Determination of polymer additives-antioxidants and ultraviolet (UV) absorbers by high-performance liquid chromatography coupled with UV photodiode array detection in food simulants

An analytical method for the quantitative determination of migration levels of polymer additives such as antioxidants and UV absorbers in food packages by high-performance liquid chromatography coupled with UV-vis photodiode array detection has been developed. The pretreatment step involved solid-phase extraction with silica C18 cartridges. The analytical method showed good linearity, presenting regression coefficients (R(2)) ≥ 0.9990 for all compounds. This optimized method was also validated with respect to precision, reproducibility, stability, and accuracy. The limits of detection and quantification were between 0.09 and 1.72 μg mL(-1) and between 0.20 and 5.64 μg mL(-1) for 12 analytes, respectively. Recoveries were in the range of 67.48 and 108.55%, with relative standard deviations between 2.76 and 9.81%. Migration levels of antioxidants and UV absorbers were determined. Butylated hydroxyanisole, 2,6-di-tert-butyl-4-methylphenol (BHT), 2,4-di-tert-butylphenol, Cyanox 2246, Irganox 1035, Tinuvin 326, Tinuvin 328, Irganox 1010, and Irganox 1330 were detected; BHT and Cyanox 2246 were at higher levels than the specific migration levels in some food simulants.     

Environmental analysis by on-line immunoextraction and reversed-phase liquid chromatography: optimization of the immunoextraction/RPLC interface

The use of antibodies in HPLC columns for on-line immunoextraction combined with reversed-phase liquid chromatography (RPLC) is of growing interest in environmental and agricultural analysis. This technique is typically performed by using a small RPLC precolumn to capture and concentrate analytes as they elute from the immunoextraction column; however, there is little information on the conditions required for optimizing this interface. This study examined the behavior of this interface by using 2,4-dichlorophenoxyacetic acid (2,4-D) and related herbicides as model analytes. It was found that analyte dissociation from immunoextraction columns followed first-order decay and that the elution of these analytes through the immunoextraction/RPLC interface gave an exponentially modified Gaussian profile. Computer simulations were used to see how analyte elution through the interface changed with different dissociation and retention conditions. Several guidelines were developed from this work that could be used for developing and optimizing on-line immunoextraction/RPLC systems for other chemicals of environmental or agricultural interest.     

Antioxidant content of and migration from commercial polyethylene, polypropylene, and polyvinyl chloride packages

Antioxidants commonly used in polyolefins were studied in commercial food packages made of low- and high-density polyethylene (LDPE and HDPE), polypropylene (PP), polyvinylchloride (PVC), and polyethylene terephtalate (PET) and in a LDPE film extruded at the laboratory. The phenolic antioxidants BHA, BHT, AO 2246, AO 425, Ethanox 330, Irganox 1010, and Irganox 1076 were studied together with the phosphite Irgafos 168 and their two degradation products, phosphate and DBP. Antioxidants were extracted from polyolefins using microwave energy and analyzed using high-performance liquid chromatography (HPLC) to determine the antioxidant content in the diverse commercial films. Irganox 1010 and Irganox 1076 were found in the majority of the samples generally together with the phosphite Irgafos 168 and its oxidized product (phosphate). Specific migration levels of each antioxidant were determined by HPLC after pretreatment with solid-phase extraction (SPE) in aqueous food simulants and after their dilution with tetrahydrofuran (THF) in fatty food simulant. These levels were much lower than limits allowed by legislation.     

Adsorption of Bisphenol A on Sediments in the Yellow River

Bisphenol A is widely used for the production of epoxy resins and polycarbonate plastics, and it has been found in many wastewaters or surface waters. Adsorption of bisphenol A on sediments sampled from several representative hydrologic stations of the Yellow River was studied, and some factors that may affect the sorption of bisphenol A were analyzed using the LC-MS/MS following solid-phase extraction. The results show that neither linear nor Freundlich isotherms is fit to the experimental data due to the high carbonate content in sediments. Bisphenol A has greater adsorption after the elimination of the carbonate in the sediment, and the adsorption of bisphenol A on the treated sediment can be described by both the linear and the Freundlich isotherms. The adsorption amount of bisphenol A is related to both the total organic carbon and dissolved organic carbon of sediments. The effects of Ca^{2 +} and K⁺ on the adsorption of bisphenol A were also studied. It is found that Ca²⁺ and K⁺ showed different effects on the adsorption of bisphenol A because of their different valences.     

Determination of oxyfluorfen herbicide and oxyfluorfen amine residues in garbanzo beans by liquid chromatography

Oxyfluorfen and oxyfluorfen amine were determined by liquid chromatography (LC) with ultraviolet (UV) and photoconductivity detection (PCD). A simple extraction procedure acceptably recovered both analytes from garbanzo beans over a wide range of fortifications (0.05 to 20 ppm) (83 +/- 4 for oxyfluorfen; 85 +/- 4 for oxyfluorfen amine). Percent recoveries decreased slightly as the fortification level decreased. Both analytes could be determined simultaneously at a concentration greater than 0.2 ppm in garbanzo beans. Detection limits were 3 ng for oxyfluorfen and 100 ng for oxyfluorfen amine using LC/UV, and 12 ng for both oxyfluorfen and oxyfluorfen amine with LC/PCD. Different knitted reaction coils and photoreactors were evaluated. Photoproduct yields and identification were determined by ion chromatography. The LC/PCD method measures oxyfluorfen and oxyfluorfen amine separately and has a shorter analysis time, while the standard method using gas chromatography measures total residues and is more sensitive.     

Determination of spinosad and its metabolites in food and environmental matrices. 2. Liquid chomatography-mass spectrometry

A selective and sensitive method utilizing liquid chromatography-mass spectrometry (LC-MS) has been developed for determining residues of the natural insect control agent spinosad in several crop matrices that are difficult to analyze by HPLC with UV detection. The method determines the active ingredients (spinosyns A and D) and three minor metabolites (spinosyns B and K and N-demethylspinosyn D) in alfalfa hay, wheat hay, wheat straw, sorghum fodder, and corn stover. The analytes are extracted from the samples with an acetonitrile/water solution, and the extracts are purified by solid phase extraction with a C(18) disk and a silica cartridge. All five analytes are determined simultaneously in a single injection using positive atmospheric pressure chemical ionization LC-MS with selected ion monitoring. The average recoveries ranged from 69 to 96% with standard deviations ranging from 4 to 15%. The method has a validated limit of quantitation of 0. 01 microgram/g and a limit of detection of 0.003 microgram/g. The LC-MS method can also provide residue confirmation in addition to quantitation.     

A mass spectrometric validated high-performance liquid chromatography procedure for the determination of folates in foods

A series of five food reference materials (RM) that had certified values of folate concentrations and five frozen food samples were analyzed for 5-methyltetrahydrofolic acid (5-MTHFA) and folic acid (FA) using a high-performance liquid chromatography (HPLC) method with fluorescence detection that was validated using an HPLC mass spectrometry (MS) method with electrospray ionization. Identical sample specimens were extracted and analyzed in triplicate using both instrumental methods, and a comparison was made of the mean values of 5-MTHFA and FA resulting from these determinations. The analytes were isolated on either a high capacity strong anion exchange solid phase extraction column (HPLC method) or a phenyl Bond Elut column (MS method) prior to analyses. For quantification of the analytes by MS, (13)C-labeled 5-MTHFA and FA were added to samples as internal standards prior to enzymatic digestion and conversion of the polyglutamate forms of 5-MTHFA to the monoglutamic acid. Quantification of FA and 5-MTHFA using the HPLC analysis was carried out using external standards. With the exception of one RM (pig liver), the values established for 5-MTHFA using these methods were highly comparable. In determining the variance associated with these two procedures, it was observed that the mean relative standard error for 5-MTHFA was 12 (range, 2-27%) and 11% (range, 5-25%) for the HPLC and MS methods, respectively. FA was detected in only three of the samples, and the values obtained for it by either method were similar. This is the first paper that describes a mass spectrometric method used in the validation of an HPLC determination of food folates across a wide range of sample matrixes. The comparable values for 5-MTHFA and FA suggest that HPLC analysis with fluorescent detection may be used to accurately quantify folates present in a variety of food matrixes.     

An evaluation of methods for measurement of pesticides in sorption experiments

Abstract

The concentration of four pesticides (2,4‐D, atrazine, phorate, and terbufos) in soil solution during sorption experiments was measured using UV spectrophotometry, Gas Liquid Chromatography (GLC), High Performance Liquid Chromatography (HPLC), and radiotracer technique. The presence of water soluble organic matter in soil solution interfered with the measurement of pesticide using the UV spectrophotometry. The use of GLC, HPLC, and radiotracer technique involving ¹⁴C gave a good estimate of the concentration of pesticide in soil solution. The pesticide remaining in soil can be quantitatively analyzed by extracting with a scintillation solution containing an organic solvent such as toluene or dioxane. Among the various centrifuge tubes glass tubes with Teflon caps sorbed negligible amount of pesticides and these tubes can be used for the sorption measurements.     

Kinetics of light-induced cis-trans isomerization of four piperines and their levels in ground black peppers as determined by HPLC and LC/MS

The pungent compounds piperine and isomers thereof, secondary metabolites present in black and white pepper fruit, undergo light-induced isomerizations. To facilitate studies in this area, an HPLC method has been developed for analysis and isolation of the following four possible piperine-derived photoinduced isomers: piperine, isopiperine, chavicine, and isochavicine. The limits of detection (LOD) estimated from calibration plots were approximately 15-30 ng for each isomer. Reproducibilities of the analyses were excellent, and recoveries of spiked samples were as follows (average +/- SD; n = 3): chavicine, 98.4 +/- 2.1%; isopiperine, 96.2 +/- 3.2%; piperine, 104 +/- 3.8%; isochavicine, 98.9 +/- 3.0%. To determine the kinetics of these isomerizations, fluorescent light, sunlight, and UV radiation at 254 nm was used to induce cis-trans geometric isomerization as a function of light intensities and time of exposure determined with the aid of high-performance liquid chromatography (HPLC) and liquid chromatography with diode array UV detection-mass spectrometry (LC-DAD/MS). HPLC was also used to determine the distribution of the isomers in four commercial ground black pepper products used as spices in culinary practice. Isomerization increased with light intensities and time of exposure and leveled off at the so-called photostationary phases. The piperine levels of the four products were quite similar, ranging (in wt %) from 10.17 to 11.68. The amounts of the other three isomers ranged from 0.01 to 0.07 of the total for chavicine; from 0.15 to 0.23 for isopiperine; and from 0.37 to 0.42 for isochavicine. The results establish the utility of the HPLC method for simultaneous analysis of the four isomers both in pure form and in black pepper extracts. The dietary significance of the results is discussed.     

High-performance liquid Chromatography/Mass spectrometry analysis of proanthocyanidins in foods and beverages.

Monomeric and oligomeric proanthocyanidins present in a range of plant-derived foods and beverages were separated by degree of polymerization and identified using a modified normal-phase high-performance liquid chromatography (HPLC) method coupled with on-line mass spectrometry (MS) analysis using an atmospheric pressure ionization electrospray chamber. In addition, ultraviolet (UV) and fluorescence detection were used to monitor the separation of proanthocyanidins, with fluorescence detection demonstrating both increased sensitivity and the ability to reduce interfering signals from other components present in the food and beverage matrices as compared to UV detection. This qualitative study demonstrates the ability of this HPLC/MS technique to separate singly and doubly linked procyanidins, prodelphinidins, and copolymer oligomers, including their galloylated derivatives, present in a range of food and beverage samples.     

Enhanced bioremediation of soil containing 2,4-dinitrotoluene by a genetically modified Sinorhizobium meliloti

The symbiotic nitrogen-fixing soil bacterium, Sinorhizobium meliloti, is well known for its ability to interact with the leguminous plant Medicago sativa L. It has, however, not been reported that this species possesses the capability to degrade toxic nitroaromatic compounds, such as 2,4-dinitrotoluene (DNT) which is commonly associated with the degradation of the explosive trinitrotoluene (TNT). In this study, the pJS1 DNT-biodegradative plasmid was genetically transferred to S. meliloti strain USDA 1936, which was confirmed by plasmid profile analysis. Several standard analytical and chemical tests including high performance liquid chromatography (HPLC), nitrite (NO₂) release assays, rhizosphere population and plant greenhouse studies were conducted to test the ability of S. meliloti to degrade 2,4-DNT. The possible presence of 2,4-DNT remaining in the treated soil was tested, and no 2,4-DNT had been absorbed by the soil. The pJS1-carrying recombinant strain DHK1 produced ‘ARC’ alfalfa plants that were almost 2-fold higher in shoot dry weight than that produced by the parent strain on soil containing 0.14 mM 2,4-DNT. The transconjugant strain DHK1 reduced significantly one-third more 2,4-DNT in both 0.14 and 0.28 mM contaminated soil, and in 0.55 mM contaminated soil it degraded 94% of the 2,4-DNT present. In liquid cultures, however, only about 4% reduction in 2,4-DNT concentrations was obtained in 10 days. We interpret the results as clearly establishing that genetic modification was successfully used, for the first time, to improve the capability of the symbiotic nitrogen-fixing soil bacterium S. meliloti DHK1 to bioremediate in situ 2,4-DNT-contaminated soil in the presence of alfalfa plants.     

One- and two-dimensional direct chiral liquid chromatographic determination of mixtures of diclofop-Acid and diclofop-methyl herbicides

Simple one- and two-dimensional high-performance liquid chromatography (HPLC) methods for the simultaneous enantiomeric determination of alkyloxyphenoxypropionic acid herbicides is presented. Compounds studied were ( R, S)-2-[4-(2,4-dichlorophenoxy)phenoxy]propionic acid (diclofop-acid) and ( R, S)-2-[4-(2,4-dichlorophenoxy)]methyl propionate (diclofop-methyl). Mobile phases necessary to separate their enantiomers on an alpha1-acid glycoprotein chiral stationary phase are different; therefore, the simultaneous separation by an isocratic mode is not possible. The chiral separation method proposed involves a one-step gradient allowing for the simultaneous determination of both racemic enantiomers. Detection limits of the method were 0.03 mg/L for both diclofop-acid enantiomers and 0.14 and 0.15 mg/L for diclofop-methyl enantiomers, respectively. The two-dimensional method involves the use of two chromatographs in one achiral-chiral coupling. The LC-LC method is more suitable for complex samples because it involves an online cleanup effect. Detection limits were 1.25 and 1.87 mg/L for diclofop-acid and 2.70 and 3.02 mg/L for diclofop-methyl enantiomers, respectively. Accuracy, repeatability, and reproducibility have been studied in standard samples and a technical product.     

Structure-activity relationship (SAR) studies of benzoxazinones, their degradation products, and analogues. Phytotoxicity on problematic weeds Avena fatua L. and Lolium rigidum Gaud

Avena fatua L. (wild oat) and Lolium rigidum Gaud. (rigid ryegrass) are highly problematic weeds affecting a wide variety of cereal crops worldwide. The fact that both of these weeds have developed resistance to several herbicide groups made them optimal candidates as target organisms for ongoing research about the potential application of allelochemicals and analogue compounds as natural herbicide models. Benzoxazinones, a family of natural allelochemicals present in corn, wheat, and rye, including 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one and 2,4-dihydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one, together with some degradation products, found in crop soils as well as in other systems, and some synthetic analogues of them were tested on wild oat and rigid ryegrass seeds; the results were statistically treated, and some structure-activity relationships, useful in further development of natural herbicide models, were elucidated. The most active compounds were the synthetic benzoxazinone 2-acetoxy-(2H)-1,4-benzoxazin-3(4H)-one and the degradation product 2-aminophenoxazin-3-one, with highly significant inhibition on the development of both weeds. The ecological role of these compounds is discussed by considering both degradability and phytotoxicity. The bioactivity of aminophenoxazines has been correlated by their aqueous solubility-lipophilicity predicted by means of computational methods.     

Determination of phenolic constituents of biological interest in red wine by capillary electrophoresis with electrochemical detection

A simultaneous determination of trans-resveratrol, (-)-epicatechin, and (+)-catechin in red wine by capillary electrophoresis with electrochemical detection (CE-ED) is reported. The effects of the potential of the working electrode, pH and concentration of running buffer, separation voltage, and injection time on CE-ED were investigated. Under the optimum conditions, the analytes could be separated in a 100 mmol/L borate buffer (pH 9.2) within 20 min. A 300 microm diameter carbon disk electrode has a good response at +0.85 V (vs SCE) for all analytes. The response was linear over 3 orders of magnitude with detection limit (S/N = 3) ranging from 2 x 10(-7) to 5 x 10(-7) g/mL for all analytes. This method has been used for the determination of these analytes in red wine without enrichment, and the assay result was satisfactory.     

On-line HPLC detection of tocopherols and other antioxidants through the formation of a phosphomolybdenum complex

An on-line method to detect and quantify antioxidant species in complex extracts has been developed as a combination of conventional HPLC separation and a postcolumn reaction with phosphomolybdenum reagent at acidic pH. Sample analytes were chromatographed by HPLC, and the postcolumn formation of a phosphate/Mo(V) complex was detected at 598 nm with an on-line absorbance detector. An optimized instrumental system was set up using pure alpha-tocopherol, and it was successfully tested with complex food extracts including lettuce, tomato, red pepper, and soybean seed, where several tocopherols and carotenoids were identified. A potential application of this detection method to quantitatively determine different antioxidants was considered, and a specific application to the determination of tocopherols was developed. The new method was characterized with respect to linearity interval, repeatability, and reproducibility, the quantitative results obtained were validated by comparison with a conventional HPLC method with fluorometric detection, and it was applied to the determination of tocopherols in different foods. The results suggest that the proposed on-line HPLC method can be a powerful instrument for the detection, purification, and characterization of natural antioxidants.     

Biotransformation of vinclozolin by the fungus Cunninghamella elegans

This study investigated the biotransformation of the dicarboximide fungicide vinclozolin [3-(3,5-dichlorophenyl)-5-methyl-5-vinyl-1,3-oxazolidine-2,4-dione] by the fungus Cunninghamella elegans. Experiments with phenyl-[U-ring-14C]vinclozolin showed that after 96 h incubation, 93% had been transformed to four major metabolites. Metabolites were separated by HPLC and characterized by mass and NMR spectroscopy. Biotransformation occurred predominantly on the oxazolidine-2,4-dione portion of vinclozolin. The metabolites were identified as the 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide, N-(2-hydroxy-2-methyl-1-oxobuten-3-yl)-3,5-dichlorophenyl-1-carbamic acid, and 3',5'-dichloro-2-hydroxy-2-methylbut-3-enanilide. The enanilide compound has been reported previously as a plant and mammalian metabolite and is implicated to contain antiandrogenic activity. The 3R- and 3S- isomers of 3',5'-dichloro-2,3,4-trihydroxy-2-methylbutyranilide are novel metabolites.     

Determination of bisphenol a and bisphenol B residues in canned peeled tomatoes by reversed-phase liquid chromatography

Bisphenol A (BPA) and bisphenol B (BPB) concentrations were determined in peeled canned tomatoes of different brands bought in Italian supermarkets. Tomato samples analyzed were packaged in cans coated with either epoxyphenolic lacquer or low BADGE enamel. A solid phase extraction (SPE) was performed on C-18 Strata E cartridge followed by a step on Florisil cartridge. Detection and quantitation were performed by a reversed phase high-performance liquid chromatography (RP-HPLC) method with both UV and fluorescence detection (FD). On the total of 42 tested tomato samples, BPA was detected in 22 samples (52.4%), while BPB was detected in 9 samples (21.4%). BPA and BPB were simultaneously present in 8 of the analyzed samples. The levels of BPA found in this study are much lower than the European Union migration limits of 3 mg/kg food and reasonably unable to produce a daily intake exceeding the limit of 0.05 mg/kg body weight, established by European Food Safety Authority.     

Unfermented rooibos tea: quantitative characterization of flavonoids by HPLC-UV and determination of the total antioxidant activity

Unfermented rooibos originates from the leaves and the stems of the indigenous South African plant, Aspalathus linearis, and it has been reported to have a higher content of flavonoids compared to that of fermented rooibos. The HPLC/UV method developed in our laboratory for the analysis of the fermented rooibos was applied to the quantitative characterization of the major flavonoids present in the unfermented rooibos. Main compounds determined were aspalathin (49.92 +/- 0.80 mg/g), isoorientin (3.57 +/- 0.18 mg/g), orientin (2.336 +/- 0. 049 mg/g), and rutin (1.69 +/- 0.14 mg/g), followed in order by isovitexin, vitexin, isoquercitrin and hyperoside, quercetin, luteolin and chrysoeryol. The identity of detected flavonoids was confirmed by comparing their retention times and UV spectra with those of corresponding standards. The total antioxidant activity (TAA) of the tea infusions was measured by the ABTS*+ radical cation decolorization assay. The TAA of unfermented rooibos (0.8 Trolox meq/g) resulted 2-fold higher than that of the fermented rooibos. When compared with different water infusions of Camellia sinensis (green and black tea), this TAA value was about 50% lower.     

Determination of benzoxazinone derivatives in plants by combining pressurized liquid extraction-solid-phase extraction followed by liquid chromatography-electrospray mass spectrometry

A new analytical method based on the use of pressurized liquid extraction (PLE) followed by solid-phase extraction with LiChrolut RP C18 cartridges was evaluated for the sample preparation, extraction, and cleanup of eight naturally occurring benzoxazinone derivatives, 2-beta-D-glucopyranosyloxy-4-hydroxy-1,4-benzoxazin-3-one, 2-beta-D-glucopyranosyloxy-4-hydroxy-7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one, 2-hydroxy-1,4-benzoxazin-3-one, 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one, benzoxazolin-2-one, and 6-methoxybenzoxazolin-2-one in plant samples. Afterward, liquid chromatography-electrospray mass spectrometry, using the selected ion monitoring mode and internal standard (2-MeO-DIBOA, indoxyl-beta-D-glucoside, and quercetin-3-O-rutinoside) quantification method was performed. This paper demonstrates the effectiveness of the PLE method, in conjunction with sensitive and specific mass spectrometric detection, for the quantitative recovery of compounds of the benzoxazinone class from plants. The recoveries of the analytes ranged from 66 to 110% with coefficients of variation ranging from 1 to 14%. This method gave detection limits between 1 and 27 microg/g. The method was applied to foliage and roots of three different wheat cultivars, and the analytes were detected in the range of 11-3261 microg/g of dry weight.     

Crocetin esters, picrocrocin and its related compounds present in Crocus sativus stigmas and Gardenia jasminoides fruits. Tentative identification of seven new compounds by LC-ESI-MS

Crocetin esters present in saffron (Crocus sativus L.) stigmas and in Gardenia jasminoides Ellis fruit are the compounds responsible for their color. Of the fifteen crocetin esters identified in this study, five new compounds were tentatively identified: trans and cis isomers of crocetin (beta-D-triglucoside)-(beta-D-gentibiosyl) ester, trans and cis isomers of crocetin (beta-D-neapolitanose)-(beta-D-glucosyl) ester, and cis crocetin (beta-D-neapolitanose)-(beta-D-gentibiosyl) ester. The most relevant differences between both species were a low content of the trans crocetin (beta-D-glucosyl)-(beta-D-gentibiosyl) ester, the absence of trans crocetin di-(beta-D-glucosyl) ester in gardenia, and its higher content of trans crocetin (beta-D-gentibiosyl) ester and cis crocetin di-(beta-D-gentibiosyl) ester. With the same chromatographic method it was possible to identify, in a single run, ten glycosidic compounds in saffron extracts with a UV/vis pattern similar to that of picrocrocin; among them, 5-hydroxy-7,7-dimethyl-4,5,6,7-tetrahydro-3H-isobenzofuranone 5-O-beta-D-gentibioside and 4-hydroxymethyl-3,5,5-trimethyl-cyclohexen-2-one 4-O-beta-D-gentibioside were tentatively identified for the first time in saffron. Of these ten glycosides, only the O-beta-D-gentibiosyl ester of 2-methyl-6-oxo-2,4-hepta-2,4-dienoic acid was found in gardenia samples, but it was possible to identify the iridoid glycoside, geniposide.