Separation and Quantitation of O-Phthalaldehyde Derivatives of Taurine and Related Compounds in a High Performance Liquid Chromatography (HPLC) System

Abstract

A sensitive specific assay for taurine using high performance liquid chromatograpy and fluorescence measurement is described. The method employs precolumn derivatization with o-phthalaldehyde in the presence of ethanethiol. Taurine is clearly separated from other amino acids including its precursors hypotaurine and cysteine sulfinic acid. The fluorescence peak height is linear between 1 and 100 picomoles of taurine. There is clear separation of taurine from a contaminant of other taurine assays, α-glycerophosphoryl ethanolamine.     

High Performance Liquid Chromatographic Separation and Fluorescent Measurement of Taurine,a Key Amino Acid

Abstract

A rapid, sensitive method for the analysis of taurine in oyster hemolymph (blood) has been developed. Highly fluorescent, substituted isoindoles formed by the reaction of taurine and other amino acids with o-phthaldialdehyde/ethanethiol reagent were separated on a reverse phase, high performance liquid chromatographic column. It was necessary to carefully control the concentration of the sodium ion in the phosphate buffer in order to maintain an adequate resolution of both taurine and tyrosine from β-alanine and arginine. Calibration plots of the fluorescent taurine derivative were linear over 2.5 orders of magnitude with a limit of detection of 0.10 nanomoles per ml of oyster hemolymph.     

Separation of Orthophthalaldehyde/Ethanethiol Derivatives of Taurine and Closely Eluting Amino Acids by High Performance Liquid Chromatography

Abstract

In studies of the reverse phase, HPLC analysis of amino acids employing precolumn derivatization with o-phthalaldehyde and ethanethiol, it was shown that α-amino-n-butyric acid, β-amino-isobutyric acid and taurine coeluted in the acetonitrile/aqueous phosphate solvent system. By using a ternary solvent system of acetonitrile/tetrahydrofuran/aqueous phosphate buffer and efficient 5- and 10-μm octadecylsilane packings, the co-elution problem has been resolved. This modified chromatographic system is now being used to quantitatively determine taurine and other closely eluting amino acids in a variety of physiological fluids in order to clarify the role of taurine in human development.     

A Method for the Measurement of total Drug Convertible to Cysteamine: Application to Pharmacokinetic Experiments with Ethiofos

Abstract

An analytical method has been developed for the quantification of cysteamine (2-aminoethanethiol) in plasma. A reductive sample pretreatment is used to convert disulfide-bound cysteamine to the free thiol which is subsequently separated by HPLC and detected by electrochemical detection (LCEC). The method was developed to follow drug disposition after administration of ethiofos (WR-2721, S-2-(3-aminopropylamino) ethyl phosphorothioic acid) and WR-1065 (2-(3-aminopropylamino)ethanethiol). Standard calibration curves were linear over the range 0.01- to 25.0 /μg/mL (0.130-324 μM) and a minimum detectable quantity of 0.01 μg/mL was calculated at a signal-to-noise ratio of 3. Assay precision over the same range averaged 2% (coefficient of variation) and relative recovery, used as a measure of accuracy, was 100Z. Stability of cysteamine in plasma, relative to an internal standard (2-aminopropanethiol, WR-186) was good; stored samples were found to contain an average of 94.6 ± 10.4z of the original cysteamine concentration following 28 days at -75 °C.

This method was successfully applied in dosing experiments with rhesus monkeys in which ethiofos was administered into the cephalic vein (IV), portal vein (P) and duodenum. In IV experiments total VR-1065 plasma concentrations greatly exceeded total cysteamine concentrations. While results indicate formation of free and bound cysteamine is not a major metabolic pathway of ments total VR-1065 plasma concentrations greatly exceeded total cysteamine concentrations. While results indicate formation of free and bound cysteamine is not a major metabolic pathway of ethiofos, increased levels of bound cysteamine were observed.     

Determination of Glutamine and Asparagine by Isocratic Elution Reverse Phase Liquid Chromatography with Fluorescent Detection

Abstract

A method was developed specifically for the determination of glutamine and asparagine in the presence or absence of other amino acids. The amino acids were derivatized by o-phthalaldehyde/ 2-mercaptoethanol and separated by isocratic elution with a mobile phase consisting of acetonitrile and sodium acetate buffer. An application of the method for the analysis of glutamine and asparagine in the enzymatic hydrolysate of cottonseed protein is described.     

Assay of N-Propylajmaline in Blood Plasma by Ion-Pair Liquid-Liquid Chromatography

Abstract

A sensitive method for assay of N-propylajmaline (prajmaline) in human plasma is described. The quaternary ammonium compound exists as a pair of stereoisomers, which are isolated and separated by ion-pair liquid-liquid chromatography on microporous silica particles. An aqueous solution containing perchloric acid and sodium perchlorate is used as stationary phase and a mixture of butanol, dichloroethane and hexane as mobile phase. The procedure involves ion-pair extraction from plasma and evaporation prior to the chromatographic separation. Selective detection is achieved by using a fluorescence detector. The method allows assay of concentrations down to 10 pmol of the two forms of prajmaline in 1 ml of plasma with a relative standard deviation below 5 %.     

Radioimmunoassay of Triazolam

Abstract

Two radioimmunoassay (RIA) assay methods (I, II) have been developed for triazolam (T, U-33,030) in serum and plasma. The parameters of equilibration time, serum blank, antibody specificity, extraction efficiency, and drug-binding to glass were studied. Of the various triazolam analogs tested for cross reactivity, only the hydroxy metabolites interfered significantly. At the levels normally found in plasma or serum, a background blank was encountered from constituents such as fatty acids, lysolecithin, lecithin, and cholesterol. However, serum or plasma samples could be analyzed with (I) by constructing standard curves in which blank serum from the same subject was used. An alternate method (II) was found which simultaneously extracted and precipitated the interferences. Both methods could be employed for analysis of plasma or serum samples. However, II detects T metabolites less efficiently than I. The within day and between day coefficients of variation for method I were found to be 5.7% and 3.9% respectively at the 8 ng/ml level. Method I is suitable for measuring large numbers of samples where blank subject serum can be obtained and where detection of T metabolites in addition to T would not present a problem.     

Simple, rapid and sensitive determination of plasma taurine by high-performance liquid chromatography using pre-column derivative formation with fluorescamine.

A simple, rapid and sensitive method for the determination of plasma taurine by high-performance liquid chromatography in the isocratic mode has been developed. The deproteinized plasma was treated with fluorescamine. These derivatives were separated on a LiChrospher 100 RP-8 column within 15 min. The detection limit for taurine was 0.2 microM. The plasma taurine contents of yellowtail fish, Seriola quinqueradiata, beef cattle, dairy cows and chickens were determined to be 125 +/- 54, 5.6 +/- 1.4, 2.2 +/- 0.7 and 20.0 +/- 9.6 micrograms/ml, respectively.     

Fractionation into Components of a Mixture of Acidic Ninhydrin-Positive Compounds of Mouse Brain Extracts with Thin-Layer and Ion-Exchange Chromatography

Abstract

A mixture of more than ten acidic or highly ionized ninhydrin-positive compounds of mouse brain extracts, occurring in ion-exchange chromatography from the beginning up to aspartic acid, was analyzed using ion-exchange and thin-layer chromatography standardized with known substances. These formed four peak groups in the chromatogram of an automatic amino acid analyzer. The first group contained cysteic acid, cysteine-sulfinic acid and phosphoserine, which could be separated from each others only by thin-layer chromatography. Only phosphoserine could be identified in the brain extract (about 0.14 mmol/kg brain wet weight), however. Taurine (4.1 mmol/kg) and phosphoethanolamine (1.05 mmol/kg) in the second group could be satisfactorily separated from each others after the hydrolysis of glycerylphosphoethanolamine (about 0.6 mmol/kg) and certain acidic peptides with 6 mol/1 HCl. Hypotaurine (0.03 mmol/kg) and urea (6.6 mmol/kg) were completely overlapped in the third peak, but urea was decomposed in the hydrolysis with 6 mol/1 HCl. The fourth group consisted of aspartic acid (2.1 mmol/kg). A number of low-molecular weight peptides also appeared in the chromatograms, above all in the phosphoserine and taurine peaks, but they were eliminated by the hydrolysis. They contained, however, some of the above-mentioned critical amino acids (phosphoserine, taurine and aspartic acid).     

Determination of neurotransmitter amino acids in mouse central nervous system by CE–LIF

An MEKC method with LIF detection has been developed for the determination of seven neurotransmitter amino acids (NAAs) using 1,3,5,7‐tetramethyl‐8‐(N‐hydroxysuccinimidyl butyric ester)difluoroboradiaza‐S‐indacene as the labeling reagent. After derivatization at room temperature for 30 min, the seven target NAAs including glycine, alanine, γ‐aminobutyric acid, taurine, glutamine, glutamic acid, and aspartic acid were separated in running buffer, which consisted of 70 mM pH 4.00 H₃PO₄/Na₃PO₄ buffer, 5.5 mM cetyltrimethyl ammonium bromide and 20% v/v acetonitrile within 17 min. The LODs were 2 ～ 14 × 10^?10 M without interference from other coexisting amino acids. The proposed method has been applied to the analysis of NAAs in the central nervous systems of healthy mice and those with Alzheimer's disease with recoveries of 92–104%.     

Development of a Specific Analytical Method for Cis-Dichlorodiammineplatinum (II) in Plasma

Abstract

An analytical method has been developed to specifically monitor cis-dichlorodiammineplatinum (II) in plasma. The drug is separated from plasma protein and other macromolecular species by centrifugal ultrafiltration, which removes components with molecular weight greater than 50,000. The ultrafiltrate was then fractionated by HPLC. Mixtures were chromatographed on a strong anion exchange column and the eluent monitored spectrophotometrically (at concentrations of analyte exceeding 1μg/ml) or by atomic absorption spectrometry. Using the latter readout system, the detected limit for the analyte was 4 0 ng/ml of plasma at the 2s level and the detector output was linearly related to the drug concentration over the range 40–1000 ng/ml. The method appears to specifically respond to parent drug and is able to differentiate between it and other platinum species (metabolites or breakdown products) which may be present in the sample.     

HPLC Analysis of Nonionic Sufactants. V. Ethoxylated Fatty Acids

Abstract

High performance liquid chromatography technique was used in order to achieve separation and identification of product composition of nonionic surfactants of ethoxylated fatty acids.

Lichrosorb SI-60 (10μm) column, under gradient elution of mixture of isopropanol, methanol and n-hexane (50°C) and UV detector at 220 nm, were used for best separation of ethylene oxide (EO) adducts of fatty acids consisting of up to 20 EO units.

No derivatization of the compounds was needed. An improved baseline, in spite of gradient elution, was achieved by adding negligible amounts of anthracene to the eluents.

Brominated ethoxylated fatty acids resulting from addition of bromine to the double bond of the hydrophobic chain were also separated without a need for change in elution conditions or derivatization.     

Simultaneous determination of thirteen substances related to NAFLD in mouse brain tissue using 3-aminobutyric acid as internal standard by HPLC–FLD

Disorders of certain branched-chain amino acids may be associated with the occurrence and development of non-alcoholic fatty liver disease. Measurement of related branched-chain amino acid levels could provide a reference for the clinical and scientific research of the non-alcoholic fatty liver disease. An established HPLC–FLD method was used to quantify aspartic acid, glutamate, glutamine, glycine, taurine, tyrosine, 4-amino butanoic acid, tryptophan, methionine, valine, phenylalanine, isoleucine and leucine in mouse brain tissue. Brain tissue samples mixed with internal standard (3-aminobutyric acid) were processed, then derivatized with 2-O-phthaldialdehyde, and finally separated on an ODS2 column through gradient elution at a flow rate of 1.0 ml·min⁻¹. The excitation and emission wavelengths were set at 340 and 455 nm, respectively. The mobile phase A was 100% methanol and the mobile phase B consisted of 30 mmol·L⁻¹ sodium acetate (pH 6.8). The injection volume was 20 μl and the single run time was 45 min. Several parameters, accuracy, precision, and stability, were verified and the results showed the established method had good sensitivity and resolution for all of the 13 compounds and internal standard in mouse brain.     

Separation and Determination of 2,5-Dimethoxy-4-Methylamphetamine Enantiomers in Plasma by High-Performance Liquid Chromatography

Abstract

A method for quantitation of 2,5-dimethoxy-4-methylamphetamine (DOM) enantiomers in plasma by high-performance liquid chromatography (HPLC) is described, d- and l-DOM were readily converted to the amides by condensation with a newly developed chiral reagent, succinimidyl ester of l-α-methoxy-α-methyl-1-naphthaleneacetic acid. The yielded diastereomers were separated on the μPorasil column with cyclohexane/ethyl acetate (3:1) exhibiting satisfactory k' and R values. The clean-up procedure by use of Sep-pak C₁₈ and carboxymethyl Sephadex LH-20 (CM-LH-20) proved to be effective for determination of the drug in biological fluids by HPLC. The plasma levels of d- and l-DOM after administration of the racemate to the rabbit were determined by the method thus established.     

A Rapid Determination of Taurine in Human Plasma by LC

Taurine is an amino acid which is not incorporated into proteins but found in the cytosol of many mammalian cells, in high concentrations (2–30 mM). Increase in plasma taurine concentration has already been reported after surgical trauma, X-radiation, muscle necrosis, carbon tetrachloride-induced liver damage, and paracetamol overdose. Plasma taurine concentration was measured using LC with fluorescence detection following derivatization by o-phtalaldehyde plus 3-mercapto-propionic acid and α-aminobutyric acid as internal standard. Under these conditions the retention time of taurine was 10 min. This method was sensitive enough, to quantify 150 pg mL^?1 and detect 50 pg mL^?1 of taurine ranging normally between 65 and 179 mmol L^?1 (8–22 μg mL^?1). The validated method allowed simple determination of human plasma taurine in pharmacokinetic and biomarker studies.     

Studies on the interaction of aquacobalamin with cysteinesulfinic and cysteic acids,hypotaurine and taurine

abstract

This study reports results of kinetic and mechanistic studies on the reactions of aquacobalamin (H₂OCbl⁺) with oxo-derivatives of cysteine, that is, cysteinesulfinic and cysteic acids, hypotaurine and taurine. The reactions with cysteinesulfinic acid and hypotaurine result in formation of S-bound complexes, whereas cysteic acid and taurine are coordinated with aquacobalamin via amino-group. Reaction of taurine with H₂OCbl⁺ produces a substantially more stable complex than with cysteic acid, whereas the affinity of cysteinesulfinic acid toward aquacobalamin is slightly higher than that of hypotaurine.     

9-Bromomethylacridine a Novel Fluorescent Labeling Reagent of Carboxylic Group for HPLC

Abstract

9-Bromomethylacridine (9-Br · Ma) gave blue fluorescent 9-acridinylmethyl (9-AM) esters by the reaction with carboxylic acids. The reactions were performed simply by mixing the 9-Br · Ma, tetraethylammonium carbonate (TEA C) and carboxylic acids in N,N-dimethylformamide at room temperature. The 9-AM derivatives of fifteen kinds of fatty acids were separated and detected with high performance liquid chromatography. By this method, fatty acids could be determined at levels down to either pmol order by fluorometry or 10 pmol order by UV method. After saponification, those in triacylglycerols were also determined. When four kinds of fatty acids were added to a butter, their recoveries were 92–101%. The coefficient of variation of myristate as a representative was 2.5% (42.8 pmol, n=6).     

Thin-Layer Chromatography of Bile Alcohols,Bile Acids and Conjugated Bile Acids

Abstract

Thin-layer chromatography of bile alcohols, bile acids and bile acid conjugates has been reviewed. Particular emphasis has been placed on the separation of the glycine and taurine conjugated bile acids as a class and as individual compounds, and on the isolation of bile alcohols and C₂₇ bile acids diastereo-isomeric at C-25.     

Rapid Amino Acid Analysis in Biotechnology: Determination of Alanine and Aspartic Acid

Abstract

A fast and sensitive chromatographic method was developed to monitor the enzymatic conversion of aspartic acid into alanine in a membrane reactor. The amino acids were converted into dansyl derivatives which were separated by ion-pair chromatography on a reversed-phase column and detected by fluorimetry using only conventional HPLC equipment.     

A Rapid Determination of Taurine in Human Plasma by LC

Taurine is an amino acid which is not incorporated into proteins but found in the cytosol of many mammalian cells, in high concentrations (2–30 mM). Increase in plasma taurine concentration has already been reported after surgical trauma, X-radiation, muscle necrosis, carbon tetrachloride-induced liver damage, and paracetamol overdose. Plasma taurine concentration was measured using LC with fluorescence detection following derivatization by o-phtalaldehyde plus 3-mercapto-propionic acid and α-aminobutyric acid as internal standard. Under these conditions the retention time of taurine was 10 min. This method was sensitive enough, to quantify 150 pg mL⁻¹ and detect 50 pg mL⁻¹ of taurine ranging normally between 65 and 179 mmol L⁻¹ (8–22 μg mL⁻¹). The validated method allowed simple determination of human plasma taurine in pharmacokinetic and biomarker studies.