Chemometric-Assisted Optimization of RP-HPLC Method for Determination of Some Bioflavonoids in Brassica oleracea Species and Their Antioxidative Activity

In the present work, the rapid RP-HPLC method with UV (DAD) detection for simultaneous quantification of bioflavonoids: quercetin, apigenin, catechin, epicatechin, kaempferol, and luteolin in Brassica oleracea species samples (cauliflower, broccoli, and Brussels sprouts) was developed with the aid of LC-Simulator (ACD Labs® suite) software. A series of extracts obtained with different extraction method were evaluated for antioxidant activity. The optimal conditions for separation and quantification were established after nine scouting runs entered to LC-Simulator software. The optimized separation was achieved on Hypersil GOLD aQ column with isocratic elution and mobile phase composition A:2 % acetic acid in water and B:acetonitrile in 91:9 (v/v %) ratio. The R _s values were in the range from 2.6 to 8.00, indicating good selectivity of the method. The obtained results generally show good agreement with published data. Low detection limits (0.02–0.055 μg/mL) were obtained with acceptable recoveries (90–109 %). Total time of analysis was less than 11 min; therefore, the proposed method represents significant improvement over existing methods. Extracts from Brassica vegetables, obtained using different extraction procedures, were studied for their radical scavenging effects. Scavenging of DPPH showed different kinetics at the beginning of the assay period and after 15 min from the initialization of reaction. Different kinetics suggested the presence of polymerized and/or less active antioxidants with different scavenging mechanisms for particular polyphenolic compounds.     

A TLC-HPLC Method for Determination of Thiamphenicol in Pig,Chicken, and Fish Feedstuffs

An effective thin-layer chromatography (TLC) purification procedure coupled to high-performance liquid chromatography (HPLC) method was developed for the determination of thiamphenicol (TAP) in pig, chicken, and fish feedstuffs. The feedstuff samples were extracted with ethyl acetate, defatted with n-hexane saturated with acetonitrile, and further purified by TLC. The chromatographic separation was performed on a Waters Symmetry C₁₈ column using an isocratic procedure with acetonitrile-water (21.7:78.3, v/v) at 0.6 mL/min. The ultraviolet (UV) detector was set at a wavelength of 225 nm. The TAP concentrations in feedstuff samples were quantified using a standard curve. Good linear correlations (y?=?162,630x???2381.7, r?>?0.9998) were achieved within the concentration range of 0.05–10.00 μg/mL. The recoveries of TAP spiked at levels of 1, 10, and 100 μg/g ranged from 82.0 to 114.9% with the intra-day and inter-day relative standard deviation (RSD) less than 9.0%. The limit of detection (LOD) and limit of quantitation (LOQ) were 0.02 and 0.03 mg/kg for pig feedstuffs, 0.02 and 0.04 mg/kg for chicken feedstuffs, and 0.02 and 0.03 mg/kg for fish feedstuffs, respectively. This reliable, simple, and cost-effective method could be applied to the routine monitoring of TAP in animal feedstuffs.     

Determination of Deoxynivalenol and Nivalenol in Wheat by Ultra-Performance Liquid Chromatography/Photodiode-Array Detector and Immunoaffinity Column Cleanup

An ultra-performance liquid chromatography (UPLC®) method has been developed for the simultaneous determination of deoxynivalenol (DON) and nivalenol (NIV) in wheat. Ground sample was extracted with water and the filtered extract was cleaned up through an immunoaffinity column containing a monoclonal antibody specific for DON and NIV. Toxins were separated and quantified by UPLC® with photodiode-array detector (λ?=?220 nm) in less than 3 min. Mean recoveries from blank wheat samples spiked with DON and NIV at levels of 100–2,000 μg/kg (each toxin) ranged from 85 to 95 % for DON and from 81 to 88 % for NIV, with relative standard deviations less than 7 %. Similar recoveries were observed from spiked samples when methanol/water (80:20, v/v) was used as extraction solvent. However, by using a wheat sample naturally contaminated with DON and NIV, the one-way analysis of variance (Student–Newman–Keuls test) between different extraction solvents and modes showed that water extraction provided a significant increase (P?<?0.001) in toxin concentrations (mean values of six replicate analyses) with respect to methanol/water (80:20, v/v). No significant difference was observed between shaking (60 min) and blending (3 min). The limit of detection (LOD) of the method was 30 μg/kg for DON and 20 μg/kg for NIV (signal-to-noise ratio 3:1). The immunoaffinity columns showed saturation of DON/NIV binding sites at levels higher than 2,000 ng in blank wheat extracts spiked with the corresponding amount of mycotoxin, as single mycotoxin or sum of DON and NIV. The range of applicability of the method was from LOD to 4,000 μg/kg, as single mycotoxin or sum of DON and NIV in wheat. The analyses of 20 naturally contaminated wheat samples showed DON contamination in all analyzed samples at level ranging from 30 to 2,700 μg/kg. NIV was detected in two samples at negligible toxin levels (up to 46 μg/kg). This is the first UPLC® method using immunoaffinity column cleanup for the simultaneous and sensitive determination of DON and NIV in wheat.     

Analysis of the isothiocyanates present in three Chinese Brassica vegetable seeds and their potential anticancer bioactivities

Epidemiological studies give evidence that high dietary intake of cruciferous vegetables has been associated with lower risk of cancer, this activity due to their content in glucosinolates (GL), which upon myrosinase hydrolysis release the corresponding isothiocyanates (ITCs). In this study, the isothiocyanates contents and the anticancer activity of ethyl acetate extracts from three kinds of Chinese Brassica vegetable seeds have been examined and analyzed. A correlation was found between the potency of anticancer activity and the amount of isothiocyanates in their extracts. Extracts followed by GC–MS analysis revealed that 3-BITC (3-butenyl isothiocyanate) was mainly found in Chinese kale contained 77.58 ± 7.28 mg/g (dry weight); AITC (allyl isothiocyanate), 3-BITC and iberverin were the predominant isothiocyanates in Oxheart cabbage contained 33.57 ± 3.94, 26.50 ± 1.92 and 22.77 ± 0.00 mg/g, respectively; 3-BITC and sulforaphane were found as the major isothiocyanates in broccoli contained 138.52 ± 3.42 and 49.77 ± 0.18 mg/g, respectively. The amount of total isothiocyanates in broccoli was twice higher than that in other two species. The anticancer activity of broccoli was the highest, and the IC₅₀ value of the extract inhibiting on the growth of A549, LAC, HELA and HepG₂ were 14.38 ± 0.35, 10.38 ± 0.34, 19.45 ± 1.72 and 26.75 ± 0.82 mg/g, respectively. The extracts of Chinese Brassica vegetable seeds have the potential in inducing cancer cells apoptosis by morphology observation. The results of this study got a new kind of Brassica vegetable seeds that could produce high content of isothiocyanates, which is important to preparation of anticancer food additives.     

Speciation of Organoarsenic Species in Food of Animal Origin Using Accelerated Solvent Extraction (ASE) with Determination by HPLC-Hydride Generation-Atomic Fluorescence Spectrometry (HG-AFS)

Concerning the residual organoarsenical feed additives, an effective method has been developed for the separation and determination of organoarsenic species including p-arsanilic acid (ASA), nitarsone (NIT) and roxarsone (ROX) in the food of animal tissue origin by high-performance liquid chromatography coupled to ultraviolet oxidation hydride generation atomic fluorescence spectrometry using a C₁₈ column with 50 mM KH₂PO₄, 0.1 %?v/v trifluoroacetic acid at pH 2.43 as the mobile phase. Accelerated solvent extraction (ASE) as an effective sample preparation method was used to deal with animal meat to extract organoarsenic species. The ASE conditions, including extraction solvent, temperature, static extraction time, flush volume and cycle time, were investigated in terms of extraction yield and species stability. In this paper, aimed to separate these species efficiently, the conditions of the mobile phase and HG system were also investigated. The methodology developed allows us limits of detection and quantification of 0.24, 0.74, 0.41 and 0.72, 2.24, 1.24 ng?mL^?1 for ASA, NIT and ROX, respectively. This method was used to separate and determine three organoarsenic species in porcine and chicken liver samples that were purchased at a supermarket in China. At the optimized conditions, the ranges of concentrations of the three arsenic species were found to be varied from 3 to 9 ng?mL^?1. The results of recovery rates and RSDs, which were higher than 94 % and lower than 5 %, respectively, approved it to be a convenient, fast and efficient method for the determination of organoarsenic species in animal tissue.     

Simultaneous Determination of Cyadox and Its Metabolites in Chicken Tissues by LC-MS/MS

A specific and sensitive LC-MS/MS method was firstly established for the simultaneous extraction and determination of cyadox and its three main metabolites—1,4-bisdesoxycyadox, 4-desoxycyadox, and quinoxaline-2-carboxylic acid—in chicken muscle, liver, kidney, and fat tissues. Samples were subjected to extraction using ethyl acetate and followed by acetonitrile–chloroform (1:4, v/v) and further purified by Oasis mixed mode anion exchange SPE cartridge. Analysis was performed on a C₁₈ column by detection with MS in multiple-reaction monitoring mode. A gradient elution program with 0.1 % formic acid solution, acetonitrile, and 1 % formic acid (adjusted to pH 8 with ammonia) was performed at a flow rate of 0.2 mL min^?1. The correlation coefficients (r) for each calibration curve are higher than 0.99 within the experimental concentration range. The recoveries of the four target analytes at three spiking levels of 2.5, 25 and 250 μg kg^?1 were between 74.5 and 93.8 %, with relative standard deviations less than 12 %. The decision limits (CCαs) of the four analytes in chicken edible tissues ranged from 0.3 to 1.5 μg kg^?1, and the detection capabilities (CCβs) were below 2.3 μg kg^?1. The developed method demonstrated a satisfactory applicability in incurred chicken tissue samples.     

Fluorescence Polarization Immunoassay for Rapid, Accurate and Sensitive Determination of Ochratoxin A in Wheat

A sensitive and accurate fluorescence polarization (FP) immunoassay has been developed for the determination of ochratoxin A (OTA) in naturally contaminated wheat samples. A fluorescein-labeled OTA tracer was synthesized, and its binding response with three monoclonal antibodies was tested. The most sensitive competitive FP immunoassay showed an IC₅₀ value of 0.48 ng/mL with a negligible cross-reactivity for ochratoxin B (1.7 %) and no cross-reactivity with other mycotoxins commonly occurring in wheat. The wheat sample was extracted with acetonitrile/water (60:40, v/v) and purified by a rapid solid-phase extraction procedure using an aminopropyl column prior to the FP immunoassay. The overall time of analysis was less than 20 min. The average recovery from spiked wheat samples (3 to 10 μg/kg) was 87 %, with relative standard deviations generally lower than 6 %. Limits of detection and quantification were 0.8 and 2.0 μg/kg, respectively. The trueness of the method was assessed by using two reference materials for OTA showing good accuracy and precision. A good correlation (r?=?0.995) was observed between OTA contamination of 19 naturally contaminated wheat samples analyzed by both FP immunoassay and high-performance liquid chromatography/immunoaffinity clean-up used as reference method. These results show that the developed FP method is suitable for high-throughput screening, as well as for reliable quantitative determination of OTA in wheat at level far below the EU regulatory limits.     

Optimization of Matrix Solid-Phase Dispersion Method for Extraction of Aflatoxins from Cornmeal

An extraction method for simultaneous determination of aflatoxins (AFLAs) G2, G1, B2, and B1 in cornmeal, based on vortex-assisted matrix solid-phase dispersion (MSPD) and high-performance liquid chromatography (HPLC) with fluorescence detection was optimized by a central composite design, validated and applied. Multivariate analysis was performed to evaluate the effect of cornmeal composition on AFLA extraction. The amount and proportion of solid support (celite and C18) and volume of elution solvent (methanol and acetonitrile) were the variables tested. The mobile phase of methanol/acetonitrile/water (24:14:62, v/v/v) in isocratic elution mode provided satisfactory AFLA separation. The best recoveries (85.7 to 114.8%) were obtained when the sample preparation contained 25 mg C18 as solid support and 10 mL of elution solvent. The limits of detection ranged from 0.01 to 0.04 ng g^?1, and the limits of quantification varied from 0.02 to 0.1 ng g^?1. The optimized method was suitable for coarse and medium grind cornmeal. Multivariate correlation analysis showed that the main interferers for AFLA recovery were proteins and sugars.     

Rapid determination of Alternaria mycotoxins in tomato samples by pressurised liquid extraction coupled to liquid chromatography with fluorescence detection

ABSTRACT

A sensitive and reliable method using pressurised liquid extraction (PLE) followed by molecularly imprinted solid phase extraction (MISPE) and high performance liquid chromatography with fluorescence detection (HPLC–FLD) has been developed for the analysis of alternariol (AOH) and alternariol monomethyl ether (AME) in tomato samples. Influence of several extraction parameters that affect PLE efficiency were evaluated for the simultaneous extraction of both mycotoxins in the selected samples. AOH and AME were optimally extracted using MeOH/water (25:75, v/v) at 70°C as solvent, a pressure of 1000 psi and a single extraction cycle. The resulting PLE extracts were pre-concentrated by molecularly imprinted solid phase extraction (MISPE) cartridges followed of analysis by HPLC with fluorescence detection (λ_exc = 258, λ_em = 440 nm). The proposed method was applied to the analysis of AOH and AME in fortified tomato samples (20–72 µg· kg–1) with recoveries of 84–97% (RSD < 8%, n = 6) for AOH and 67–91% (RSD < 13%, n = 6) for AME. The detection limit for AOH and AME were 7 and 15 µg· kg^–1, respectively. The ensuing PLE–MISPE–HPLC–FLD method was validated for the analysis of both mycotoxins in tomato samples in accordance with European Commission Decision 2002/657/EC.     

Effect of the Storage Conditions (Light and Temperature) on the Detection of Thiamethoxam and Clothianidin Content in Rapeseeds by LC-DAD

A new method has been developed and validated to determine potential differences in thiamethoxam and its metabolite (clothianidin) contents in treated rapeseed samples, which were stored under different conditions of light exposure and temperature (protected at 20 and 30 °C; unprotected at 30 °C), using liquid chromatography coupled to a diode array detector. An efficient extraction procedure has also been proposed (average analytes recoveries were between 82 and 104%); this involved a solvent extraction using a mixture of acetonitrile and sodium chloride (60:40, v/v), centrifugation, and a concentration step in a rotary evaporator. The chromatographic analysis of the compounds was achieved using a core-shell technology-based column (Kinetex C₁₈, 150 × 4.6 mm, 2.6 μm, 100 Å). The mobile phase consisted of 0.1% formic acid in water and 0.1% of formic acid in acetonitrile (25:75, v/v), with a flow rate of 0.5 mL/min in isocratic elution mode. The method was fully validated in terms of selectivity, limits of detection, and quantification, as well as matrix effect, linearity, precision, and trueness. Finally, the developed methodology was applied to determine thiamethoxam and clothianidin content in rapeseed samples, which were stored under different conditions of light exposure and temperature during 100 days. The results showed that rapeseeds should be stored at 20 °C and protected from light exposure, and that the loss of thiamethoxam was directly related to the formation of clothianidin.     

Simultaneous Determination of Abietic Acid and Dehydroabietic Acid Residues in Duck Meat by HPLC-PAD-FLD

Abietic acid (AA) and dehydroabietic acid (DHAA) are major components of rosin, and both of them are well-recognized causes of skin allergy. Rosin was once widely used for removal of duck feathers in China. In the present study, an analytical method was developed for simultaneous determination of AA and DHAA in duck meats. AA and DHAA were extracted with acetonitrile and cleaned by solid-phase extraction, followed by chromatographic separation on C18 column with methanol/2 mM phosphoric acid (86/14, v/v) as mobile phase. AA was detected by photodiode array detection (PAD) at 240 nm, while DHAA was detected by fluorescence detection (FLD) with excitation and emission at 225 and 287 nm, respectively. Limits of quantification for AA and DHAA were 50 and 15 μg/kg, respectively. The method was applied to analysis of AA and DHAA residues in meats of rosin-defeathered ducks and commercial ducks. The results indicated that removal of duck feathers by means of rosin could lead to residues of AA and DHAA in duck skins, and 5 out of 32 commercial ducks were found to be contaminated by AA and DHAA, ranging from 1,556.1 to 9,638.7 μg/kg and 476.4 to 2,623.8 μg/kg, respectively. The proposed method could be used to identify those rosin-defeathered ducks and to monitor the illegal use of rosin in duck processing.     

Enhancing Fluorescence LC Analysis of Biogenic Amines in Fish Tissues by Precolumn Derivatization with Naphthalene-2,3-dicarboxaldehyde

This work reports a sensitive high-pressure liquid chromatography method for the simultaneous determination of biogenic mono- and diamines in fish tissues. Highly fluorescent derivatives of the amines were obtained by precolumn derivatization with naphthalene-2,3-dicarboxaldehyde, in the presence of cyanide ion as the nucleophile and of heptylamine as the internal standard. The chromatographic separation was performed using a mobile phase of acetonitrile/methanol/10 % (v/v) acetone in water, delivered in gradient mode on an Inertsil ODS-3 column (250?×?4 mm i.d., 5 μm). The method was successfully applied to the analysis of fresh and canned tuna fish tissues, subjected to ultrasound-assisted liquid–liquid extraction. The variables affecting the derivatization yield as well as the extraction recovery of the analytes from the sample matrix were investigated. Results were quantified against the internal standard, according to the matrix-matched approach. Limits of detection between 2.5 and 330 mg kg^?1 sample were achieved. The precision and accuracy of assay in samples was satisfactory, yielding relative standard deviation and recovery values between 0.3 and 4.2 % and 81.0 and 102.5 %, respectively.     

Determination of Baclofen Residue in Muscle,Liver, Kidney and Fat of Swine by Liquid Chromatography-Tandem Mass Spectrometry

Baclofen was illegally used in veterinary clinical medicine as a growth-promoting agent. To date, few methods have been developed for the monitoring of baclofen in animal tissues. In this study, a sensitive and efficient liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to identify and quantify baclofen in the muscle, liver, kidney, and fat of swine was developed and validated. Baclofen was extracted from tissues with ammonium acetate buffer (pH 5.2) and isolated with isopropyl-ethyl acetate (4:6, v/v). Then, a solid phase extraction using MCX cartridge was used to clean up the extracts. The elution was evaporated to dryness and reconstituted with water/methanol (90:10 v/v). All samples were determined by LC-MS/MS system through positive ionization in a multiple reaction monitoring (MRM) mode. The proposed method was validated by evaluation of specificity, linearity, recovery, accuracy, precision, LOD, and LOQ values according to Commission Decision 2002/657/EC. Estimated limit of quantification for baclofen in the muscle, liver, kidney, and fat of this method was 1.00 μg/kg, respectively. The mean intra- and inter-day assay accuracies fell within a range 88.5–93.9% and 86.2–93.2%, respectively. The mean intra- and inter-day precisions were 1.78 and 4.95% (RSD < 15%), respectively. The proposed method has proved to be suitable for accurate quantitative determination of baclofen for residue analysis.     

Assay of Citrus Flavonoids, Troxerutin, and Ascorbic Acid in Food Supplements and Pharmaceuticals by Capillary Zone Electrophoresis

Capillary electrophoretic method suitable for the assay of citrus flavonoids (hesperidin, diosmin, and rutin), troxerutin, and ascorbic acid in food supplements and pharmaceutical formulations was devised and validated. The separation was carried out at 25 kV in uncoated fused-silica capillary (50 μm i.d.; total length, 32 cm (21 cm to detector); and spectrophotometric detection at 280 nm) maintained at 25 °C with 40 mM sodium tetraborate buffer of pH 9.5 containing 25 % (v/v) of methanol as the background electrolyte. The samples were injected hydrodynamically (50 mbar, 6 s). The calibration curves were linear (correlation coefficients r?=?0.9994–0.9998) for 0.05–0.50 mg/mL of hesperidin, diosmin, rutin, and troxerutin and 0.10–1.00 mg/mL of ascorbic acid when using cinnamic acid (0.05 mg/mL) as internal standard (IS). The LOQ values (S/N?=?10) ranged between 0.02 and 0.04 mg/mL of an analyte. The intra- and interday repeatability of migration times and corrected peak areas was characterized by RSD?≤?3.6 %. Single CE analysis of a standard mixture containing all five analytes and the IS took less than 11 min. Accuracy of the method was evaluated by the added/found analyte recovery experiments (recoveries, 95.5–99.8 %). The method was applied to the analysis of five commercially available food supplements and pharmaceuticals.     

Simple and Rapid Method for the Simultaneous Determination of Cholesterol and Retinol in Meat Using Normal-Phase HPLC Technique

A direct saponification and normal-phase high-performance liquid chromatography (NP-HPLC) procedure was developed for simultaneous determination of cholesterol and retinol in meat. A normal-phase silica column fitted with diode array detection at 208 nm for cholesterol and fluorescence detection (λ-excitation 344 nm/λ-emission 472 nm) for retinol, with mobile phase consisting of 2% (v/v) 2-propanol in n-hexane was used. Cholesterol was eluted at 10 min and retinol at 12.66 min. High linearity (R ² > 0.9996 for both compounds) in calibration range was obtained. The LOD and LOQ values show the high sensitivity of the developed methodology for simultaneous determination of cholesterol and retinol in meat. Recovery results obtained in this study (98.67–102.14% for cholesterol and 91.72–98.27% for retinol) were between AOAC recommendations to validated method and were comparable to most recent studies in precision and accuracy. In addition, the present method showed high repeatability and reproducibility. As a general conclusion, the results indicate that the direct saponification, extraction, and HPLC analysis is an adequate method for cholesterol and retinol analysis in meat samples.     

Design of Sonotrode Ultrasound-Assisted Extraction of Phenolic Compounds from <Emphasis Type="Italic">Psidium guajava</Emphasis> L. Leaves

Psidium guajava L. has gained a special attention as health plant due to the presence of phenolic compounds. Box-Behnken design (BBD) has been applied for the extraction of target compounds from guava leaves via sonotrode ultrasound-assisted extraction (UAE). Different extraction times (5, 30, and 55 min), ratios of ethanol/water (50, 75, and 100% (v/v)), and ultrasound (US) power (80, 240, and 400 W) were tested to find their effect on the sum of phenolic compound (SPC), flavonols and flavan-3-ols via HPLC-ESI-QqQ-MS, and antioxidant activity (DPPH and TEAC assays). The best process conditions were as follows: 40 min, 60% ethanol/water (v/v), and 200 W. Established method has been used to extract phenolic compounds in two guava leaves varieties (pyrifera and pomifera). Pyrifera var. showed greater values of the SPC via HPLC-ESI-QqQ-MS (49.7 mg/g leaf dry weight (d.w.)), flavonols (12.51 mg/g d.w.), flavan-3-ols (7.20 mg/g d.w.), individual phenolic compounds, and antioxidant activity (8970 ± 5 and 465 ± 6 μmol Trolox/g leaf d.w, respectively) than pomifera var. Conventional extraction showed lower amounts of phenolic compounds (7.81 ± 0.03 and 4.64 ± 0.01 mg/g leaf d.w. for flavonols and flavan-3ols, respectively) in comparison to the ultrasound-assisted ones.     

Enhancement of Functional Properties of Wami Tilapia (Oreochromis urolepis hornorum) Skin Gelatin at Different pH Values

The effects of several agents in two different concentrations and pH values (5.0 and 8.0) on the functional properties of tilapia (Oreochromis urolepis hornorum) skin gelatin were evaluated and compared using a control tilapia skin gelatin and a commercial mammalian gelatin. The addition of the agents (sucrose 4 % and 8 % (w/v), glycerol 5 % and 10 % (v/v), NaCl 0.3 and 0.8 mol/L, MgCl₂ 0.3 and 0.8 mol/L, MgSO₄ 0.3 and 0.8 mol/L, KCl 0.3 and 0.8 mol/L, and transglutaminase 10 and 15 mg/mL) slightly increased the turbidity. There were different ratios of rheological properties depending on the agent, concentration, and pH. The addition of all agents increased the viscosity of the gelatin solution, mainly at pH 5.0. The addition of glycerol (10 % (v/v)) raised viscosity up to 7.45 cP. The setting time was prolonged by incorporating the agents. The gelatin samples with the addition of MgSO4 0.8 mol/L showed higher gel strength than the mammalian gelatin, exhibiting values of 298 and 295g_f at pH 5.0 and 8.0, respectively.     

No de novo sulforaphane biosynthesis in broccoli seedlings

The isothiocyanate sulforaphane, present in significant amounts in broccoli (Brassica oleracea L.) seedlings in the form of its precursor glucoraphanin, has been identified as an inducer of quinine reductase, a phase-II detoxification enzyme known for its anticarcinogenic properties. Its concentration in broccoli seedlings usually decreases during the first 7–14 days after germination. No conclusive data on sulforaphane metabolism in seedlings are available in the literature. Here, we unambiguously demonstrate in ¹²C/¹³C-cross experiments that sulforaphane is not biosynthesised de novo during the first week of seedling development. Both ¹²C (99 atom% ¹²C) and ¹³C (98 atom% ¹³C) broccoli seeds were produced and subsequently germinated and grown either in a ¹³CO₂ or a ¹²CO₂ environment. Afterwards, the labelling degree of sulforaphane in seeds and in seedlings was analysed by HPLC–MS. We conclude that sulforaphane exclusively originates from seed reserves and that de novo biosynthesis is not detectable (<1%) in broccoli seedlings.     

Fast and Selective Determination of Ochratoxin A in Wines Using an Optimized and Validated Liquid Chromatographic Method

Determination of ochratoxin A (OTA) in wines requires a cleanup step using solid phase extraction (SPE). Immunoaffinity columns are commonly the columns of choice but due to its high cost, other SPE columns have been assayed without optimal results. The present work reports an optimized and validated liquid chromatographic method for a fast and selective quantification of OTA in wines using C₁₈ columns for cleanup. Chromatographic conditions were optimized using a central composite design, establishing the following optimal conditions: acetonitrile/water/acetic acid (59.5:39.5:1.0 v/v/v) as mobile phase, flow rate of 1.2 mL min^?1, and column temperature of 30 °C. With these conditions, OTA had a retention time (~4 min) up to five times lower than those reported earlier. Regarding validation, calibration data (n?=?8) fitted a linear regression model with a determination coefficient (R ²) of 0.9992. Repeatability (relative standard deviation (RSD)) and intermediate precision (RSD) in matrix showed values of 1.3 % (n?=?6) and 0.8 % (n?=?3), respectively. Recoveries at five levels ranged from 87.2 to 118.9 % (mean RSD of 7.4 %). Fifty-three samples from different origins were analyzed, finding only seven samples (13 %) with quantifiable OTA content (0.15–0.26 μg L^?1). According to the levels found, the contribution of wine consumption to OTA daily intake was at least 58 times lower than the current tolerable daily intake. In view of optimization and validation results as well as its applicability to real samples, this method could be considered a good alternative for routine analysis of OTA in wines.     

Multivariate optimisation of an ultrasound assisted-matrix solid-phase dispersion method combined with LC-fluorescence detection for simultaneous extraction and determination of aflatoxins in pistachio nut samples

This paper describes the application of ultrasound-assisted matrix solid-phase dispersion as an extraction and clean-up procedure for aflatoxins (B₁, B₂, G₁ and G₂) and subsequent determination by LC-fluorescence detection. A Box–Behnken design was used to determine the parameters influencing the extraction procedure through response surface methodology and experimental design. The influence of different variables including type of dispersing phase, sample-to-dispersing phase ratio, type and quantity of clean-up phase, ultrasonication time, ultrasonication temperature, nature and volume of the elution solvent were investigated in the optimisation study. C18, graphitic carbon black and acetonitrile were selected as dispersing phase, clean-up phase and elution solvent, respectively. The optimised values were sample-to-dispersing phase ratio of 1:1, 50 mg of graphitic carbon black, 11 min ultrasonication time, 30°C ultrasonication temperature and 3 ml acetonitrile. Under the optimal conditions the limits of detection (LODs) were ranged from 0.04–0.11 µg kg^?1 and the relative standard deviations (RSDs) of the extraction method were less than 8.6%. The recoveries of the matrix solid-phase dispersion process ranged from 74% to 78% with relative standard deviation lower than 9% in all cases. Finally, the matrix solid-phase dispersion was successfully applied to extraction of trace amounts of aflatoxins in pistachio samples.