UV-absorbing Derivatives Research Articles

Simultaneous determination of 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid and main plasma aminothiols by HPLC–UV based method

The report presents the first method for simultaneous determination of plasma 2-(3-hydroxy-5-phosphonooxymethyl-2-methyl-4-pyridyl)-1,3-thiazolidine-4-carboxylic acid (HPPTCA), an adduct of cysteine (Cys) and active form of vitamin B6 pyridoxal 5′-phosphate (PLP), as well as total low molecular-weight thiols content, including Cys, homocysteine (Hcy), cysteinyl-glycine (Cys-Gly), and glutathione (GSH). The assay is based on high performance liquid chromatography coupled with ultraviolet detection (HPLC–UV) and involves disulfides reduction with tris(2-carboxyethyl)phosphine (TCEP), derivatization with 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) followed by sample deproteinization with perchloric acid (PCA). The chromatographic separation of obtained stable UV-absorbing derivatives is achieved on ZORBAX SB-C18 (150 × 4.6 mm, 5.0 µm) column using gradient elution with eluent consisted of 0.1 mol/L trichloroacetic acid (TCA), pH 1.7 and acetonitrile (ACN), delivered at a flow rate 1 mL/min. Under these conditions, the analytes are separated within 14 min at room temperature, and quantified by monitoring at 355 nm. Regarding HPPTCA, the assay linearity was demonstrated within a 1–100 µmol/L in plasma and the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). The accuracy ranged from 92.74 to 105.57% and 95.43 to 115.73%, while precision varied from 2.48 to 6.99% and 0.84 to 6.98% for intra- and inter-day measurements, respectively. The utility of the assay was proved by application to plasma samples delivered by apparently healthy donors (n = 18) in which the HPPTCA concentration ranged from 19.2 to 65.6 µmol/L. The HPLC–UV assay provides complementary tool for routine clinical analysis, facilitating further studies on the role of aminothiols and HPPTCA in living systems.

A derivatization strategy for the detection and identification of volatile trialkylphosphites using liquid chromatography-online solid phase extraction and offline nuclear magnetic resonance spectroscopy

We demonstrate herein the application of selective derivatization method that converts volatile and labile trialkylphosphites (TAPs) into virtually non-volatile, thermally stable, and UV absorbing derivatives. After simple sample preparation, purification/enrichment of the derivatives was achieved by using high performance liquid chromatography (HPLC) coupled to on-line post column solid phase extraction (SPE) system. These derivatives were subjected to 31P{1H} NMR and 1D-selTOCSY experiments. Conclusive identification was achieved on the basis of their HPLC retention time and NMR spectral signatures (δH1,δpE1, nJH–H, and 3JP–H). This method was tested for the unambiguous identification of a mixture containing low concentrations (∼10μgmL−1) of trimethylphosphite (TMP), triethylphosphite (TEP), triisopropylphosphite (TIP), and tributylphosphite (TBP) along with a high concentration of irrelevant background chemicals. It offered a high dynamic range and good detection limit and recovery (>75%) without the need for special NMR probe heads or exotic NMR experiments.

On-Line Derivatization of N-acetylcysteine Using Ethyl-Propiolate as a Novel Advantageous Reagent and Sequential Injection Analysis

This study reports a novel automated assay for the determination of N-acetylcysteine (NAC). The method is based upon the on-line derivatization of the analyte with a new reagent, ethyl-propiolate (EPL), to form a UV-absorbing derivative. The reaction is fast, proceeds in aqueous solutions under mild conditions and is, therefore, ideal for automation using sequential injection analysis. All major chemical and instrumental variables were examined thoroughly. The assay was validated for linearity, range, LOD-LOQ, within and day-to-day precision, selectivity, and accuracy. The experiments confirmed its suitability for the reliable quality control of NAC-containing formulations at a sampling rate of 80 h−1.

Aracyl triflates for preparing fluorescent and UV absorbing derivatives of unreactive carboxylates, amines and other metabolites

Aracyl trifluoromethanesulfonate (triflate) reagents alkylate the unreactive carboxyl groups of betaines. The reagents also derivatise tertiary amines, amine oxides, phosphates and thioethers. A general derivatisation procedure is to dilute the sample with a polar aprotic organic solvent containing a trace of water, followed by adding an appropriate base and the aracyl triflate reagent. Limits of detection were 40 nM for glycine betaine (GB) and carnitine, 0.14 μM for N, N-dimethylglycine, 70 nM for trimethylamine (TMA), and 5.9 μM trimethylamine- N-oxide (TMAO). Limits of detection were improved when derivatisation yield was optimised for different analytes by changing solvent, base and water content of the reaction mixture. Solvents used include acetonitrile, acetone, butanone, dimethoxyethane and dimethoxyethyl ether, with the presence of some water or alcohol tolerated. Suitable bases include the inorganic bases magnesium hydroxide, silver oxide and lithium phosphate. These bases are sparingly soluble in the reaction solvent so sustain weakly basic conditions during derivatisation and can be removed by centrifugation to stop the reaction. This is particularly important for betaine assays since the derivatives are base-sensitive. The information presented here can be used to develop assays for many metabolites using these versatile reagents.

Normal spectrophotometric and stopped-flow spectrofluorimetric sequential injection methods for the determination of alendronic acid, an anti-osteoporosis amino-bisphosphonate drug, in pharmaceuticals

A normal spectrophotometric and a stopped-flow (SF) spectrofluorimetric method have been developed and optimized for the determination of alendronic acid (ALD) in its pharmaceutical formulations. Both methods are automated using the sequential injection analysis (SIA) principle. The spectrophotometric assay is based on the reaction of the analyte with Cu(II) ions in acidic medium to form an UV-absorbing derivative ( λ max = 240 nm). The SF spectrofluorimetric method is based on the reaction of ALD with o-phthalaldehyde (OPA) in the presence of 2-mercaptoethanol at basic medium ( λ ex = 340 nm/ λ em = 455 nm). Linear calibration curves were obtained in the range 1.0–60.0 mg l −1 ALD for the UV method, and in the range 0.13–10.0 mg l −1 ALD for the SF spectrofluorimetric one. The sampling rates were 60 and 30 h −1, respectively. The developed assays are critically compared and their advantages are discussed. Both methods were applied to the analysis of an ALD containing pharmaceutical formulation with satisfactory accuracy and precision.

Functional Demonstration of Reverse Transsulfuration in the Mycobacterium tuberculosis Complex Reveals That Methionine Is the Preferred Sulfur Source for Pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.

Continuous on-line derivatization and determination of amino acids by a microfluidic capillary electrophoresis system with a continuous sample introduction interface

An automatic, rapid and continuous on-line derivatization system coupled to microfluidic capillary electrophoresis (CE) for the determination of amino acids using o-phthaldialdehyde/ N-acetyl- l-cysteine (OPA/NAC) as the derivative agents has been developed. By on-line derivatization, amino acids were automatically and reproducibly converted to the UV-absorbing derivatives, which were separated by capillary zone electrophoresis (CZE). Optimization of derivatization and separation condition was carried out to achieve both good sensitivity and separation efficiency. The separation could be achieved within 4 min and sample throughput rate can reach up to 16 h −1. The repeatability (defined as relative standard deviation, R.S.D.) was 2.56, 2.85, 3.24 and 3.60% with peak area evaluation and 2.93, 3.12, 4.20 and 4.91% with peak height evaluation for arginine (Arg), phenylalanine (Phe), serine (Ser) and glycine (Gly), respectively. The limits of detection (S/N=3) were 10.46, 13.14, 34.39 and 44.79 μmol/l for Arg, Phe, Ser and Gly, respectively. Major advantages of the proposed method include improved precision and efficient automation of the derivatization by the FI system and the enhanced sampling frequencies by the combined FI–CE system.

Analysis of trace levels of toxic sodium monochloroacetate in alkylamidobetaines by HPLC

Trace levels of sodium monochloroacetate (SMCA) in an amphoteric surfactant cocoamidopropylbetaine (CAPB), have been analyzed by HPLC. SMCA is isolated from the surfactant matrix by solid phase extraction and then converted into an UV-absorbing derivative by quaternizing p-methoxycinnamidopropyldimethylamine.

The determination of pamidronate in pharmaceutical preparations by ion-pair liquid chromatography after derivatization with phenylisothiocyanate.

An analytical method was developed for the determination of pamidronate [(3-amino-1-hydroxypropylidene)bisphosphonate] by ion-pair liquid chromatography. The analyte was derivatized with phenylisothiocyanate into an UV-absorbing derivative. The reaction product was cleaned-up by a double ion-pair extraction and treated with hydrogen peroxide prior to injection. Both, the detection limit and the lower limit of quantification of pamidronate in water were 0.1 μg ml −1 disodium pamidronate. The intra-day precision was 3% for a 5-μg ml −1 pamidronate standard solution and the inter-day precision 6% for a 3-μg ml −1 solution. The method was applied in the quality control of pamidronate injection concentrates and tablets.

Separation of carnitine enantiomers as the 9-anthroylnitrile derivatives and high-performance liquid chromatographic analysis on an ovomucoid-conjugated column

9-Anthroylnitrile was used as an achiral reagent for the derivatization of carnitine. The reagent forms UV-absorbing derivatives with the hydroxyl groups of carnitine enantiomers under very mild conditions. The derivatives were separated by high-performance liquid chromatography on an ovomucoid-conjugated column with a mobile phase of acetonitrile-20 mM KH 2 PO 4 (adjusted to pH 4.5 with phosphoric acid) (17:83, v/v). The separation factor (α) and resolution ({ce:inline-formula}R s{/ce:inline-formula}) of the enantiomers were 1.44 and 5.05, respectively. The calibration plots indicated good linearity over a sample concentration ranging from 0.2 to 1.0 mg ml −1, and the detection limit at 254 nm was 0.05 mg ml −1 for each carnitine enantiomer. The reproducibility in the analysis of 1 mg ml −1 of each enantiomer was within 2.0%. The method was applied successfully to the determination of carnitine enantiomers in pharmaceutical preparations.

High-performance liquid chromatographic assay for l-glyceric acid in body fluids. Application in primary hyperoxaluria type 2

We describe a liquid Chromatographic technique to determine L-glycerate in body fluids. The method is based on the derivatisation of the L-glycerate by incubation with lactate dehydrogenase and nicotinamide-adenine dinucleotide in the presence of phenylhydrazine. Oxidation of l-glycerate forms β-hydroxypyruvate which is converted in turn into the related phenylhydrazone. The UV-absorbing derivative is determined using reversed-phase high performance liquid chromatography. The sensitivity was 5 μmol/1 and 50 μl of sample were required. The imprecision relative standard deviation was 4.5% and the recovery was 96.5 ± 6.8% for L-glycerate in plasma. L-Glycerate concentrations in urine and plasma were less than 5 μmol/1 in both normal individuals and patients with glycolic aciduria. In a patient with systemic oxalosis and normal plasma glycolate, plasma L-glyceric acid was 887 /gmmol/1.

Separation of the molecular species of diacyl and alkenylacyl subclasses of the methyl ester of dinitrophenylethanolamine glycerophospholipid by high-performance liquid chromatography

A high-performance liquid chromatographic (HPLC) procedure for the separation of the molecular species of alkenylacyl and diacyl subclasses after the derivatization of ethanolamine glycerophospholipids (EGP) to the methyl ester of the dinitrophenylated lipids is described. Methyl esters of dinitrophenylethanolamine glycerophospholipids were first separated into alkenylacyl, alkylacyl and diacyl subclasses by thin-layer chromatography, then each subclass was separated into individual molecular species by reversed-phase HPLC. By converting the EGP into UV-absorbing derivatives, it proved possible to utilize the specificity of spectrophotometric detection for the determination of the individual molecular species. Alkenylacyl and diacyl derivatives were resolved into fifteen to twenty different species in a single HPLC run. The method should facilitate studies on the metabolism of the polar head group of both molecular species of alkenylacyl and diacyl glycerophosphoethanolamine in a variety of tissues using radioactive precursors.

New ultraviolet labelling agents for high-performance liquid chromatographic determination of monocarboxylic acids

New UV-labelling agents have been synthesized, which are designed to convert monocarboxylic acids into their highly UV-absorbing derivatives for enhancement of the sensitivities of UV detection in high-performance liquid chromatography. The reagents are p-nitrobenzyl, 3,5-dinitrobenzyl and 2-(phthalimino)ethyl p-toluenesulphonates. Each has been prepared by reaction of p-toluenesulphonyl chloride with p-nitrobenzyl alcohol, 3,5-dinitrobenzyl alcohol or N-(hydroxyethyl)phtalimide, respectively, in the presence of sodium hydroxide, and they are stable in the solid state for at least 6 months. Monocarboxylic acids were derivatized to their p-nitrobenzyl, 3,5-dinitrobenzyl or 2-(phthalimino)ethyl esters with each of the above reagents, respectively, then determined by high-performance liquid chromatography with UV detection. In the UV-labelling with each reagent, 18-crown-6 was used as the catalyst. The effects of the reaction solvent, reaction temperature and time and the concentrations of each reagent and the catalyst were also examined. bl]

High-performance liquid chromatographic method for the separation and estimation of choline, glycinebetaine aldehyde and related compounds

Procedures are described for the separation and quantitative estimation of choline and glycinebetaine aldehyde in plant saps and extracts. Glycinebetaine aldehyde in the plant material, and a suitable internal standard, were converted to their p-nitrobenzyl oximes during extraction. The extracts was purified on Sep-Pak C 18 cartridges and on ion-exchange columns, after which the choline fraction was benzoylated to yield UV-absorbing derivatives. High-performance liquid chromatography was performed on an aliphatic carboxylic acid or an aromatic sulphonic acid cation-exchange column eluted with the phosphate salt of choline in aqueous acetonitrile. The detection limit for both compounds was less than 1 ng. This procedure may also be applied to the analysis of choline in hydrolysates of phospholipids.

Determination of sorbitol and galactitol at the nanogram level in biological samples by high-performance liquid chromatography

Aldose reductase is an enzyme that plays an important role in diabetic complications such as cataract and neuropathy. The best way to estimate the enzymatic activity of this enzyme in vivo consists of measuring the accumulation of sorbitol or galactitol in various types of cells or tissues. A sensitive method to measure the polyols in biological samples by high-performance liquid chromatography has been developed. This method is based on the fact that polyols like sorbitol and galactitol react with phenylisocyanate to yield uv-absorbing derivatives at 240 nm. Applications to the separation and determination of polyols in biological samples of various origins, such as lenses, sciatic nerves, human skin fibroblasts, and red cells, are described and illustrated.

Structural characterization of oligosaccharides by high-performance liquid chromatography, fast-atom bombardment-mass spectrometry, and exoglycosidase digestion

A method for structural characterization of oligosaccharides after preparing uv-absorbing derivatives is described. The derivatives can be rapidly analyzed and purified by high-performance liquid chromatography, with separation of various structures determined primarily by size and sugar composition. Derivatization requires as little as 0.5–1.0 nmol of oligosaccharide, and detection of down to 50 pmol of oligosaccharide is possible by monitoring absorbance at 229 nm. In addition, the carbohydrate portion of the derivative was found to retain its sensitivity to exoglycosidases, allowing sequential enzymatic digestions for determination of sugar sequencc and anomerity to be performed. The derivatives also possessed a site of potential positive charge, making them amenable to analysis by fast-atom bombardment-mass spectrometry. Permethylation of the derivatives permitted their separation by capillary gas chromatography, thus allowing investigation of their structures by gas chromatography-mass spectrometry. The combination of these techniques will alow almost the complete structure of small amounts of oligosaccharides to be determined.

Separation and determination of phenyl isocyanate-derivatized carbohydrates and sugar alcohols by high-performance liquid chromatography with ultraviolet detection

The free hydroxyl groups of saccharides and sugar alcohols react with phenyl isocyanate to yield very stable and strongly UV-absorbing derivatives, which possess good chromatographic properties in a reversed-phase system. UV monitoring at ca. 240 nm allows detection down to the 0.5–10 ng level.Reducing sugars yield a peak for each enantiomer, while non-reducing sugars and sugar alcohols yield a single peak. The chromatograms resemble those obtained by gas-liquid chromatography after silylation.Oligomers containing eight glucose units have been separated with good resolution by this method. Applications to the separation and determination of sugar alcohols and wood hydrolysates are described.

Separation and detection of uv-absorbing derivatives of fecal bile acid metabolites by high-performance liquid chromatography

Major fecal bile acid metabolites related to lithocholic acid were resolved by high-performance liquid chromatography (hplc). The uv-absorbing p-nitrobenzyl ester derivatives of lithocholic, isolithocholic, 3-keto-5β-cholanic, and 5β-cholanic acids were prepared using the reagent o,p-nitrobenzyl- N,N′-diisopropylisourea. Separation was achieved in less than 20 min on a microparticulate silica column using isocratic elution with 2% isopropanol in isooctane as the mobile phase. The p-chlorobenzoyl esters of methylated lithocholic and isolithocholic acids were also prepared but required purification by thin-layer chromatography before separation by hplc. These derivatives were eluted from a Porasil T column using 5% diisopropyl ether in isooctane as the mobile phase. Lithocholic and isolithocholic acids produced by microbial metabolism of [ 14COOH]taurolithocholic acid were separated and identified by preparing p-nitrobenzyl derivatives and monitoring the column effluent for both uv and radioactivity. This technique is a rapid and sensitive method for isolating bile acid metabolites.