Rapid screening method for quinolone residues in livestock and fishery products using immobilised metal chelate affinity chromatographic clean-up and liquid chromatography-fluorescence detection

An efficient LC method was developed for screening the presence of quinolones (QLs)--comprising fluoroquinolones (FQs) and acidic quinolones (AQs)--residues in various livestock and fishery products. Targeted analytes were for nine FQs of marbofloxacin (MAR), ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR), danofloxacin (DAN), orbifloxacin (ORB), difloxacin (DIF) and sarafloxacin (SAR), and three AQs of oxolinic acid (OXA), nalidixic acid (NAL) and flumequine (FMQ). Samples comprised ten different food products covering five matrices: muscle (cattle, swine and chicken), liver (chicken), raw fish (shrimp and salmon), egg (chicken), and processed food (ham, sausage and fish sausage). This method involved a simple extraction with (1:1) acetonitrile-methanol, a highly selective clean-up with an immobilised metal chelate affinity column charged with Fe(3+), a fast isocratic LC analysis using a short column (20 mm × 4.6 mm, 3 μm) with a mobile phase of (15:85:0.1) methanol/water/formic acid, and fluorescence detection (excitation/emission wavelengths of 295 nm/455 nm for FQs (495 nm for MAR), and 320 nm/365 nm for AQs). Among FQs, pairs of NOR/OFL, ORB/DIF and ENR/DAN were incompletely resolved. A confirmatory LC run with a Mg(2+) containing methanolic mobile phase was also proposed for the samples suspected of being positive. The optimised method gave satisfactory recoveries of 88.5% (56.1-108.6%) and 78.7% (44.1-99.5%) for intra- and inter-day assays with relative standard deviations of 7.2% (0.7-18.4%) and 6.8% (1.4-16.6%), respectively. Limits of quantitation ranged from 0.8 μg kg(-1) (DAN) to 6.5 μg kg(-1) (SAR). This method was successfully employed to analyse 113 real samples and two positive samples were found: fish sausage (CIP 990 μg kg(-1)) and shrimp (ENR 20 μg kg(-1)).     

Rapid screening method for quinolone residues in livestock and fishery products using immobilised metal chelate affinity chromatographic clean-up and liquid chromatography-fluorescence detection

An efficient LC method was developed for screening the presence of quinolones (QLs) – comprising fluoroquinolones (FQs) and acidic quinolones (AQs) – residues in various livestock and fishery products. Targeted analytes were for nine FQs of marbofloxacin (MAR), ofloxacin (OFL), norfloxacin (NOR), ciprofloxacin (CIP), enrofloxacin (ENR), danofloxacin (DAN), orbifloxacin (ORB), difloxacin (DIF) and sarafloxacin (SAR), and three AQs of oxolinic acid (OXA), nalidixic acid (NAL) and flumequine (FMQ). Samples comprised ten different food products covering five matrices: muscle (cattle, swine and chicken), liver (chicken), raw fish (shrimp and salmon), egg (chicken), and processed food (ham, sausage and fish sausage). This method involved a simple extraction with (1:1) acetonitrile–methanol, a highly selective clean-up with an immobilised metal chelate affinity column charged with Fe³⁺, a fast isocratic LC analysis using a short column (20?mm?×?4.6?mm, 3?µm) with a mobile phase of (15:85:0.1) methanol/water/formic acid, and fluorescence detection (excitation/emission wavelengths of 295?nm/455?nm for FQs (495?nm for MAR), and 320?nm/365?nm for AQs). Among FQs, pairs of NOR/OFL, ORB/DIF and ENR/DAN were incompletely resolved. A confirmatory LC run with a Mg²⁺ containing methanolic mobile phase was also proposed for the samples suspected of being positive. The optimised method gave satisfactory recoveries of 88.5% (56.1–108.6%) and 78.7% (44.1–99.5%) for intra- and inter-day assays with relative standard deviations of 7.2% (0.7–18.4%) and 6.8% (1.4–16.6%), respectively. Limits of quantitation ranged from 0.8?µg?kg^?1 (DAN) to 6.5?µg?kg^?1 (SAR). This method was successfully employed to analyse 113 real samples and two positive samples were found: fish sausage (CIP 990?µg?kg^?1) and shrimp (ENR 20?µg?kg^?1).     

Determination of residues of oxolinic acid and flumequine in freeze-dried salmon muscle and skin by HPLC with fluorescence detection

A procedure for the determination of residues of oxolinic acid (OA) and flumequine (FLU) in freeze-dried salmon muscle with attached skin, using reversed-phase high-performance liquid chromatography, is described. OA and FLU were extracted by a solid-liquid extraction procedure: after addition of hydrochloric acid, extraction used successively ethyl acetate, sodium hydroxide and chloroform. Liquid chromatography was performed on a 5 μm PuroSpher RP-18E ® cartridge using acetonitrile and 0.02 M aqueous orthophosphoric acid solution as mobile phase, with fluorescence detection. The performance of the method was established by spiking tissues with OA and FLU before the freeze-drying step. The method was linear over the concentration range 50-2000 ng/g freeze-dried tissue. Limits of detection and quantitation were 3.2 and 16 ng/g wet weight tissue respectively both for OA and FLU. Mean extraction recoveries of OA and FLU from freeze-dried tissue were 85.5 and 85.2% respectively. The method is suitable as a regulatory one for determination of residues of OA and FLU in freeze-dried salmon tissue.     

A novel quantum dot-based fluoroimmunoassay method for detection of Enrofloxacin residue in chicken muscle tissue

A rapid indirect competitive fluorescence-linked immunosorbent assay (cFLISA) based on quantum dots (QDs) as the fluorescent marker has been developed for the detection of Enrofloxacin (ENR) in chicken muscle tissue. The end-point fluorescent detection system was carried out using QDs conjugated with goat anti-mouse secondary antibody. The cFLISA method allowed for ENR determination in a liner working range of 1–100 ng mL⁻¹ with the 50% inhibition value (IC₅₀) of 8.3 ng mL⁻¹ and the limit of detection (LOD) of 2.5 ng mL⁻¹. The recoveries for chicken muscle samples spiked with ENR at levels of 50–200 μg kg⁻¹ ranged from 81% to 94% with coefficients of variation (CV) of 10–13%. In real chicken tissue sample analysis, the results of cFLISA were similar to those obtained from an indirect competitive enzyme-linked immunosorbent assay (cELISA) to a high performance liquid chromatography method (HPLC), which indicated that cFLISA is suitable as screening method for the monitoring of veterinary drug residues.     

Speciation of selenium in vitamin tablets using spectrofluorometry following cloud point extraction

Selenium is one of the trace and essential elements for good health but required only in a very narrow range. Hence, determination of selenium in trace level is important. In this work, cloud point extraction (CPE) with non-ionic surfactant Triton X-114 and the fluorometric ligand, 2,3-diaminonaphtalene (DAN) were used for extraction of trace amounts of organic and inorganic selenium species prior to their determination by spectrofluorometry. CPE parameters affecting complexation and phase separation were optimised. The limit of detection calculated by using nine replicate measurements of 0.020 mg/L Se solution after complexing with DAN and 10-fold CPE preconcentration was 2.1 μg/L. Accuracy of the method was checked using EnviroMat Waste Water, EU-L-2 as CRM and result was found to be in good agreement with the certified value. The suggested method can be used for selenium species of selenite, selenate, and total organic selenium at μg/L level.     

Habituation to organic acid anions induces resistance to acid and bile in Listeria monocytogenes

We evaluated the intrinsic and inducible resistance of four human pathogenic strains of Listeria monocytogenes to acid and bile, factors associated with virulence. Cells were grown in media at pH 7.4, or in media at pH 6.0 containing 0 (HCl control) or 4.75 mM of different organic acids, harvested at stationary or mid log phase, and challenged for 1 h in acid or bile. Stationary phase cells were intrinsically more resistant to either challenge than log phase cells, and large differences between strains were evident among the latter. Compared to the HCl control, habituation to log phase with organic acids induced significant (p < 0.05) and meaningful (≥ 1 log) increases in acid resistance of three of four strains tested, and in bile resistance of two strains suggesting that exposure to organic acid anions may enhance virulence in L. monocytogenes.     

Smart porous microparticles based on gelatin/sodium alginate polyelectrolyte complex

Porous microparticles of different sizes were prepared by polyelectrolyte complexation of biopolymers gelatine A and sodium alginate for microencapsulation of food bioactives. The optimum pH and ratio between the polymers sodium alginate and gelatine for maximum complexation was found as 3.7 and 1:3.5 respectively. Effect of various factors like amount of surfactant, concentration of polymer and crosslinker on the formation, size and porous/nonporous nature of the microparticles were investigated. The particles’ diameter on swelling at pH = 7.4 was twice that at pH = 1.2 indicating the pH responsiveness. These microparticles were used as carrier for ascorbic acid. The surface morphology and sizes of the microparticles were investigated by scanning electron microscope (SEM). Fourier transform infrared spectroscopy (FTIR) study indicated the formation of polyelectrolyte complex between gelatine and sodium alginate and successful encapsulation of ascorbic acid into the microparticles. The microparticles were further characterized by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) study.     

A rapid quantitative determination of phenolic acids in Brassica oleracea by capillary zone electrophoresis

A simple and rapid capillary zone electrophoresis method to quantitatively determine the phenolic acid contents in brassica vegetables is described. Phenolic compounds were extracted from broccoli, broccolini, Brussels sprouts, cabbage and cauliflower and the main hydroxycinnamic acids (sinapic, ferulic, p-coumaric and caffeic acids) were isolated by solid phase extraction with C18 cartridges. Using an optimised method, the four analytes were separated in less than 7 min in a 50 μm × 60 cm capillary with a 15 mM borate buffer (pH = 9.13) and a separation voltage of 30 kV at 30 °C. A linear relationship was observed for the method (r = 0.9997–0.9999) with detection limits ranging from 1.1 to 2.3 mg/kg of vegetables for the analytes. This method demonstrated good reproducibility with coefficients of variation of less than 5% for peak area and less than 1% for migration time (n = 7). The method was successfully applied to quantitatively determine the phenolic acid contents in a range of brassica vegetables and the results were in good agreement when compared to those from high performance liquid chromatography analysis.     

Analysis of ginsenosides in Panax ginseng in high pressure microwave-assisted extraction

High pressure microwave assisted extraction (HPMAE) was applied to extract the ginsenosides from Panax ginseng root. The influences of extraction solvent, extraction pressure and extraction time were individually investigated. HPMAE has been compared with other extraction methods, including Soxhlet extraction, ultrasound-assisted extraction and heat reflux extraction. The determination of ginsenosides was performed by HPLC–ESI-MS. The results indicated that the HPMAE not only took a shorter time but also afforded higher extraction yields of ginsenosides, especially ginsenoside Rb₁, Rc, Rb₂ and Rd. Furthermore, the neutral ginsenosides and malonyl ginsenosides in Panax ginseng root extracts by HPMAE were investigated. The malonyl ginsenoside m-Rb₁, m-Rc, m-Rb₂ and m-Rd degraded in HPMAE at 400 kPa (109–112 °C) in 70% (v/v) ethanol–water and at 600 kPa (112–115 °C) in methanol, and transformed into corresponding neutral ginsenoside Rb₁, Rc, Rb₂ and Rd. Using water as extraction solution, the neutral ginsenosides degraded under HPMAE at 400 kPa (135–140 °C), and transformed into less polarity rare ginsenosides.     

Comparison of graphite furnace and hydride generation atomic absorption spectrometry for the determination of selenium status in chicken meat

Four different procedures for the determination of selenium in chicken meat by atomic absorption spectrometry were investigated. They consisted on conventional ambient pressure acid digestion carried out before and after sample drying, associated or not with fat extraction. For all procedures muscle and skin were analyzed separately. Drying was carried out in a conventional oven at 65 °C for 24 h. For fat extraction different solvents and solvent mixtures were investigated considering both extraction yield and sample adequacy for further AAS measurement. Acid digestions were carried out with mixtures of HNO₃ and HClO₄. After digestion, selenium was measured either by Hydride Generation (HGAAS) or by Graphite Furnace Atomic Absorption Spectrometry (GFAAS). For the reduction of Se(VI) prior to the HGAAS determination, 8% (w/v) NaBr, 6 mol/l HCl (both with and without sulfamic acid), as well as UV radiation were investigated. Tests with spiked samples have shown that either UV radiation (pH 8) or NaBr/sulfamic acid presented good recoveries. In this way the HGAAS determination of selenium in tissue was carried out without interference whereas for the fatty fraction the results were satisfactory only if GFAAS was used. The results showed that drying the sample and extracting the fat prior to digestion is advantageous once the amount of acid necessary can be significantly reduced. The precision, expressed as relative standard deviation, was about 6.5% and 0.8% for GFAAS and HGAAS measurements, respectively. The limits of detection for HGAAS and GFAAS, based on three times the standard deviation of the blanks were 1 μg/l and 0.6 μg/l, respectively. The results have shown that in chicken meat 59% of the selenium is found in the muscle tissue while the skin responds for 41%.     

Pan-frying salmon in an eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) enriched margarine prevents EPA and DHA loss

Salmon is a major dietary source of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in North America, but the impact of pan-frying with various culinary fats on fatty acid content is not comprehensive. The fatty acid composition of Atlantic salmon after pan-frying with a novel EPA+DHA enriched margarine was examined. Pan-frying without oil, with canola oil and with stick margarine resulted in significantly lower levels of EPA+DHA (738 ± 181, 723 ± 94 and 704 ± 75 mg per 100 g salmon, respectively) as compared with raw salmon (1202 ± 191 mg per 100 g salmon). Pan-frying with EPA+DHA margarine prevented the decrease of EPA+DHA in salmon (924 ± 162 mg per 100 g salmon). Pan-frying salmon results in decreases of EPA+DHA, but a novel EPA+DHA enriched margarine can attenuate the decrease and possibly increase EPA and DHA intakes in North Americans.     

VIS/NIR spectroscopy for non-destructive freshness assessment of Atlantic salmon (Salmo salar L.) fillets

Visible/near-infrared spectroscopy has been evaluated for use in freshness prediction and frozen-thawed classification of farmed Atlantic salmon fillets, where fresh samples were stored as whole fish in ice. A handheld interactance probe for performing rapid measurements of single fillets and an imaging spectrometer for online analysis at an industrial speed of one fillet per second, have been used. Freshness as storage days in ice is predicted with an accuracy of 2.4 days for individual fillets, whereas frozen-thawed salmon fillets are completely separated from fresh fillets. The prediction results are comparable to previous results using the Quality Index Method with trained panelists. The region between 605 and 735 nm, which excludes interference by carotenoids and water, is appropriate for both frozen-thawed classification and freshness prediction of salmon fillets. The results indicate that the spectral changes are explained mainly by oxidation of heme proteins during the freeze–thaw cycle and during chilled storage in ice.     

Effect of different acids on the extraction of pepsin-solubilised collagen containing melanin from silky fowl feet

Four acid treatments: acetic acid (FA), citric acid (FC), hydrochloric acid (FH) and lactic acid (FL) were used in this study to extract the pepsin-solubilised collagen (PSC) containing melanin from silky fowl feet. The collagen content, melanin content, electrophoresis and ultraviolet (UV) absorption spectra (200–400 nm) were determined in order to develop a collagen with UV protection. Among four acid extractions, the acetic acid treatment had both the highest extracting yield (7.3%) and collagen content (516.6 mg/g). PSCs obtained from silky fowl feet using four acid extractions exhibited melanin content; whereas no similar observations were found in that of broiler feet by acetic acid extraction (FB). Moreover, the PSC via acetic acid extraction resulted in the significantly highest melanin content (210 mg/g) (P < 0.05), which contributed to UV absorption. In electrophoresis, three subunits were found in PSCs from silky fowl feet using the four acid extractions, which were different from typical type I collagen and assumed that the PSCs obtained from silky fowl feet consisted of more than two types of collagens.     

Anthocyanin and glucosinolate occurrences in the roots of Chinese red radish (Raphanus sativus L.), and their stability to heat and pH

Roots of three unique Chinese radish cultivars were evaluated as potential sources for anthocyanin-type colourants or value-added products. These cultivars showed high variation in anthocyanins (63.77–160.74 mg/100 g FW). Seventeen pigments were tentatively identified by mass spectroscopy as pelargonidin-3-sophoroside-5-glucoside derivatives with multiple acylation of hydroxycinnamic acids. A bright colour (CIELab) of radish anthocyanins has been shown at a wide pH range, comparably stable at pH < 4.2. Those anthocyanins also showed a remarkable thermal stability, following a zero-order kinetics at pH 2.5 with half-lives of 14.5 or 8.7 h at 90 or 100 °C, respectively. Additionally, those cultivars varied in glucosinolate contents (59.69–163.91 mg/100 g FW), whereas their degradation was sensitive to pH and followed a first-order kinetics at pH 5.8 with half-lives of 11.44 or 7.05 h at 90 or100 °C, respectively. However, the stable pH ranges for anthocyanins and glucosinolates were different: pH < 4.2 and pH > 3.6, respectively. In a radish juice model (pH 5.8/2.5), thermal degradation of anthocyanins or glucosinolates was associated closely with media pH values. In conclusion, cultivar selection, and thermal and pH conditions during processing or storage should be taken into account for quality, stability, and health benefits of radish derived natural colourants or nutraceutical products.     

Determination of catechins in green tea infusions by reduced flow micellar electrokinetic chromatography

A sulfated-β-cyclodextrin (s-β-CD) modified reduced flow micellar electrokinetic chromatography (RF-MEKC) method was developed and validated for the determination of catechins in green tea. The optimal electrolyte consisted of 0.2% triethylamine, 50 mmol/L SDS and 0.8% s-β-CD (pH = 2.9), allowing baseline separation of five catechins in 4 min. The samples and standards were injected at 0.6 psi for 5 s under constant voltage of −30 kV. Sample preparation simply involved extraction of 2 g of tea with 200 mL water at 95 °C under constant stirring for 5 min. The method demonstrated excellent performance, with limits of detection (LOD) and quantification (LOQ) of 0.02–0.1 and 0.1–0.5 μg/mL, respectively, and recovery percentages of 94–101%. The method was applied to six samples of Brazilian green tea infusions. Epigallocatechin gallate (23.4–112.4 μg/mL) was the major component, followed by epigallocatechin (18.4–78.9 μg/mL), epicatechin gallate (5.6–29.6 μg/mL), epicatechin (4.6–14.5 μg/mL) and catechin (3.2–8.2 μg/mL).     

Effects of binary solvent extraction system,extraction time and extraction temperature on phenolic antioxidants and antioxidant capacity from mengkudu (Morinda citrifolia)

An investigation into the effects of ethanol concentration (0–100%, v/v), extraction time (20–120 min) and extraction temperature (25–65 °C) on the extraction of phenolic antioxidants from mengkudu (Morinda citrifolia) was performed using a single-factor experiment. Total phenolic content (TPC) and total flavonoid content (TFC) assays were used for determination of phenolic compounds. Antioxidant capacity was evaluated by measuring the scavenging effect on 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radicals. Experimental results showed that extraction conditions had significant effect on extraction of phenolic compounds and antioxidant capacities. The optimised conditions were 40% ethanol for 80 min at 65 °C, with values of 919.95 mg GAE/100 g DW for TPC, 472.73 mg CE/100 g DW for TFC, 791.71 μmol TEAC/100 g DW for ABTS and 1928.5 μmol TEAC/100 g DW for DPPH. TPC was significantly correlated with DPPH under the effects of ethanol concentration (r = 0.932) and extraction time (r = −0.938).     

Salt effect on heat-induced physical and chemical changes of salmon fillet (O. gorbuscha)

This research studied the effect of salt on kinetics for quality changes in pink salmon fillet, during commercial sterilisation. Sample cuts from salmon fillets were placed in sealed aluminum containers and heated at 121.1 °C for 10, 30 and 60 min. Samples with 1.5% (w/w) salt addition were compared with those without added salt. Salt addition reduced cook loss, area shrinkage and shear force of the heated fillet and resulted in a slightly darker colour. Effect of salt addition in thiamin loss, degree of lipid oxidation and fatty acid profile was not significant. Peroxide (PV) and thiobarbituric acid (TBA) values slightly increased within the first 10 min of heating, followed by a significant reduction as heating progressed. No measurable loss of polyunsaturated fatty acids (PUFA) was observed. Thermally processed shelf-stable salmon investigated should be a valuable source of ω-3 PUFA, with EPA values ranging from 52 to 71 mg/100 g product and DHA ranging from 258 to 340 mg/100 g of product.     

One-step microwave-assisted headspace solid-phase microextraction for the rapid determination of synthetic polycyclic musks in oyster by gas chromatography–mass spectrometry

A rapid, simple and solvent-free procedure was developed for the determination of synthetic polycyclic musks in oyster samples by using one-step microwave-assisted headspace solid-phase microextraction (MA-HS-SPME) and gas chromatography–mass spectrometry (GC–MS). Two commonly used synthetic polycyclic musks, galaxolide (HHCB) and tonalide (AHTN), were selected in the method development and validation. The parameters (microwave irradiation power, extraction time, amount of water added, pH value and addition of NaCl) affecting the extraction efficiency of analytes from oyster slurry were systematically investigated and optimised. The best extraction conditions were achieved when the oyster tissue mixed with 10-mL deionised water (containing 3 g of NaCl in a 40-mL sample-vial) was microwave irradiated at 80 W for 5 min. The limit of quantification (LOQ) was 0.1 ng/g in 5-g of wet tissue. The good precision and accuracy of one-step MA-HS-SPME coupled with GC–MS for the determination of trace level of AHTN in oyster samples was also demonstrated.     

Supercritical fluid extraction of fish oil from fish by-products: A comparison with other extraction methods

Fish and fish by-products are the main natural source of omega-3 polyunsaturated fatty acids, especially EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), both of them with a great importance in the food and pharmaceutical industries. Comparing to conventional fish oil extraction processes such as cold extraction, wet reduction or enzymatic extraction, supercritical fluid extraction with carbon dioxide under moderate conditions (25 MPa and 313 K) may be useful for reducing fish oil oxidation, especially when fish oil is rich in omega-3 such as salmon oil, and the amount of certain impurities, such as some species of arsenic. Furthermore, taking profit of the advantages of supercritical carbon dioxide as extractive solvent, a coupled extraction-fractionation process is proposed as a way to remove free fatty acids and improve fish oil quality, alternatively to physical and chemical refining procedures.     

Production of soy protein isolates with low phytic acid content by membrane technologies: Impact of the extraction and ultrafiltration/diafiltration conditions

The content of the antinutrient, phytic acid, of soy protein was analyzed during their extraction and purification by a series of ultrafiltration and diafiltration steps. The phosphorus content of the extracts was used as an indication of their phytic acid content and their ash content as an indication of their mineral content. The extraction of soy proteins was conducted by using a 2³ factorial experimental design, pH (7.5 or 9), solvent (0.06 M KCl or water), and temperature (25 °C or 50 °C). The most promising extraction conditions were 0.06 M KCl/pH 9.0/25 °C for the lowest phosphorus to protein ratio (12.2 ± 0.1 mg P/g protein) and H₂O/pH 9.0/50 °C for the combination of low phosphorus to protein ratio and the lowest ash content (13.9 ± 1.2 mg P/g protein, 9.6 ± 0.8% w/w ash content). After extraction, soy proteins were purified by sequential ultrafiltration (UF) with a volume concentration ratio (VCR) of 5 and diafiltration (DF) with volume diafiltration ratio (VD) of 4. Extracts were purified with no pH adjustment or with pH adjustment to 6.5 between the UF and the DF steps. The extraction conditions 0.06 M KCl/pH 9.0/25 °C and the purification conditions UF pH 9.0/DF pH 6.5 showed the lowest phosphorus to protein ratio (4.4 ± 0.3 mg P/g protein) and reduced membrane fouling when compared to extraction conditions with water.