Methanol-acetic Acid Research Articles

Comparison of different sample treatments for the analysis of ochratoxin A in must, wine and beer by liquid chromatography

Ochratoxin A (OTA) is a mycotoxin produced by some species of Aspergillus and Penicillium verrucosum. It has been found in foods and feed all over the world. There is a great concern about OTA because it is nephrotoxic and probably, carcinogenic to humans. Most of analytical methods developed for OTA in wine, beer and other products are based on LC with fluorescence detection (LC–FLD). In the present work, various procedures for extraction and/or clean-up for determination of OTA in musts, wine and beer by LC–FLD were compared: (1) dilution with polyethylen glycol 8000 and NaHCO 3 solution and clean-up an on immunoaffinity column (IAC); (2) extraction with chloroform and IAC clean-up; solid-phase extraction (SPE) on (3) reversed-phase (RP) C 18; (4) RP phenylsilane and (5) Oasis HLB cartridges. SPE on phenylsilane and Oasis HLB have not been reported for OTA analysis in beverages. The same LC–FLD conditions and concentration ratio were used. The former procedure was simple, rapid and provided flat baselines, free from most impurity peaks, high OTA recoveries and quite repeatable results. RP C 18 using methanol–acetic acid (99.5:0.5) as elution solvent provided good recoveries and precision, thus becoming a cheaper but interesting alternative at 0.1–1 ng/ml spiking levels. Oasis HLB cartridges were usually better than phenylsilane. Possible binding of OTA to proteins or other components was tested by acid treatment before extraction but no significant differences with controls appeared.

Some novel N-(carboxyacyl)chitosan filaments

A suspension of chitosan filaments in methanol was treated with each of maleic ( 1), (2-octen-1-yl)succinic ( 2), citraconic ( 3), phthalic ( 5), glutaric ( 4), and succinic ( 9) anhydrides to give rise to novel six N-(carboxyacyl)chitosan filaments ( F1– F5 and F9) at d.s. 0.09–0.75 for N-carboxyacyl. However, no N-(carboxyacyl)chitosan filaments were obtained by treatment with each of trimellitic ( 6), 4-methylphthalic ( 7) and pyromellitic ( 8) anhydrides due to a steric hindrance in the chitosan filaments. On the other hand, chitosan was N-carboxyacylated in a solution of 2% aqueous acetic acid–methanol (1:1, v/v) with each of 1– 9 to give rise to N-(carboxyacyl)chitosan derivatives C1– C9 (d.s. 0.39–0.80 for N-acyl) in 69–96% yields. Each of C1, C4– C7, C9 and F1 was soluble in water and in 1% aqueous sodium hydroxide solutions.

Arsenic speciation in marine certified reference materials

This study describes the analysis of arsenicals in three marine based certified reference materials (CRMs), DORM-2, DOLT-2 and TORT-2, all produced by the National Research Council of Canada. Extraction protocols involving accelerated solvent extraction (ASE) and sonication were investigated and arsenicals were determined on-line by anion-exchange and cation-exchange high-performance liquid chromatography (HPLC). The major species in all three tissues was found to be arsenobetaine (AsB), corresponding to 90%, 67% and 73% of the water-soluble arsenic in DORM-2, DOLT-2 and TORT-2, respectively. Other species quantified were arsenocholine (AsC+), tetramethylarsonium ion (TMAs+), trimethylarsonioproprionate (TMAP), dimethylarsenic acid (DMA), monomethylarsenic acid (MMA), arsenosugar D (As-sug D), arsenate (AsV) and arsenosugar A (As-sug A). The major species detected in all three materials were AsB and DMA, which together accounted for 97.4% (DORM-2), 95.4% (DOLT-2) and 91.7% (TORT-2) of the sum of the water soluble As species. The agreement for the determination of AsB by the different methods was good for all three materials, whereas some discrepancy was evident in the results for DMA. When an ASE approach with 50% acetic acid in methanol was used for the extraction, between 22% (DOLT-2) and 58% (TORT-2) more DMA was determined compared with extraction by sonication with water. Some discrepancy was evident between the sum of the species extracted and both the total As determined in the extracts and the certified As content based on complete digestion of the materials. The sum of the species recovered by sonication corresponded to 84% (DOLT-2) to 94% (DORM-2) of the total As determined in the aqueous extract. In comparison with the certified As content of the three materials, the sums of the extracted species range from 46% (DOLT-2) to 76% (TORT-2) and 102% (DORM-2).

Determination of Clenbuterol, Salbutamol, and Cimaterol in Bovine Retina by Electrospray Ionization–Liquid Chromatography–Tandem Mass Spectrometry

Abstract An electrospray ionization–liquid chromatography–tandem mass spectrometry (ESI/LC/MS/MS) method was developed for the simultaneous determination of the β-agonists clenbuterol, salbutamol, and cimaterol in bovine retina. The tissue was homogenized in alkaline buffer and spiked to give 10, 15, and 20 ng/g each of the 3 analytes together with the internal standards d6-salbutamol and d6-clenbuterol. The mixture was incubated with protease enzyme to release any protein-bound analytes and then made alkaline before extraction with isobutanol. The extract was dissolved in water and transferred to a clenbuterol immunoaffinity column. After washing, the analytes were eluted and analyzed by ESI/LC/MS/MS using a C18 column with acetic acid–methanol as mobile phase. No interferences were observed from the spiked retina extract at the various single-reaction monitoring modes. Average recoveries for clenbuterol, salbutamol, and cimaterol were 94, 85, and 87% with coefficients of variation (CVs) of 9.4, 9.9, and 8.6%, respectively. A correlation coefficient of r2 = 0.9999 was obtained for all analytes. The limits of detection for clenbuterol, salbutamol, and cimaterol, determined from 3 times the standard deviation of 7 replicates of the lowest spike, were 2.5, 3.5, and 2.0 ng/g with CVs of 8.9, 11.6, and 7.2%, respectively.

Simultaneous co-extraction of organometallic species of different elements by accelerated solvent extraction and analysis by inductively coupled plasma mass spectrometry coupled to liquid and gas chromatography.

This paper describes the development of an accelerated solvent extraction methodology that is capable of simultaneously extracting organometallic species of As, Sn and Hg in a semi-automated manner. Accelerated solvent extraction (ASE) methods based on previous research on the separate extraction of organotin and -arsenic species in our laboratory were adapted for the co-extraction of six different species from an oyster tissue certified reference material (BCR 710). For the first time, the extraction of MeHg by this technique is also investigated. The proposed ASE conditions employed 50% acetic acid in methanol at a temperature of 100 degrees C with up to five consecutive extraction cycles of 3 minutes. Extraction efficiencies for organoarsenic species ranged from 80% (dimethylarsinic acid, DMA) to 99% for arsenobetaine (AsB). Species of toxicological interest, such as dibutyltin (DBT), tributyltin (TBT) and methylmercury (MeHg), were extracted with mean recoveries of 81, 84 and 76%, respectively. The extracted species were analysed by gas chromatography/inductively coupled plasma mass spectrometry (GC/ICPMS; DBT, TBT and MeHg) and liquid chromatography/inductively coupled plasma mass spectrometry (LC/ICPMS; MMA, DMA, AsB) after ethylation with sodium tetraethylborate or dilution with water, respectively. In addition to those species for which the extraction efficiency was assessed during this study, a further five arsenic species (arsenite, arsenate and three unidentified species), as well as monobutyltin (MBT) and mono-, di- and triphenyltin, could also be extracted from other matrices. The developed ASE method provides a significant improvement over many currently available routine monitoring methods for trace element speciation due to the fact that it is capable of extracting several species of toxicological interest simultaneously and quantitatively.

Genome size of Adriatic seagrasses

Genome size (C-value) was measured in four species of Adriatic seagrasses with interphase-peak DNA image cytometry. The estimated 2C-value was 1.5 pg DNA for Zostera noltii (2 n=12), 1.2 pg for Zostera marina (2 n=12), 1.1 pg for Cymodocea nodosa (2 n=28) and 6.2 pg for Posidonia oceanica, using Pisum sativum (2C-value=8.84 pg) as the calibration standard. Seagrass leaves were fixed in 4% buffered formaldehyde to mitigate stoichiometric error due to tannins and post-fixed in 3:1 methanol:acetic acid (MAA). DNA was stained with the Feulgen reaction after hydrolysis in 5 M HCl for 90 min at 20 °C. Comparison of genome size of seagrasses with the data for other species of Alismatidae indicated that the ancestral genome of Alismatidae was relatively small.

Capillary electrophoresis/mass spectrometry: a promising tool for the control of some physiologically hazardous compounds. I-derivatives of 3-quinuclidinol.

A method based on the coupling of capillary electrophoresis with mass spectrometry (CE/MS) was developed for the monitoring of 3-quinuclidinol and its four N-alkyl derivatives (methyl, ethyl, propyl and isopropyl derivatives). A fragmentation study (collision-induced dissociation of ions in an ion trap) and optimization of the ion optics set-up for CE/MS experiments using direct infusion of a methanolic solution of the standards into the mass spectrometer were carried out in advance. Molecular ions of all quaternary compounds and the quasi-molecular ion [M + H]+ of free 3-quinuclidinol prevail in the mass spectra. In the MS/MS of propyl and isopropyl derivatives, the elimination of the alkyl chain dominates, leading to the ion at m/z 128. The fragmentation of the other compounds is more complex. Previous CE separation of the mixture of isobaric propyl and isopropyl derivatives is necessary for their unambiguous identification. A 10 mM ammonium acetate buffer (pH 4.0) is the optimum running electrolyte, allowing the CE separation of methyl, ethyl, propyl and isopropyl derivatives. A 0.5% (v/v) solution of acetic acid in methanol provides sufficient detection sensitivity when used as the sheath liquid. Limits of detection of 0.1 ppm for 3-quinuclidinol and 0.05 ppm for quaternary derivatives were achieved under the optimum conditions. The optimized method was applied to the determination of 3-quinuclidinol and related quaternary derivatives spiked into a sample of pond water. The experimental set-up for CE/MS/MS was investigated, which strongly increases the identification capability of the technique.

An approach to estimate the chemical structure of melanoidins

A blue pigment (Blue-M1, C27H31N4O13) was isolated from the reaction mixture of d-xylose and glycine in 60% ethanol (starting pH 8.1) stored at 26.5 °C for 48 h or 2 °C for 96 h under nitrogen. Its novel chemical structure, two pyrrolopyrrole rings combined with methine bridge, was elucidated. Involvement of 3-deoxyxylosone, xylosone and N-substituted pyrrole-2-aldehyde to the formation of Blue-M1 were proposed. Blue-M1 kept five hydroxyl groups belonging to 3-deoxyxylosone and xylosone. The chemical character of Blue-M1 was compared to that of a nondialyzable melanoidin preparation (Melanoidin-1) obtained from the reaction mixture of d-xylose and butylamine neutralized with acetic acid in methanol incubated at 50 °C for 7 days. The results indicated strong resemblance between both substances, suggesting that Blue-M1 would be a key intermediate in melanoidin formation. The possibility of occurrence of pyrrolopyrrole ring formation in colored advanced glycation end products (AGEs) was also discussed.

HPLC and spectrophotometric determination of annatto in cheese

A rapid and simple method for the detection of E 160b (annatto) in cheese and other milk products is described. After extraction with acetone, the two main components bixin and norbixin are separated from fat and β-carotene by solid-phase extraction (SPE). Both sample preparation and determination are made without halogenated solvents. For quantification of the annatto content, high-performance liquid chromatography (HPLC) and a spectrophotometric method were used. For the spectrophotometric method, extinction values of bixin and norbixin were determined in a mix of methanol and glacial acetic acid. The spectrophotometry is sufficient for the quantitative determination of the entire content of annatto. If the colorings E 100 (curcumine) or E 160f (β-apo-8'-carotenal) are present beside annatto, then HPLC is favored. Other food colors and emulsifiers do not disturb the determination. The correlation coefficient between the different methods was R2=0.9989. The recovery rates determined by spiking cheese, processed cheese, butter, and ice cream, were between 80 and 100%.

Direct chiral separation of troglitazone stereoisomers using reversed-phase high-performance liquid chromatography

A simple HPLC method for the direct chiral separation of troglitazone stereoisomers was developed. The separation was performed on a reversed-phase cellulose-derivertized chiral column (Chiralcel OJ-R) using a mobile phase consisting of methanol–acetic acid (1000:1, v/v) at a flow rate of 0.5 ml/min. The peak areas of stereoisomers separated from 0.13 to 0.75 mg/ml of troglitazone had good linearity, with correlation coefficients >0.999 in the reversed-phase mode. The repeatability of the ratios of stereoisomers isolated from 0.5 mg/ml of troglitazone had a relative standard deviation of 0.1–0.2%. The relative sensitivities of the four isomers at UV 285 nm were similar, as each response factor was within the range of 0.99–1.01. Troglitazone racemized at the chiral center of the thiazolidine ring in methanol solution, but was found to be stable for 24 h in methanol–acetic acid (1000:1, v/v). This method was applied to the stereoisomeric analysis of troglitazone in pharmaceutical formulations and used to evaluate the constancy of the stereoisomer ratio in the manufacturing process and stability testing.

ChemInform Abstract: Direct C‐Nitration of Cyclic α,β‐Unsaturated Oximes under the Action of Sodium Nitrite and Acetic Acid in Methanol.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Improved sample extraction and clean-up for the GC-MS determination of BADGE and BFDGE in vegetable oil

A straightforward method was established for the determination of migration contaminants in olive oil with a special focus on the two can-coating migration compounds bisphenol A diglycidyl ether (BADGE) and bisphenol F diglycidyl ether (BFDGE). The preferred sample preparation was a single liquid-liquid extraction of compounds from the oil into 20% (v/v) methanol in acetonitrile, followed by clean-up with solid-phase extraction on aminopropyl bonded to silica. This purification procedure selectively removed all free fatty acids from the extracts without removing phenolic compounds of interest. The solid-phase extraction columns were used many times by implementing a procedure of washing out the strongly retained fatty acids with 2% acetic acid in methanol. Gas chromatography coupled with full scan (m/z 33-700) electron ionization mass spectrometry was used for the determination of several model compounds in olive oil samples. BADGE and BFDGE could be determined in the 0.05-2 mg kg−1 range in oil samples with a relative SD of <6% (six replicates). The method was used in an enforcement campaign for the Norwegian Food Control Authority to analyse vegetable oil samples from canned fish-in-oil.

SEMIQUANTITATIVE ANALYSIS BY THIN‐LAYER CHROMATOGRAPHY (TLC) OF BIOGENIC AMINES IN DRIED, SALTED AND CANNED FISH PRODUCTS

AbstractA thin‐layer chromatography (TLC) method was tested using dried, salted and canned fish products for the identification and semiquantification of biogenic amines: histamine, cadaverine, putrescine, tryptamine, and tyramine. The solvent system was acetone: NH4OH (95:5) on a precoated silica gel plate, and the developing reagent was 0.2% ninhydrin with 2% acetic acid in methanol. The TLC results were compared with liquid chromatography (LC). For LC levels between 6–9 mg%, TLC gave close to 5 mg%, and for samples with biogenic amines of 10–60 mg%, LC and TLC results are more similar to each other. In case of upper values (&gt; 60 mg%) other methods have to be used or sample dilution is suggested. The proposed TLC was suitable to estimate: cadaverine (5‐40 mg%), and tyramine (10‐20 mg%) in dried salted, and cadaverine (10‐20 mg%), tryptamine (5‐10 mg%), and histamine (10‐60 mg%) in canned fish. With this TLC methodology is possible to screen samples with these amines levels, for routine quality control in laboratories.

Quantification of cholesterol in foods using non-aqueous capillary electrophoresis

A simple method for the rapid quantification of cholesterol in egg yolk and milk by non-aqueous capillary electrophoresis (NACE) is described in this paper. The samples were treated with saponification and then quantified by NACE, in which 100 m M sodium acetate–acetic acid in methanol was employed as the running buffer. The correlation coefficient between the cholesterol concentration and the corresponding peak area was 0.999. The detection limit of cholesterol was 5 μg/ml (twice the signal-to-noise ratio). This method can be used as a routine method for the rapid and sensitive determination of cholesterol in foods.

Homogeneous ZnO Nanoparticles by Flame Spray Pyrolysis

Zinc oxide (ZnO) nanoparticles were made by flame spray pyrolysis (FSP) of zinc acrylate–methanol–acetic acid solution. The effect of solution feed rate on particle specific surface area (SSA) and crystalline size was examined. The average primary particle diameter can be controlled from 10 to 20 nm by the solution feed rate. All powders were crystalline zincite. The primary particle diameter observed by transmission electron microscopy (TEM) was in agreement with the equivalent average primary particle diameter calculated from the SSA as well as with the crystalline size calculated from the X-ray diffraction (XRD) patterns for all powders, indicating that the primary particles were rather uniform in diameter and single crystals. Increasing the solution feed rate increases the flame height, and therefore coalescence and/or surface growth was enhanced, resulting in larger primary particles. Compared with ZnO nanoparticles made by other processes, the FSP-made powder exhibits some of the smallest and most homogeneous primary particles. Furthermore, the FSP-made powder has comparable BET equivalent primary particle diameter with but higher crystallinity than sol–gel derived ZnO powders.

Evaluation of extraction techniques for the determination of butyltin compounds in sediments using isotope dilution-GC/ICPMS with 118Sn and 119Sn-enriched species.

Different liquid-solid extraction techniques, including room-temperature leaching with mechanical shaking, ultrasonic, and microwave-assisted extractions, have been evaluated for the quantitative speciation of tin for mono-, di-, and tributyltin (MBT, DBT, and TBT, respectively) in PACS-2 and BCR-646 certified reference materials. A methanol-acetic acid mixture was used as the extractant reagent in all cases. For this purpose, a mixed spike containing 119Sn-enriched MBT (79.7 At%), 118Sn-enriched DBT (86.7 At%), and 119Sn-enriched TBT (83.1 At%), was synthesized, characterized, and used for isotope dilution analysis. The isotopic composition of the mixed spike was determined by gas chromatography/ICPMS after aqueous ethylation using sodium tetraethylborate, and the determination of the concentration of the different species in the spike was performed by means of reverse isotope-dilution analysis using natural MBT, DBT, and TBT standards. In the analysis of the certified sediments, the sample was spiked with the mixed spike, extracted under different conditions, derivatized with sodium tetraethylborate, and extracted into hexane, and the isotope ratios 120/118 and 120/119 were measured as peak area ratios for all butyltin species after GC/ICPMS. Mass bias was corrected using a derivatized natural standard every three sample injections. Sequential degradation reactions during extraction (from TBT to DBT, from DBT to MBT, and from MBT to inorganic tin) were assumed, and mathematical equations were developed that allowed the determination of the correct species concentration and the decomposition factor for each of the transformation reactions. For ultrasonic extraction and mechanical shaking, negligible degradation reactions were observed. However, for microwave assisted extractions, degradation factors up to 7% (TBT to DBT) and 16% (DBT to MBT) were obtained for both reference materials when high-MW energy was applied in the extraction step. For the three extraction techniques tested, the DBT and TBT concentration values obtained for PACS-2 closely matched the certified values. However, for MBT the concentrations found by microwave and ultrasonic extraction were much higher than the certified value. This was not the case for mechanical shaking. The results obtained for BCR-646 using microwave assisted extraction were in good agreement with the certified values for all tin species.

Rapid HPLC screening of jasmonate-induced increases in tobacco alkaloids, phenolics, and diterpene glycosides in Nicotiana attenuata.

A rapid, HPLC-based screening procedure for the main classes of secondary metabolites in Nicotiana attenuata leaves (alkaloids, phenolics, and diterpene glycosides) is reported. In a single step, leaves are extracted in aqueous acidified (0.5% acetic acid) methanol, and the extracted compounds are separated by reversed-phase HPLC with an acidic water/acetonitrile gradient in <30 min. The utility of the method in quantifying changes in the secondary metabolites after methyl jasmonate treatment of the plants, a treatment known to elicit resistance to herbivores in nature, is illustrated. Methyl jasmonate treatment elicited dramatic increases in some secondary metabolites (caffeoylputrescine, nicotine, and diterpene glycosides increased 12.5-, 1.4-, and 1.9-fold, respectively) but left others, such as rutin, unchanged. Such broad-based analytical screens will help characterize environmental and genetic changes in secondary metabolite profiles.

Chromosomes of the Lancelet Branchiostoma belcheri Gray

We characterized the chromosomes of the lancelet Branchiostoma belcheri Gray. Testes were removed from adult males, cut up, treated with colchicine (25–50 μg/ml) for 3–6 hr, and dissociated into single cells by pippeting. The dissociated cells were swollen with 5.0–6.5% sodium citrate for 10 min and fixed with methanol-acetic acid (3:1). The fixed cells were dropped onto a glass slide and air-dried. The haploid chromosome number was 18 and diploid number was 36.

Preparative separation of isoflavone components in soybeans using high-speed counter-current chromatography

Four isoflavone components were purified from soybean extract by high-speed counter-current chromatography (HSCCC). Two types of multilayer coil separation columns were used: a small column made of standard 2.6-mm I.D. PTFE (polytetrafluoroethylene) tubing with a 260-ml capacity and a large column of convoluted PTFE tubing of 5.7-mm average I.D. with a 1200-ml capacity. Separation was performed with a two-phase solvent system composed of hexane–ethyl acetate–1-butanol–methanol–acetic acid–water (1:2:1:1:5:l, v/v) by eluting the lower aqueous phase at 2 ml/min (small column) and 5 ml/min (large column) at a revolution speed of 700 rpm. From 500 mg of crude sample the small column yielded 33 mg of daidzin, 41 mg of genistin, 27 mg of 6″- O-malonyldaidzin and 24 mg of 6″- O-malonylgenistin. The large convoluted column separated, from 3 g of crude sample, 203 mg of daidzin, 241 mg of genistin, 158 mg of 6″- O-malonyldaidzin and 135 mg of 6″- O-malonylgenistin all at over 90% purity. The convoluted tubing facilitated preparation of a large multilayer coil due to its high flexibility.

Analysis of black tea theaflavins by non-aqueous capillary electrophoresis

In this study a new capillary electrophoresis (CE) method was developed to quantify the four major theaflavins occurring in black tea. Where aqueous based CE methods showed poor selectivity and considerable band broadening, non-aqueous CE achieved baseline separation of the theaflavins within 10 min. The effects of the organic solvent composition and background electrolyte concentration on the separation selectivity and electrophoretic mobilities were investigated. Our optimized separation solution consisted of acetonitrile–methanol–acetic acid (71:25:4, v/v) and 90 mM ammonium acetate. This method was used to analyze three black tea samples.