Acetic Acid In Acetonitrile Research Articles

Rapid high-performance liquid chromatographic method of determining malondialdehyde for evaluation of rancidity in edible oils

A simple HPLC method for the determination of malondialdehyde (MDA) in oxidised vegetable oils was developed and the results were compared with those given by a spectrophotometric method. Vegetable oil was steam-distilled in a Kjeldahl distillation apparatus and the MDA was determined in the aqueous distillates by HPLC, using a µ-Bondapak C18 column, with a mobile phase of 1% acetic acid–acetonitrile (85 + 15, v/v). A total time of 2 min was necessary to assay each distillate and only MDA was detected. MDA can be determined at a level of 1.5 × 10–9 mol l–1.

Speciation of organo-tin compounds using liquid chromatography–atmospheric pressure ionisation mass spectrometry and liquid chromatography–inductively coupled plasma mass spectrometry as complementary techniques

A liquid chromatographic method for the determination of dibutyltin (DBT), tributyltin (TBT), diphenyltin (DPhT) and triphenyltin (TPhT) in sediments has been developed, which is compatible with both atmospheric pressure ionisation (API) mass spectrometry and inductively coupled plasma (ICP) mass spectrometry. As a result of this development both techniques may be used for the complementary speciation of organo-tin compounds. The chromatographic system comprises of a Kromasil-100 5 μm C18 (150×2.1 mm) column and a mobile phase of 0.05% triethylamine in acetonitrile–acetic acid–water (65:10:25), at a flow-rate of 0.2 ml min−1. The optimisation of the LC–API-MS conditions is discussed, together with the analysis of a real sediment sample for DBT and TBT using selected ion monitoring (SIM).

Derivatization and high-performance liquid chromatographic analysis of pentaazapentacosane pentahydrochloride.

A rapid high-performance liquid chromatographic method for determination of the dansyl derivative of pentaazapen-tacosane (PAPC) pentahydrochloride has been developed. The chromatographic system uses a reversed-phase C8 column, a mobile phase of acetic acid buffer and acetonitrile and UV detection. The dansylation conditions were optimized with a pH of 11.0 and a 20-fold dansyl chloride excess. The yield of dansyl PAPC increased 10-fold as the reaction pH was changed from 9.5 to 10.5. Under derivatization conditions of pH 8.5-11.0 and 1-30-fold excess dansyl chloride only perdansyl PAPC was found.

Preliminary Evaluation of a Molecular Imprinted Polymer for Solid-phase Extraction of Tamoxifen

A molecular imprinted polymer, prepared around a tamoxifen template, has been synthesised. The feasibility of using this polymer for solid-phase extraction of tamoxifen from biological samples has been investigated. The results show that the drug can be quantitatively retained and eluted from the polymer using acetonitrile–acetic acid. Experiments with biological fluids indicate that clean traces are obtained for HPLC with UV detection using this type of protocol.

Isolation, Purification, and Liquid Chromatographic Determination of Stilbene Phytoalexins in Peanuts

Abstract A liquid chromatographic (LC) method for determination of stilbene phytoalexins (SPs) in peanuts has been developed. SPs were extracted with acetonitrile–water (90 + 10, v/v) by high-speed blending. An aliquot of extract was applied to a minicolumn packed with AI2O3–ODS (C18) mixture and eluted with acetonitrile-water (90 + 10, v/v). Eluate was evaporated under nitrogen, and residue was dissolved in LC mobile phase. SPs in an aliquot of purified extract were quantitated by normal-phase partition LC on silica gel with n-heptane–2-propanol–water–acetonitrile–acetic acid (1050 + 270 + 17 + 5 + 1, v/v) as mobile phase. Recoveries of SPs [frans-4-(3-methyl-but-1-enyl)-3,5,4′-trihy-droxystilbene (trans-arachidin-3; t-Ar-3), trans-3-isopentadienyl-4,3′,5′-trihydroxystilbene (t-IPD), and trans-3,5,4′-trihydroxystilbene (trans-resveratrol; t-Res)] from peanuts spiked at 300 ppb were 96.05 ± 2.8%. Limits of quantitation for t-Ar-3 and t-IPD were about 100 ppb. t-Ar-3, t-IPD, and t-Res were found in all parts of the peanut plant as major SPs produced in response to fungal invasion.

Antitumour imidazotetrazines. Part 33. New syntheses of the antitumour drug temozolomide using ‘masked’ methyl isocyanates

Ethyl 8-carbamoyl-4-oxo-3,4-dihydroimidazo[5,1-d]-1,2,3,5-tetrazin-3-ylacetate 6a can be prepared by treating 5-diazoimidazole-4-carboxamide 3 with ethyl isocyanatoacetate or by diazotisation of N-(5-amino-4-carbamoylimidazol-1-ylcarbonyl)glycine ethyl ester 5. Barton radical decarboxylation of the tetrazin-3-ylacetic acid 6b affords temozolomide 1(26%) whereas deprotection of the 3-trimethylsilylmethyl-imidazotetrazine 6g with TBAF in acetonitrile–acetic acid yields 1 in 78% yield. 3-Benzylimidazotetrazinones 10a–c are stable to hydrogenolytic or oxidative debenzylation reactions.

Determination of azosemide and its metabolite in plasma, blood, urine and tissue homogenates by high-performance liquid chromatography

High-performance liquid chromatographic methods were developed for the determination of azosemide and its metabolite, M1, in human plasma and urine and rabbit blood and tissue homogenates. The methods involved deproteinization of the biological samples: 2.5 volumes of acetonitrile were used for the determination of azosemide and 1 volume of saturated Ba(OH) 2 and ZnSO 4 for that of M1. A 50-μl aliquot of the supernatant was injected onto a C 18 reversed-phase column in each instance. The mobile phases employed were 0.03 M phosphoric acid—acetonitrile (50:40, v/v) for azosemide and 0.03 M phosphoric acid/0.2 M acetic acid—acetonitrile (83:17, v/v) for M1. The flow-rate was 1.5 ml/min in both instances. The column effluent was monitored by ultraviolet detection at 240 and 236 nm for azosemide and M1, respectively. The retention times for azosemide and M1 were 6.0 and 8.3 min, respectively. The detection limits for both azosemide and M1 in both human plasma and urine were 50 ng/ml. The coefficients of variation of the assay were generally low (below 11.0%) for plasma, urine, blood and tissue homogenates. No interferences from endogenous substances or other diuretics tested were observed.

Determination of Malachite Green Residues in the Eggs, Fry, and Adult Muscle Tissue of Rainbow Trout (Oncorhynchus mykiss)

Malachite green, an effective antifungal therapeutant used in fish culture, is a known teratogen. We developed a method to simultaneously detect both the chromatic and leuco forms of malachite green residues in the eggs, fry, and adult muscle tissue of rainbow trout (Oncorhynchus mykiss). Homogenates of these tissues were fortified with [14C] malachite green chloride and extracted with 1% (v/v) acetic acid in acetonitrile or in methanol. The extracts were partitioned with chloroform, dried, redissolved in mobile phase, and analyzed by liquid chromatography (LC) with postcolumn oxidation of leuco malachite green to the chromatic form. LC fractions were collected every 30 s for quantitation by scintillation counting. Recoveries of total [14C] malachite green chloride residue were 85 and 98% in eggs fortified with labeled malachite green at concentrations of 0.5 and 1.00 microgram/g, respectively; 68% in fry similarly fortified at a concentration of 0.65 microgram/g; and 66% in muscle homogenate similarly fortified at a level of 1.00 microgram/g. The method was tested under operational conditions by exposing adult rainbow trout to 1.00 mg/L [14C] malachite green chloride bath for 1 h. Muscle samples analyzed by sample oxidation and scintillation counting contained 1.3 and 0.5 microgram/g total malachite green chloride residues immediately after exposure and after a 5-day withdrawal period, respectively.

High-performance liquid chromatographic assay for antiinflammatory agents diclofenac and flurbiprofen in ocular fluids

An HPLC assay for diclofenac (DC) and flurbiprofen (FP) in a 100-μl sample of aqueous humour is presented. After acetonitrile extraction, the residue is analyzed using a reversed-phase octyle column and ultraviolet detection. The method is simple and reproducible. Excellent selectivity and resolution are achieved using an acetic acid—acetonitrile—triethylamine mobile phase. The lower limit of detection, defined as the amount of drug required to produce a peak twice the threshold, was ca. 0.3 ng DC on column and ca. 0.4 ng on column FP. The utility of the method is demonstrated by determining drug levels in aqueous humour of normal rabbits and of patients undergoing cataract surgery. The assay should be applicable to other antiinflammatory agents.

Liquid Chromatographic Method for Analysis of the Naphthalene Dicarboxaldehyde Derivative of Fumonisins

Abstract A liquid chromatographic (LC) method for determining fumonisins in corn, feed, and laboratory cultures was optimized. We investigated the efficiency of extraction solvents, the performance of cleanup columns, the stability of fluorescent derivatives, and the efficiency of LC columns to separate deri-vatized fumonisins from interferences so that we could develop a sensitive, reliable assay for fumonisins. Maximum extraction efficiency was accomplished by shaking contaminated samples in acetonitrile–water (50 + 50, v/v) for 1 h; methanol–water mixtures and other ratios of acetonitrile–water were less efficient. Solid-phase extraction cleanups on reversed-phase Cia and strong anion exchange (SAX) columns were compared. The SAX columns were very efficient in isolating fumonisins from crude extracts; however, only the C18 columns could be used to partially purify fumonisin hydrolysis products. A highly fluorescent and stable derivative, prepared from naphthalene-2,3-dicar-boxaldehyde, proved to be suitable for LC analysis. Both parent compounds and hydrolysis products were separated in a simple gradient systjem of acetonitrile-acetic acid and water-acetic acid in less than 30 min.

Determination of pilocarpine in aqueous humour by liquid chromatography—atmospheric pressure chemical ionization mass spectrometry

A new method has been developed for rapid analysis and determination of pilocarpine in aqueous humour using liquid chromatography—atmospheric pressure chemical ionization mass spectrometry. The chromatography was carried out on a reversed-phase phenyl column with 0.1% acetic acid—acetonitrile (95:5, v/v). Pilocarpine and its analogues, isopilocarpine, pilocarpic acid and isopilocarpic acid, were separated. An aqueous humour sample was deproteinized with methanol. After evaporation, the residue was dissolved in the mobile phase. The method was applied to the analysis of the metabolite in aqueous humour after the topical application of 2% pilocarpine (w/v) eye-drops. The main metabolite, pilocarpic acid, was easily identified. The protonated molecular ion of pilocarpine was used for the determination. The calibration curve had a good linearity within the concentration range investigated (2 ng to 10 μg/ml). The limit of determination was estimated to be an aqueous humour concentration of ca. 2 ng/ml. The method was applied to the determination of unchanged pilocarpine after the topical application of 2% pilocarpine (w/v) eye-drops.

Development and validation of a high-performance liquid chromatographic method for the quantitation of warfarin enantiomers in human plasma

An HPLC method for the quantitation of warfarin enantiomers in human plasma has been developed and validated. Baseline separation of S- and R-warfarin was achieved on a silica-bonded β-cyclodextrin column with a mobile phase of acetonitrile—acetic acid—triethylamine (1000:3:2.5, v/v/v). The detection was performed at 320 nm. The established linearity range was 12.5–2500 ng ml −1 ( r > 0.99). Tle limit of quantitation was 12.5 ng ml −1 for each enantiomer. Inter-day precision and accuracy of 12.5 ng ml −1 standards were 12.1% relative standard deviation (RSD) and +0.67% bias for S-warfarin and 9.7% RSD and +10.8% bias for R-warfarin. The low quality control samples at 37.5 ng ml −1 showed 6.9% RSD and 0.0% bias for S-warfarin, 7.2% RSD and +0.5% bias for R-warfarin. S-Naproxen was used as internal standard. Potential metabolites of warfarin were well resolved from S- and R-warfarin, the internal standard ( S-naproxen) and each other. The run time was 25 min. The silica-bonded β-cyclodextrin column showed excellent stability; over 1000 samples were injected without significant loss of performance. The column variability test showed that the method can be applied on several batches of β-cyclodextrin columns but not all the β-cyclodextrin columns were suitable for this method.

Thin-layer chromatography of urinary 17-oxosteroids using dansylhydrazine as a prelabelling reagent

This paper describes a modification of a high-performance liquid chromatographic method for measurement of 17-oxosteroids in biological fluids for use with thin-layer chromatography and fluorometric scanning detection. After extraction from urine samples with Separon-C 18 microcolumns, free oxosteroids were labelled with dansylhydrazine in acetonitrile-acetic acid and chromatographed on silica gel F-254 plates with the solvent system chloroform—methanol (97:3). Linearity of fluorescence detection (Shimadzu CS-9000 densitometer) was obtained between 30 and 1000 ng.

Photochemical transformations. 49. Solvent effects on singlet ionic and triplet photoreactions of some bridged polycyclic chlorides: Comparison with ground-state reactions

Direct irradiations of 2-chloro-6,7 : 8,9-dibenzotricyclo [3.2.2.02,4] nona-6,8-diene (5-Cl) were conducted in cyclohexane, acetic acid and wet acetonitrile. The products are 1-methylfluoranthene (8) in all three solvents, the allylic chlorides 8-chloro-7-methylene-2,3 : 5,6-dibenzobicyclo [2.2.2] octa-2,5-diene (3-Cl) and 7-chloromethyl-2,3 : 5,6-dibenzobicyclo [2.2.2] octa-2,5,7-triene (2-Cl) in acetic acid and acetonitrile and solvolysis product amides in wet acetonitrile. Compound 5-Cl had previously been shown to be the product of triplet sensitization of 2-Cl and 3-Cl, so that the singlet reaction reverses that of the triplet. The formation of 8 from 5-Cl was quenched with piperylene, whereas that of the allylic chlorides was not. Quantum yields of products and singlet lifetimes in the three solvents were measured. The solvent effects are discussed. Deuterium-labeling results on the formation of 5-Cl from 2-Cl and of 8 from 5-Cl are reported.

Alternative mobile phases for the reversed-phase high-performance liquid chromatography of peptides and proteins

The use of a high content of acetic acid as mobile phase additive for the reversed-phase high-performance liquid chromatography (RP-HPLC) of several proteins and extracts of biological tissues was evaluated for a divinylbenzene (DVB)-based stationary phase, and the separations obtained with acetic acid gradients in acetonitrile, isopropanol or water were compared with classical polypeptide RP-HPLC on silica C 4 with trifluoroacetic acid (TFA)acetonitrile. The separation patterns for recombinant derived interleukin-1β (IL-1β) on the C 4 column eluted with TFA—acetonitrile and the DVB column eluted with acetic acid—acetonitrile were similar, but only the polymeric column was able to separate the components present in an iodinated IL-1β preparation. Neither eluent has any harmful effect on the biological activity of IL-1β isolated after RP-HPLC. Several standard proteins could be separated when the polymeric column was eluted with acetic acid gradients in acetonitrile, isopropanol of water and, although the separation efficiency with acetic acid in water was lower than that in combination with classical organic modifiers, insulin, glucagon and human growth hormone (hGH) were eluted as sharp, symmetrical peaks. The recoveries of insulin and hGH were comparable for all three mobile phases (80–90%). The separation patterns obtained from a crude acetic acid extract of a normal and a diabetic, human pancreas analysed using acetic acid gradients with or without orgaic modifiers were found to be similar and comparable to those obtained on a silica C 4 column eluted with an acetonitrile gradient in TFA. The principal differences resulted from the use of different UV wavelengths (215 nm for TFA—acetonitrile, 280 nm for acetic acid). Acetic acid extracts of recombinant derived hGH-producing Escherichia coli were separated on the DVB column eluted with an acetic acid gradient in water. Although the starting material was a highly complex mixture, the hGH isolated after this single-step purification was suprisingly pure (as judged by sodium dodecyl sulphate—polyacrylamide gel electrophoresis). Consequently several (pure) polypeptides and complex biological samples were separated on a polymeric stationary phase eluted with acetic acid gradients in water without the use of organic modifiers.

High-performance liquid chromatographic—mass spectrometric assay of high-value compounds for pharmaceutical use from plant cell tissue culture: Cinchona alkaloids

The use of high-performance liquid chromatography (HPLC) interfaced with thermospray (TSP) mass spectrometry is described for the separation and identification of various alkaloids from Cinchona ledgeriana extracts. The use of water—acetonitrile—acetic acid (71:25:4) with 0.01 M ammonium acetate (pH 3.0) as the mobile phase gave good HPLC separation and good TSP sensitivity. The specificity obtained by single-ion monitoring allowed the analysis of commercially important alkaloids such as quinine and quinidine in plant material, transformed roots and in cells from tissue culture, with relatively simple extraction and work-up procedures. TSP gave protonated species with few fragment ions but collision-induced dissociation offers the promise of increased analytically specificity from the fragment ion data. This work has important implications for the biotechnological production of pharmaceuticals normally obtained from plant sources.

Preparative-scale synthesis and reversed-phase purification of a gonadotropin-releasing hormone antagonist

The preparation of “Nal—Glu” antagonist (Ac— d-Nal— d-Cpa— d-Pal—Ser—Arg— d-2-amino-5-oxo-5-(4-methoxyphenyl)pentanoic acid—Leu—Arg—Pro— d-Ala—NH 2) was accomplished in two steps: (i) preparation of [Ac— d-Nal 1, d-Cpa 2, d-Pal 3, Arg 5, d-Glu 6, d-Ala 10]-GnRH via standard solid-phase synthetic techniques and (ii) acylation of anisole (Friedel—Crafts) by the glutamic acid residue in position 6. The preparative-scale, reversed-phase high-performance liquid chromatographic (HPLC) purification of the crude “Nal—Glu” antagonist employs first a triethylammonium phosphate (TEAP) (Ph 2.25)—acetonitrile solvent system, followed by an HPLC-based desalting procedure, yielding the acetate salt of the peptide. This repetitive process of purification in TEAP—acetonitrile, followed by counter-ion exchange with 0.5% acetic acid—acetonitrile is highly reproducible and allows large amounts of a given peptide to be purified efficiently in a batchwise fashion. The procedure described for the synthesis, purification and characterization of the “Nal—Glu” antagonist is presented as a model for the multi-gram synthesis and purification of peptides to be used in clinical investigations.

Determination of Acidic Herbicides and Related Compounds in Water and Soil by Capillary Gas Chromatography Using a Nitrogen-Phosphorus Detector

Methods are described for the determination of acidic herbicides and related compounds in water and soil. Eight acidic herbicides and related compounds were extracted from water using either dichloromethane or an XAD-2 resin column. The acidic moieties were derivatized with 2-cyanoethyldimethyl(diethyl)aminosilane. The derivatized compounds were separated using capillary gas chromatography and quantitated using a nitrogen-phosphorus detector. Extraction from water using dichloromethane or an XAD-2 resin column resulted in recoveries greater than 90% at 0.1 ppb with an average coefficient of variation (CV) of 6%. In soils extracted with aqueous acetonitrile-acetic acid and partitioned into dichloromethane, recoveries at 500 ppb were greater than 75% with an average CV of 3.3%. The methods are rapid and there are few interferences.

Titrations in non-aqueous media. Part XIII. Potentiometric and conductimetric titrations of alpha-amino acids with perchloric acid in acetic acid and acetonitrile-acetic acid solvents.

Of the 22 α-amino acids studied, all but five (tyrosine, cystine, cysteine, glutamic acid and aspartic acid) have been titrated potentiometrically with perchloric acid in two solvents, glacial acetic acid and 90% acetonitrile-acetic acid. The half-neutralisation potentials and the pKa′ values of the amino acids in these solvents have been calculated. The half-neutralisation potentials of the monoaminomonocarboxylic acids were ca. 390 mV in acetic acid and ca. 290 mV in 90% acetonitrile-acetic acid. The titrations of binary mixtures of histidine with monoaminomonocarboxylic α-amino acids have also been carried out in these solvents. The titration curves of binary mixtures of histidine with diaminomonocarboxylic acids showed only one stoicheiometric end-point, corresponding to the sum of the four amino groups. The data obtained from the titrations in glacial acetic acid suggested that this solvent has a strong levelling effect on the basicity of the amino groups of the α-amino acids. All the amino acids titrated potentiometrically could be determined with an error of less than ±3%.Seventeen of the 22 α-amino acids have also been titrated conductimetrically in acetic acid. All the α-amino acids gave one stoicheiometric end-point except for histidine and arginine which gave two.No reliable conductimetric titration curves or results could be obtained in 90% acetonitrile-acetic acid. In those conductimetric determinations of α-amino acids that were possible, the relative errors never exceeded ±3%.

Photochemistry of 5-methylpyrimidin-4-ones in acetic acid solution: thermal rearrangements of Dewar pyrimidinones and 4-acetoxyazetidin-2-ones

Irradiation of 3,6-dialkyl-5-methylpyrimidin-4-ones (1a–f) in acetic acid-acetonitrile (1 : 2, v/v) solution at 0 °C gave 4-acetoxy-3-(1-imino-2,2-dimethylpropyl)-1,3,4-trimethylazetidin-2-one (3a), 6-acetoxy-7-(1-imino-2,2-dimethylpropyl)-7-methyl-1-azabicyclo[4.2.0]octan-8-one (3b), 4-acetoxy-3-acetyl-1,3,4-trialkylazetidin-2-ones (4c–e), 4-acetoxy-3-acetyl-1,3-dimethylazetidin-2-one (4f), 3-acetyl-4-alkylidene-1,3-dimethylazetidin-2-ones (5d) and (5e) as the major products. Reaction of 1,4,6-trimethyl-3-t-butyl-2,6-diazabicyclo[2.2.0]hex-2-en-5-one (2a)(Dewar pyrimidinone) in acetic acid–acetonitrile (1 : 14, v/v) solution at 0 °C and thermolysis of (2a) without solvent at 80 ° C gave the imine azetidin-2-one (3a) and 2,3,5-trimethyl-6-t-butylpyrimidin-4(3H)-one (1a) respectively as the major products. Reactions of the imino azetidin-2-ones (3a) and (3b) and the 4-acetoxyazetidin-2-ones (4c–e) in the presence of acetic acid at 21–40 °C and without solvent at 100–110 °C were carried out to elucidate the rearrangements. The major products from (3a) and (3b) in acidic solutions at 40 °C were N-acetyl-3-acetamido-2,4,4,N-tetramethylpent-2-enamide (6a), N-methylacetamide (9a), 2,5-dimethyl-4-t-butyl-1,3-oxazin-6-one (10a), 2-piperidone (9b), and 1,3-oxazin-6-one (10b)=(10a), and from the thermolysis of (3a) and (3b) at 100–110 deg; C were the pyrimidin-4-ones (1a) and (1b), 1,3-dimethyl-3-pivaloyl-4-vinylazetidin-2-one (5a) and 7-methyl-7-pivaloyl-1-azabicyclo[4.2.0]octa-5-en-8-one (5b). Both reactions of (4d,e) at 23–24 °C in acidic solutions and of (4c,d) without solvent at 100–110 °C gave the 3-acetyl-4-alkylidene-1,3-dimethylazetidin-2-ones (5c–e) as the major products. The reaction mechanisms and intermediates of these reactions are discussed.