Determination of patulin in apple juice by liquid chromatography.
ABSTRACT A method was developed and validated in-house for the determination of patulin (PAT), a toxic mold metabolite, in apple juice. The sample was extracted with ethyl acetate-hexane and analyzed by liquid chromatography equipped with a C18 column and diode array detector. The mobile phase used for the quantification was water-ethanol, at a flow rate of 0.5 mL/min. The method showed a mean recovery of 84.8%, the relative standard deviation obtained in the precision study was <7.7%, the quantification and detection limits were 7 and 3 microg/L, respectively, and the linear range for PAT in apple juice was 2.6-650 microg/L. The ruggedness was evaluated by an intralaboratory experiment, in which 5 factors were studied, and only one was found to influence the observed results. The developed method is fast, practical, and simple; the solvents (except hexane) and reagents used were nontoxic. The results of the validation confirmed the efficiency of the method, which is sensitive enough to be used in studies required to quantify PAT in apple juice.
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ABSTRACT: A method for the simultaneous determination of 33 pesticides or degradation products together with patulin in apples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed. The method involved homogenization of the apples, extraction with ammonium acetate-acetic acid solution in methanol-water by ultrasonication, filtration, and determination by LC-MS/MS. The repeatability and within-laboratory reproducibility for the three spiking levels 0.02, 0.04 and 0.2 mg kg(-1) were between 4% and 35%. In general, the repeatability and reproducibility were about 10-20%. The limits of quantification (LOQs) were between 0.01 and 0.14 mg kg(-1). The method was used on incurred samples from parts of the ISAFRUIT project financed by the European Commission under the 6th Framework Programme. Samples were analysed at four different stages: after harvest, after storage (controlled), after a water bath, and after 28 days at room temperature. Pesticide residues were found at all stages, but no significant differences in the concentration were seen between the stages analysed. The concentration decreased significantly only for tolylfluanid after storage at room temperature for 28 days when only 0-6% of the original amount of tolylfluanid remained in the apples. No patulin was found in the apples stored for 28 days at room temperature and no growth of Penicillium expansum was observed on these apples. However, when the apples were inoculated with a spore suspension of P. expansum, high concentrations of patulin were found.Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment 08/2009; 26(7):1013-23.
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ABSTRACT: Patulin is a mycotoxin produced by species of Penicillium and Aspergillus and is toxic to a wide range of organisms, including humans and livestock. To produce large amount of pure patulin for research purposes, high-speed counter-current chromatography (HSCCC) and preparative high-performance liquid chromatography (prep-HPLC) were applied to the purification of patulin. Apple juice was inoculated with P. expansum and containing 0.5 mg patulin per ml was used as a starting material for separation. For HSCCC, a biphasic solvent system consisted of ethyl acetate-hexane-pH 4 acetic acid (7.5:2.5:10, v/v/v) was used. For prep-HPLC, the separation was carried out in a C18 reversed-phase preparative column with a mobile phase containing acetonitrile-pH 4 acetic acid (5:95, v/v). Fractions containing patulin were collected and analysed by analytical HPLC and identified by congruent retention time and ultraviolet/visible (UV-VIS) spectrum of the standard. The structure of the purified patulin was confirmed by mass spectrometry and nuclear magnetic resonance. HSCCC produced 21.9 mg of patulin from 50 ml apple juice culture whereas the prep-HPLC yielded 18.1 mg. HSCCC also produced purer patulin than the prep-HPLC (98.6 versus 96.3%) and higher recovery (86.2 versus 71.3%). In addition, the HSCCC method is advantageous for its lower cost and a simpler procedure compared with the prep-HPLC. This one-step HSCCC method can potentially provide a simple, effective and environmentally friendly tool for obtaining gram-level pure patulin for toxicology, detoxification and many other patulin-related studies.Food Additives and Contaminants - Part A Chemistry, Analysis, Control, Exposure and Risk Assessment 02/2009; 26(1):101-7.
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ABSTRACT: Although, Patulin and Ochratoxin are produced by the same genera of molds, however, Patulin was the most extensively studied mycotoxins in apple juice and no reports have explored the presence of Ochratoxin A in the apple juice. Therefore, the objective of this study was to explore the presence of Patulin and Ochratoxin A in apple juice in Saudi Arabian market of Jeddah. Potato dextrose agar(PDA) was used to detect fungal contamination. Patulin was determined using HPLC equipped with a UV detector set at 276 nm. Also, HPLC with fluorescence detector was set at 333 and 420 nm as excitation and emission wavelength, respectively,was used for Ochratoxin A separation. All samples of apple juice were free from fungi and yeasts. The Patulin (PAT) was detected in only one type out of 17 types (5.88%) with a concentration of 152.5 ppb, (305%) increased compared with the maximum permitted level (50 ppb). However the occurrence of Ochratoxin A (OTA) in apple juice samples was discovered in 5 types out of 17 types (29.41%). The concentration of OTA ranged from 100 to 200 ppb reaching 5–10-folds compared with the permissible limits (20 ppb).Saudi Journal of Biological Sciences. 01/2010;