Anti-oxidative capacity of enzymatically released peptides from soybean protein isolate

Shelf-life of dietary products and the need for health supporting functional ingredients often require components with high anti-oxidative properties. The study aimed at the characterisation of peptides with antioxidant properties from enzymatically hydrolysed soybean protein isolate. Soybean protein isolate was hydrolysed with a food grade pancreatic trypsin/chymotrypsin for industrial applications, ultrafiltered and the permeate was fractionated by size exclusion chromatography. Anti-oxidative properties of these fractions were tested in a Trolox^®-equivalent-anti-oxidative capacity assay, which is based on the absorbance suppression of 2,2′-azinobis(3-ethyl benzothiazoline-6-sulfonate) radical cations by anti-oxidative substances at λ 414 nm. The amino acid compositions and sequences of the peptide fractions were determined by liquid chromatography and mass spectrometry analysis. Compared to the intact soybean protein isolate a peptide fraction with an anti-oxidative capacity of 113 mg TEAC/g was obtained. This fraction was shown to be enriched in peptides smaller than 1 kDa with prevailing aromatic amino acid residues. Nine peptides derived from glycinin and β-conglycinin were sequenced by tandem mass spectrometry. The particular sequences were synthesized and assayed. The synthetic peptide analogue TTYY (β-conglycinin fragment 291–294) revealed significant radical scavenging properties of 13.6 up to 59.6% with 0.18–18 mM in a concentration dependent manner. Peptides from soybean protein isolate were shown to possess anti-oxidative capacities which might be linked to the presence of carboxy terminal tyrosine. These findings offer new functional application possibilities in nutrition, cosmetics and pharmaceuticals.     

Liquid Chromatography Orbitrap Mass Spectrometry Method for Analysis of Homocysteine and Related Aminothiols in Cereal Products

Liquid chromatography Orbitrap mass spectrometry method for quantification of biological aminothiols (cysteine, homocysteine, and glutathione) in cereal products has been developed. The assay is based on preliminary derivatization with N-(2-acridonyl)maleimide and high resolution accurate mass spectrometry with utilization of dl-Homocystine-3,3,3′,3′,4,4,4′,4′-d₈ (homocystine-d₈) as internal standard. The limits of quantification for homocysteine, cysteine, and glutathione are 19.44, 40.78, and 338.93 pg, respectively, per 10 μl injection. Intra- and inter-day precision expressed as relative standard deviations are in the range of 1.76 to 2.94 % and 1.06 to 4.13 %, respectively. The average recoveries were 98 % for Hcy, 87 % for Cys, and 92 % for GSH. Wheat, maize, and bakery products with different origin were analyzed. The content of Hcy in the investigated samples was found to be in range of 9–436 μg/100 g, Cys in range of 29–6,895 μg/100 g and GSH in range of 259–14,795 μg/100 g.     

Moniliformin analysis in maize samples from North-West Italy using multifunctional clean-up columns and the LC-MS/MS detection method

A fast clean-up method has been developed to purify maize extracts and to detect moniliformin (MON) in maize samples from North-West Italy over a four-year period (2008–2011). The method is based on the use of MycoSep® 240 Mon clean-up columns (Romer Labs^®). Samples were extracted using acetonitrile/water (84:16, v/v), and the extracts were purified with previously described clean-up columns. The liquid chromatography–tandem mass spectrography (LC-MS/MS) analysis has been carried out by means of hydrophilic interaction chromatography (HILIC), combined with negative electrospray mass spectrometry. The method has a recovery of 76–91% (relative standard deviation, RSD%: 6–14%), a limit of detection (LOD) of 1 µg kg^–1 and a limit of quantification (LOQ) of 4 µg kg^?1. Naturally contaminated maize (108 samples) was analysed for MON content. The average percentages of positive samples was 93% with the following ranges (µg kg^?1): 33–2606 (2008); <LOD–527 (2009); <LOD–920 (2010); <LOD–409 (2011).     

Simultaneous determination of kasugamycin and validamycin-A residues in cereals by consecutive solid-phase extraction combined with liquid chromatography–tandem mass spectrometry

Two polar aminoglycosides, kasugamycin and validamycin-A, were determined in cereals (brown rice, wheat and corn) by high-performance liquid chromatography–tandem mass spectrometry. The analytes were extracted from samples using methanol and water (70:30, v/v) at pH 5.5, purified using both a hydrophilic–hydrophobic-balanced cartridge and a strong cation-exchange cartridge, and then analysed using multiple reaction monitoring in positive electrospray ionisation mode with a special ReproSil 100 C₁₈ high-performance liquid chromatography column. This newly proposed method yielded good sensitivity and excellent chromatographic performance. The limits of quantification for kasugamycin and validamycin-A were 4.1 µg/kg and 1.0 µg/kg, respectively. The recoveries for both compounds at three fortification levels (4, 100 and 500 µg/kg for kasugamycin; 1, 10 and 100 µg/kg for validamycin-A) ranged from 75% to 110%, and the relative standard deviations were below 15%.     

3-Monochloro-1,2-propandiol (3-MCPD) in soy sauce from the Bulgarian market

The 3-monochloro-1,2-propandiol (3-MCPD) levels in soy sauces which contained hydrolysed vegetable protein were evaluated for the Bulgarian market. For analysis of 3-MCPD, a gas chromatography–mass spectrometry (GC-MS) method was applied with a linear range of 0.03–2.00 μg mL^?1 and a limit of detection (LOD) of 2.3 μg kg^?1 and a limit of quantification (LOQ) of 3.4 μg kg^?1. At these levels, the standard deviation was 5.1%, with recoveries between 81% and 102%. The method was applied to the analysis of 21 samples of soy sauce from the Bulgarian market. Results ranged from 3.7 to 185.6 μg kg^?1. Soy sauces produced from hydrolysed soy protein contained higher levels of 3-MCPD than naturally fermented sauces. In 38.4% of samples of Bulgarian origin, the 3-MCPD content was above the EU limit of 20 μg kg^?1. In all analysed samples, 33.3% had a 3-MCPD content above the EU limit.     

Determination of Five Alkaloids of Pericarpium Papaveris in Hot Pot Broth Using Ultra-Performance Liquid Chromatography Coupled to Triple Quadruple Mass Spectrometry

Simultaneous determination of five alkaloids (morphine, codeine, noscapine, thebaine, and papaverine) of pericarpium papaveris in hot pot broth was established by ultra-performance liquid chromatography (UPLC) coupled to triple quadruple mass spectrometry (QqQ-MS). Water containing 0.1 mol/L hydrochloric acid was used as extraction solution and petroleum ether were used for fat removal. Then, the water-phase extracts were purified by an Oasis MCX cartridge. After separation by a BEH C18 (50?×?2.1 mm, 1.7 μm) column, five alkaloids were detected by mass spectrometry in the positive electrospray ionization with multiple reaction monitoring (MRM). The linear ranges were 0.1–10.0 μg/kg for morphine, codeine, and thebaine, as well as 0.01–1.0 μg/kg for papaverine and noscapine, respectively. The limits of quantification (LOQs) for morphine, codeine, and thebaine were 0.1 μg/kg and for papaverine and noscapine were 0.01 μg/kg. Mean recoveries at three spiked concentrations for the objectives were varied from 72.1 % to 124 %. The method was employed in real samples and demonstrated its rapidness and high sensibility.     

Rapid Screening and Determination of the Residues of Hormones and Sedatives in Milk Powder Using the UHPLC-MS/MS and SPE

A method based on the ultra-high-performance liquid chromatography tandem mass spectrometry for determination of the residues of sex hormones, glucocorticoids, and sedatives in milk powder was developed. The sample was extracted with the acetic acid-acetonitrile (1:99, v/v) twice, purified by the PRiME hydrophilic-lipophilic balance (HLB) cartridges and analyzed by the ultra-high-performance liquid chromatography-tandem mass spectrometry. The analytes were separated by the Waters Acquity UPLC? BEH C18 column (50 mm?×?2.1 mm, 1.7 μm) and determined using the electrospray ionization in the positive mode with the multiple reaction monitoring (MRM). The developed method was validated with the specificity, linearity and range, matrix effects, recovery, and precision. The results showed that the analytes were linear with the correlation of determinations (R²) higher than 0.991 in the corresponding ranges. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.1–1.1 μg kg^?1 and 0.3–3.8 μg kg^?1, respectively. The average recoveries of the analytes ranged from 78.5 to 107.0% with the relative standard deviations lower than 15%. The practical applicability was tested by analyzing real samples and the progesterone was observed in two samples.     

Investigation of Levels and Fate of Pyridalyl in Fruit and Vegetable Samples by Fast Gas Chromatography–Mass Spectrometry

The search on pyridalyl residues, the novel insecticide, in strawberries and spring onions was evaluated. The QuEChERS technique was used for sample preparation. A fast gas chromatography–mass spectrometry (GC–MS) method was developed and validated for the analysis of pyridalyl in both matrices. Fast GC–MS was performed with a narrow-bore capillary column and a quadrupole mass detector with electron ionization and negative chemical ionization, both operating in selected ion monitoring mode. Fortification studies at 1, 5, 10, and 250 μg/kg for fruit and vegetable matrices were performed. Recoveries for all fortification levels, two ionization modes ranged from 72 to 114 %. Matrix effects were discussed. Limits of quantification were established at 1 μg/kg. Field experiments to investigate the pre-harvest interval were carried out. The proposed method was applied successfully to the determination of pyridalyl residues in samples available on Slovak market, and none of the samples contained detectable amounts of pesticides. The developed method is simple, efficient, and easy to adopt in laboratories engaged in pesticide residue analysis method.     

A Simple and Rapid Method for Determination of Patulin in Juice by High Performance Liquid Chromatography Tandem Mass Spectrometry

A simple and rapid method was developed for analysis of patulin in juice using ultra performance liquid chromatography coupled with tandem mass spectrometry. The samples were purified by an Oasis MAX 96-Well Plate. Chromatographic separation was performed on an ACQUITY UPLC® HSS T3 column with gradient elution using acetonitrile and water as mobile phase. Analytes was quantified by external calibration curves over the ranges of 0.50–50 μg/L, with correlation coefficients > 0.9986. The LOD and LOQ in juice sample were 0.30 and 1.0 μg/L, respectively. Recoveries of the method (spiked at levels of 1, 5, 25 and 50 μg/L) ranged from 91.6% to 95.7%, while intraday and interday relative standard deviations were 6.87–11.0% and 6.53–11.6%, respectively.     

Development of a Headspace-Solid Phase Microextraction-Gas Chromatography/Mass Spectrometry (HS-SPME-GC/MS) Method for the Determination of Benzene in Soft Drinks

A method based on headspace-solid phase microextraction and gas chromatography with mass spectrometry has been developed and validated for the determination of benzene in soft drinks. The extraction step was optimized using a rotatable central composite design including the following experimental variables: extraction temperature, extraction time, sample weight, and salt concentration. The optimized procedure, which was carried out at 30 °C during 30 min by using a 75 μm carboxen-polydimethylsiloxane fiber, showed good linearity within the concentration range 0–25 μg?kg^?1 (r ² ?>?0.999), mean recoveries from 97.5 to 103.1 %, and coefficients of variation from 1.5 to 13.4 % for repeatability and from 1.5 to 15.7 % for within-laboratory reproducibility. Limits of detection and quantification were calculated at 0.02 and 0.08 μg?kg^?1, respectively. The method was applied to determine the concentrations of benzene in 77 samples of beverages from the Brazilian market. Levels from <0.08 to 10.84 μg?kg^?1 were obtained, which are comparable to those verified in other countries. Most of the samples (72.2 %) contained benzene up to 1 μg?kg^?1.     

Determination of Underivatized Glyphosate Residues in Plant-Derived Food with Low Matrix Effect by Solid Phase Extraction-Liquid Chromatography-Tandem Mass Spectrometry

A method was developed for the determination of glyphosate residues in plant-derived food using a two-step solid phase extraction (SPE) combined with mixed-mode liquid chromatography-tandem mass spectrometry (LC-MS/MS). The samples were extracted with water. Then, the extracting solution was pretreated by a C18 cartridge to remove protein and weak-polar interferences and further directly extracted using a strong anion exchange (SAX) cartridge to remove neutral and basic substances. The obtained glyphosate residues from the SPE were separated on a hydrophilic interaction/weak anion-exchange (HILIC/WAX) column and detected by mass spectrometry with negative electrospray ionization (ESI-) in multiple reaction monitoring (MRM) mode. This approach was evaluated by five different kinds of plant-derived food (soybean, corn, carrot, apple, and spicy cabbage) matrices in terms of matrix effect and recovery. Results showed that two-step SPE and mixed-mode chromatography separation provided the method with a very low matrix effect, and the spiked recoveries of glyphosate were satisfied in the range of 83.1 to 100.8 % at three spiked levels. The limit of quantification (LOQ) and detection (LOD) of the method in different matrices were 0.016–0.026 and 0.005–0.008 mg kg^?1, respectively. The procedure was validated and showed good accuracy and precision over a large linear range of 0.02–10 mg kg^?1.     

Simultaneous Determination of Trimethoprim and Diaveridine in Tissues of Chicken, Porcine, and Fish by Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry

A sensitive liquid chromatography–tandem mass spectrometry method for simultaneous determination of trimethoprim and diaveridine in edible tissue samples (chicken and porcine muscle; liver, kidney, and fat tissues as well as fish muscle) was developed and validated in this study. Trimethoprim, diaveridine, and the internal standard trimethoprim-D₃ were extracted from samples using acetonitrile. The Oasis MCX Solid-Phase Extraction Cartridge was selected for cleaning up the extracts. Chromatographic separation was performed on a hydrophilic interaction liquid chromatography column with gradient elution. The analytes were determined using triple–quadrupole mass spectrometry in positive electrospray ionization and multiple reaction monitoring mode. The relative recoveries from spiked samples ranged from 82.5 to 117.2 %, with the relative standard deviations generally being below 15.4 %. Limits of detection and quantification for analytes were within 0.3–1 and 1–2 μg kg^?1, respectively. The proposed method was successfully applied to detect trimethoprim and diaveridine in real samples.     

Determination of Ochratoxin A in Cereals and Feeds by Ultra-performance Liquid Chromatography Coupled to Tandem Mass Spectrometry with Immunoaffinity Column Clean-up

This paper describes the preparation of reusable immunoaffinity columns and the development of an ultra-performance liquid chromatography tandem mass spectrometry method combined with immunoaffinity column clean-up (IAC-UPLC-MS/MS) for the determination of ochratoxin A (OTA) in cereals and feeds. The monoclonal antibody (mAb) was produced from a stable hybridoma cell line (4H10), which belongs to the immunoglobulin G1 (κ-light chain) isotype. A competitive indirect enzyme-linked immunosorbent assay was used to characterize the mAb. The concentrations causing 50 % inhibition of binding of mAb to OTA-ovalbumin by free OTA, ochratoxin B, and ochratoxin C were 1.29, 4.78, and 0.94 ng mL^?1, respectively. The IAC-UPLC-MS/MS method offers a limit of quantification (LOQ, S/N >10) ranging from 0.5 to 1.0 μg kg^?1 and a limit of detection (LOD, S/N >3) ranging from 0.2 to 0.3 μg kg^?1 in cereal and feed samples. The IAC-UPLC-MS/MS method offers a good LOQ and LOD for OTA in cereal and feed samples. The accuracy and precision at this level fall within the EU regulatory limit. This methodology has been validated in four different matrices (millet, maize, soybean, and swine finisher diet) with highly satisfactory results and applied to the analysis of samples collected from the markets.     

Application of Fast Gas Chromatography–Mass Spectrometry in Combination with the QuEChERS Method for the Determination of Pesticide Residues in Fruits and Vegetables

A fast gas chromatography–mass spectrometry method has been developed for multiresidue determination of up to 56 pesticides in fruits and vegetables in a chromatographic run time of <10 min, using a single quadrupole mass spectrometer operating in selected ion monitoring mode. The well-known acetate-buffering version of the QuEChERS method has been used for sample preparation. Programmable temperature vaporizer injection of 3 μL allowed reaching limits of detection between 0.15 and 15 μg/kg for most compounds in the sample matrices tested. The applicability of the method has been evaluated in apple, orange, carrot, and tomato. Recoveries at three fortification levels (0.01, 0.1 and 0.5 mg/kg) ranged from 70 to 120 % for most compounds, with relative standard deviations below 20 % in all cases. The developed method has been applied to fruit and vegetable samples from different Spanish provinces.     

Trace Analysis of Seven Neonicotinoid Insecticides in Bee Pollen by Solid–Liquid Extraction and Liquid Chromatography Coupled to Electrospray Ionization Mass Spectrometry

A new, fast, simple, and efficient sample treatment to extract seven neonicotinoid insecticides (acetamiprid, clothianidin, dinotefuran, imidacloprid, nitenpyram, thiacloprid, and thiamethoxam) in corbicular bee pollen has been developed. This procedure consisted of a solid–liquid extraction of the neonicotinoids from pollen with dichloromethane, followed by evaporation and reconstitution. Once the neonicotinoids were extracted, they were determined using a liquid chromatography coupled to an electrospray ionization mass spectrometry method. The method was validated in terms of selectivity, linearity, precision and recovery. The limits of detection and quantification were 0.4–2.8 μg/kg and 1.2–9.1 μg/kg, respectively, and the extraction recoveries were between 86 and 106 % in all cases. Finally, the proposed method was applied to analyze bee pollen samples collected from apiaries located close to fruit orchards in two Spanish regions, and traces of acetamiprid and imidacloprid were found in two samples.     

Rapid Quantification Method of Three <Emphasis Type="Italic">Alternaria</Emphasis> Mycotoxins in Strawberries

Validation of simple and rapid method for three Alternaria mycotoxins determination including alternariol (AOH), alternariol methyl ether (AME) and tentoxin (TEN) in strawberries is described. The extraction procedure was based on a simple liquid–liquid extraction with ethyl acetate, which provided the highest recoveries and the lowest matrix effect. Analytes were determinated by liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS). The obtained relative recoveries were higher than 63 % for the studied mycotoxins in strawberries at the limit of quantification (LQ). Good linearity (r ²?>?0.993) and quantification limits (3–14 ng/g) were obtained. Repeatability, expressed as relative standard deviation, was always lower than 6 %, whereas interday precision was lower than 13 % for the developed method. The method was applied to analyse 24 strawberry samples commercialized in markets of the Valencia city (Spain). Analysed samples were only contaminated with AOH and AME.     

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.     

Development and Validation of RP-HPLC Method for the Simultaneous Quantification of Seven Flavonoids in Pericarpium Citri reticulatae

In this study, a reversed phase high performance liquid chromatography (RP-HPLC) method was developed and validated for simultaneous quantification of seven flavonoids including nobiletin, tangeretin, naringin, naringenin, hesperidin, neohesperidin, and hesperetin in Pericarpium Citri reticulatae (PCR). Ultrasonic-assisted extraction (UAE) conditions were optimized using response surface methodology (RSM) to obtain maximum extractive contents of the seven flavonoids. A Box–Behnken design (BBD) was employed to study the main effects and interactions of independent variables. Following UAE, chromatographic separation was accomplished on a C₁₈ column with a linear gradient elution. Full validation of the assay was implemented. It was found that this method had good linearity and specificity. Limit of detection (LOD) and limit of quantification (LOQ) for the tested flavonoids were 0.17–0.49and 0.75–1.75 μg/mL, respectively. Precisions (relative standard deviation, RSD) for intra- and interday were better than 4.54 %. Reproducibility was less than 4.91 %. Analyzed samples were stable for at least 48 h. Analysis had a good robustness (RSD?<?8.00 %). Recoveries for the flavonoids were 98.00–101.32 %. The established method was successfully applied to determine the seven components in real samples from different locations and under different stress conditions. Results demonstrated that this analytical method was rapid, comprehensive, and cost effective for quality control of Pericarpium C. reticulatae.     

Determining pesticide residues in wheat flour by ultrahigh-performance liquid chromatography/quadrupole time-of-flight mass spectrometry with QuEChERS extraction

ABSTRACT

Pesticides are used to increase crop yields and preserve quality by protecting crops against pests; however, their overuse can adversely affect human health and the environment. Herein, we report the development of a multi-pesticide screening method using optimized QuEChERS coupled with liquid chromatography/quadrupole time-of-flight (QTOF) mass spectrometry for the analysis of 13 pesticides in wheat flour. Mass accuracies with errors of less than 2.4 ppm were obtained for all analysed pesticides, and the method provided satisfactory recovery and linearity. Repeatabilities of 0.3–12.7% and reproducibilities of 2.5–15.2% were observed in full-scan TOF mode. The performance of the developed full-scan TOF method was compared to that obtained in high-resolution multiple reaction monitoring (MRM-HR) mode. The limits of quantification for the full-scan TOF and MRM-HR modes ranged from 2 to 10, and 3 to 9 μg kg^?1, respectively. The two quantification methods exhibited high sensitivities (limit of detections: 1–3 μg kg^?1 in full-scan TOF, and 1–3 μg kg^?1 for MRM-HR mode). No pesticide residues were detected when the developed method was applied to 22 real wheat flour samples.     

Multiresidue method for detection of pesticides in beef meat using liquid chromatography coupled to mass spectrometry detection (LC-MS) after QuEChERS extraction

Beef meat is an important food that can be contaminated by pesticides. This study aimed to optimize a multiresidue method for identification and quantification of pesticides in beef meat by liquid chromatography coupled to mass spectrometry detection (LC-MS). The extraction and clean-up procedures were adapted from the QuECHERS method. From the 188 analytes tested, the method was validated as qualitative method for 19 compounds and as quantitative method for 152 compounds. The results were satisfactory, yielding coefficients of variation of less than 20% and recoveries ranging from 70% to 120% and expanded uncertainty of less than 50%. The quantification limit was typically 10 µg kg^?1 (but 25 µg kg^?1 for 12 of the compounds) and the detection limit was 5.0 µg kg^?1. Thirty-two real samples of commercialized beef meat were analyzed without any residual pesticide being found. Thus, the results showed that the multiresidue method for detecting 171 pesticides, using adapted QuECHERS for extraction and LC-MS for detection, is suitable for analyzing beef meat.