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 simple,rapid and novel method based on salting-out assisted liquid–liquid extraction for ochratoxin A determination in beer samples prior to ultra-high-performance liquid chromatography coupled to tandem mass spectrometry

A novel, simple, easy and cheap sample treatment strategy based on salting-out assisted liquid-liquid extraction for ochratoxin A (OTA) ultra-trace analysis in beer samples using ultra-high-performance liquid chromatography-tandem mass spectrometry determination was developed. The factors involved in the efficiency of pre-treatment were studied employing factorial design in the screening phase and the optimal conditions of the significant variables on the analytical response were evaluated using a central composite face-centred design. Consequently, the amount of salt ((NH₄)₂SO₄), together with the volumes of sample, hydrophilic (acetone) and nonpolar (toluene) solvents, and times of vortexing and centrifugation were optimised. Under optimised conditions, the limits of detection and quantification were 0.02 µg l^?1 and 0.08 µg l^?1 respectively. OTA extraction recovery by SALLE was approximately 90% (0.2 µg l^?1). Furthermore, the methodology was in agreement with EU Directive requirements and was successfully applied for analysis of beer samples.     

RP-HPLC-DAD Determination of Phenolics in Industrial Apple Pomace

Processing of apple fruits for juice/cider production generates large volume of pomace at industrial scale. This biomass (cell wall material and pulp tissues) is rich in array of nutrients, especially dietary fiber and polyphenols. Apple fruits confront various processing conditions at industrial level, influencing the biochemical composition of generated pomace, including its phenolic profile. In present study, a simple, fast and reproducible reversed-phase high-performance liquid chromatography method using diode array detector was developed and validated for separation of different phenolics present in industrial apple pomace. The present method showed reliable and reproducible intraday (0.2–4.0 % RSD) and interday (3.7–8.1 % RSD) precision with limits of detection and quantification values in range of 0.14–0.58 and 0.48–1.95 μg ml^?1, respectively, for all the phenolics. Different solvent-mediated extraction of dried pomace powder was also performed to evaluate its total antioxidant potential using standard spectrophotometric assays. The major phenolics found in industrial apple pomace extracts were quercetin (1.4–10.3 μg mg^?1), phloretin (1.1–9.3 μg mg^?1) and phloridzin (0.62–2.0 μg mg^?1).     

Thermal degradation kinetics of anthocyanins from Chinese red radish (Raphanus sativus L.) in various juice beverages

Thermal and storage stabilities of red radish anthocyanins (RRAs) in various juice beverages (apple, grape, peach, pear, pomegranate and lemon) were studied over temperature range 70–90 °C and 4–25 °C. RRAs degradation in all juice beverages followed first-order reaction kinetics. RRAs showed a much faster degradation rate during storage at room temperature (t _1/2 value ≤84.0 days) than did in refrigerated temperature (t _1/2 ≥value 130.9 days). The rate constant (k), E _a and Q ₁₀ values for RRAs in juice beverages varied from 1.33 to 0.33, 47.94 to 14.77 kJ mol^?1 and 1.16 to 1.89 at 70–90 °C. During heating, RRAs in peach and pomegranate showed higher stability than others at these temperatures. There was a positive correlation (R ² > 0.9128) between ascorbic acid content of juice beverages (8–36 mg/100 mg) and stability of RRAs at 70–90 °C. It was found that RRAs in apple and pear juice beverage were more stable than in other juice beverages.     

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 %.     

Comparison of Simple and Rapid Extraction Procedures for the Determination of Pesticide Residues in Fruit Juices by Fast Gas Chromatography–Mass Spectrometry

Three sample treatment methods, based on QuEChERS, solid-phase extraction (SPE) and solid-phase microextraction (SPME), were compared and evaluated in order to obtain the best conditions to determine pesticide residues in fruit juice by fast gas chromatography–mass spectrometry (single quadrupole GC-MS). Analysis were performed under selected ion monitoring, acquiring the three most abundant and/or specific ions for each analyte and using their relative intensity ratios as a confirmatory parameter. The 3 methodologies (QuEChERS, SPE and SPME) were validated taking 15 selected pesticides as model compounds, using commercial apple juice. QuEChERS procedure was based on the AOAC Official Method 2007.01, using acetonitrile (containing 1 % acetic acid) as extraction solvent and primary–secondary amine during the dispersive solid-phase extraction. Oasis hydrophilic–lipophilic balance cartridges were used for SPE, and polyacrylate fibers were used for direct immersion SPME procedure. Three isotopically labeled standards were added to the samples before extraction and used as surrogate standards. Validation parameters as recoveries, limits of detection, and limits of quantification (LOQ), as well as matrix effects and sample throughput, were obtained and compared for the three extraction procedures. QuEChERS was considered faster and led to the best quantitative results. In this way, validation was extended to up to 56 pesticides by applying QuEChERS in multi-fruit juice samples, obtaining LOQs ranging from 2 to 20 μg/L for most compounds. Accuracy and precision were evaluated by means of recovery experiments at two concentration levels (10 and 100 μg/L), obtaining recoveries between 70 and 120 % in most cases and relative standard deviations below 15 %. Finally, the QuEChERS method was applied to the analysis of commercial juices, including mango–apple, pineapple, grapefruit and orange.     

Determination of Total Carbohydrate Content in Beer Using Its Pre-column Enzymatic Cleavage and HPLC-RI

Beside ethanol, carbohydrates are the main source of total energy in beer. While analyses of fermentable carbohydrates are important from the technological point of view, the total content of carbohydrates is relevant in terms of nutrition. A high-performance liquid chromatography (HPLC) method with refractometric (RI) detection was developed for determination of total carbohydrate content in beer. Using enzymatic reaction with amyloglucosidase, the carbohydrates were cleaved to yield glucose and short glucose oligomers of less than 10 units, and separated on HPLC ion exchanger Rezex RSO-Oligosaccharide column in Ag⁺ mode. Optimum parameters were established for the enzymatic sample treatment and for the HPLC separation of reaction products. Calibration curves of glucose, fructose, maltose and simultaneously analyzed glycerol ranged from 0.001 to 0.5 g/100 ml, correlation coefficients of all calibration curves were 0.9999. The instrumental limits of quantification were 0.001 g/100 ml and they were verified using repetitive injections, with coefficient of variation (CV) less than 10 % in five replicates. The method limit of quantification was 0.01 g/100 ml since it was necessary to dilute the beer samples before chromatographic analysis. Recovery of the method in non-alcoholic and alcoholic beer was 98.5 %, and 92.3 %, respectively. Finally, ten non-alcoholic and 15 alcoholic beers from Czech market were analyzed using the method, the average content of total carbohydrates in non-alcoholic and alcoholic beers being 4.21 and 3.70 g/100 ml, respectively. These results are in a good correlation with the real extract of beer, which is on average 4.58 and 4.27 g/100 ml.     

Identification and Quantification of Dityrosine in Grain Proteins by Isotope Dilution Liquid Chromatography-Tandem Mass Spectrometry

A novel liquid chromatography-tandem mass spectrometry method was developed and validated for identification and quantification of dityrosine in zein protein. The zein samples spiked with a dityrosine standard and 3,3-¹³C₆-dityrosine, as the internal standard were hydrolysed in a mixture of 6 N hydrochloric acid and propionic acid followed by cleaning-up using Sep Pak C₁₈ cartridges. Good linear regression (R ² = 0.999) was obtained in the range 1–1000 ng/mL. Method limit of detection and method limit of quantification were 42.1 and 140 ng/g, respectively. Recoveries were from 92 to 95.2%. Precision and inter-day reproducibility expressed as relative standard deviation were from 3.9 to 22.1% and from 4.4 to 16.6%, respectively. The validated method was applied to quantify dityrosine in various grain proteins. The levels of dityrosine ranged from 0.38 to 1.92 ng/mg. This validated method will be of great value for understanding the role of dityrosine in grain food texture.     

Dispersive Liquid–Liquid Microextraction Followed by Capillary High-Performance Liquid Chromatography for the Determination of Six Sulfonylurea Herbicides in Fruit Juices

In this study a simple, rapid, and efficient method has been developed for the determination of six sulfonylurea herbicides (SUHs): triasulfuron, metsulfuron-methyl, chlorsulfuron, flazasulfuron, chlorimuron-ethyl, and primisulfuron-methyl in commercial grape and apple juice samples, using dispersive liquid–liquid microextraction coupled with capillary high-performance liquid chromatography with diode array detection. Various parameters that influence the extraction efficiency, such as the type and volume of extraction and disperser solvents, sample pH, and salt addition, were investigated and optimized. Under the optimum conditions, limits of detection and quantification of the method were in the ranges of 2–9 and 8–29 μg L^?1, respectively, lower than the maximum residue limits set by the European Union for the raw fruits, such as grape and apple. The intra- and inter-day relative standard deviations varied from 1.0 to 8.2 and 1.8 to 9.8 %, respectively, with recoveries between 72.0 and 109.5 % for commercial grape (both white and red) and apple juice samples, showing satisfactory accuracy for the determination of SUHs in fruit juices.     

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.     

Biomass and Patulin Production by Byssochlamys nivea in Apple Juice as Affected by Sorbate, Benzoate, SO2 and Temperature

The effects of potassium sorbate, sodium benzoate, SO₂ and incubation temperature on biomass and patulin production by Byssochlamys nivea in apple juice were determined. Growth at 21, 30 and 37°C over a 25-day incubation period was significantly retarded by 75 ppm SO₂, 150 ppm potassium sorbate and 500 ppm sodium benzoate. Biomass accumulated to approximately 500 mg/100 ml in control samples of apple juice. Patulin was produced in the highest concentrations at 21°C after 20 days incubation. After reaching a maximum concentration at 30 and 37°C, a rapid decline in patulin content was observed. Patulin production was also observed at 12°C. On the basis of concentration, SO₂ had the most significant effect on the rate of biomass and patulin production by B. nivea followed by potassium sorbate and sodium benzoate, respectively.     

One-Step Synthesis of Zirconia and Magnetite Nanocomposite Immobilized Chitosan for Micro-Solid-Phase Extraction of Organophosphorous Pesticides from Juice and Water Samples Prior to Gas Chromatography/Mass Spectroscopy

In this research, a magnetic sorbent was prepared by immobilizing zirconia and magnetite (Fe₃O₄) nanoparticles in chitosan, which is characterized and used as an effective nanosorbent in magnetic dispersive micro-solid-phase extraction (MDMSPE) of organophosphorous pesticides (OPPs) from juice and water samples prior to gas chromatography-mass detection (GC-MS). The properties and morphology of synthesized sorbent were characterized by scanning electron microscopy (SEM), Fourier transform-infrared spectroscopy (FT-IR), vibrating sample magnetometry (VSM), and differential scanning calorimetric (DSC) analysis. The main experimental parameters including pH level, extraction time, sorbent mass, salt concentration, and desorption conditions were investigated and optimized to maximize extraction efficiency. Under optimized conditions, the calibration curves were obtained in the concentration range of 0.1–500 ng mL^?1 with correlation coefficients between 0.9993 and 0.9999. The limits of detection (signal-to-noise ratio (S/N) = 3) and limits of quantification (S/N = 10) of the method ranged from 0.031 to 0.034 ng mL^?1 and 0.105–0.112 ng mL^?1, respectively. The intra-day and inter-day RSDs were 2.2–5.7 and 2.5–7.5%, respectively. The method was successfully applied to the analysis of OPPs in fruit juices (apple, peach, and cherry) and water (mineral, tap, and river) real samples, with recoveries in the range of 86.0–106.0% for the spiked juice and water samples. The results showed that with combination of high selectivity of zirconia and magnetic property of magnetite as well as immobilizing ability of chitosan, the fabricated sorbent exhibited exceptional extraction ability toward the OPPs.     

Quantitative analysis of patulin in apple juice by thin-layer chromatography using a charge coupled device detector

A method was developed and validated in-house for the detection and quantification of patulin in apple juice concentrate using a charge coupled device (CCD) on thin-layer chromatography (TLC) plates. Samples were extracted with ethyl acetate and then cleaned-up by extraction with a sodium carbonate solution. The method showed a mean recovery of 95%. The quantification and detection limit were 14 µg l^?1 and 0.005 µg per spot, respectively. The CCD camera is sufficiently sensitive to detect changes in spot fluorescence intensity caused by small differences in mycotoxin concentration under homogeneous illumination from a UV light source. The results of validation confirmed the efficiency of the method, which is sensitive enough to be used to quantify patulin in apple juice by producers or for government monitoring/survey programs. The method was applied to the analysis of 16 apple juice concentrate samples and patulin levels ranged from 15 to 46 µg l^?1. This study demonstrated the applicability of the TLC–CCD technique as a tool for monitoring patulin in apple juice.     

Development of an Indirect Competitive ELISA for Analysis of Alternariol in Bread and Bran Samples

Alternariol (AOH) is one of the major mycotoxins produced by various species of Alternaria fungi. Natural occurrences of AOH have been reported in various foods, including fruits; processed fruit products such as apple juice, tomato products; wheat and other grains; oilseeds and products thereof, such as sunflower seeds, oilseed rape meal, and flax seed/linseed; and pecans. In this study, AOH-specific polyclonal antibodies were generated and developed an indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) for monitoring AOH in bread and bran samples. The assay was very sensitive with a limit of detection (LOD) of 2.4?±?0.6 ng/g and a half maximal inhibitory concentration (IC₅₀) of 15.2?±?2.6 ng/g in bread and a LOD of 8.4?±?1.2 ng/g and IC₅₀ of 52.8?±?10.8 ng/g in bran extract. The assay was very specific to AOH and showed no cross-reactivity to alternariol monomethyl ether, altertoxin, altenuene, tentoxin, or tenuazonic acid. The effect of organic solvents on the assay was tested. The ELISA system tolerated methanol and acetonitrile as co-solvents at up to 5% content without significant loss of IC₅₀ value. Recoveries in all cases were greater than 75%, and the results using this method were comparable to those obtained from mass spectrometry methods. We conclude that this method is suitable for rapid detection of AOH in bread and bran samples, without expensive analytical equipment or time-consuming sample preparation.     

Aflatoxin B1 and sterigmatocystin survey in beer sold in China

ABSTRACT

A total of 101 samples of beer from the Chinese market were analysed for the presence of aflatoxin B₁ (AFB₁) and sterigmatocystin (STC), using methods based on liquid chromatography–tandem mass spectrometry. The limit of quantification and the limit of detection in beer were 0.1 and 0.03 µg/kg, respectively. Recoveries of AFB₁ and STC from spiked beer samples were 97.8–103.6% and 92.7–102.1%, respectively. None of the beer purchased samples were contaminated with AFB₁ or STC.     

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.     

Inactivation Kinetics of Pectin Methylesterase,Polyphenol Oxidase,and Peroxidase in Cloudy Apple Juice under Microwave and Conventional Heating to Evaluate Non-Thermal Microwave Effects

Continuous-flow microwave pasteurization provides important advantages over conventional heat exchangers such as fast volumetric heating, lower tube surface temperature, and possible non-thermal effects that enhance enzymatic and bacterial inactivation. Conventional and microwave-assisted inactivation of pectin methylesterase (PME), polyphenol oxidase (PPO), and peroxidase (POD) in cloudy apple juice were investigated to evaluate non-thermal effects. Experiments were conducted to provide uniform heating with accurate temperature acquisition and similar temperature profiles for conventional and microwave treatments. A two-fraction first-order kinetic model was successfully fitted to the data in a procedure that took into account the whole time-temperature profile instead of assuming isothermal conditions. Predicted inactivation curves for pasteurization at 70 and 80 °C of the cloudy apple juice showed that PME has the highest thermal resistance (residual activity of 30% after 250 s at 80 °C) and that there was no evidence of non-thermal microwave effects on the inactivation of these enzymes.     

Comparison of Various Preparation Methods for Determination of Organic Acids in Fruit Vinegars with a Simple Ion-Exclusion Liquid Chromatography

An ion-exclusion liquid chromatography with mobile phase 0.005 mol L^?1 H₂SO₄ and step flow rate gradient (0.2 mL min^?1 in the first 40 min and 0.5 mL min^?1 from 41 to 60 min) was used to determine 20 organic acids simultaneously at 17 °C within 51 min. The peak resolutions (Rs) were 0.45??3.02 and separation factors (??) were all higher than 1. Impurities in fruit vinegar executed with direct injection or C18 cartridge clean-up for analysis would influence the glutaric and oxalic acid measurement; however, SAX cartridge extraction could reduce the interferences (organic acid recoveries were 93.93??99.98%). Acetic, ascorbic, citric, malic, and malonic acids were the major organic acids in fruit vinegars (apple, apple sparkling, plum, cranberry, and grape).     

Synthesis of octadecyl acrylate-co-ethylene glycol dimethacrylate resin for removal of dark-colored compound from apple juice and comparison with polyvinylpolypirrolidone (PVPP)

Adsorption process has an importance for improving the color of juice and also stabilizing the final product during the shelf life. In this study, polyvinylpolypirrolidone (PVPP) and octadecyl acrylate-co-ethylene glycol dimethacrylate (ODA-EGDMA) polymeric resins were used as adsorbents for improving the color properties of apple juice. The ODA-EGDMA resin was prepared by classical suspension polymerization technique and characterized by electron microscopy. The obtained spherical particles diameters were between 20 and 140 μm. PVPP was selected as reference polymeric material for its importance in the juice industry. The adsorption kinetics of dark-colored compounds on adsorbent polymeric resins were studied at different adsorbent resins concentrations (1, 2, 4, and 8 g adsorbent resin per liter of apple juice) at the constant temperature (20 °C) in batch reactor. Langmuir adsorption model was applied for both PVPP and ODA-EGDMA polymeric adsorbents. The Langmuir isotherms were plotted for both polymeric adsorbents by evaluation of the absorbance data of apple juice at 420 nm. Langmuir isotherm's empirical constants known as K _ad and Q ₀ were calculated from the equilibrium data. Numerical value of K _ad and Q ₀ were determined as 5.0578 and 0.3089 for the ODA-EGDMA polymeric resin, 2.4824 and 0.5268 for the PVPP adsorbent, respectively. The scope of this study included comparison of reusability and regeneration properties of the each polymeric adsorbent. For this purpose, series of experiment were done in pack bed column application. Pressure drop measured throughout the PVPP pack bed column was 4.8 times higher than ODA-EGDMA pack bed column at same flow rate (2.5 mL/min) during the this group experiment. ODA-EGDMA resin and PVPP were regenerated with NaOH (4% w/v, 50 °C) after each run. After regeneration, average removal of dark color (relative absorbance change at 420 nm) of apple juice was determined as 76.47±0.90% for ODA-EGDMA and 91.51±0.86% for PVPP adsorbent resin.