High-performance liquid chromatographic determination of histamine in biological samples after derivation with o-phthalaldehyde

A high-performance liquid chromatographic (HPLC) method for the determination of histamine in tissues, based on precolumn derivation with o-phthalaldehyde, is described. Trichloroacetic acid extracts of rat brain, but not of rat stomach or of rat peritoneal mast cells, had to be cleaned-up by a chromatographic step before HPLC. The extracts were allowed to react with o-phthalaldehyde at pH 12.5 and -20 degrees C for 12 h, followed by acidification to pH 2.0. HPLC was performed on a reverse-phase column with isocratic elution using sulfuric acid in methanol as solvent system. A fluorescence detection system was used; excitation was set at 353 nm and emission was read at 451 nm. One chromatographic run was completed in 20 min. The detection limit with the conventional procedure was 1.5 ng histamine per sample, with a scaled-down procedure it was 250 pg per sample. With extracts of rat gastric mucosa the within-run variation was 2.7% and the day-to-day variation 4.5%.     

Determination of cholesterol using o-phthalaldehyde

A simple, rapid method for the determination of cholesterol in plasma and tissue using o-phthalaldehyde is presented. Comparison of this method with the FeCl(3) method gave identical results. However, the o-phthalaldehyde determination is three times more sensitive than the FeCl(3) determination (molar extinction coefficients of 11,610 and 33,440 for FeCl(3) and o-phthalaldehyde, respectively), it takes less time to complete, and the color developed is more stable. The o-phthalaldehyde method can be used to assay free and esterified cholesterol directly after thin-layer chromatographic separation.     

The differential reaction of histamine and N tau-methylhistamine with Pauly's diazo reagent: application to assay of histamine N-methyltransferase activity

A simple nonradioisotopic fluorescent method for assay of histamine N-methyltransferase (HMT) activity was developed. After termination of the HMT reaction, the remaining excess substrate, histamine, was degraded by Pauly 's diazo reagent, whereas the product, N tau-methylhistamine (N- MeHA ), was not degraded by the reagent. Then the mixture was applied to high-performance liquid chromatography under conditions in which N- MeHA was not separated from histamine, and N- MeHA was measured fluorometrically by condensation with o-phthalaldehyde. The method would be convenient for measurement of HMT activity during enzyme purification.     

Distribution of histamine in the thallus ofFurcellaria lumbricalis

Histamine occurs in all regions of the thallus of male, female and tetrasporophyte plants ofFurcellaria lumbricalis (Huds.) Lamour. The mean values varied from 60 μg to 500 μg (g fresh weight)^?1. There were few consistent differences either between the same region of different types of plant or different regions of the thallus of one type of plant. Fluorescence staining of sections of the thalli ofF. lumbricalis, with o-phthalaldehyde, revealed that cells of the cortex and medulla have histamine associated with them. The histamine appears to be intracellular since only intact cells exhibit fluorescence. Cells in sections of female plants ofPolyides rotundus (Huds.) Grev., which do not contain histamine, do not exhibit fluorescence dependent upon o-phthalaldehyde.     

Stain for histamine in mast cells in fixed tissue

Summary A technique is described for staining histamine in mast cells of tissues fixed in alcohol. The method employs cold ethanol fixation, xylene clearing, paraffin embedding, and staining of the sections with 1% o-phthalaldehyde in ethyl benzene in a humid chamber. This study was supported by a grant from the John A. Hartford Foundation.     

High-performance liquid chromatographic method for the evaluation of possible interferences in basophil-histamine release measurements.

Basophil activation studies commonly rely on the measurement of histamine following extraction and condensation to o-phthalaldehyde (OPT) as a quantitative measurement of degranulation. Specificity has long been recognized as a problem with this method. We have described a new high-performance liquid chromatographic method that allows for both a qualitative and a quantitative check of the purity of the OPT-histamine adduct. This method was sensitive (limit of detection = 2.55 pmol) and linear over a wide range (5 to 1000 ng/ml).     

A Highly Sensitive Assay for Histamine Using Ion-Pair HPLC Coupled with Postcolumn Fluorescent Derivatization: Its Application to Biological Specimens

A simple and highly sensitive method for the determination of histamine (HA) was developed using ion-pair, reversed-phase HPLC coupled with postcolumn o-phthalaldehyde derivatization fluorometry, and it was applied to the unpurified extracts of human and rat plasma, and brains of rats and mice. The HA concentrations both in the plasma and brains determined by the present method were well consistent with the values obtained by cation-exchange HPLC with postcolumn fluorescent derivatization currently in use. The present method was more advantageous than the assay using cation-exchange HPLC: (1) it was three to four times more sensitive (the detection limit was 0.5 pg of HA), and (2) it enabled the measurement of HA in samples containing (R)alpha-methylhistamine, a potent and specific H3-receptor agonist, which could not be separated from HA by cation-exchange chromatography. Using the present method coupled with intracerebral microdialysis, we found in the rat hypothalamus that (R)alpha-methylhistamine (5 mg/kg i.p.) markedly decreased the extracellular concentration of HA with a maximal effect (83% reduction) during 30-60 min after injection, suggesting that most of HA in the microdialysate fraction is neuronal in origin.     

Use of water-soluble 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide for the fluorescent determination of uronic acids and carboxylic acids

Reaction between glucuronic acid and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was monitored by the o-phthalaldehyde (OPA) method, which was developed for the fluorescent assay of compounds containing an amino group. About 1 nmol of glucuronic acid was detected by this method. This EDC-OPA method was effective in detecting not only acidic sugar but also carboxylic acid. Although the sensitivity of the EDC-OPA method was somewhat lower than that of amino acid determination by OPA, a very simple and convenient assay was attained for compounds containing a carboxyl group.     

A reliable and sensitive method for the simultaneous determination of dopamine, noradrenaline, 5-hydroxytryptamine and 5-hydroxy-indolacetic acid in small brain samples

A sensitive and reliable fluorometric method for the simultaneous determination of dopamine, noradrenaline, 5-hydroxytryptamine and 5-hydroxy-indol acetic acid in small samples of brain tissues is described. The procedure is based on solvent extraction; catecholamines are oxidized by the Chang's method and 5-hydroxytryptamine and 5-hydroxy-indol acetic acid determined by reaction with o-phthalaldehyde, alpha-methyl-p-tyrosine causes a negligible interference with the procedure. Results of determination of these amines in different brain areas are reported.     

Kinetics of protease hydrolysis of extended peptide substrates: measurement by flow-injection analysis

A flow-injection analysis (FIA) system was developed to study the enzyme-catalyzed hydrolysis of synthetic peptides, each of which contained one scissile bond. The concentrations of alpha-amino groups in reactions mixtures were determined by FIA with o-phthalaldehyde as a fluorescence reagent. The method allows a rapid, precise, and sensitive determination of kinetic constants for proteases acting on extended peptide substrates.     

Determination of boronophenylalanine in biological samples using precolumn o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography

A reversed-phase high-performance liquid chromatographic method for the detection of boronophenylalanine is described. Determination was obtained by precolumn reaction of o-phthalaldehyde with a mixture of standard amino acids containing boronophenylalanine and separating the corresponding o-phthalaldehyde derivatives, using a Kromasil C-18, 250 x 4.6 mm, 5-microm particle size column, a step gradient with two buffers, a flow rate of 1.2 ml/min, a column temperature of 23 degrees C, and fluorimetric detection (excitation and emission wavelengths of 330 and 430 nm, respectively). The use of such a method for assaying boronophenylalanine in biological samples was tested in neutralized perchloric acid blood and cerebral tissue extracts of rats treated with intracarotid administration of 300 mg/kg of body weight boronophenylalanine. Results of these experiments showed that the present HPLC method represents a valid alternative to currently available analytical techniques for assaying boronophenylalanine based on boron determination in terms of reproducibility, recovery, or sensitivity. Therefore, it is suggested that the present method may routinely be used in all preclinical and clinical studies in which quantification of circulating and tissue concentrations of boronophenylalanine is critical for the application of boron neutron capture therapy.     

Validation of a reversed-phase HPLC method for quantitative amino acid analysis.

A semi-automated method for amino acid derivatization and analysis has been validated for use in analysis of protein biopharmaceuticals. The method includes protein hydrolysis, o-phthalaldehyde derivatization, and reversed-phase high-performance liquid chromatography analysis in a general-purpose UV-visible high-performance liquid chromatography system. Amino-acid derivatization is performed automatically by the high-performance liquid chromatography autosampler right before injection. The required validation parameters, i.e., specificity, linearity, accuracy, precision, limit of detection, and limit of quantification, were studied for bovine serum albumin and for a recombinant human Fab fragment. The method can be employed as an absolute quantification method for determination of extinction coefficients of recombinant proteins.     

Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids

A simple and rapid high performance liquid chromatographic method for the determination of plasma amino acids was developed. The method uses minimal sample volume and automated online precolumn derivitization of amino acids with o-phthalaldehyde and fluorescent detection. Amino acids are separated by a simplified gradient without column heating. The assay is linear from 5 to 1000 micromol/L for all amino acids. Recovery of amino acids was between 91 and 108%, intra-assay coefficient of variation (CV) was 1-7%, and inter-assay CV was 2-12%. The simple sample preparation and minimal sample volume make the method useful for the quantitation of amino acids in both patient and experimental animal samples.     

A new spectrofluorimetric method for determination of trace amounts histamine in human urine and serum

A new spectrofluorimetric method was developed for the determination of trace amounts of histamine in human urine and serum samples. In NaAc–HAc buffer solution of pH 4.0, histamine can react with the acetylacetone–formaldehyde system to produce a fluorescent derivative which emits yellow‐green fluorescence at 476 nm, according to the Hantzsch reaction, and the enhanced fluorescence intensity is in proportion to the concentration of histamine. Optimum conditions for the determination of histamine were also investigated. The dynamic range and detection limit for the determination of histamine is 5.96 × 10^–8–1.50 × 10^–5 mol/L and 4.35 × 10^–8mol/L, respectively. This method is practical and can be successfully applied to determination of histamine in human urine and serum samples. A proposal of the reaction pathway is suggested. Copyright © 2009 John Wiley & Sons, Ltd.     

Fluorometric assay for polyamines in urine and tissues using electrophoresis on Titan III cellulose acetate

A simple, rapid assay method for polyamines (putrescine, spermidine, and spermine) in urine and tissues using electrophoresis on Titan III cellulose acetate was developed. In this procedure, polyamines are preliminarily extracted from a hydrolysate of urine or from supernatants of tissue homogenates by use of a Bio-Rex 70 minicolumn. After electrophoretic separation, polyamines are fluorometrically detected by the reaction with o-phthalaldehyde and 2-mercaptoethanol. Six extracts and two external standards of polyamines can be separated and detected in 11 min on a cellulose acetate strip. This method permits the determination of polyamines in a range of 0.1 mM (25 pmol) to 1.0 mM (250 pmol).     

High-performance liquid chromatographic method for the determination of histamine and 1-methylhistamine in biological buffers

A method for the determination of histamine and its catabolite 1-methylhistamine (1-MH) was developed, using HPLC with fluorescence detection. Derivatization of both compounds occurred on-column with o-phthaldialdehyde dissolved in an alkaline borate buffer, followed by separation on a reversed phase C18 column. Histamine and 1-MH could be detected with comparable sensitivity (limit of quantification, 50 nM). The method was proven suitable to investigate catabolism of histamine by epithelia of pig colon. The method should be useful in research on histamine metabolism.     

Enzymatic assay of histamine by amperometric detection of H2O2 with a peroxidase-based sensor

A method for an enzymatic assay of histamine by using histamine oxidase from Arthrobacter globiformis in combination with amperometric determination of H2O2 is described. Histamine could be quantified at a level as low as 10(-7) M. The assay is adaptable to determine histamine in food samples including tuna fish with good sensitivity and selectivity.     

A direct and sensitive determination of histamine in acid-deproteinized biological samples by high-performance liquid chromatography

A convenient method for the routine measurement of histamine (HA) in biological samples was developed. This method does not require any preliminary purification or concentration of HA, and features high sensitivity, specificity, and reliability. The method consists of the direct application of the acid-deproteinized sample to high-performance liquid chromatography on a sulfonated polystyrene column with detection by means of a postcolumn fluorogenic reaction with o-phthaladehyde. The detection limit was found to be 0.1 pmol (signal-to-noise ratio = 3). The coefficient of variation for measurements of 10 pmol of standard histamine was 1.1%. Each chromatography takes only 10 min and therefore more than 50 samples can be measured in a day. The high sensitivity of the method allows it to be applied even to samples of very low HA concentration such as human plasma without any procedure for concentration of the sample, and further, only 0.1 ml of the sample is necessary for determination. The method was applied to compare the HA levels of the whole blood and plasma of man and various animals. Applications of the method to the supernatant of rat peritoneal mast cell incubates and to extracts of mouse brain and stomach are also described.     

An apparatus for continuous analysis of protease-catalyzed acyl transfer reactions

An apparatus that allows continuous analysis of protease-catalyzed acyl transfer reactions is described. Hydrolysis reaction is assayed using automatic titration. A continuous determination of amino group concentration by reaction with o-phthalaldehyde gives the rate of peptide bond formation. The apparatus allows the determination of the partition constant for the nucleophile at various nucleophile concentrations from one run.     

Liquid chromatography method for detecting native fluorescent bioamines in urine using post-column derivatization and intramolecular FRET detection

Liquid chromatography (LC) with fluorescence detection is described for simultaneous determination of native fluorescent bioamines (indoleamines and catecholamines). This is based on intramolecular fluorescence resonance energy transfer (FRET) in an LC system following post-column derivatization of native fluorescent bioamines' amino groups with o-phthalaldehyde (OPA). OPA fluorescence was achieved through an intramolecular FRET process when the molecules were excited at maximum excitation wavelength of the native fluorescent bioamines. Bioamines separated by reversed-phase LC on ODS column were derivatized with OPA and 2-mercaptoethanol. This method provides sufficient selectivity and sensitivity for the determination of normetanephrine, dopamine, tyrosine, 5-hydroxytryptamine, tryptamine, and tryptophan in healthy human urine without prior sample purification.