L significantly suppressed HIF-1α expression in three cell lines (P < 0.05); and these compounds at test doses failed to affect bFGF expression (P > 0.05). Three triterpenic acids dose-dependently decreased production and expression of VEGF and IL-8, retained glutathione level, lowered ROS and NO levels, and declined cell invasion and migration in test cell lines (P < 0.05). These compounds also dose-dependently reduced uPA production and expression in Hep3B and Huh7 cell lines (P < 0.05); but these agents only at 4 μmol/L significantly suppressed uPA production and expression in HA22T cells (P < 0.05). These findings suggest that these triterpenic acids are potent antiangiogenic agents to retard invasion and migration in liver cancer cells.     

Simultaneous HPLC analysis of biogenic amines, amino acids, and ammonium ion as aminoenone derivatives in wine and beer samples

A method has been developed for the simultaneous analysis of biogenic amines, amino acids, and the ammonium ion in wine and beer. Aminoenones formed by the reaction of amino acids, biogenic amines, and the ammonium ion with the derivatization reagent diethyl ethoxymethylenemalonate are separated by HPLC. Reaction takes place in methanolic alkaline medium for 30 min in an ultrasonic bath. Further heating at 70 degrees C for 2 h produces complete degradation of excess derivatization reagent and byproducts. Comparison of the results of ammonium analysis and enzymatic analysis showed a good correlation (r = 0.953). The proposed analytical method has the following advantages: easy derivatization of wines and beers; quantification of 24 amino acids, nine biogenic amines, and the ammonium ion in a single injection; use of the photodiode array detector; complete degradation of excess derivatization reagent during sample preparation; and detection limits below 0.40 mg/L for amino acids and below 0.06 mg/L for biogenic amines.     

Organic acids from leaves,fruits, and rinds of Garcinia cowa

Organic acids in fresh leaves, fruits, and dried rinds of Garcinia cowa (G. cowa) were determined by high-performance liquid chromatography. Fresh leaves, fruits, and dried rinds were extracted with water at 120 degrees C for 20-30 min under 15 lbs/in(2) pressure. Also, dried rinds were extracted with solvents (acetone and methanol) using a Soxhlet extractor at 60 degrees C for 8 h each. The samples were injected to HPLC under gradient elution with 0.01 M phosphoric acid and methanol with a flow rate of 0.7 mL/min using UV detection at 210 nm. The major organic acid was found to be (-)-hydroxycitric acid present in leaves, fruits, and rinds to the extent of 1.7, 2.3, and 12.7%, respectively. (-)-Hydroxycitric acid lactone, and oxalic and citric acids are present in leaves, fruits, and rinds in minor quantities. This is the first report on the composition of organic acids from G. cowa.     

Capillary gas chromatographic analysis of amino acids by enantiomer labeling

The optical isomers of amino acids can be easily separated by gas chromatography using capillary columns coated with the chiral polysiloxane peptide, Chirasil-Val. Quantitative trace amino acid analysis in complex mixtures such as biological fluids, sea water, or protein hydrolysates can be achieved by enantiomer labeling: The D-amino acid enantiomers, which do not occur naturally, are added to the sample prior to analysis as internal standards. Because the D-enantiomers show the same physical and chemical properties as the natural L-enantiomers, they are ideal standard references. In routine analysis, the derivatization is achieved with a new automated derivatization robot. The D-standard serves as overall internal standard for the whole analytical procedure from sample enrichment to derivatization, chromatography, and response of the detector.     

Quantitative amino acid analysis of feedstuff hydrolysates by reverse phase liquid chromatography and conventional ion-exchange chromatography

Corn, soybean meal, and isolated soybean protein samples were acid-hydrolyzed and analyzed for amino acid content by reverse phase liquid chromatography (LC) and by conventional ion-exchange chromatography (IEC) using an amino acid analyzer. The former method employed pre-column derivatization with orthophthalaldehyde (OPTA)/ethanethiol and fluorescence detection. In the LC procedure, glycine and threonine were not resolved, and proline and cyst(e)ine were not detected. In general, amino acid values obtained by LC and IEC compared closely within and across feedstuffs, and both agreed well with published amino acid composition data. The notable exceptions were aspartic acid, glutamic acid, and alanine. Results of this study suggest that reverse phase LC with pre-column OPTA derivatization can be applied to accurately measure primary amino acids in individual feedstuffs.     

GC-MS determination of flavonoids and phenolic and benzoic acids in human plasma after consumption of cranberry juice

A GC-MS method was developed for the determination of various flavonoids and phenolic and benzoic acids in human plasma. The procedure involved the extraction of flavonoids and phenolic and benzoic acids with ethyl acetate, followed by the derivatization of the phenolic and benzoic compounds with BSTFA (N,O-bis(trimethylsilyl) trifluoroacetamide) + TMCS (trimethylchlorosilane) reagent. The trimethylsilyl derivatives formed were separated and quantitated using GC-MS. Twenty flavonoids and phenolic and benzoic compounds have been well separated in the spiked human plasma without any interference. The average recovery was 79.3%. Several phenolic acids such as o-hydroxybenzoic, p-hydroxyphenylacetic, 2,3-dihydroxybenzoic, 2,4-dihydroxybenzoic, ferulic, sinapic, and benzoic acid were identified and quantified in human plasma after consumption of a cranberry juice. This developed method provides a simple, specific, and sensitive technique for the simultaneous determination of flavonoids and phenolic and benzoic acids in human plasma and is suitable for bioavailability and pharmacokinetic studies.     

Mass spectral investigations on trichothecene mycotoxins. II. Detection and quantitation of macrocyclic trichothecenes by gas chromatography/negative ion chemical ionization mass spectrometry

A general, sensitive gas chromatographic/negative ion chemical ionization mass spectrometric (GC/NICIMS) method of analysis was developed for the detection and quantitation of several polar, thermally labile, toxic macrocyclic trichothecenes. The procedure involves the conversion of the molecules to their corresponding alcohols (verrucarols) by alkaline hydrolysis, followed by derivatization of the hydrolysate with heptafluorobutyrylimidazole and analysis by GC/MS technique under negative ion chemical ionization conditions. Nanogram (250 ng) quantities of several macrocyclic trichothecenes with different verrucarol and ester moieties were analyzed successfully with good precision by this procedure. The method was applicable for the accurate determination of at least low ppb levels of these macrocyclic trichothecenes in environmental samples, such as fungal products, fermentation broths, and plant samples. This is the first reported, well developed, sensitive, and applicable method for the detection and quantitation of these compounds in naturally occurring samples.     

Quantitative HPLC analysis of acids in Brazilian cachaças and various spirits using fluorescence detection of their 9-anthrylmethyl esters

An analytical procedure for the separation and quantification of 17 short-chain, medium-chain, and long-chain acids in cacha?as and various spirits has been developed involving C18 solid phase extraction, derivatization with 9-anthryldiazomethane, and reverse phase HPLC using fluorescence detection. The limit of detection was between 5 and 15 fmol, whereas the recovery of nonanoic acid as internal standard was >95%. Relative standard deviation values for reproducibility were between 0.09 and 20.4%, and repeatability was between 0.05 and 11.3%.     

Monitoring of the fermentation media of citric acid by the trimethylsilyl derivatives of the organic acids formed

In this approach, a derivatization method is described for monitoring of organic acids in fermentation media without any separation step. The aqueous phase of fermentation media was evaporated and heated in a silylation reagent to form trimethylsilyl (TMS) derivatives. The silylated compounds are analyzed by 29Si nuclear magnetic resonance (29Si NMR) and gas chromatography-mass spectrometry (GC-MS). 29Si NMR can qualitatively monitor the components produced in the Krebs cycle. Quantification of these compounds is investigated by using selected ion monitoring mode of mass spectrometry. In this mode, mass to charge (m/z) values of their [M - 15]+ ions, which are 465, 275, 247, 221, 335, 251, and 313 of TMS derivatives of citric, alpha-ketoglutaric, succinic, fumaric, l-malic, oxaloacetic, and palmitic (as an internal standard), acids, respectively, are used. The limit of detection and the linear working range for derivatized citric acid were found to be 0.1 mg L(-1) and 10-3 x 10(4) mg L(-1). The relative standard deviation of the method for five replicates was 2.1%. The average recovery efficiency for citric acid added to culture media was approximately 97.2%. Quantitative results of GC-MS are compared with those obtained by an ultraviolet-visible method.     

Determination of organic acids by high-performance liquid chromatography with electrochemical detection during wine brewing

Voltammetric determination of acids by means of the electrochemical reduction of quinone was applied to high-performance liquid chromatography (HPLC) with electrochemical detection (ED) for determining organic acids in fruit wines. A two-channel HPLC-ED system was fabricated by use of an ion-exclusion column and an electrochemical detector with a glassy carbon working electrode. Aqueous solution of 0.1 mM HClO(4) and ethanol containing 2-methyl-1,4-naphthoquinone served as a mobile phase and reagent solution, respectively. Determination of acetic, citric, lactic, malic, succinic, and tartaric acids was made by measuring the peak areas of the flow signals due to the reduction current of quinone caused by the eluted acids. The peak area was found to be linearly related to the acid amount ranging from 0.1 to 40 nmol per 20 microL injection. The present method was characterized by reproducibility with the simple and rapid procedure without derivatization of analytes. The method was shown as an effective means for following acid contents in fruit juices during fermentation with wine yeast.     

Modified 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (abts) method to measure antioxidant capacity of Selected small fruits and comparison to ferric reducing antioxidant power (FRAP) and 2,2'-diphenyl-1-picrylhydrazyl (DPPH) methods

The measurement of antioxidant capacity in fruits differs from that of other biological samples due to their low pH and very low lipophilic antioxidant capacity. In this report, we present a modified 2,2-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) method for fruits and compare its performance with the other commonly used antioxidant methods of 2,2'-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP). The antioxidant capacity and reaction kinetics of four phenolic compounds, two antioxidant standards, and five fruits were also investigated. The modified ABTS method prepared at a pH of 4.5 with sodium acetate buffer is highly stable and easily applied to fruit samples as compared to the standard (pH 7.4) version. The measured antioxidant capacity of samples varied with the assay method used, pH, and time of reaction. Traditional antioxidant standards (trolox, ascorbic acid) displayed stable, simple reaction kinetics, which allowed end point analysis with all of assays. Of the phenolic compounds examined, chlorogenic and caffeic acids exhibited the most complex reaction kinetics and reaction rates that precluded end point analysis while gallic acid and quercetin reached stable end points. All fruit extracts exhibited complex and varied kinetics and required long reaction times to approach an end point. Because the antioxidant capacity of fruit extracts is a function of the array of individual antioxidants present, accurate comparisons among fruit samples require that reaction times be standardized and of sufficient length to reach steady state conditions and that more than one assay be used to describe the total antioxidant activity of fruit samples.     

Influence of yeast strain and aging time on free amino acid changes in sparkling ciders

An analytical method for the determination of free amino acids in ciders is reported. It is based on high-performance liquid chromatography with an automatic precolumn derivatization with o-phthaldehyde and 3-mercaptopropionic acid and diode array detection. The method was applied to monitor the amino acids during second fermentation of sparkling ciders. This paper reports the influence of yeast strains and aging time on the amino acid composition of sparkling ciders. The application of principal component analysis enables the ciders to be differentiated on the basis of the two factors considered (yeast strain and aging time). The first principal component, which accounts for 58% of the total variance, achieved the separation according to aging time with serine, glycine, alanine, valine, ornithine, leucine, and lysine as the most important variables. The second principal component, accounting for 28% of the explained variance, is closely related to aspartic acid and asparagine and separates the samples according to the yeast strain.     

Phenolic acids in foods: an overview of analytical methodology

Phenolic acids are aromatic secondary plant metabolites, widely spread throughout the plant kingdom. Existing analytical methods for phenolic acids originated from interest in their biological roles as secondary metabolites and from their roles in food quality and their organoleptic properties. Recent interest in phenolic acids stems from their potential protective role, through ingestion of fruits and vegetables, against oxidative damage diseases (coronary heart disease, stroke, and cancers). High performance liquid chromatography (HPLC) as well as gas chromatography (GC) are the two separation techniques reviewed. Extraction from plant matrixes and cleavage reactions through hydrolysis (acidic, basic, and enzymatic) are discussed as are the derivatization reagents used in sample preparation for GC. Detection systems discussed include UV-Vis spectroscopy, mass spectrometry, electrochemical, and fluorometric detection. The most common tandem techniques are HPLC/UV and GC/MS, yet LC/MS is becoming more common. The masses and MS fragmentation patterns of phenolic acids are discussed and tabulated as are the UV absorption maxima.     

Capillary electrophoretic analysis of gamma-aminobutyric acid and alanine in tea with in-capillary derivatization and fluorescence detection

The aim of this study was to investigate an in-capillary derivatization capillary electrophoresis (CE) technique that was performed to determine the concentration of gamma-aminobutyric acid (GABA) and alanine (Ala) in tea after being derivatized with o-phthaldialdehyde/2-mercaptoethanol (OPA/2-ME) to form fluorescence-labeled products. The conditions of labeled derivatization and CE separation were optimized and then applied to real sample analysis. The labeled derivatization with 20 mM OPA and 26.67 mM 2-ME (mol ratio=0.75) at pH 10 offered the most sensitive detection, and the separation with 30 mM sodium tetraborate buffer (pH 10.0) under 21 kV achieved good selectivity within 14 min. The detections were linear in the range of 0.05-5 microM with correlation coefficients (R2) of 0.9995 and 0.9964 and with detection limits of 0.004 and 0.02 microM for GABA and Ala, respectively. The recoveries were 94.22% (3.58% RSD) and 93.54% (6.46% RSD) for five determinations of GABA and Ala, respectively. This method is a fast, convenient, sensitive, and eco-friendly way to determine the GABA and Ala in tea samples from different manufacturing processes.     

Liquid chromatographic determination of taurine in vitamin premix formulations

A liquid chromatographic (LC) method is described for the determination of taurine in vitamin and vitamin-mineral premix formulations. The method involves extraction of taurine with 0.1 M bicarbonate buffer, followed by precolumn derivatization with dansyl chloride and LC using fluorescence detection. 6-Aminocaproic acid is used as an internal standard. A reverse phase analytical column and a mobile phase of 0.1 M acetate buffer solution (pH 7.2)-acetonitrile (75 + 25) are used. Vitamins, minerals, and other excipients in the premix formulations do not interfere in the determination. The method is simple, precise, and accurate.     

Simultaneous determination of five plant growth regulators in fruits by modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) extraction and liquid chromatography-tandem mass spectrometry

An effective method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed and optimized to obtain a complete separation of five representative plant growth regulators (PGRs) [gibberellic acid, 2,4-dichlorophenoxyacetic acid (2,4-D), thidiazuron, forchlorfenuron, and paclobutrazol] in fruits. Extraction was performed with acetonitrile containing 0.1% (v/v) acetic acid, applying modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) methodology. LC-MS/MS conditions including composition of mobile phases and mass spectrometry (MS) conditions were evaluated to achieve the highest sensitivity in MS detection. All of the data acquisition was employed in the segmented multiple-reaction monitoring mode for the selected negative and positive transition ions. The octadecylsilyl (C18) dispersive solid-phase extraction (SPE) sorbent was found to provide the more satisfied recoveries than primary secondary amine (PSA) and graphitized carbon black (GCB) for five target PGRs. The optimized method allowed for recoveries of 76-112% for the five PGRs from fruit samples with relative standard deviation (RSD) values less than 10%. Limits of quantification (0.5-16.5 μg/kg) were lower than the maximum limit of residues established for PGRs. The results demonstrated that the developed LC-MS/MS and QuEChERS extraction method is highly effective for analyzing trace amounts of target PGRs in fruit samples. Finally, the method was successfully used to detect residual PGRs in Beijing, China, in 2010. The concentrations of 2,4-D (5.1-1503 μg/kg) and paclobutrazol (1-1381 μg/kg) found in orange and peach, respectively, suggesting that the use of these PGRs in these fruits should be regulated in China in the future.