Application of an enzyme immunoassay for the quantitative determination of azo dye (Orange II) in food products

This paper reports the preparation of polyclonal antibodies against a synthetic azo dye, Orange II, and the development of an indirect ELISA to detect Orange II in foods. The sulfonic group of Orange II was modified and linked with carrier protein to synthesise an artificial antigen. Based on the checkerboard titration, the method showed excellent sensitivity (IC₅₀?=?0.61 ng?g^?1) to Orange II in the linear range of 0.05–10?ng?g^?1. The antibody had little cross-reactivity with Chromotrope FB, Gardenia Yellow, Ponceau 4R, Sunset Yellow and Sudan dyes. The ELISA had limits of detection (LOD) of 0.22, 0.97 and 0.74?ng?g^?1 in chilli powder, chilli oil and braised pork, respectively. The limits of quantification (LOQ) of the assay were 0.91?ng?g^?1 in chilli powder, 1.48?ng?g^?1 in chilli oil and 1.10?ng?g^?1 in braised pork. For food products fortified with 1–10?ng?g^?1 Orange II, the inter- and intra-assay variations were all less than 24.0% and 18.0%, respectively. Therefore, the proposed test could be used as a rapid screening method for Orange II detection in food samples.     

Development of enzyme-linked immunosorbent assay (ELISA) for the detection of neomycin residues in pig muscle,chicken muscle,egg, fish,milk and kidney

A colorimetric competitive direct enzyme-linked immunosorbent assay (ELISA) method was developed using polyclonal antibody to determine neomycin residues in food of animal origin. No cross-reactivity of the antibody was observed with other aminoglycosides. The limit of detection of the method was 0.1 μg/kg. A simple and efficient sample extraction method was established with recoveries of neomycin ranged from 75% to 105%. The detection limits were 5 μg/kg(l) in pig muscle, chicken muscle, fish and milk, 10 μg/kg in kidney and 20 μg/kg in egg, respectively. Chemiluminescence assay was developed for detecting neomycin residues in pig muscle and chicken muscle. The limit of detection of the method was 0.015 μg/kg, and the detection limits were 1.5 μg/kg in pig muscle and 6 μg/kg in chicken muscle. The ELISA tests were validated by HPLC, and the results showed a good correlation (r²) which was greater than 0.9.     

A HPLC-UV method for the determination of angiotensin I-converting enzyme (ACE) inhibitory activity

To determine the angiotensin-converting enzyme (ACE) inhibitory activity of a fish hydrolysate, different methods were tested. Finally, a sensitive, extraction-free HPLC method using N-(3-[2-furylacryloyl)-Phe-Gly-Gly (FAPGG) as substrate was preferred. This method relies on the UV-titration of the peptide 2-furylacryloyl-l-Phe (FAP) resulting from the hydrolysis of the FAPGG after a chromatographic separation on a reverse phase column. The experimental conditions (enzyme/substrate ratio, incubation time, NaCl concentration) were optimised for linearity, sensitivity and precision. The assay was adequate for the study of ACE inhibition by Captopril, used as reference, and several peptides. Captopril and the fish hydrolysate had IC₅₀ values, respectively of 0.19 ng and 43 μg with standard deviations of 0.09 ng and 5 μg. Afterwards, the determination of the Hill coefficient sustained the hypothesis that active peptides present in the fish hydrolysate were low-molecular weight molecules. This result was confirmed by the activity measurement of the fish hydrolysate fractions obtained by gel filtration.     

A rapid and simple fluorometric method for quantifying isoeugenol in seawater and in plasma and white muscle from Australasian snapper (Pagrus auratus)

Isoeugenol residues in Australasian snapper (Pagrus auratus) white muscle, blood plasma and seawater were accurately and precisely quantified after extraction with acetonitrile using fluorometric detection (ex. 260 nm, em. 340 nm) without chromatographic separation. Isoeugenol residues in Australasian snapper (P. auratus) muscle tissue following 30 min exposure to 58.2 μmol L⁻¹ isoeugenol (ca. 20 ppm of the aquatic anaesthetic AQUI-S™) reached a maximum of 134.37 ± 8.13 μmol kg⁻¹ (±SEM; n = 6). Blood plasma isoeugenol concentrations following this harvesting regime were 253.2 ± 25.1 μmol L⁻¹. After 7 h recovery, fillet isoeugenol residues reduced to 7.89 ± 1.67 μmol kg⁻¹. Storage of fillets from fish harvested with AQUI-S™ at 3.87 ± 0.54 °C for 5 days resulted in a rate of isoeugenol decay in the fillets of 6.51 ± 1.19 μmol kg⁻¹ day⁻¹. The method reported can be used for measuring isoeugenol residues in food products or to further study the physiological and biological effects of isoeugenol in fish.     

Multi-residue and multi-class method for the determination of antibiotics in bovine muscle by ultra-high-performance liquid chromatography tandem mass spectrometry

A multi-residue quantitative screening method covering 41 antibiotics from 7 different families, by ultra-high-performance–liquid-chromatography tandem mass spectrometry (UHPLC–MS/MS), is described. Sulfonamides, trimethoprim, tetracyclines, macrolides, quinolones, penicillins and chloramphenicol are simultaneously detected after a simple sample preparation of bovine muscle optimized to achieve the best recovery for all compounds. A simple sample treatment was developed consisting in an extraction with a mixture of acetonitrile and ethylenediaminetetraacetic acid (EDTA), followed by a defatting step with n-hexane. The methodology was validated, in accordance with Decision 2002/657/EC by evaluating the required parameters: decision limit (CCα), detection capability (CCβ), specificity, repeatability and reproducibility. Precision in terms of relative standard deviation was under 20% for all compounds and the recoveries between 91% and 119%. CCα and CCβ were determined according the maximum residue limit (MRL) or the minimum required performance limit (MRPL), when required.     

Fluorescence immunoassay through histone-ds-poly(AT)-templated copper nanoparticles as signal transductors for the sensitive detection of Salmonella choleraesuis in milk

The rapid and sensitive detection of foodborne pathogens is one of the most important issues in food safety control. In this work, we developed a novel fluorescence immunoassay method for the sensitive detection of Salmonella choleraesuis. The method uses the fluorescent signals of histone-ds-poly(AT)-templated copper nanoparticles (His-pAT CuNP) as signal transducers and glucose oxidase as an alternative for horseradish peroxidase for the generation of hydrogen peroxide (H₂O₂) through the catalysis of glucose. The H₂O₂ is then further converted into hydroxyl radical (·OH) by Fenton reagents. Owing to the ultrahigh sensitivity of His-pAT CuNP synthesis toward ·OH, the proposed fluorescence immunoassay method exhibited excellent sensitivity for S. choleraesuis, with a limit of detection of 8.04 × 10¹ cfu/mL, which is 3 orders of magnitude lower than that of the tetramethylbenzidine-based traditional immunoassay. The reliability of the proposed method was evaluated by using spiked milk samples with S. choleraesuis concentration ranging from 8.8 × 10¹ to 8.8 × 10⁴ cfu/mL. The average recoveries for the intra- and inter-assay ranged from 73.52 to 96.59% and from 66.99 to 98.24% with a coefficient of variation from 6.85 to 31.26% and 5.46 to 17.99%, respectively. These results indicated that the proposed fluorescence immunoassay possesses a great potential for ultra-sensitive detection of foodborne pathogens in food safety control.     

A simple and highly sensitive spectrophotometric determination of Cr(VI) in food samples by using 3,4-dihydroxybenzaldehydeisonicotinoylhydrazone (3,4-DHBINH)

A simple, rapid, sensitive and inexpensive method has been developed for the determination of trace amounts of chromium(VI) using 3,4-dihydroxybenzaldehydeisonicotinoylhydrazone (3,4-DHBINH). The metal ion gives a yellow coloured complex with 3,4-DHBINH in acetate buffer of pH 5.5 with 1:1 (metal:ligand) composition. The complex shows a maximum absorption at 400 nm. Beer’s law is obeyed in the range 0.5–7.7 ppm of Cr(VI). The molar absorptivity, Sandell’s sensitivity and detection limit were found to be 1.35 × 10⁴ L mol⁻¹ cm⁻¹, 0.0075 μg cm⁻² and 0.0045 μg mL⁻¹, respectively. The correlation co-efficient and regression co-efficient of the Cr(VI)–3,4-DHBINH complex were 0.99 and 0.12, respectively. Major cations and anions did not show any interference. The developed method has been successfully applied for the analysis of Cr(VI) in food samples (leafy vegetables), comparing the results simultaneously with those obtained using an Atomic Absorption Spectrophotometer, whereby the validity of the method has been tested.     

Changes of hormone-sensitive lipase (HSL), adipose tissue triglyceride lipase (ATGL) and free fatty acids in subcutaneous adipose tissues throughout the ripening process of dry-cured ham

Changes in adipose triglyceride lipase (ATGL) and hormone-sensitive lipases (HSL) in subcutaneous adipose tissue of Xuanwei ham at different ripening stages were studied. Green hams were salted for 40 d and then ripened in a ventilated chamber for 15 months.     

Conjugated linoleic acid (CLA) and polyunsaturated fatty acids in muscle lipids of lambs from the Patagonian area of Argentina

The concentrations of fatty acids were measured in total lipids, triacyglycerol and phospholipid fractions of intramuscular fat (IMF) from the Longissimus dorsi (LD) muscle of 10 lambs reared to approximately 30kg live weight on natural pasture with their dams. Fatty acid composition was also measured in 25 (five of each) Semitendinosus (ST), Semimembranosus (SM), Rectus femoris (RF), Gluteus (GLU) and Tensor fascia latea (TFL) muscles. Intramuscular fat percentages were similar for all muscles. Aspects of the fatty-acid patterns of relevance to human nutrition tended to favor the leg muscles with lower saturated fatty acids (SFA %), n-6/n-3 fatty acid ratios (p<0.01) and higher concentrations of the conjugated linoleic acid (CLA) (p<0.05). The estimated fatty acid concentrations (mg/100g of meat) showed higher contribution of arachidonic (C20:4 n-6), eicosapentanoic (C20:5 n-3), docosapentanoic (C22:5 n-3) and docosahexanoic (C22:6 n-3) acids in leg compared to LD lipids.     

A sensitive and selective method for the quantitative determination of fatty acid tryptamides as shell indicators in cocoa products

 Tetracosanoyl-2-(3-indolyl)ethane amide (lignocerinic acid tryptamide; LAT) and docosanoyl-2-(3-indolyl)ethane amide (behenic acid tryptamide; BAT) were identified as the most prominent tryptamides in cocoa shells based on electrospray ionisation mass spectrometry and ¹H NMR measurements. The structure of LAT, which is reported for the first time in cocoa shells, and also that of BAT were confirmed by synthesis. By using synthesised heptadecanoyl-2-(3-indolyl)ethane amide as the internal standard, a sensitive and reproducible method was developed for the quantification of LAT and BAT in the picogram range by means of HPLC/fluorescence detection. The detection limit was determined to be 30 pg/run. In authentic shell samples, 50-fold higher concentrations of both tryptamides were determined compared to the cocoa cotyledons. In 15 commercial chocolate samples, concentrations of 23.1–63.0 μg of the tryptamides (sum of both) per gram of fat were found. A first experiment attempted to correlate the tryptamide content with the amounts of shells in a model chocolate showed that the method is a promising tool to determine the shell content in the quality assessment of cocoa products. Received: 8 May 1998     

植物油中苯并(a)芘含量的快速检测方法研究

建立一种快速灵敏测定植物油中苯并(a)芘含量的方法。采用四氢呋喃作为萃取溶剂,样品溶解过0.22 μm滤膜后直接注入高效液相色谱仪进行测定。结果表明：方法在苯并(a)芘质量浓度0.1~20 ng/mL的范围内线性关系良好,相关系数R2为0.999 9,检出限为0.1 μg/kg,定量限为0.3 μg/kg,样品加标回收率在93.5%~102.5%之间。该方法重现性好,灵敏度高,可对6类植物油中苯并(a)芘含量进行测定,具有较强的实际应用价值。     

A simplified X-ray fluorescence (XRF) procedure for the determination of sulphur in graminaceous materials

An automated, rapid and much simplified XRF procedure for assessing total sulphur in graminaceous plant materials is described. Sample preparation has been refined and microprocessor control incorporated to provide considerable savings in analytical time. The procedure has been tested using samples from two grass-silage cuts, taken from an ADAS (Agricultural Development and Advisory Service) trial studying responses, under intensive grassland management, to sulphur and nitrogen applications. Good correlation between XRF and wet chemical analysis was achieved, and sample turnround was markedly increased. The method produced accurate results for both cuts with high precision and reproducibility where careful attention was paid to sample homogeneity and disc preparation. The potential for wider application of XRF techniques to elemental analysis of agricultural materials is discussed.     

A monoclonal antibody-based sensitive enzyme-linked immunosorbent assay (ELISA) for the analysis of the organophosphorous pesticides chlorpyrifos-methyl in real samples

Chlorpyrifos-methyl hapten, O-methyl-O-(3,5,6-trichloro-2-pyridinyl)-N-(2-carboxyethyl)-phosphoramidothionte (H1), was synthesized and conjugated with bovine serum albumin (BSA) and ovalbumin (OVA) by the active ester method. Then H1–OVA conjugate was used as coating antigen, while H1–BSA conjugate was used as immunogen for producing monoclonal antibody. After optimisation, a monoclonal antibody-based effective competitive indirect enzyme-linked immunsorbent assay (ELISA) was developed and applied for determination of chlorpyrifos-methyl with a novel combination of antibody/antigen, I₅₀ of which was 75.22 ng/ml, limit detection (LD) was 0.32 ng/ml, and there was relative high cross-reactivity (CR) only with chlorpyrifos (1.4%), and CRs with other tested pesticides were all below 1% and regarded as negligible. The recoveries obtained by standard chlorpyrifos-methyl addition to real samples, including grape, Chinese cabbages, water and soil were all from 82.4% to 110.2%. Therefore, the optimised ELISA might become a convenient and satisfied analytical tool for monitoring chlorpyrifos-methyl residues in agriculture ecosystem.     

Sensitive gas chromatographic-mass spectrometric (GC-MS) method for the determination of bisphenol A in rice-prepared dishes

Certain chemicals possess the potential to modulate endocrine systems, and thereby interfere with reproductive and developmental processes. Bisphenol A is suspected to be one of them. The compound is widely used as a plastic additive, lacquer, resin, or plastic and can usually be found in food samples. An accurate and reproducible gas chromatographic-mass spectrometric (GC-MS) method to detect and measure trace amounts of the compound in rice-prepared dishes samples is proposed. Solid–liquid extraction with acetonitrile was carried out in order to isolate and pre-concentrate the analyte. The solvent was removed and a silylation step using N,O-bis(trimethylsilyl)trifluoro acetamide/pyridine (BSTFA/PYR) was carried out. The silylated compound was identified and quantified by GC-MS using a DB-5 MS column. Bisphenol F was used as a surrogate internal standard. The detection limit was 2.0 ng g^?1 while inter- and intra-day variability was less than 6%. Due to the absence of reference materials, the method was validated using standard addition calibration and a recovery assay. Recoveries for spiked samples were between 90% and 105%.     

Occurrence of mycotoxins (ochratoxin A, deoxynivalenol) and toxigenic fungi in Moroccan wheat grains: impact of ecological factors on the growth and ochratoxin A production

The aim of the present work was to evaluate the contamination of some samples, taken from Moroccan wheat grains, by ochratoxin A (OTA), deoxynivalenol (DON) and the associated toxigenic fungi. Moreover, we focused on the influence of environmental factors on both the growth and OTA production by three strains of Aspergillus. The results showed that only few samples were contaminated by the two mycotoxins (2 samples for OTA and 7 for DON). The main isolated fungi belong to the Aspergillus, Penicillium and Fusarium genus; 74 Aspergillus and 28 Penicillium isolates were tested for their ability to produce OTA. Only 2 A. alliaceus and 14 A. niger were able to synthesize OTA. However, none of Penicillium isolates can produce this toxin under the conditions mentioned. In respect of the effects of the temperature and water activity (aw), the optimal conditions for the growth and OTA production were different. While the optimal conditions of growth for A. alliaceus and A. terreus are 30 degrees C and 0.98 aw, A. niger preferred 0.93-0.95 aw at 25 degrees C, whereas the optimal production of OTA was observed at 30 degrees C for both A. alliaceus and A. niger at 0.93 and 0.99 aw, respectively.     

A simple method for the determination of fluoroquinolone residues in tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus) employing LC-MS/MS QToF

The use of antimicrobials in livestock production is a powerful resource applied throughout the world to guarantee high yield and control bacterial diseases in aquaculture. However, residues of these substances in animal products represent a potential risk to consumer health when residue levels are above the established maximum residue limits (MRLs). Fluoroquinolones (FQs) are antimicrobials commonly used worldwide in aquaculture. The aim of this work was to develop and validate a simple analytical method for the simultaneous determination of norfloxacin, danofloxacin, enrofloxacin and ciprofloxacin levels in tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus) fillets using liquid chromatography–tandem mass spectrometry (LC-MS/MS) quadrupole time of flight (QToF). The FQs were extracted from the fillets with 1% acetic acid–methanol and 1% acetic acid–acetonitrile solutions using ultrasonic assistance. The clean-up was performed with hexane. Chromatographic separation was conducted in an XTerra RP18 column (2.1 × 150 mm, 5 µm) at 25 °C with a flow of 0.2 mL min^?1. The mobile phase consisted of 0.1% aqueous formic acid and acetonitrile, with gradient elution. The validation parameters for all FQs were linearity (>0.99), intra-day precision (CV of 1%–9%), inter-day precision (CV of 3%–17%), decision limit (63–126 ng g^?1), detection capability (76 –152 ng g^?1) and accuracy (90%–111%). The limit of quantification was lower than the MRL for each FQ, indicating that the method is suitable for the determination of the FQ levels in the fish fillets. The mass analyser of the QToF type was able to confirm the identities of the FQs with an error of the accuracy of the mass (reasons m/z) of less than 10 ppm.     

A new potassium tetrabromoaurate (III)-luminol chemiluminescence system for the determination of folic acid in milk powder

Abstract: A new chemiluminescence (CL) system based on the CL‐emitting reaction between potassium tetrabromoaurate (III) [Au(III)] and luminol in alkaline medium is described in this paper. On the basis of this study, folic acid (FA) could dramatically enhance CL intensities, and incorporated with flow injection (FI) and solid‐phase extraction (SPE), the novel CL system has been applied for the determination of FA in infant formula milk powder. Under optimum conditions, the CL intensities were linearly related to the concentration of FA in the range of 8.0 × 10^?8 to 4.0 × 10^?5 g/L with a correlation coefficient of 0.999, and the detection limit was 2.0 × 10^?8 g/L. The relative standard deviation was 3.5% for 1.0 × 10^?6g/L FA. The optimal conditions for the detection of FA were evaluated, and the interferences from some common inorganic ions and a couple of relevant organic compounds were also investigated. Practical Application: The new Au(III)–luminol chemiluminescent system is proposed for the determination of FA; it will be an adequate technique for the analysis of low FA concentrations in pharmaceutical preparations and food samples, and it will have potential analytical applications for other substances.