Pre-column O-phthalaldehyde Derivatization Research Articles

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 × 4.6 mm, 5-μm particle size column, a step gradient with two buffers, a flow rate of 1.2 ml/min, a column temperature of 23°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.

SPECIFICITY OF CYSTEINE SULFINATE DECARBOXYLASE (CSD) FOR SULFUR-CONTAINING AMINO-ACIDS

Cysteine sulfinate decarboxylase (CSD) which decarboxylates cysteine sulfinic acid (CSA) to form hypotaurine is thought to be involved in the biosynthesis of taurine. It was recently localized in astrocytes in the cerebellum and hippocampus by immunocytochemistry. Another sulfur-containing aminoacid (SCAA), homocysteic acid (HCA), was also found in astrocytes in these regions. We therefore investigated the specificity of CSD vs CSA and HCA as well as the related analogs homocysteine sulfinic acid (HCSA) and cysteic acid (CA). CSD was immunotrapped from brain and liver tissue supernatant using a specific CSD antiserum and Protein-A Sepharose. It was then incubated with the l-form of the various SCAA. Reaction products were identified and quantified by pre-column o-phthalaldehyde derivatization HPLC. CA and HCA from 2.5 to 25 mM inhibited the formation of hypotaurine from CSA (0.25 mM). Moreover, the inhibition curves were parallel for liver and brain CSD. CA or HCA (25 mM) elicited a near-total inhibition. HCSA did not produce a significant inhibition up to 25 mM. Incubation with 25 mM CSA or CA led to the formation of hypotaurine and taurine, respectively. The ratio of formation of taurine to that of hypotaurine was similar for CSD from liver and brain. In contrast no homotaurine, the decarboxylated reaction product of HCA, could be detected following incubation with 25 mM HCA. According to the sensitivity of the HPLC analysis this indicates that the decarboxylation of HCA, if any, was 130-fold and 50-fold less than that of CSA by CSD from liver and brain, respectively, in our experimental conditions. Similarly, following incubation with HCSA, no new peak appeared on the chromatogram when compared to a blank sample. These results show that CSD from either brain or liver has a high specificity for CSA and CA, which are the SCAA involved in the biosynthesis of taurine. HCA is an inhibitor of CSD but does not appear to be a substrate for CSD in vitro. HCSA is neither a substrate nor an inhibitor of CSD in vitro. Accordingly, CSD is unlikely to play a role in the metabolism of HCA or HCSA in vivo. Copyright © 1996 Elsevier Science Ltd.

Whole blood analysis of gluconeogenic amino acids for estimation of de novo gluconeogenesis using pre-column o-phthalaldehyde derivatization and high-performance liquid chromatography

By measuring the potential glucose precursors entering and exiting the liver, an estimate of the maximal rate of de novo gluconeogenesis can be made. Tradiotionally, measurements of gluconeogenic amino acids have been extracted from full amino acid profiles using conventional ion-exchange chromatography. These methods are labor intensive, costly procedures that do not focus on gluconeogenic amino acids. The present paper describes a method that provides an accurate whole blood gluconeogenic amino acid profile (intra-assay coefficients of variation from 0.8 to 1.1% and inter-assay coefficients of variation from 2.9 to 4.3%) using high-performance liquid chromatography with o-phthalaldehyde chemistry. This automated method is relatively fast (injection to injection time = 30 min), and linear ( r 2>0.996) for both standards and deproteinized whole blood. Furthermore, it is economical and capable of assessing gluconeogenic amino acids across a broad physiological range of concentrations using small sample volumes.

Analysis of Dezocine in Serum and Urine by High Performance Liquid Chromatography and Pre-Column Derivatizationxs

Abstract We present a reverse phase liquid chromatographic assay involving pre-column o-phthalaldehyde derivatization and fluorescence detection with the capability of measuring dezocine in human serum and urine for pharmacokinetic or forensic purposes. It features a simple liquid-liquid extraction scheme using hexane, conservative detection limits of 1 ng/ml, linearity to 1000 ng/ml, precision of 10% (CV%) in the low ng/ml region, approximate chromatography time of 25 minutes and the use of a new internal standard, WY-19083.

Precolumn 𝘖-Phthalaldehyde Derivatization and Reversed-Phase Liquid Chromatography of S-Methylmethioninesulfonium in Satsuma Mandarin Juice

Abstract Experiments were conducted to determine the amount of S-methylmethlonlnesulfonium (MMS) in Satsuma mandarin Juice using a precolumn 𝘖-phthalaldehyde (OPA) derivatization and reversedphase liquid chromatography. Favorable analytical conditions that allowed MMS analysis were achieved by using OPA/3-mercaptopropionlc acid (MPA) as a derivatization agent with fluorescence detection (excitation at 330 nm, emission at 450 nm). A C18 reversed-phase column with 5 μm particle size and a length of 250 mm was used. Resolution of MMS/OPA/MPA derivative was accomplished with a linear gradient eluent (30mM sodium acetate buffer solution, pH 7.3, and 70% (v/v) methanol). Quantitative analysis of MMS by the internal standard method using β-alanine gave highly reproducible result with a coefficient of variation less than 3%. Recovery of MMS added to Juice samples was 105%. MMS content in Satsuma mandarin Juice was 28.2μM.

Comparison of automated pre-column and post-column analysis of amino acid oligomers

It has been shown that various amino acids will polymerize under plausible prebiotic conditions on mineral surfaces, such as clays and soluble salts, to form varying amounts of oligomers ( n = 2–6). The investigation of these surface reactions required a quantitative method for the separation and detection of these amino acid oligomers at the picomole level in the presence of nanomole levels of the parent amino acid. In initial high-performance liquid chromatography (HPLC) studies using a classical postcolumn o-phthalaldehyde (OPA) derivatization ion-exchange HPLC procedure with fluorescence detection, problems encountered included lengthy analysis time, inadequate separation and large relative differences in sensitivity for the separated species, expressed as a variable fluorescent yield, which contributed to poor quantitation. We have compared a simple, automated, pre-column OPA derivatization and reversed-phase HPLC method with the classical post-column OPA derivatization and ion-exchange HPLC procedure. A comparison of UV and fluorescent detection of the amino acid oligomers is also presented. The conclusion reached is that pre-column OPA derivatization, reversed-phase HPLC and UV detection produces enhanced separation, improved sensitivity and faster analysis than post-column OPA derivatization, ion-exchange HPLC and fluorescence detection.

Pre-column o-phthalaldehyde derivatization of amino acids and their separation using reversed-phase high-performance liquid chromatography : I. Detection of the imino acids hyroxyproline and proline

Description d'une methode utilisant la chloramine T et le borohydrure de sodium pour former des derives de l'hydroxyproline et de la proline reagissant avec le phtalaldehyde et le mercapto-2 ethanol pour donner des composes fluorescents separables par chromatographie liquide

Pre-column o-phthalaldehyde derivatization of amino acids and their separation using reversed-phase high-performance liquid chromatography : II. Simultaneous determination of amino and imino acids in protein hydrolysates

Pre-column o-phthalaldehyde derivatization of amino acids and their separation using reversed-phase high-performance liquid chromatography : II. Simultaneous determination of amino and imino acids in protein hydrolysates

Electrochemical detection of sympatomimetic drugs, following pre-column o-phthalaldehyde derivatization and reversed-phase high-performance liquid chromatography

In order to assay sympatomimetic drugs (heptaminol and related compounds), a derivatization procedure with the use of o-phthalaldehyde (OPA) and various thiols was developed. Some structural assignments of the OPA-amino drug adducts are proposed and the electroactive properties of these substituted isoindolic products were investigated by voltamperometry. High-performance liquid chromatographic separations were carried out on a reversed-phase column (LiChrosorb RP-18) with phosphate buffer (pH 7.2)-methanol mixtures as mobile phase. For detection , either UV spectrophotometry (λ = 340 nm) or amperometry (+ 0.9 V) was used. By the latter levels in the picomole range were detectable.

A Method for Quantitative Amino Acid Analysis Using Precolumn o-Phthalaldehyde Derivatization and High Performance Liquid Chromatography

A fluorometric procedure for quantitative amino acid analysis of a typical peptide hydrolysate is presented. The amino acids were derivative with o-phthalaldehyde (OPA) prior to injection into the high performance liquid chromatographic (HPLC) apparatus. The OPA-amino acid adducts were resolved using a linear gradient of acetonitrile against an initial solvent consisting of 0.01875 M triethylamine acetate (TEA-A), pH 7.5. Complete analysis required 46 min, with an injection-to-injection time of 56 min. The lower limit of quantitation using the equipment specified was found to be 100 pmoies, with a lower detection limit of 25 pmoles.