Aqueous Citrate Buffer Research Articles

Microextraction by packed sorbent (MEPS) to analyze catecholamines in innovative biological samples

A new microextraction by packed sorbent (MEPS) procedure coupled to a high-performance liquid chromatographic method has been developed for quantitation of catecholamines (i.e. norepinephrine, epinephrine and dopamine) in innovative biological samples, namely dried plasma and urine spots. Analyses were carried out on a C18 reversed-phase column using a mobile phase composed of 2.5% methanol and 97.5% aqueous citrate buffer, containing octanesulfonic acid. Coulometric detection was used, setting the first analytical cell at −0.350V and the second analytical cell at +0.400V. Dried matrices were purified by means of a fast and feasible MEPS procedure, optimized on C18 sorbent and requiring only a small amount of biological sample. The availability of miniaturized procedures allowed satisfying specific requirements that ought to be considered during pre-treatment intended for catecholamine analysis. The extraction yield values were always higher than 85% and sensitivity was good, with a limit of quantitation of 100pgmL−1 for all the analytes. Satisfactory results were also obtained in terms of linearity, precision and accuracy. The method was successfully applied to dried plasma and urine spots derived from two socially diversified groups, namely psychiatric patients and poly-drug abusers, in comparison to healthy volunteers.

Mild and selective labeling of malondialdehyde with 2-aminoacridone: assessment of urinary malondialdehyde levels

Malondialdehyde (MDA) has become a well-established biomarker for oxidative stress. The most commonly used way to determine urinary MDA levels is the thiobarbituric acid (TBA) assay, which suffers from several drawbacks. In this manuscript, we describe a novel derivatization strategy for the highly sensitive and selective fluorescence-based determination of MDA in urinary samples. The methodology is based on the mild labeling of MDA with 2-aminoacridone, which can be carried out in aqueous citrate buffer at 40 °C, yielding a highly fluorescent substance. No further sample preparation than mixing with the necessary chemicals is necessary. The formed MDA derivative can conveniently be separated from the label itself and matrix constituents by gradient LC in less than 5 minutes on a cyano-based reversed-phase material. The method was validated with respect to matrix effects, linearity, selectivity and sensitivity (values as low as 1.8 nM for the LOD and 5.8 nM for the LOQ could be achieved). Standard addition quantitation was applied for the determination of MDA in human urine samples. Additionally, the protocol was applied to the measurement of a stability indicating analysis of MDA in urine at different storage conditions.

ChemInform Abstract: Asymmetric Sulfoxidation of a β-Carbonyl Sulfide Series by Chloroperoxidase.

Abstract The chloroperoxidase (CPO)-catalyzed oxidation of a series of β-carbonyl sulfides to sulfoxides has been studied at room temperature in aqueous citrate buffer. For dialkyl β-carbonyl sulfides, the products with methyl and ethyl substituents are obtained in ca. 100% yield. However when the alkyl group is n- propyl or i -propyl the yield drops dramatically (25%). An aryl sulfide derivative afforded product in very low yield (4%), but when the phenyl group bears a carbonyl, and the sulfur substituents are methyl or ethyl, the oxidation occurs with high yields (91–95%). Steric control of the sulfoxidation reaction is also confirmed with cyclohexanone derivatives, where a low product yield is observed even at high enzyme concentrations. Noteworthy are the yields obtained with cyclopentanone sulfide (65%) and an unexpected quantitative yield obtained with the γ-butyrolactone sulfide.

Chromatographic analysis of serotonin, 5-hydroxyindolacetic acid and homovanillic acid in dried blood spots and platelet poor and rich plasma samples

An isocratic high-performance liquid chromatographic method has been developed for the measurement of serotonin, 5-hydroxyindolacetic and homovanillic acids in dried blood spots and in platelet poor and rich plasma samples. Analyses were carried out on a C18 reversed-phase column using a mobile phase composed of 13% methanol and 87% aqueous citrate buffer, containing octanesulfonic and ethylendiaminotetracetic acids. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at −0.200 V and the second analytical cell at +0.400 V. For the pre-treatment of biological samples a novel solid-phase extraction procedure, based on mixed-mode reversed-phase – strong anion exchange Oasis cartridges, was implemented. Extraction yields of the analytes from all these matrices were satisfactory, being always higher than 89.0%. The calibration curve was linear over the on-column concentration range of 0.1–22.5 ng mL −1 for serotonin and 5-hydroxyindolacetic acid and of 0.25–22.5 ng mL −1 for homovanillic acid. The sensitivity was good with a limit of detection of 0.05 ng mL −1 for serotonin and 5-hydroxyindolacetic acid and 0.12 ng mL −1 for homovanillic acid. Results were also satisfactory in terms of precision, selectivity and accuracy. The analytical method was successfully applied to human platelet poor and rich plasma samples and to dried blood spots from volunteers and psychiatric patients.

Fast analysis of catecholamine metabolites MHPG and VMA in human plasma by HPLC with fluorescence detection and a novel SPE procedure

A fast and sensitive high-performance liquid chromatographic method has been developed for the determination in human plasma of MHPG (3-methoxy-4-hydroxyphenylethylenglycol) and VMA (vanillyl mandelic acid), the main metabolites of epinephrine and norepinephrine. Analyses were carried out at 325 nm while exciting at 285 nm on a reversed-phase column (Atlantis C18, 150 mm × 4.6 mm I.D., 5 μm) using a mobile phase composed of 2% methanol and 98% aqueous citrate buffer at pH 3.0. A careful solid-phase extraction procedure, based on mixed-mode reversed-phase – strong anion exchange Oasis cartridges (MAX, 30 mg, 1 mL), was developed for the pre-treatment of plasma samples. Extraction yields were satisfactory, always higher than 90%. Calibration curves were linear over the 0.2–40.0 ng mL −1 concentration range for MHPG and over the 0.5–40.0 ng mL −1 concentration range for VMA. The method was successfully applied to plasma samples of former drug users undergoing detoxification therapy and subjects “at risk” of developing drug addiction.

A Comparative Study of Complexation of β-Cyclodextrin, Calix[4]arenesulfonate and Cucurbit[7]uril with Dye Guests: Fluorescence Behavior and Binding Ability

The complexation behaviors of acridine red (AR), neutral red (NR) and rhodamine B (RhB) dye guest molecules by three kinds of supramolecular hosts, including β-cyclodextrin (β-CD), calix[4]arene tetrasulfonate (C4AS) and cucurbit[7]uril (CB[7]), have been investigated by means of fluorescence spectra in aqueous citrate buffer solution (pH 6.0). The results obtained show that the three hosts, possessing different types of cavity, lead to various complexation-induced fluorescence of dye guests, and present different binding ability and molecular selectivity. The complexation stability constants decrease in the order of NR > AR > RhB for C4AS and CB[7] hosts, while in the order of RhB > AR > NR for β-CD host. Particularly, CB[7] displays the strongest binding ability with NR (K S = 33300 M− 1), and provides the molecular selectivity of 4.8 for NR/AR pairs. Although the binding ability of C4AS for present dye guests is weaker than CB[7], but the molecular selectivity of the two hosts are nearly equivalent. β-CD shows stronger binding ability with RhB (K S = 5880 M− 1) as comparison with CB[7] and C4AS. Furthermore, the solvent effects and salt effects during the course of complexation have also been investigated.

Determination of homovanillic acid (HVA) in human plasma by HPLC with coulometric detection and a new SPE procedure

A sensitive high-performance liquid chromatographic method has been developed for the determination of homovanillic acid (HVA), the main metabolite of dopamine, in human plasma. Analyses were carried out on a reversed-phase column (C8, 250 mm × 4.6 mm i.d., 5 μm) using a mobile phase composed of 10% methanol and 90% aqueous citrate buffer, containing octanesulfonic acid and EDTA at pH 4.8. Coulometric detection was used, setting the guard cell at +0.100 V, the first analytical cell at −0.200 V and the second analytical cell at +0.500 V. A careful solid-phase extraction procedure, based on strong anion exchange (SAX) cartridges (100 mg, 1 mL), was implemented for the pre-treatment of plasma samples. Extraction yield was satisfactory, being the mean value 98.0%. The calibration curve was linear over the concentration range of 0.2–25.0 ng mL −1 of homovanillic acid. The limit of quantitation (LOQ) was 0.2 ng mL −1 and the limit of detection (LOD) was 0.1 ng mL −1. The method was successfully applied to plasma samples from former alcohol, cocaine and heroin addicts. Results were satisfactory in terms of precision and accuracy. Hence, the method is suitable for the determination of homovanillic acid in human plasma.

Substrate specificity of mutants of the hydroxynitrile lyase from Manihot esculenta.

Several tryptophan128-substituted mutants of the hydroxynitrile lyase from Manihot esculenta (MeHNL) are constructed and applied in the MeHNL-catalyzed addition of HCN to various aromatic and aliphatic aldehydes as well as to methyl and ethyl ketones to yield the corresponding cyanohydrins. The mutants (especially MeHNL-W128A) are in most cases superior to the wild-type (wt) enzyme when diisopropyl ether is used as the solvent. Substitution of tryptophan128 by an alanine residue enlarges the entrance channel to the active site of MeHNL and thus facilitates access of sterically demanding substrates to the active site, as clearly demonstrated for aromatic aldehydes, especially 3-phenoxybenzaldehyde. These experimental results are in accordance with the X-ray crystal structure of MeHNL-W128A. Aliphatic aldehydes, surprisingly, do not demonstrate this reactivity dependence of mutants on substrate bulkiness. Comparative reactions of 3-phenoxybenzaldehyde with wtMeHNL and MeHNL-W128A in both aqueous citrate buffer and a two-phase system of water/methyl tert-butyl ether again reveal the superiority of the mutant enzyme: 3-phenoxybenzaldehyde was converted quantitatively into a cyanohydrin nearly independently of the amount of enzyme present, with a space-time yield of 57 g L(-1) h(-1).

High-performance liquid chromatographic determination of some anthraquinone and naphthoquinone dyes occurring in historical textiles

A reversed-phase HPLC method has been developed for identification and quantitation of nine natural quinone dyes and applied to historical textile fibres. A Purospher RP18e column was used with a convex gradient of methanol in a mobile phase of 0.1 M aqueous citrate buffer (pH 2.5) and spectrophotometric diode-array detection at 270 nm. For identification of alizarin, purpurin and xanthopurpurin, occurring together in the madder plant, an isocratic method was used with a methanol–0.2 M acetate buffer (pH 4.3) (75:25) as the mobile phase. After an acid extraction of textile fibres and the analysis of the extracts, alizarin and purpurin were identified and quantitated in three fibres.

Asymmetric sulfoxidation of a β-carbonyl sulfide series by chloroperoxidase

The chloroperoxidase (CPO)-catalyzed oxidation of a series of β-carbonyl sulfides to sulfoxides has been studied at room temperature in aqueous citrate buffer. For dialkyl β-carbonyl sulfides, the products with methyl and ethyl substituents are obtained in ca. 100% yield. However when the alkyl group is n-propyl or i-propyl the yield drops dramatically (25%). An aryl sulfide derivative afforded product in very low yield (4%), but when the phenyl group bears a carbonyl, and the sulfur substituents are methyl or ethyl, the oxidation occurs with high yields (91–95%). Steric control of the sulfoxidation reaction is also confirmed with cyclohexanone derivatives, where a low product yield is observed even at high enzyme concentrations. Noteworthy are the yields obtained with cyclopentanone sulfide (65%) and an unexpected quantitative yield obtained with the γ-butyrolactone sulfide.

(R)-Oxynitrilase catalyzed synthesis of (R)-ketone cyanohydrins

(R)-Oxynitrilase from almonds (Prunus amygdalus) catalyzes the enantioselective addition of HCN to ethyl alkyl ketones 1 in diisopropyl ether yielding (R)-ethyl alkyl ketone cyanohydrins (R)-2, which are hydrolyzed under acid catalysis to give the α-hydroxy acids (R)-3. This (R)-oxynitrilase also catalyzes the enantioselective addition in aqueous citrate buffer (50 mM, pH 4.0), as demonstrated for the preparation of (R)-methyl alkyl ketone cyanohydrins (R)-5 which are obtained in high enantiomeric excesses comparable to those in diisopropyl ether as solvent.

Nitro radical anion formation from nifurtimox II: electrochemical evidence

The cyclic voltammetric (CV) behaviour of nifurtimox was studied. The addition of aprotic solvent (DMF) to nifurtimox in an aqueous citrate buffer system was examined. Qualitatively this results in separation of the initial irreversible four-electron reduction into two stages, the RNO 2/RNO 2 .− and RNO 2 .−,4H +/RNHOH,H 2O couples respectively. Particul attention was directed to the one-electron RNO 2/RNO 2 .− couple as measured by the CV mode in mixed media. Analysis of the CV response as a function scan rate and non-aqueous solvent content yields information on the stability of the radical anion. The chemical forward reaction of the radical anion follows a second-order kinetic with a stability constant of 954 L mol −1 s −1 and a half-life of 1.05 s for 1 mM nifurtimox in aqueous citrate buffer, pH 10.5, + DMF, 40:60.

Photolysis, thermolysis, and protolytic decomposition of a triazene polymer in solution

AbstractThe depolymerization of a triazeno‐group containing photopolymer, poly[oxy‐1,4‐phenyleneazo(methylimino)hexamethylene(methylimino)azo‐1,4‐phenylene], is investigated in a tetrahydrofuran (THF) solution. Irradiation of the material leads to a clean decomposition of the photolabile polymer, as monitored by UV spectroscopy and gel‐permeation chromatography (GPC). As compared to the photolysis in THF solution, the light‐induced decomposition rate of a polymer in a polymer film is shown to be much slower. The highly photosensitive triazeno group also decomposes thermally at temperatures above approx. 220°C. The kinetics of thermal degradation of the polymer in substance was investigated at a temperature of 256°C, and monitored by GPC measurements. During this decomposition, one first observes the development of higher molar‐mass fractions, which result from grafting reactions of primary radicals. Upon further thermolysis the triazene polymer is completely degraded to low‐molar‐mass products. The volatile decomposition products were identified by gas chromatography/mass spectrometry (GC/MS) analysis. The protolytic decomposition, which represents the retrosynthesis of the triazene polymer, was studied in a 9:1 mixture of tetrahydrofuran and an aqueous citrate buffer solution. Although the decomposition rate in this solvent mixture is slow, as compared to the depolymerization in diluted hydrochloric acid, a clean decomposition of the triazene polymer is obtained.

A nitro radical anion formation from nifedipine: an electrochemical approach

The cyclic voltammetric behaviour of nifedipine was studied. The addition of three aprotic solvents to nifedipine in an aqueous citrate buffer system was examined. Qualitatively they result in separation of the initial irreversible 4 electron reduction into two stages, the NO 2/RNO . 2 − and RNO . 2 −, 4H +/RNHOH, H 2O couples, respectively. Particular attention was directed to the 1-electron RNO 2/RNO . 2 − couple as measured by the cyclic voltammetric mode in mixed media. Analysis of the cyclic voltammetric response as a function of scan rate and non-aqueous solvent content yields information on the stability of the radical anion. The chemical forward reaction of the radical anion follows a second order kinetics with a stability constant of 1.1 × 10 −3 l mol −1 s −1 and a half-life time of 0.09 s for 1 mM of nifedipine in aqueous citrate buffer, pH 7.4/DMF; 50:50.

The Application of Solid Phase Extraction to the Analysis of Tobacco-Specific Nitrosamines

A new isolation and separation method has been developed for the alkaloid-related nitrosamines in tobacco, called tobacco-specific nitrosamines (TSNA). The improved isolation procedure involves the extraction by sonication of TSNA from cured tobacco with toluene and an aqueous citrate buffer solution, while purification is achieved by solid phase extraction on small silica cartridges. Interfering extract materials are eluted from the silica cartridges with the toluene and a chloroform-methylene chloride solvent, while subsequent elution with chloroform yields the TSNA in a purified fraction. Gas chromatographic analyses on an SE-54-coated glass capillary column and detection by a nitrogen-phosphorous detector produce very good data on the four TSNAs, which include N-nitrosanatabine (NATB), N-nitrosoanabasine (NAB), 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone (called NNK), and N-nitrosonornicotine (NNN). The method is shown to be quantitative, reproducible, and applicable to the determination of TSNA in various tobacco samples, such as stems and lamina of cured tobacco leaves, as well as to smokeless tobacco.

Electrochemical investigation on prostaglandin F2α derivatives. I. Anodic iodination of prostaglandin F2α and its methylesther

Studies of the anodic iodination of prostaglandin F2α were carried out under a variety of electrolytic conditions. The effect of anode material, potential, pH of the electrolyte, initial substrate concentration and charge passed were investigated. A preparative technique for the iodination, in an undivided cell, has been developed. The iodinated compound-in the form of its 6-endo-(H)isomer-can be obtained in a two-electron process, with 100% conversion and nearly a quantitative product and current yield, without by-products, when using a platinum anode and cathode, +1.5 V vs SCE potential and 30 vol% methanolic 1 M aqueous citrate buffer solution (pH 3.4–3.6) containing 1–2×10−2 M of the substrate and KI as electrolyte. The method can be extended to other prostaglandin derivatives. On the basis of the experimental results a mechanism is proposed involving the iodine radical and the iodinated substrate radical as intermediates.

Quantitative analysis of total thiamine in human blood, milk and cerebrospinal fluid by reversed-phase ion-pair high-performance liquid chromatography

Whole blood hemolysed by freezing, serum, cerebrospinal fluid, and milk of human origin were deproteinized by perchloric acid. Thiamine pyrophosphate and thiamine monophosphate were hydrolysed to thiamine by acid phosphatase. Chromatography was performed on C 18-coated silica using an n-octanesulfonate containing mobile phase methanol—aqueous citrate buffer pH 4.0 (45:55, v/v). In a post-column reaction K 3Fe(CN) 6 is used to oxidise thiamine to thiochrome, which is detected by fluorometry. Two ml blood is needed. The minimum detectable amount is 60 femtomol of thiamine. The intra-assay coefficient of variation (C.V.) is 2.3% and the inter-assay C.V. is 3.9%. The recovery of added thiamine pyrophosphate to blood samples was 98.7%. The reference range was found to be 88–157 nmol/l whole blood. Examples of the analysis of cerebrospinal fluid, serum and milk are given.

Determination of Sulfadiazine and N4-Acetylsulfadiazine in Biological Fluids by Liquid Chromatography on Silica Gel with an Aqueous Buffer as Mobile Phase

Sulfadiazine and N4-acetylsulfadiazine were determined in biological fluids by the direct injection (plasma after protein precipitation and urine after dilution 100 times) of 20 μl on a silica gel column. The mobile phase was an aqueous citrate buffer (pH 4.0) and UV detection was at 264nm. Chromatographic selectivity was optimized by the silica gel surface and pH of the mobile phase. Detection limits were ~0.4 μg/ml for sulfadiazine in plasma and ~5 and 7 μg/ml for sulfadiazine and N4-acetylsulfadiazine in urine, respectively. In quantitations by peak heights relative to an internal standard (sulfamerazine), within-run precisions (srel%) for sulfadiazine were 1.7 and 4.0% at 40 and 2 μg/ml, respectively, in plasma and 0.76 and 1.7% at 750 and 25 μg/ml, respectively, in urine.