Anion-exchange high-performance liquid chromatography with conductivity detection for the analysis of phytic acid in food

A sensitive method for the accurate determination of phytic acid in food samples is described. The proposed procedure involves the anion-exchange liquid chromatography with conductivity detection. Initially, two methods of determination of phytic acid were compared: absorptiometry and high-performance ion chromatography (HPIC) with chemically suppressed conductivity detector. Unlike most conventional methods involving precipitation by FeCl3, the simpler and more reliable HPIC assay avoids the numerous assumptions inherent in the iron precipitation and the accuracy is independent of the phytate content. The protocol was also applied to a survey of phytic acid concentration in some cereal, oil and legume seeds.     

Separation of phytic acid and other related inositol phosphates by high-performance ion chromatography and its applications

A high-performance anion-exchange chromatographic method was developed for the separation of phytic acid and other inositol phosphates (myo-inositol bis-, tris-, tetrakis-, and pentakisphosphates) with gradient elution and ultraviolet absorbance detection after post-column derivatization. With the acidic eluents, the combination of anion-exchange and ion suppression retention mechanisms led to the separation of 35 inositol phosphates (excluding enantiomers) into 27 peaks for the first time, and the retention behaviors of all myo-inositol bis- to hexakisphosphate isomers were studied. The whole separation procedure was completed within 65 min. Based on the investigations of nonenzymatic hydrolysis of phytic acid under different conditions by using this method, an in-house reference standard solution was produced, which can be used for method development. In addition, by applying this method to in vitro kinetic studies, at least one new enzymatic hydrolysis pathway of phytic acid was found, and one rule of enzymatic dephosphorylation of inositol phosphates (position effect) was proposed and another one (neighboring effect) was confirmed. The principle of the proposed identification approach for several inositol phosphate isomers based on hydrolysis products study will be applicable to other natural products analysis, for which standards are very expensive or not available.     

Preparation of samples for high-performance liquid chromatography of inositol phosphates.

A simple method is described for the removal of extraneous material from tissue extracts prior to anion-exchange high-performance liquid chromatography of inositol phosphates. Samples are prepared by extraction with trichloroacetic acid or perchloric acid followed by removal of the excess acid. The extracts are then passed through small Dowex-50 cation-exchange columns and eluted with water. Dowex-50 pretreatment removes most of the ultraviolet absorbing material and cations from the samples but does not alter the content of inositol phosphates. This treatment results in improved reliability of chromatography, especially with respect to weakly retained molecules such as adenosine 5'-phosphate and the isomers of inositol monophosphate. In addition, sample pretreatment improves the useful lifetime of the analytical anion-exchange columns.     

On-line post-column fluorescence detection for N-terminal tyrosine-containing peptides in high-performance liquid chromatography

A detection system based on on-line post-column fluorescence derivatization is described for the determination of N-terminal tyrosine-containing peptides by reversed-phase high-performance liquid chromatography. The peptides are automatically converted into fluorescent derivatives by reaction with hydroxylamine, cobalt (II) and borate after peptide separation on a reversed-phase column (TSKgel ODS-120T) followed by passage through an ultraviolet absorbance detector. The reaction system permits the fluorescence detection at 435 nm (emission) with excitation at 335 nm for N-terminal tyrosine-containing synthetic peptides in as little as picomole amounts. The facile fluorescence detection of N-terminal tyrosine-containing fragments produced from methionine-enkephalin by enzymatic degradation using a rat brain homogenate was achieved by comparison with the ultraviolet absorption detection at 215 nm.     

Scale-up potential of ion-pair high-performance liquid chromatography method to produce biologically active inositol phosphates

This study was undertaken to evaluate the possibility that an analytical ion-pair HPLC procedure used to determine phytic acid (IP6) and its degradation products (IP3-IP5) can be transformed to a preparative purification method. A commercial phytic acid (CPA) preparation was separated into its component fractions of IP3, IP4, IPS, and IP6 on two C18 columns (1.8 and 4.2 ml) using 51% methanol containing 0.6-1% tetrabutylammonium hydroxide as ion-pair reagent and 0-0.025 M formic acid (pH 4.3) as eluent at 1.7 and 3.0 cm/min linear velocity, respectively, and 40 degrees C. Elution was monitored at 40 degrees C by a refractive index detector. Reproducible separation of CPA into four well-resolved peaks on these columns was possible after optimizing method variables, particularly the concentration of ion-pair reagent in the injected sample (>1.5%). The same separations were obtained after CPA loads were scaled up 25 times on a steel column (15 cm x 19 mm I.D.), packed with Ethyl C2 sorbent (10 microm) and on a 25 cm x 21.2 mm I.D. C18 column, but at a reduced linear velocity to increase the resolution. Therefore, this optimization of separation not only is useful for analysis of phytic acid and its degradation products but also it provides key parameters for scale up for further fractionation and characterization.     

Determination of alkylamines by high-performance liquid chromatography with post-column fluorescence derivatization

A styrene/divinylbenzene polymer column and an amino column are compared for the non-aqueous separation of primary, secondary and tertiary alkylamines. Post-column derivatization with o-phthalaldehyde/2-mercaptoethanol is selective for primary amines and derivatization with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is selective for secondary amines after on-line masking of primary amines. This procedure can tolerate 0.4 M butylamine. The limit of detection is 18.5 mM for dioctylamine (with NBD-Cl) and 0.18 mM for decylamine and tetraethylenepentamine (with o-phthalaldehyde/2-mercaptoethanol).     

Body fluid analysis of a phosphonic acid angiotensin-converting enzyme inhibitor using high-performance liquid chromatography and post-column derivatization with o-phthalaldehyde

A method is described for the extraction of a phosphonic acid angiotensin-converting enzyme inhibitor from either urine or plasma, and subsequent quantitation using high-performance liquid chromatographic (HPLC) analysis and post-column o-phthalaldehyde reagent derivatization. The compound cannot be quantitatively extracted from the body fluids, but use of a fluorinated internal standard allowed for the computation of accurate results. With the use of an internal standard, excellent precision, linearity, and recovery were obtained for analyte response in both urine and plasma. In urine a working range of 0.2-10 micrograms/ml was found, with a limit of detection of 0.1 micrograms/ml. For plasma the working range was found to be 2-500 ng/ml, and the limit of detection was established as 1 ng/ml. Due to the non-polar character of the analyte at low pH values, it was possible to use novel extraction (solid-phase C8 column) and HPLC [poly(styrenedivinyl benzene) HPLC column] conditions to separate and quantitate the compound from plasma and urine.     

Assay for enkephalin-degrading peptidases in rat brain tissues by high-performance liquid chromatography with on-line post-column fluorescence detection

The activities of enkephalin-degrading peptidases such as enkephalinases A and B in rat brain tissues were simultaneously assayed by a high-performance liquid chromatographic method with fluorimetric detection with an automatic reaction system. Tyrosine and tyrosine-containing peptides produced enzymatically from the substrate, methionine-enkephaline, were separated by gradient elution on a reversed-phase column (TSK gel ODS-120T), and then converted into fluorescent derivatives for detection by reaction with hydroxylamine, cobalt(II) and borate reagents. The method permits the simple and sensitive detection of N-terminal tyrosine-containing fragments of the enkephalin peptide. The limits of detection are 5-20 pmol per assay tube for the N-terminal tyrosine-containing fragments. The enzyme activities in the regionally separated tissues were 54-191 pmol/min.mg protein for enkephalinase A and 79-153 pmol/min.mg protein for enkephalinase B, which were calculated from the formation of Tyr-Gly-Gly and Tyr-Gly, respectively, during the enzyme reaction.     

Determination of citrulline and homocitrulline by high-performance liquid chromatography with post-column derivatization

A high-performance liquid chromatographic method was developed for the determination of citrulline and homocitrulline using a post-column colorimetric reaction with o-phthaladehyde and N-(1-naphthyl)-ethylenediamine. Citrulline and homocitrulline were determined with no interferences from protein amino acids. The results show that the level of citrulline in the plasma of patients with uremia on intermittent hemodialysis is higher than that in healthy human plasma, and that homocitrulline is excreted into the urine of healthy adults.     

Determination of inositol polyphosphates from human T-lymphocyte cell lines by anion-exchange high-performance liquid chromatography and post-column derivatization.

The intracellular amounts of several inositol tris-, tetrakis- and pentakisphosphates and inositol hexakisphosphate were determined in resting and stimulated cells from human T-lymphocyte lines. The inositol polyphosphates were separated by anion-exchange high-performance liquid chromatography and were detected on-line by a recently developed post-column dye system. In the human T-lymphocyte cell line Jurkat, basal intracellular concentrations ranged between 25 +/- 10 pmol per 10(9) cells for inositol 1,4,5-trisphosphate to 6380 +/- 355 pmol per 10(9) cells for inositol hexakisphosphate. Similar basal concentrations were observed in the human T-lymphocyte cell line HPB.ALL, with the exception that inositol hexakisphosphate was approximately 665 +/- 10 pmol per 10(9) cells. Stimulation of the human T-lymphocyte cell line Jurkat via the T-cell receptor by a monoclonal antibody directed against the T-cell receptor-CD3 complex induced time-dependent changes in the intracellular concentrations of multiple inositol polyphosphate isomers, including inositol 1,3,4-trisphosphate, inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4,6-tetrakisphosphate, an as yet unidentified inositol tetrakisphosphate isomer, inositol 1,3,4,5,6-pentakisphosphate, inositol 1,2,3,4,6-pentakisphosphate and DL-inositol 1,2,4,5,6-pentakisphosphate. Inositol 1,4,5-trisphosphate increased only transiently after 5 min, whereas DL-inositol 1,4,5,6-tetrakisphosphate (determined as the enantiomeric mixture) increased after 20 min.     

Improved measurement of formaldehyde in water-soluble polymers by high-performance liquid chromatography coupled with post-column reaction detection

An improved methodology for the analysis of free formaldehyde in water-soluble polymers used for industrial water treatment is reported. Previously, derivatization prior to HPLC or colorimetric techniques has been used. The data generated by these approaches are suspect in that the derivatizing agent can react with the polymer or other sample components to produce high results. Post-column reaction derivatization is applied after separation of the free formaldehyde from the product interferences. The type of polymer product analyzed influences the choice of column(s). The degree of high bias of the commonly used 2,4-dinitrophenylhydrazine pre-column derivatization is reported and the results are compared to those with the post-column reaction for two polymer products. This method, being more selective, should be applicable to any polymer containing formaldehyde.     

Analysis of alkyl organoiodide mixtures by high-performance liquid chromatography using electrochemical detection with a post-column photochemical reactor

A method for the analysis of six alkyl organoiodides (iodomethane, iodoethane, 1-iodopropane, 1-iodobutane, 1-iodopentane, 1-iodohexane) commonly found in acetic acid process was developed. In this method the target analytes were determined by high-performance liquid chromatography (HPLC) using a post-column photochemical reactor with electrochemical detection (ED) in less than 30 min. HPLC was performed in ODS C18 reversed-phase column (5 microm, 250 x 4.6 mm I.D.) under isocratic conditions with methanol-0.067 M acetate buffer (70:30, v/v), pH 6.2 as mobile phase at flow-rate 1.1 ml/min. Alkyl organoiodides, which are electrochemically inactive, were made oxidizable at potential of 120 mV after post-column irradiation with low-pressure mercury lamp in a knitted PTFE tube. The photoreactor was placedin an aluminum housing full of nitrogen in order to prevent from the interference of oxygen. The detection limit for most analytes was of the order of 1-2 microg/l. The HPLC-ED method with a post-column photochemical reactor has good precision and linearity and can be readily applied to the routine determination of alkyl organoiodides in real acetic acid samples.     

Determination of nine pyrethroid insecticides by high-performance liquid chromatography with post-column photoderivatization and detection based on acetonitrile chemiluminescence

An HPLC method was developed to determine pyrethroids, including fenpropathrin, beta-cyfluthrin, lambda-cyhalothrin, deltamethrin, fenvalerate, permethrin, acrinathrin, tau-fluvalinate, and bifenthrin, by coupling HPLC, post-column irradiation with UV light and chemiluminescence detection of the resulting photoproducts. It is based on the observation that photolyzed pyrethroids take part in a chemiluminescent reaction in presence of K3Fe(CN)6 and NaOH, whose signal increases with the percentage of acetonitrile in the reaction medium. As the yield of the photoderivatization process and the chemiluminescent signals depend on the percentage of acetonitrile, the chromatographic column (a Gemini C18, Phenomenex 150 mm x 4.6 mm, 5 microm particle size) was chosen with the aim of using high percentages of this organic solvent in the mobile phase. Previous studies showed that the rate of the chemiluminescent reaction was very fast. Therefore, a modification was carried out in the detector in order to mix the analytes and reactives as near as possible to the measure cell. The optimised method was validated with respect to linearity, precision, limits of detection and quantification accuracy. Under the optimised conditions, linear working range extends three orders of magnitude with the relative standard deviation on intra-day precision below 10% and detection limits between 0.013 and 0.049 microg mL(-1), according to the compound. The proposed method has been successfully applied to the determination of pyrethroids in tomato with good results.     

Direct, sensitive and selective detection of free fatty acids by high-performance liquid chromatography with post-column ion-pair extraction and absorbance detection

Free fatty acids (C8-C18) are separated by reversed-phase liquid chromatography and detected using a simple post-column dynamic extraction system in which the acids are extracted as ion pairs with chloroform from the aqueous acetonitrile (gradient: 79-99% acetonitrile) mobile phase after the post-column addition of aqueous Methylene Blue solution. The chloroform phase containing the ion pairs is monitored with an absorbance detector at 651 nm. The detection limits ranged from 26 to 83 ng, depending upon the acid, with coefficients of variation of 1.2-14%. Application of the method to butter and margarine samples permitted detection of free fatty acids down to 35 ppm and in orange juice, down to 0.5 ppm using only an organic solvent extraction without further sample clean-up for isolation of the fatty acids.     

Novel quartz flow-cell as a post-column photochemical reactor for high-performance liquid chromatography

The construction of a new post-column photochemical reactor with quartz flow cells in series for high-performance liquid chromatography (HPLC) is described. The performance of the new reactor was compared with a conventional open tubular PTFE coil reactor. The sensitivity, accuracy and precision obtained with both reactors are comparable. The new reactor has the obvious advantages of smaller cell volume as well as inertness and resistance to not only light and heat produced by the UV lamp, but also to organic solvents in the mobile phases, which results in greatly improved durability, reduced peak broadening and shorter chromatographic run times. Application of the new reactor to the fluorescence detection of DU-6859a, a new fluoroquinolone antimicrobial agent, in human serum is reported.