Liquid chromatographic determination of multiple sulfonamide residues in bovine milk

A liquid chromatographic method has been developed for simultaneous determination of residues of 10 sulfonamide drugs at 10 ppb and above in raw bovine milk. The method is based on a chloroform-acetone extraction, evaporation of organic phase, dissolution of residues in an aqueous potassium phosphate solution, and extraction of fatty residue into hexane. The aqueous layer is collected, filtered, injected onto an LC system, and detected by ultraviolet absorption at 265 nm. To elute all 10 sulfonamides isocratically, 2 chromatographic conditions are required. Seven sulfonamides can be quantitated with 12% methanol in the mobile phase; 4 sulfonamides can be quantitated with 30% methanol. Sulfamethazine, the most widely used sulfonamide, is detected on both systems. Recoveries are 44-87% for individual sulfonamides, with only 2 below 60%. Coefficients of variation are 3-13% at 10 ppb.     

Bovine serum albumin produces a synergistic increase in the antioxidant activity of virgin olive oil phenolic compounds in oil-in-water emulsions

Virgin olive oil is valued for its flavor, but unacceptable off-flavors may develop on storage in food products containing this oil due to oxidation. The oxidative stability of oil-in-water emulsions containing bovine serum albumin (BSA) and virgin olive oil phenolic compounds was studied. Four oil-in-water emulsions with and without BSA and phenols isolated from virgin olive oil were prepared. These model systems were stored at 60 degrees C to speed up lipid oxidation. Primary and secondary oxidation products were monitored every three days. Peroxide values and conjugated diene contents were determined as measures of the primary oxidation products. p-Anisidine values and volatile compounds were determined as measures of the secondary oxidation products. This latter determination was carried out by headspace solid-phase microextraction coupled with gas chromatography. Although olive oil phenolic compounds and BSA contributed some antioxidant activity when present as individual additives, the combination of BSA with phenols in an emulsion showed 58-127% synergy, depending on which analytical method was used in the calculation. The emulsion containing phenolic compounds and BSA showed a low level of deterioration after 45 days of storage at 60 degrees C.     

Residue levels of ethoxyquin, imazalil, and iprodione in pears under cold-storage conditions.

A gas chromatographic method is presented for the simultaneous determination of the antiscald ethoxyquin and the fungicides imazalil and iprodione in peel and pulp of Blanquilla pears. Fruits were cold-stored in commercial chambers in normal atmosphere and in controlled atmosphere with low oxygen content (oxygen and carbon dioxide were held at 2.5% and 1.5%, respectively). The method uses gas-liquid chromatography (GLC) with an alkaline flame ionization detector (detector of N-P, NPD) and allows the detection of the mentioned compounds to minimum levels of 0.08-0.12 mg/kg in fresh fruit. With this system the evolution of residues in fruit was monitored throughout the period of cold storage. In the surveys carried out the residue levels of these compounds were found to be below the limits allowed by the legislation of European Union. For the three studied products residues in pulp are lower and disappear more quickly than in peel.     

Simple multiresidue method for monitoring of trimethoprim and sulfonamide residues in buffalo meat by high-performance liquid chromatography

A simple, specific, and rapid analytical method for the determination of trimethoprim (TMP) and three sulfonamide (SA) antimicrobial drug residues in buffalo meat is developed and validated. This method is based on a solid-phase extraction technique followed by high-performance liquid chromatography (HPLC)-photodiode array (PDA) detection. Target compounds were extracted from the meat by acetonitrile and water, cleaned up on a Bond Elute C 18 cartridge column, and separated on a RP-C 18 column during HPLC analysis. Acetonitrile along with water appears to be an excellent extractant as recovery of the analytes at maximum residues levels (MRLs) in spiked sample was in the range of 75-108%, with coefficient of variations (CVs) ranging between 1.34 and 22%. The limit of detection (LOD) and the limit of quantification (LOQ) were 0.031 and 0.062 microg/g, respectively, for all of the compounds. Intra- and interday assay precisions of the method at 0.125 microg/g concentrations for any drug ranged between 3 and 4%. The linearities of the TMP, sulfadimidine (SDM), sulfadoxine (SDO), and sulfamethoxazole (SMX) were 0.9989, 0.9999, 0.9998, and 0.9997, respectively. For robustness, the analytical method was applied to 122 buffalo meat samples obtained from export meat processing plants.     

HPLC method validated for the simultaneous analysis of cichoric acid and alkamides in Echinacea purpurea plants and products

A reversed-phase high-performance liquid chromatography (HPLC) method has been developed to determine caffeic acid derivatives, for example, cichoric acid, and alkamides in plant parts and herbal products of Echinacea purpurea. The method consists of an extraction procedure whereby the hydrophilic phenolics as well as the lipophilic alkamides are released from the samples, followed by the analytical HPLC procedure for quantitative determination of these compounds. The method is the first one validated for the determination of these two groups of compounds in the same procedure. Naringenin has been used as an internal standard, as no other flavanones are present in the extract and it does not interfere with any of the compounds under investigation. Analysis of Danish-grown plant material shows that it is possible to raise plants of a very high chemical quality in Denmark. A selection of international herbal products available on the Danish market show surprisingly variable quality, not necessarily reflecting the product information given on the labels.     

Investigation of the reaction between 4-hydroxy-5-methyl-3(2H)-furanone and cysteine or hydrogen sulfide at pH 4.5

Reaction of 4-hydroxy-5-methyl-3(2H)-furanone (HMF) with cysteine or hydrogen sulfide at pH 4.5 for 60 min at 140 degrees C produced complex mixtures of volatile compounds, the majority of which contained sulfur. Sixty-nine compounds were identified, some tentatively, by GC/MS. These included disulfides (26), thiols (7), dithiolanones (6), thiophenones (4), dithianones (3), and thienothiophenes (6). The main non-sulfur compounds were 2, 3-pentanedione, 2,4-pentanedione, and 3,4-hexanedione. Both systems produced approximately the same total quantity of volatile compounds, but the reaction containing cysteine gave the larger number of individual compounds, with thiols quantitatively the dominant components. By comparison, the major products formed in the reaction with hydrogen sulfide were the dithiolanones. Reaction pathways are presented for the major products and, where applicable, possible reasons for the differences in composition of the two systems are discussed. The contribution of these reactions, and their products, to the flavor of roasted foods is considered.     

Reversed phase ion-pair liquid chromatographic determination of nicotine in commercial tobacco products. 1. Moist snuff.

The official methods for the determination of nicotine in commercial tobacco products (AOAC, CORESTA) are based on approaches that are not selective for nicotine (colorimetric measurement, steam distillation, perchloric acid titration), and the availability of published methods based on state-of-the-art chromatographic methods is limited. Reversed phase ion-pair liquid chromatography has been established as a viable alternative for the analysis of basic analytes. A reversed phase ion-pair liquid chromatographic method for the determination of nicotine in commercial tobacco products was developed and optimized in separate experiments (Ciolino, L. A.; Turner, J. A.; McCauley, H. A.; Smallwood, A. W.; Yi, T. Y. J. Chromatogr. 1999a, 852 (2), 451-463). An extensive within-laboratory performance study of the optimized method was subsequently conducted, and results are presented here for the determination of nicotine in commercial moist snuff. Results for the determination of nicotine in commercial cigarettes are presented in a subsequent paper.     

Gas-liquid chromatographic determination of individual sugars in confectionery products.

A gas-liquid chromatographic (GLC) procedure is presented for the separation and quantitative determination of sucrose, lactose, maltose, and glucose in commercial confectionery products. By converting reducing sugars to oximes and then forming trimethylsilyl ethers of these compounds and separating them on a 6 ft X 4 mm id glass column packed with 2% OV-17 on 100--120 mesh Supelcoport, single peaks were obtained for each of the sugars. Results for sugars present in samples at levels of 5% or more are within 2.8%, on the average, of results obtained by polarization measurements. The data also compare favorably with others in the literature on similar products analyzed by other GLC procedures that do not involve oxime formation.     

Second derivative spectrophotometric determination of acetaminophen and phenacetin in presence of their degradation products

A rapid and accurate method for determining acetaminophen and phenacetin in presence of their degradation products is presented. Solutions of these drugs in 0.1N HCl were analyzed by measuring their second derivative spectral response at 295 nm where the degradation products do not interfere. The mean percent recoveries for mixtures of acetaminophen and/or phenacetin with the corresponding degradation products were 100.2 +/- 0.6 and 100.6 +/- 1.1, respectively. The method can be used for assessing the stability of the 2 drugs. The proposed method is also applied to the determination of acetaminophen in tablets and syrups.     

Determination of total N-nitroso compounds by chemical denitrosation using CuCl

A method for the determination of total N-nitroso compounds (NOC) by chemical denitrosation and subsequent chemiluminescence detection of evolved NO is described. Denitrosation was accomplished with CuCl in HCl at 70 degrees C. The detection limit for N-nitrosoproline (NPRO) was 1 pmol. NO formation from NPRO was linear (R(2) = 0.999) from 4 pmol to 2 nmol. Among the possible interfering compounds tested, only S-nitroso compounds contribute any significant interference. This method had several advantages over other similar methods: (1) A commercially available one-piece reaction vessel and a NO analyzer with software were used. (2) NO release occurred rapidly and was easily measured and quantified. (3) Compared to HBr or HI, CuCl was more convenient to work with and safe. (4) CuCl was suitable for samples in aqueous and most organic solvents. The application of this method to food, personal care products, and human body fluids demonstrates its utility.     

Comparison of two analytical methods for the evaluation of the complexed metal in fertilizers and the complexing capacity of complexing agents

The aim of this research is to develop an analytical methodology for the determination of complexed element in fertilizers and, then, to obtain an adequate criteria for the inclusion of these products in European Regulations on Fertilizers. This paper compares the CEN method EN 13366:2001, based on the retention of the cations into a sulfonated resin, and an AOAC modified method, based on the precipitation of the inorganic forms at pH 9. A limited interlaboratory trial was carried out to demonstrate the applicability of the AOAC modified method and to study the effect of the removal of organic compounds and the addition of a matrix modifier solution before the element quantification. Then, a global interlaboratory trial was developed to evaluate the validation and quality parameters of the method. As a second objective, the AOAC modified method was applied to the determination of the complexing capacity of complexing agents based on lignosulfonates and amino acids. The AOAC modified method was the choice methodology because it is adequate for the determination of complexing capacity of micronutrients in fertilizer.     

Determination of gibberellic acid in fermentation broth and commercial products by micellar electrokinetic chromatography

Micellar electrokinetic chromatography (MEKC) was developed as a method for quantitative determination of gibberellic acid (GA3) in fermentation broth and commercial products, using 25 mM disodium tetraborate as a buffer at pH 9.2 and 100 mM sodium dodecyl sulfate as a micellar phase. The baseline resolution (Rs of GA3 from other compounds in fermentation broth was achieved with Rs > 2.5. The addition of methanol or acetonitrile in the MEKC buffer did not give a better resolution. Advantages of this MEKC method include high accuracy and precision and no sample preparation except for dilution and filtration.     

Solid phase extraction and liquid chromatography-tandem mass spectrometry for the evaluation of 4-hydroxy-2-nonenal in pork products

This research was aimed at setting up an analytical method for the determination in pork products of 4-hydroxy-2-nonenal (4-HNE), an aldehyde produced from the oxidation of omega-6-polyunsaturated fatty acids. Such a compound mediates various biological effects, but it is considered to be very toxic to mammalian cells at levels higher than physiological ones. The methods used for the determination of 4-HNE in biological fluids, such as blood, were found to be unsuitable for meat samples because both the repeatability and the recovery in spiked samples were unsatisfactory. A new method, based on solid phase extraction and HPLC-MS/MS, was therefore developed and validated. The limit of detection of 4-HNE in spiked samples was 0.043 mg/kg, and the recovery was approximately 60% depending on the concentration. Good linearity was observed in the range of 0.1-10 mg/kg, and repeatability and interday and intraday precision expressed as relative standard deviation were <10%. The method has been successfully applied to the determination of the aldehyde in samples of various pork products. 4-HNE was present in some products, especially the smoked and/or cooked ones, at levels that might not be a real risk for human health.     

Enzymatic assay for the determination of olive oil polyphenol content: assay conditions and validation of the method

A new spectrophotometric assay for the determination of the polyphenolic content of olive oil is presented. It is a substrate-recycling assay for phenolic compounds that employs tyrosinase in the presence of excess NADH. The reaction of various phenols with the enzyme produces an o-quinone, which is detected by recycling between reactions with the enzyme and NADH. The method offers some advantages over the classical methods employed to determine the polyphenolic content of olive oil, that is, ease and reproducibility of the analysis, highly increased sensitivity, and selectivity toward phenolic compounds. The amount of total polyphenols was determined in virgin olive oils both with the Folin-Ciocalteu reagent and with the proposed enzymatic method. The results suggest a better estimation of the polyphenol content, as compared with the colorimetric method. This has to be attributed to the different reactivities of the two methods toward phenols and catechols. Finally, the enzymatic method demonstrates that there is a linear relationship between the olive oil phenolic content and the antioxidative capacity of oil extracts.     

Improving broad specificity hapten recognition with protein engineering

Sulfa antibiotics (sulfonamides) are derivatives of p-aminobenzenesulfonamide that are widely used in veterinary medicine. Foods derived from treated animals may be contaminated with these drugs. However, current immunobased sulfonamide detection methods are unfit for screening of products because they are either too insensitive or specific for a few compounds only. An immunoassay capable of detecting all sulfas in a single reaction would be ideal for screening. For development of a binder capable of binding all sulfas, a protein engineering approach was chosen and the properties of monoclonal antibody 27G3 were improved with mutagenesis followed by selection with phage display. Several different mutant antibodies were isolated. The cross-reaction profile of the best mutant antibody was significantly improved over that of the wild-type antibody: it was capable of binding 9 of the tested 13 sulfonamides within a narrow concentration range and also bound the rest of the sulfas, albeit within a wider concentration range.     

Analysis of metsulfuron-methyl in soil by liquid chromatography/tandem mass spectrometry. Application to a field dissipation study.

An analytical method is described for the extraction of metsulfuron-methyl from soil at sub-parts per billion levels (LOQ = 0.2 microgram kg(-1)). The herbicide was quantitatively determined and identified by ESI LC/MS/MS. The method has been applied to a field dissipation study in which metsulfuron-methyl was applied to spring barley at three dosage rates: 4, 8, and 16 g of active ingredient ha(-)(1). The results of 2 years are presented. The dissipation rate of metsulfuron-methyl in topsoil was very rapid, with a calculated half-life of 6.5 days. Laboratory mineralization studies with native soils in contrast to autoclaved soils indicated that microbial degradation of (14)C-labeled metsulfuron-methyl and (14)C-labeled 2-amino-4-methoxy-6-methyl-1,3,5-triazine in soil microcosms is an important factor for the complete degradation of metsulfuron-methyl in the field. However, the mineralization rate of the sulfonamide was much higher.     

Simultaneous determination of terbuthylazine and its major hydroxy and dealkylated metabolites in wetland water samples using solid-phase extraction and high-performance liquid chromatography with diode-array detection

A method based on high-performance liquid chromatography with diode-array detection was developed and validated aiming at the simultaneous determination of terbuthylazine (TER) and its five major metabolites, desisopropyl-hydroxy-atrazine, desethyl-hydroxy-terbuthylazine, desisopropyl-atrazine, hydroxy-terbuthylazine, and desethyl-terbuthylazine. Although s-triazines are used worldwide as herbicides for agricultural and nonagricultural purposes, there is limited information on the environmental impact of TER degradation products. The proposed method includes a solid-phase extraction procedure (using MCX cartridges) with adequate recovery efficiency (70-80%). The statistical evaluation of the method reveals good linearity, accuracy, and precision for the compounds determined, with RSD values less than 14.6%, while the detection limit was found to be 0.05 microg L(-1) for DIHA and 0.01 microg L(-1) for the other substances. This method can be employed in biodegradation studies of TER and its metabolites in water samples from constructed wetlands, thus assisting the evaluation of their environmental impact.     

Rapid assay for analyzing biogenic amines in cheese: matrix solid-phase dispersion followed by liquid chromatography-electrospray-tandem mass spectrometry

A new rapid and sensitive method based on matrix solid-phase dispersion (MSPD) followed by liquid chromatography-electrospray-tandem mass spectrometry was devised for the determination of biogenic amines at trace levels in cheese samples. The method required 0.25 g of sample, CN-bonded silica as a dispersant sorbent, and a formic acid aqueous solution/methanol mixture as an eluting solvent. Extraction recoveries from soft cheese products were calculated in the 98 +/- 4-110 +/- 6% range. A procedure based on solid-phase extraction was also evaluated for the extraction of these compounds in cheese. Chromatographic separation was performed using a C18 column with an aqueous ammonium acetate/methanol mixture as the mobile phase under gradient conditions. The method was validated in terms of detection limits (LOD), quantitation limits (LOQ), linearity, recovery, precision, and trueness. Results in the 0.05-0.25 mg kg(-1) range were obtained for the LOD of histamine, tyramine, and beta-phenylethylamine in soft cheese samples. Linearity was established over 2 orders of magnitude. Excellent precision in terms of intra-day repeatability was calculated (RSD% < 5). The applicability of the method to the determination of biogenic amines in cheese products was demonstrated.     

Flameless atomic absorption spectrophotometric determination of Vendex, an organic tin miticide, in apples, oranges, and tea leaves.

A method is described for the determination of an organic tin insecticide, Vendex or hexakis(2-methyl-2-phenylpropyl)distannoxane (HMPD). Tin compounds are extracted from the sample homogenate with ethyl ether containing 1% acetic acid. HMPD and its degradation products are separated through a silica gel column, and analyzed by an atomic absorption spectrophotometer coupled with a heated graphite atomizer. By this technique, as little as 5 ng HMPD and its degradation products can be determined with an average recovery of 67-96%.     

New capillary electrophoresis method for the determination of furosine in dairy products

A new capillary electrophoresis (CE) method was established for the quantitative determination of furosine in dairy products. Sample preparation and suitable electrophoretic conditions allowed accurate and reproducible quantitation of furosine in dairy products. Sample preparation consisted of drying hydrolyzed samples, redissolving them in 0.2 M NaOH, and purifying them by solid-phase extraction. The electrophoretic separation was carried out in an uncoated capillary maintained at 30 degrees C using 0.1 M phosphate buffer containing the additive hexadecyl trimethylammonium bromide (HDTAB, 1.2 mM) (pH 7.0) under 10 kV voltage and reverse polarity. Coefficients of variation of less than 2.25% for migration time and 5.80% for peak areas indicated that the technique was reproducible. The calibration curve followed a linear relationship with a highly significant (p < 0.01) coefficient of multiple determination (R (2) = 0.997). The limit of quantitation was 0.5 ppm, a concentration that corresponds to 4.5 mg/100 g of protein in milk samples. Furosine concentration (mg/100 g of protein) ranges of different dairy products (raw, pasteurized, UHT, and evaporated milks and yogurt) agreed with ranges previously reported. Therefore, the CE method presented is a suitable technique for the routine assessment of furosine in dairy products.