Rapid liquid chromatographic determination of patulin in apple juice

A rapid method is described for the quantitative determination of patulin in apple juice. The mycotoxin is extracted from the sample with ethyl acetate and the extract is cleaned up by extraction with a sodium carbonate solution. Patulin is determined by reverse phase liquid chromatography using a muBondapak C18 column and a 254 nm ultraviolet detector. The lower detection limit in patulin standard solution is 0.32 ng and recovery is greater than 75%.     

Development of liquid chromatography-electrospray mass spectrometry for the determination of patulin in apple juice: investigation of its contamination levels in Japan

Patulin is a mycotoxin produced by mainly Penicillium species, for example, P. expansum, and Aspergillus species. There are several reports of patulin contamination in apple juice. Last year, the Ministry of Health, Labour and Welfare of Japan set the maximum allowable level of patulin in apple juice at 50 ppb and decided that the measurement of patulin levels in apple juice products should be conducted. To this end, a simple, accurate, and selective analytical method for the detection of patulin at levels lower than 5 ppb, the detection limit, is desired. This paper reports the development of an analytical method that employs solid-phase extraction-liquid chromatography-mass spectrometry (SPE-LC-MS). When MS measurements were conducted with the selected ion monitoring (SIM) mode, the pseudomolecular ions at m/z 153 and 156 were used to monitor patulin and (13)C(3)-labeled patulin, respectively. The detection limit (S/N = 3) and the quantification limit (S/N = 10) of patulin at injection levels into LC-MS were 12.5 and 25 pg, respectively. However, when the actual sample was applied for the analysis based on the developed method including the sample preparation, the detection limit (S/N = 3) and quantification limit (S/N = 10) were 2.5 and 5 pg in sample, respectively. The calibration curve obtained for concentrations ranging from 5 to 500 ppb showed good linearity with a coefficient of determination (r (2)) of 0.999. In addition, the recovery was >95% when an internal standard was used. The method was applied to the analysis of 76 apple juice samples from Japan, and as a result, patulin levels ranging from <1.0 to 45 ppb (detection frequency = 15/76) were detected. In this study, it was found that patulin was a greater contaminant in concentration/reduction than in "not from concentrate" apple juice.     

Patulin in Italian commercial apple products

Patulin is a mycotoxin produced by microscopic fungi belonging to the Penicillium and Aspergillus genera. The natural occurrence of patulin in four apple products marketed in Italy and purchased from the supermarket, herbalist, and retail shops was studied. Thirty-three samples of the four products had no detectable patulin contamination. The 11 positive samples had a concentration ranging between 1.4 and 74.2 microg/L with a mean of 26.7 microg/L. All vinegar samples were negative for patulin; of 10 apple-based baby foods, two samples were contaminated with 17.7 and 13.1 microg/L and both were labeled as "organic food". Comparing organic and conventional agricultural practices, no significant differences were found. Finally, optimization of extraction protocol more general and useful for juices, clarified juices, baby foods, vinegars, and purees was performed. The low incidence of the patulin level in Italian apple products is a clear parameter to judge the quality of the fruit, and the process is of a high standard.     

Analysis of patulin in apple juice by diphasic dialysis extraction with in situ acylation and mass spectrometric determination.

A procedure combining diphasic dialysis extraction with in situ acylation and gas chromatography/mass spectrometry (GC/MS) determination was developed for detection and quantification of the mycotoxin patulin in apple juice. Apple juice samples spiked with 4-N,N-dimethylaminopyridine were dialyzed using methane chloride and acetic anhydride inside dialysis tubing. Patulin was derivatized into its acetate and collected in the tubing after diphasic dialysis and was directly determined using GC/MS with the selective ion monitoring mode without further concentration and cleanup steps. Quantification was carried out by a calibration curve with an internal standard of correlation. The appropriate parameters of both dialysis and derivatization were examined. The linear range of the calibration curve was found to be 10-250 microg/L for patulin, and the limit of quantification was 10 microg/L. Levels of patulin ranging from 0 to 107.2 microg/L with 77-109% recovery were found in 10 apple samples. The technique combining diphasic dialysis extraction and acylation was demonstrated and showed potential for other applications.     

Volatility of patulin in apple juice

Patulin is a mycotoxin produced by certain fungi, such as those found commonly on apples. The patulin content of apple juice is a regulatory concern because patulin is a suspected carcinogen and mutagen. A simple model of the apple juice concentration process was carried out to examine the possible contamination of patulin in apple aroma, a distillate produced commercially in the concentration of apple juice. The results show no evidence for patulin volatility, and document a reduction in patulin content by at least a factor of 250 in the apple distillate obtained from apple juice. Furthermore, a survey of several commercial apple aroma samples found no evidence of patulin content.     

High-pressure liquid chromatographic method for the determination of patulin in apple butter.

Patulin is extracted from apple butter samples with ethyl acetate and the extract is cleaned up on a silica gel column, using benzene-ethyl acetate (75+25) as the eluant. High-pressure liquid chromatography, using a 25 cm ZorbaxSil column, isooctane-ethyl ether-acetic acid (750+250+0.5) as the mobile solvent, and a 254 nm ultraviolet detector, is used for the determinative step. Under these conditions, patulin is eluted before 5-hydroxymethylfurfural, a component of apple butter which interferes with other liquid chromatographic and thin layer chromatographic methods. Recoveries of patulin added at levels of 34.6, 138.4, and 276.8 mug/kg ranged from 89.0 to 112.1%.     

Capillary column gas chromatographic determination of dicamba in water, including mass spectrometric confirmation

A sensitive method is described for determining dicamba at low micrograms/L levels in ground waters by capillary column gas chromatography with electron-capture detection (GC-EC); compound identity is confirmed by gas chromatography-mass spectrometry (GC-MS) using selected ion monitoring. Dicamba residue is hydrolyzed in KOH to form the potassium salt. The sample is then extracted with ethyl ether which is discarded. The aqueous phase is acidified to pH less than 1 and extracted twice with ethyl ether. The combined ethyl ether extracts are concentrated, and the residue is methylated using diazomethane to form the corresponding dicamba ester. The derivatized sample is cleaned up on a deactivated silica gel column. The methylated dicamba is separated on an SE-30 capillary column and quantitated by electron-capture or mass spectrometric detection. Average recoveries (X +/- SD) for ground water samples fortified with 0.40 microgram/L of dicamba are 86 +/- 5% by GC-EC and 97 +/- 7% by GC-MS detections. The EDL (estimated detection limit) for this method is 0.1 microgram dicamba/L water (ppb).     

Flavonoid and hydroxycinnamate profiles of english apple ciders

Seventeen phenolic compounds in 23 English apple ciders were identified and quantified by HPLC-PDA-MS (2). The total phenolic content of the ciders varied greatly ranging from 44 to 1559 mg/L. Four groups of compounds were identified, flavan-3-ols, hydroxycinnamates, flavonols, and dihydrochalcones. Hydroxycinnamates were the predominant group of phenolics in the majority of the ciders. Procyanidins were analyzed by HPLC after thiolysis, and total procyanidin content ranged from 8 to 722 mg/L and an average degree of polymerization of 2.5-3.5. This investigation of a wide range of ciders has shown a substantial variation in the profile and quantity of the phenolics. The analysis of single variety ciders highlighted the importance of using an apple cultivar with a high phenolic content to produce a phenolic-rich cider. Adaptations to the cider-making process could be used to increase the phenolic content with potential health benefits.     

Penicillium expansum: consistent production of patulin, chaetoglobosins, and other secondary metabolites in culture and their natural occurrence in fruit products

Penicillium expansum is known for its destructive rot and patulin production in apple juice. According to the literature, P. expansum can, among other compounds, produce citrinin, ochratoxin A, patulin, penitrem A, and rubratoxin B. In this study the qualitative production of metabolites was examined using TLC (260 isolates), HPLC (85 isolates), and MS (22 isolates). The results showed that none of the 260 isolates produced ochratoxin A, penitrem A, or rubratoxin B. However, chaetoglobosin A and communesin B were produced consistently by all 260 isolates. Patulin and roquefortine C were produced by 98% of the isolates. Expansolides A/B and citrinin were detected in 91 and 85% of the isolates, respectively. Chaetoglobosins and communesins were detected in naturally infected juices and potato pulp, whereas neither patulin nor citrinin was found. Because most P. expansum isolates produce patulin, citrinin, chaetoglobosins, communesins, roquefortine C, and expansolides A and B, foods contaminated with this fungus should ideally be examined for chaetoglobosin A as well as patulin.     

Prevention of patulin toxicity on rumen microbial fermentation by SH-containing reducing agents

Patulin, a toxic fungal metabolite, negatively affects rumen fermentation. This mycotoxin has also been associated with intoxication cases in cattle. This study investigates the use of SH-containing reducing compounds to prevent patulin's negative effects on the rumen microbial ecosystem. The effect of 50 microg/mL patulin on the fermentation of alfalfa hay was measured in batch cultures with and without reducing agents. Sulfhydryl-containing cysteine and glutathione prevented the negative effects of the toxin on dry matter degradation, gas, and volatile fatty acid production (P < 0.01). However, non-sulfhydryl-containing ascorbic and ferulic acids did not protect against patulin's toxicity (P > 0.01). Patulin was unstable in buffered rumen fluid as the concentration decreased by half after 4 h of incubation. In the presence of sulfhydryl groups, the toxin disappeared rapidly and was not detected after 1 h of incubation. The utilization of sulfhydryl-containing compounds such as cysteine to avert patulin toxicity could have practical implications in ruminant nutrition.     

Parallel synthesis: a new approach for developing analytical internal standards. Application to the analysis of patulin by gas chromatography-mass spectrometry

The polymer-assisted reaction of 4-(hydroxymethyl)furan-2(5H)-one (4HM2F) with 21 carboxylic acids using polystyrene-carbodiimide (PS-carbodiimide) yielded an ester library. Four of the esters, (5-oxo-2,5-dihydrofuran-3-yl)methyl acetate (IS-1), (5-oxo-2,5-dihydrofuran-3-yl)methyl butyrate (IS-2), (5-oxo-2,5-dihydrofuran-3-yl)methyl 2-methylpropanoate (IS-3), and (5-oxo-2,5-dihydrofuran-3-yl)methyl chloroacetate (IS-4), were tested as internal standards for the quantification of patulin in apple juice by gas chromatography-mass spectrometry in the selected ion monitoring mode (GC-MS-SIM). The developed method combines an AOAC official extractive step and a GC-MS-SIM analysis. Using a chromatographic column containing trifluoropropylmethylpolysiloxane as the stationary phase and IS-1 as the internal standard, it was possible to perform an accurate and precise quantification of underivatizated patulin in apple juice at concentrations down to 6 microg/L. A detection limit of 1 microg/L was established.     

Cider proteins and foam characteristics: a contribution to their characterization

A capillary sieving electrophoretic method for protein analysis and molecular weight determination was used to characterize ciders from Asturias, northern Spain. The total protein content (Bradford method) and the foam parameters (Bikerman method) were also analyzed to complete this characterization. The polypeptide profile, based on the molecular weight, together with exploratory and classification techniques, that is, principal component analysis (PCA) or linear discriminant analysis (LDA), allowed ciders to be differentiated on the basis of their foam assessment and the apple juice extraction technology used in the cidermaking process. In addition, the application of correlation analysis, that is, canonical correlations (CCA) or partial least-squares (PLS), revealed that the proteins with higher molecular weight were more relevant with respect to cider foamability. PLS analysis also provided a mathematical equation that is able to predict the stabilization time of foam from the polypeptide profile of the cider, because this is the foam parameter most influenced by these compounds.     

Development of a solid-phase extraction method for phenoxy acids and bentazone in water and comparison to a liquid-liquid extraction method

A rapid solid-phase extraction (SPE) method was developed for the determination of bentazone and the phenoxy acids 2,4-D, dichlorprop, MCPA, and mecoprop in Norwegian environmental water samples. Cartridges with a high-capacity cross-linked polystyrene-based polymer were used for off-line preconcentration. The effects of elution solvent, elution volume, sample volume, sorbent mass, pH, and flow rate on the recoveries of the pesticides were investigated using HPLC. Average recovery of >90% was achieved with 500 mg sorbents using 2 mL of methanol with 5% NH3 as elution solvent. The recoveries were independent of sample pH in the tested range of pH 1-7. Using a sample volume of 200 mL, the limits of determination for the phenoxy acids and bentazone are 0.02 microg/L. Sample volumes up to 2000 mL at a flow rate of 60 mL/min could be handled without any loss of analytes, which makes it possible to lower the limits of determination. The SPE method was compared to a routinely used liquid-liquid extraction method. Three different water matrices spiked at 1.0 and 0.05 microg/L were extracted, and the quantification was performed by GC-MS. Both methods permitted the determination of phenoxy acids and bentazone in distilled water, creek water, and well water down to a level of 0.05 microg/L with recoveries >80% for 200 mL samples. Important advantages of the SPE method compared to the liquid-liquid extraction method were the short extraction times, lack of emulsions, use of disposable equipment, and reduced consumption of organic solvents.     

Development of a monoclonal antibody-based enzyme-linked immunosorbent assay for the herbicide chlorimuron-ethyl

Hybridomas secreting a monoclonal antibody (mAb) against the herbicide chlorimuron-ethyl (CE) were produced by fusing the mouse myeloma cell line (SP2/0) with splenocytes from a mouse immunized against the conjugate of the sulfonamide moiety of CE and bovine serum albumin (BSA). The mAb, designated 1F5C5A10, had very weak affinity with metsulfuron, ethametsulfuron, pyrazosulfuron, bensulfuron, and chlorsulfuron. Two mAb-based indirect competitive enzyme-linked immunosorbent assays (icELISA) were developed. A conventional icELISA (icELISA-I) showed a concentration of half-maximum inhibition (IC(50)) of 11.6 ng/mL with a dynamic range of 1.6-84 ng/mL. A simplified icELISA (icELISA-II) had an IC(50) of 28.7 ng/mL and a dynamic range of 2.2-372 ng/mL. The two assays were tested on spiked water and soil samples. CE (1-500 ng/mL) fortified in water samples could be analyzed directly without any sample preparation by both immunoassays with an average recovery between 74 and 114%. icELISA-II, but not icELISA-I, was able to accurately analyze the herbicide residues in the crude soil extracts with recoveries between 99 and 129% without obvious matrix effects due to its lesser amount of sample used. In contrast to icELISA-I, icELISA-II is more convenient, whereas it consumes more reagents of coating antigen and goat anti-mouse IgG-peroxidase.     

Analysis of patulin in pear- and apple-based foodstuffs by liquid chromatography electrospray ionization tandem mass spectrometry

A liquid chromatography electrospray ionization tandem mass spectrometry method for the determination of patulin in apple- and pear-based foodstuffs was developed. The sample preparation is based on the QuEChERS procedure involving an initial extraction step with water and acetonitrile, followed by a partitioning step after the addition of magnesium sulfate and sodium chloride. The cleanup was performed by using dispersive solid-phase extraction with a mixture of magnesium sulfate, primary secondary amine sorbent, and n-octadecylsiloxane sorbent added together to the extract. The cleaned extract was finally evaporated and reconstituted in water prior to injection. Quantitation was performed by isotope dilution using ((13)C(7))-patulin as internal standard. The method was first fully validated in three different baby food products including apple-pear juice, apple-pear puree, and infant cereals. Then the scope of application of the method was extended to pear concentrate, raw apples, apple flakes (naturally contaminated), dried apples, and yogurt. The sensitivity achieved by the method in all matrices gave limits of detection (LOD) and quantitation (LOQ) of ≤0.5 and ≤10 μg/kg, respectively, which was compliant with maximum levels settled in Commission Regulation (EC) No. 1881/2006. Method performances for all matrices also fulfilled the criteria established in the CEN/TR 16059:2010 document. Indeed, recoveries were within the 94-104% range; relative standard deviations of repeatability (RSD(r)) and intermediate reproducibility (RSD(IR)) were ≤7.5 and ≤13.0%, respectively, and trueness in an infant apple drink (FAPAS 1642) was measured at 99%.     

Interlaboratory study of blood selenium determinations

Fifty-one laboratories from 14 countries participated in a survey on the determination of selenium (Se) in 8 bovine blood samples with Se concentrations ranging from 0.2 mumol/L (0.016 microgram/mL) to 14 mumol/L (1.1 micrograms/mL). The methods used (and the percentage of participants using each method) were fluorometry (61), hydride-generation atomic absorption spectrophotometry (AAS) (23), graphite-furnace AAS (6), gas chromatography (4), neutron activation analysis (4), and X-ray fluorometry (2). There was little difference in the mean Se results obtained by fluorometry or hydride-generation AAS (P greater than 0.05). Mean intralaboratory coefficients of variation (CVs) from known replicates ranged from 4 to 14% for all samples. Interlaboratory CVs were related to blood Se concentration and increased to 55% at Se levels below 0.4 mumol/L (0.032 microgram/mL). Laboratories that used quality control (QC) schemes had lower interlaboratory CVs than those that did not, but the advantage began to diminish at blood Se concentration below 0.4 mumol/L (0.032 microgram/mL). The high interlaboratory CVs, coupled with the false assurance from the low intralaboratory CVs and the ineffectiveness of the QC schemes at blood Se concentrations below 0.4 mumol/L (0.032 microgram/mL), are of concern in diagnosis of marginal Se deficiency in livestock where the concentrations of interest are in the range 0.15-0.5 mumol/L (0.012-0.039 microgram/mL).     

果汁护色剂JPX对苹果汁中棒曲霉素的降解作用及护色效果

为了考察果汁护色剂JPX在护色同时对苹果汁中棒曲霉素的降解作用与条件，该文研究处理温度、添加剂用量及处理时间对棒曲霉素降解率和果汁色值的影响。结果表明：在苹果浓缩汁生产过程中，用JPX控制棒曲霉素的较佳方法是，在榨汁阶段加入0.8g/L，酶解阶段加入1.6 g/L，棒曲霉素的降解率可以达到70%～80%，且对果汁有很好的护色效果。     

二氧化氯对苹果表面和鲜榨苹果汁中酿酒酵母1450的杀灭效果

利用二氧化氯(ClO₂)对水、苹果汁中和苹果表面的酿酒酵母1450(Saccharomyces cerevisiae)进行杀菌处理。结果表明：6、12 mg/L的ClO₂处理15 min使水中酵母分别减少(5.81±0.12)和(6.20±0.05)lg cfu/mL;100 mg/L的ClO₂对苹果汁中酵母的杀菌作用不明显,200 mg/L的ClO₂处理1 h可将果汁中(3.79±0.04)lg cfu/mL的酵母完全杀死,使果汁中(6.48±0.03)lg cfu/mL的酵母减少(3.67±0.05)lg cfu/mL,300 mg/L的ClO₂可将苹果汁中(6.52±0.06)lg cfu/mL的酵母完全杀死;10、20、30和40 mg/L的ClO₂处理1 h使苹果表面接种量为(4.95±0.02)lg cfu/mL的酵母数分别减少(3.41±0.02)、(3.64±0.08)、(3.80±0.04)、(4.47±0.09)lg cfu/mL,50 mg/L的ClO₂处理1 h,可将接种于苹果表面的酵母全部杀死,接种量进一步增加,ClO₂对苹果表面酵母的杀灭效果将会减弱;ClO₂处理对苹果汁的理化指标影响较小。     

Characterization of aroma compounds in apple cider using solvent-assisted flavor evaporation and headspace solid-phase microextraction

The aroma-active compounds in two apple ciders were identified using gas chromatography-olfactometry (GC-O) and GC-mass spectrometry (MS) techniques. The volatile compounds were extracted using solvent-assisted flavor evaporation (SAFE) and headspace solid-phase microextraction (HS-SPME). On the basis of odor intensity, the most important aroma compounds in the two apple cider samples were 2-phenylethanol, butanoic acid, octanoic acid, 2-methylbutanoic acid, 2-phenylethyl acetate, ethyl 2-methylbutanoate, ethyl butanoate, ethyl hexanoate, 4-ethylguaiacol, eugenol, and 4-vinylphenol. Sulfur-containing compounds, terpene derivatives, and lactones were also detected in ciders. Although most of the aroma compounds were common in both ciders, the aroma intensities were different. Comparison of extraction techniques showed that the SAFE technique had a higher recovery for acids and hydroxy-containing compounds, whereas the HS-SPME technique had a higher recovery for esters and highly volatile compounds.