Quantification of patulin in fruit leathers by ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA)

Patulin is a mycotoxin commonly found in certain fruit and fruit products. For this reason many countries have established regulatory limits pertaining to, in particular, apple juice and apple products. Fruit leathers are produced by dehydrating fruit puree, leaving a sweet product that has a leathery texture. A recent report in the literature described the detection of patulin at substantial levels in fruit leathers. To investigate this further, an ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA) method was developed for the sensitive detection of patulin in fruit leathers. Investigations were also made of the suitability of direct analysis in real time-mass spectrometry (DART-MS) for detection of patulin from the surface of fruit leathers. Results indicated DART-MS was insufficiently sensitive for quantification from the surface of home-style apple leathers, although patulin spiked onto the surface of leather or peel could be detected. The UPLC-PDA method was used to determine the fate of patulin during the preparation of home-made fruit leathers. Interestingly, when a home-style process was used, the patulin was not destroyed, but rather increased in concentration as the puree was dehydrated. The UPLC-PDA method was also used to screen for patulin in commercial fruit leathers. Of the 36 products tested, 14 were above the limit of detection (3.5 μg kg^–1) and nine were above the limit of quantification (12 μg kg^–1). Positive samples were confirmed by UPLC-MS/MS. Only one sample was found above the US regulatory limit for single-strength apple juice products (50 μg kg^–1). These results suggest patulin can be concentrated during preparation and can be found in fruit leathers. The limited survey suggests that patulin is fairly prevalent in such commercial products, but that the levels are usually low.     

Determination of patulin in apple juice using magnetic solid-phase extraction coupled with high-performance liquid chromatography

ABSTRACT

An efficient magnetic sorbent consisting of benzofuran-2-carboxylic acid-loaded magnetic nanocomposite was successfully synthesised for pre-concentration of patulin from apple juice. The prepared magnetic nanocomposite was characterised by scanning electron microscopy, transmission electron microscopy and Fourier-transform infrared spectroscopy. Determination of enriched patulin was performed by high-performance liquid chromatography. The best adsorption conditions were 40 mg of sorbent, 50 ml of apple juice sample, pH 5, ambient temperature and 25 min; the elution conditions were 500 μl methanol, pH 5, ambient temperature, and 4 min. Under optimised conditions, pre-concentration factor was 100, linearity range was 1–400 μg l^–1 of patulin, limit of detection was 0.15 μg l^–1 and limit of quantification was 0.5 μg l^–1. When samples were determined 20 times, the recovery was 93.9–102.6% and the relative standard deviation was below 5.3%. In terms of proposed procedure, the developed method was successfully applied for patulin detection in apple juice samples.     

Optimization and Validation of a Method Without Alkaline Clean-Up for Patulin Analysis on Apple Puree Agar Medium (APAM) and Apple Products

A sensitive high-performance liquid chromatography-UV (HPLC-UV) method, based on the Association of Analytical Communities (AOAC) Official method 2000.02, was developed and validated for the high-throughput analysis of patulin in in vitro experiments on apple puree agar medium (APAM). The importance of repeating the ethyl acetate extraction step at liquid-liquid extraction (LLE) was examined, as well as the extent of patulin degradation during the sodium carbonate clean-up. In addition to this alkaline clean-up, the efficiency of using an Oasis HLB or C₁₈ cartridge as solid-phase extraction (SPE) clean-up was compared. This resulted in a two-step ethyl acetate LLE, followed by an Oasis HLB SPE clean-up, without alkaline clean-up conditions. The method was fully validated for APAM, cloudy apple juice, and apple puree. Average patulin recoveries at levels of 100, 500, and 1000 μg kg^?1 of APAM varied between 95 and 113 % over 3 independent days, with an interday precision (RSD_R) of 5 to 10 %. Recovery experiments carried out with the spiked apple juice (at 50 μg kg^?1) and apple puree (10 μg kg^?1) showed average recovery rates laying between 80–101 % (RSD_R?=?12 %) and 77–100 % (RSD_R?=?9 %), respectively. This method offered a detection limit of 3–4 μg kg^?1 and a quantification limit of 5–8 μg kg^?1 for APAM, apple juice, and puree.     

A Fast and Reliable UHPLC-PDA Method for Determination of Patulin in Apple Food Products Using QuEChERS Extraction

A fast and reliable method for the quantification of patulin using ultra high performance liquid chromatography coupled to a photodiode array detector was developed and validated for the analysis of several apple-based foodstuffs. Sample preparation was based on the QuEChERS procedure. Samples were extracted with acetonitrile and partitioned with a mixture of sodium citrate, NaCl, and Mg₂SO₄. The cleanup step was performed using dispersive SPE mixed with Mg₂SO₄ and PSA. This step was carried out twice in order to improve chromatographic results. The method was validated in spiked cloudy apple juice, apple puree, apple yogurt, beer with apple juice, and cider and applied to 24 commercial samples. The limits of detection and limits of quantification were ≥0.4 and ≥2 ng/g, respectively. Both were below the maximum legal limit established by the European Union. Recoveries for all the matrices were between 78.4 and 94.7 % while relative standard deviations were between 3.8 and 10.4 %. Patulin was detected on four apple juices from concentrate, one cloudy apple juice from an eco-store, and one beer. Nevertheless, the highest concentration found was 25.4 ng/g.     

Novel Binary Solvents-Dispersive Liquid—Liquid Microextraction (BS-DLLME) Method for Determination of Patulin in Apple Juice Using High-Performance Liquid Chromatography

A simple and rapid binary solvents-based dispersive liquid–liquid microextraction (BS-DLLME) method has been developed for determination of patulin (PAT) in apple juice followed by high-performance liquid chromatography. This method involves the use of an appropriate mixture of miscible binary extraction solvents and disperser solvent to form fine droplets of extractant in a sample solution. Parameters affecting extraction efficiency such as the type and volume of high-density extraction solvent, the volume of ethyl acetate, the kind and volume of disperser solvent, and salt addition were investigated and optimized. The detection and quantification limits were 2.0 and 10.0 μg L^?1, respectively. The relative standard deviation for five measurements of 25 μg L^?1 of PAT was 3.8 %. The relative recoveries of PAT from apple juice samples at spiking levels of 25, 50, and 75 ng mL^?1 were in the range of 91.3–95.2 %.     

Occurrence of benzene as a heat-induced contaminant of carrot juice for babies in a general survey of beverages

A survey of benzene contamination of 451 beverage samples, using headspace sampling combined with gas chromatography and mass spectrometry (HS-GC/MS) with a quantification limit of 0.13 µg l^?1, was conducted. Artefactual benzene formation during headspace sampling was excluded by gentle heating at 50°C only and adjustment of sample pH to 10. The incidence of benzene contamination in soft drinks, beverages for babies, alcopops and beer-mixed drinks was relatively low, with average concentrations below the EU drinking-water limit of 1 µg l^?1. Significantly higher concentrations were only found in carrot juice, with the highest levels in carrot juice specifically intended for infants. About 94% of 33 carrot juice for infants had detectable benzene levels, with an average concentration of 1.86?±?1.05 µg l^?1. Benzene contamination of beverages was significantly correlated to iron and copper concentrations, which act as catalyst in benzene formation. The formation of benzene in carrot juice was predominantly caused by a heat-induced mechanism, which explains the higher levels in infant carrot juices that are subject to higher heat-treatment to exclude microbiological contamination.     

固相萃取-高效液相色谱法测定苹果汁及其饮料中展青霉素的含量

建立固相萃取-高效液相色谱法测定苹果汁及其饮料中展青霉素含量的检测方法。样品经预处理后,采用固相萃取柱净化处理,以乙腈-水作为流动相,C18色谱柱分离,二极管阵列检测器进行检测。结果表明：方法检出限为8 μg/kg,方法定量限为25 μg/kg;展青霉素标准溶液在50.0～500.0 μg/L质量浓度范围内,决定系数R2为0.999 6;样品中展青霉素含量为25～100 μg/kg时,回收率在90.2%～102.8%之间,精密度（变异系数）为0.57%～1.44%。此方法准确度较高,成本适中,操作较简便,适合苹果浓缩汁生产企业用于产品安全自控。     

Survey of patulin occurrence in apple juice and apple products in Catalonia,Spain, and an estimate of dietary intake

This study was conducted to assess patulin exposure in the Catalonian population. Patulin levels were determined in 161 apple juice samples, 77 solid apple-based food samples and 146 apple-based baby food samples obtained from six hypermarkets and supermarkets from twelve main cities of Catalonia, Spain. Patulin was analysed by a well-established validated method involving ethyl acetate extraction and direct analysis by high-performance liquid chromatography (HPLC) with ultraviolet light detection. Mean patulin levels for positive samples in apple juice, solid apple-based food and apple-based baby food were 8.05, 13.54 and 7.12 µg kg^?1, respectively. No samples exceeded the maximum permitted levels established by European Union regulation. Dietary intake was separately assessed for babies, infants and adults through a Food Frequency Questionnaire developed from 1056 individuals from Catalonia. Babies were the main group exposed to patulin, however no risk was detected at these levels of contamination. Adults and infants consumers were far from risk levels. Another approach to determine estimated exposure was conducted through Monte Carlo simulation that distinguishes variability in exposures from uncertainty of distributional parameter estimates.     

Incidence of patulin in apple juices marketed in Turkey.

The purpose of this study was to investigate the patulin contamination of apple juices consumed by the Turkish population. Patulin was detected using high-performance liquid chromatography (HPLC) with a UV detector at 280 nm, and the identification of patulin was further confirmed by thin-layer chromatography (TLC). Using HPLC, the recoveries were 79.9 +/- 6.7% and 83.7 +/- 4.6%, and the coefficients of variation were 8.4 and 5.5% for apple juices spiked with the known amounts of patulin (60 and 120 microg/liter. respectively). The minimum patulin level detected was 5 ng in a standard solution and 5 microg/liter in apple juices. The TLC method was used only to confirm patulin levels higher than 20 microg/liter (100 ng/spot) in apple juices. The total number of samples was 45. Patulin was present in detectable levels in 60% of apple juices at concentrations ranging from 19.1 to 732.8 microg/liter. Forty-four percent of the apple juice samples had patulin contamination levels higher than 50 microg/ liter, which is the allowable upper limit in Turkey.     

Rapid determination of picogram levels of patulin in apple juice using a flow injection chemiluminescence procedure

BACKGROUND: A sensitive, rapid and simple chemiluminescence procedure, based on the enhancing effect of patulin on the chemiluminescence (CL) reaction between luminol and hydrogen peroxide in a flow injection (FI) system, has been proposed for the determination of patulin. RESULT: The increment of CL intensity was linear over the concentration of patulin ranging from 0.05 to 80 ng mL^?1, with a detection limit (3S/N) of 0.01 ng mL^?1. At a flow rate of 2.0 mL min^?1, a complete analysis could be performed in 25 s with a relative standard deviation of less than 4.0%. CONCLUSION: The proposed method was successfully applied to the measurement of apple juice samples obtained from the Shaanxi Entry–Exit Inspection and Quarantine Bureau. The recoveries were between 93.4 and 107.9% Copyright © 2008 Society of Chemical Industry     

Ochratoxin A in wines,musts and grape juices from Spain

A survey on the occurrence of ochratoxin A (OTA) in 240 grape‐based beverages was carried out. Red and white wines from four different Spanish Designations of Origin (n = 160), musts (n = 20), grape juices (n = 10), ordinary wines (n = 20), special wines (Malaga, muscatel, sherry, vermouth, etc) (n = 20) and sparkling wines (n = 10) were assayed for OTA content using immunoaffinity column clean‐up and high‐performance liquid chromatography with fluorimetric detection (detection limit 0.05 µg l^?1). Forty‐three (17.9%) of the samples tested contained detectable levels of OTA. The overall mean OTA concentration in red and white wines of Designations of Origin was 0.30 and 0.18 µg l^?1 respectively (ranges 0.05–3.19 and 0.05–1.13 µg l^?1 respectively). The percentage of wine samples with detectable amounts of OTA was higher for red (18.3%) than for white (10%) wines. OTA was also found in two of 10 red ordinary wines (0.68 and 4.24 µg l^?1), whereas none of 10 white ordinary wines contained OTA. The mean OTA amount detected in sparkling wines was 0.44 µg l^?1 (range 0.14–0.71 µg l^?1). Two of 20 must samples contained OTA at low levels (0.08 and 0.18 µg l^?1), while none of 10 grape juice samples contained OTA. Highest amounts of OTA were found in special wines (45%), with a maximum of 15.25 µg l^?1 in a muscatel sample. Copyright © 2004 Society of Chemical Industry     

Rapid multi-class multi-residue method for the confirmation of chloramphenicol and eleven nitroimidazoles in milk and honey by liquid chromatography-tandem mass spectrometry (LC-MS)

A confirmatory method was developed to allow for the analysis of eleven nitroimidazoles and also chloramphenicol in milk and honey samples. These compounds are classified as A6 compounds in Annex IV of Council Regulation 2377/90 (European Commission 1990) and therefore prohibited for use in animal husbandry. Milk samples were extracted by acetonitrile with the addition of NaCl; honey samples were first dissolved in water before a similar extraction. Honey extracts underwent a hexane wash to remove impurities. Both milk and honey extracts were evaporated to dryness and reconstituted in initial mobile phase. These were then injected onto a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system and analysed in less than 9 min. The MS/MS was operated in multiple reaction monitoring (MRM) mode with positive and negative electrospray ionization. The method was validated in accordance with Commission Decision 2002/657/EC and is capable of analysing metronidazole, dimetridazole, ronidazole, ipronidazole and there hydroxy metabolites hydroxymetronidazole, 2-hydroxymethyl-1-methyl-5-nitroimidazole, and hydroxyipronidazole. The method can also analyse for carnidazole, ornidazole, ternidazole, tinidazole, and chloramphenicol. A recommended level of 3 µg l^?1/µg kg^?1 for methods for metronidazole, dimetridazole, and ronidazole has been recommended by the Community Reference Laboratory (CRL) responsible for this substance group, and this method can easily detect all nitroimidazoles at this level. A minimum required performance level of 0.3 µg l^?1/µg kg^?1 is in place for chloramphenicol which the method can also easily detect. For nitroimidazoles, the decision limits (CCα) and detection capabilities (CCβ) ranged from 0.41 to 1.55 µg l^?1 and from 0.70 to 2.64 µg l^?1, respectively, in milk; and from 0.38 to 1.16 µg kg^?1 and from 0.66 to 1.98 µg kg^?1, respectively, in honey. For chloramphenicol, the values are 0.07 and 0.11 µg l^?1 in milk and 0.08 and 0.13 µg kg^?1 in honey. Validation criteria of accuracy, precision, repeatability, and reproducibility along with measurement uncertainty were calculated for all analytes in both matrices.     

Some factors influencing patulin production by Penicillium expansum in pome fruits

BACKGROUND: The objective of our study was to examine the effects of Penicillium expansum on patulin production in relation to isolates, species and cultivar type, incidence and severity of decay. In addition, patulin production at different incubation times and its diffusion were also investigated. These factors were evaluated in pome fruits inoculated with P. expansum and kept at 20 °C for short periods of time. RESULTS: The ability of five P. expansum isolates to grow and produce patulin in inoculated Golden Delicious apples varied among the strains from below the limit of quantification to 662 µg kg^?1. Variety and species of pome fruits influenced patulin production. P. expansum isolate PE97.IT produced a higher patulin content in apples than in pears. The highest patulin production was 386 µg kg^?1 in Golden Delicious. No blue mould symptom appeared in pears inoculated with P. expansum and no patulin was detected after 3 days at 20 °C. However, patulin increased with incubation time after 6 and 8 days. No patulin was detected in healthy pear tissue but it was high in the decayed area. CONCLUSION: Since patulin production is associated primarily with infected rotten tissue, patulin control is possible by using healthy fruits, sorting damaged and rotten fruits before processing. Copyright © 2010 Society of Chemical Industry     

A quantification and confirmation method of patulin in apple juice by GC/MS

A sensitive and selective method for quantification and confirmation of patulin in apple juice by GC/MS was developed. By this method, patulin was precisely determined and confirmed down to the level of 1 and 5 microg/kg in samples, respectively. Patulin was extracted with ethyl acetate from a sample and then hexane was added to the concentrated extract solution. Significant amounts of insoluble impurities were filtered off, followed by further clean-up by solid-phase extraction with combined silica gel and Florisil cartridges. The filtration step in a low-polarity condition was very effective to remove the impurities in the sample extract solution. The eluate from the cartridges was evaporated to dryness under reduced pressure and patulin was determined and confirmed by GC/MS after derivatization with 2.5% N,O-bis(trimethylsilyl)trifluoroacetamide ethyl acetate solution. Patulin was determined in the selected ion monitoring mode (m/z 226) and confirmed in the SCAN mode (m/z 40-340). The recovery from apple juice spiked with 10-500 microg/kg ranged from 93.4 to 100%. The limits of detection and quantification were 0.1 (S/N = 3) and 1 microg/kg (S/N = 30) of patulin in samples, respectively. Levels down to 5 microg/kg of patulin in sample were readily confirmed.     

浓缩苹果汁加工链中棒曲霉素的动态分析研究

研究了浓缩苹果汁加工过程中各工序对棒曲霉素含量的影响。结果表明,喷淋、拣选、清洗是去除棒曲霉素的关键步骤,去除率为60.18％;吸附树脂对棒曲霉素去除效果显著。此外,研究了加工季节中棒曲霉素的变化规律,为果汁加工厂家建立科学完善的HACCP管理体系提供了理论依据。     

In‐house validation of a high‐performance liquid chromatography analytical method for quantification of ochratoxin A in unfermented grape juice

A validated high‐performance liquid chromatography (HPLC) method with fluorescence detection for the quantitative analysis of ochratoxin A (OTA) in unfermented grape juice is described. Five millilitres of unfermented grape juice was mixed with 45 mL of PBS, and the pH was adjusted to 7.2. Then the mixture was filtered under vacuum through a glass microfibre filter and cleaned up with immunoaffinity columns prior to analysis by HPLC. Validation of the analytical method was based on the following criteria: selectivity, linearity, limit of detection and quantification, precision (within‐day and between‐day variability) and recovery and uncertainty estimation. Detection and quantification limits obtained were 0.02 µg L^?1 and 0.05 µg L^?1 respectively. The percentage recovery was 91.5% (RSD = 3.9). This method was applied to the measurement of 30 veraison stage unfermented grape juice samples. Copyright © 2007 Society of Chemical Industry     

The effects of processing technology on the patulin content of juice during commercial apple juice concentrate production

 The effects of different treatments on the patulin content of apple juice during the production of industrial apple juice concentrate were investigated. Conventional clarification using a rotary vacuum precoat filter was found to be more effective than using ultrafiltration for the removal of patulin from apple juice. The average losses of patulin were 39% and 25% for conventional clarification and filtration, and ultrafiltration, respectively. Washing and handling appeared to be the most critical steps in removing patulin from apples since up to 54% could be removed using high-pressure water spraying. Received: 22 January 1998 / Revised version: 21 April 1998     

Analysis of underivatizated patulin by a GC-MS technique

An alternative approach based on the use of gas chromatography-mass spectrometry (GC-MS) is used to confirm the presence of patulin in apple juice. In the gas chromatography (GC) methods previously described, derivatization of patulin was always necessary in order to achieve good chromatographic detection. The use of electronic pressure control (EPC) and on-column injection avoids the need for patulin derivatization and allows a sensitive analysis of patulin. A detection limit of 4 microg/liter in apple juice can be attributed to the method.     

The effects of processing technology on the patulin content of juice during commercial apple juice concentrate production

 The effects of different treatments on the patulin content of apple juice during the production of industrial apple juice concentrate were investigated. Conventional clarification using a rotary vacuum precoat filter was found to be more effective than using ultrafiltration for the removal of patulin from apple juice. The average losses of patulin were 39% and 25% for conventional clarification and filtration, and ultrafiltration, respectively. Washing and handling appeared to be the most critical steps in removing patulin from apples since up to 54% could be removed using high-pressure water spraying. Received: 22 January 1998 / Revised version: 21 April 1998     

Occurrence and stability of inorganic and organic arsenic species in wines,rice wines and beers from Central European market

We investigated in total 80 wine samples of different types and seven grape juice and 23 beer samples purchased from markets in Central Europe in order to understand the arsenic (As) speciation and help assess the potential As toxicity via intake of alcoholic beverages. Generally, total As concentrations in most samples investigated were below the drinking water limit 10?µg?l^?1 published by the World Health Organization (WHO); ranging from 0.46 to 21.0?µg?l^?1 As in red and white wines and from 0.75 to 13.4?µg?l^?1 As in beers. In addition, concentrations of total As in rice wine and in rice beer were 0.63–6.07 and 3.69–8.23?µg?l^?1 As, respectively. The total As concentrations in ice wine ranged from 7.94 to 18.8?µg?l^?1 As, significantly higher than in white and red wine. Arsenite predominated as the As species in most of the wine samples, whereas arsenate was the dominant species in rice wine, beer and rice beer. Methyl As components were usually minor components in all wine and beer samples. Monomethylarsonic acid, dimethylarsinic acid and two additional unknown As species were frequently found in grape juice, late harvest and ice wine with higher sweetness. After air exposure, arsenite, arsenate, monomethylarsonic acid and dimethylarsinic acid were stable at 4°C for months, probably due to the acidic conditions of wine and beer samples. The presence of sulfite had little influence on As speciation in wine. Despite the predominance of more toxic arsenite and arsenate in wine and beer, the estimated weekly exposure to As (via consumption of beer, wine and rice wine) is low. The As intake per capita is 6.81?µg from beer, <1.93?µg from wine and 0.88?µg from rice wine, estimated using the median of total As concentration multiplied by the average consumption per capita of the corresponding beverage.