苏丹红Ⅰ直接竞争ELISA检测试剂盒的研制

直接包被多克隆抗体建立了苏丹红ⅠELISA检测技术,研制苏丹红ⅠELISA检测试剂盒。结果表明,制备的多克隆抗体对苏丹红Ⅰ亲合力强、特异性高,试剂盒标准曲线呈典型的S型,符合4参数logit曲线拟合,线性范围为1.55～100μg/L,50%抑制率(IC50)为13.52μg/L,灵敏度(IC10)为1.95μg/L,平均批内与批间变异系数分别为7.6%与10.6%,与苏丹红Ⅱ、Ⅲ的交叉反应率分别为4.40%和1.70%,与苏丹红Ⅳ及其他违禁食品染色剂交叉反应率低于0.01%;干辣椒、辣椒粉、辣椒油与辣椒酱等4种样品的苏丹红平均添加回收率分别为88.1%、91.4%、78.2%和84.3%,最低检测限分别为1.82、1.71、2.19和2.12μg/kg;与HPLC检测方法具有较高的相符性,4种样品相关系数分别为0.95,0.94,0.86和0.87;该试剂盒在4℃下至少可保存180d,可用于辣椒及其制品中苏丹红Ⅰ的批量快速低成本筛查。     

Fast determination of Sudan dyes in chilli tomato sauces using partial filling micellar electrokinetic chromatography

A new method based on partial filling micellar electrokinetic chromatography (MEKC) for the quantitative determination of Sudan dyes (I, II, III, and IV) in chilli sauces is presented. The separation is achieved filling 25% of the capillary with a MEKC buffer composed of 40 mM NH(4)HCO(3), 25 mM sodium dodecyl sulfate, and 32.5% (v/v) acetonitrile (ACN). The rest of the capillary is filled using a capillary zone electrophoresis (CZE) buffer composed of 40 mM NH(4)HCO(3) and 32.5% (v/v) ACN. Under optimized conditions, the azo dyes are baseline separated in less than 8 min with limits of detection ranging from 0.57 to 0.71 μg mL(-1) (S/N > 3). Using an internal standard, the repeatability of the quantitative determination is improved almost four times. The applicability of the method for rapid screening and determination of Sudan dyes is corroborated by analyzing spiked chilli sauce samples with recoveries from 85 to 99%. The reported conditions are demonstrated to be compatible with mass spectrometry detection.     

Synthesis and characterization of a molecularly imprinted silica gel sorbent for the on-line determination of trace Sudan I in Chilli powder through high-performance liquid chromatography

A highly selective imprinted polymer was synthesized by a surface molecular imprinting technique in combination with a sol-gel process. The imprinted polymer was evaluated by FT-IR and static and kinetic adsorption experiments. The results showed that the imprinted sorbent exhibited good recognition and selective ability, offered a faster kinetics for the adsorption and desorption of Sudan I than the non-imprinted sorbent, a saturated binding capacity (Qmax) that reached 33.47 mg g-1. The prepared sorbent was applied for the determination of trace Sudan I through on-line solid-phase coupled with high-performance liquid chromatography (SPE-HPLC). With a loading flow rate of 1.5 mL min-1 for sampling 50 mL, an enrichment factor of 1266 was achieved. The detection limit (S/N = 3) was 1.2 ng L-1, and the peak area precision (RSD) for five replicate detections of 0.01 microg L-1 Sudan I was 3.66%. The Sudan I in the chilli powder from the local market was determined at three levels (0.25, 0.5, and 1.0 ng g-1) with recoveries ranging from 80.31 to 94.02%.     

Advanced solid phase extraction using molecularly imprinted polymers for the determination of quercetin in red wine

Solid phase extraction (SPE) based on molecularly imprinted polymers (MIPs) is a novel approach for sample preparation and preconcentration, gaining increased interest in the fields of environmental, clinical, and food analysis. The first application combining MIPs with SPE for advanced beverage analysis is reported. MIPs for the flavonoid quercetin have been generated, using quercetin as a template molecule in a self-assembly approach and yielding imprinting of 1% of the used template. The MIP achieved a capacity of 0.4 g quercetin per gram polymer and a recovery rate of 98.2%. The application of these synthetic receptors as SPE material for the selective extraction and preconcentration of quercetin from synthetic and red wine samples was investigated. Red wine samples from a French Merlot were directly applied onto the SPE cartridge. The collected fractions were analyzed by high-pressure liquid chromatography. For verification of the obtained results, a similarly prepared nonimprinted polymer and a classical octadecyl silane reversed-phase cartridge were applied as the SPE matrix during control experiments. The MIP enabled the selective extraction of quercetin from a complex matrix, such as red wine, spiked with 8.8 mg per liter quercetin, demonstrating the potential of molecularly imprinted solid phase extraction for rapid, selective, and cost-effective sample pretreatment.     

Flow injection chemiluminescence determination of sudan I in hot chilli sauce

A chemiluminescence method based on the luminol-H2O2 system with flow injection technology was proposed for the determination of sudan I in hot chilli sauce. It was found that sudan I could enhance chemiluminescence intensity generated from the luminol-H2O2 system. The increment of chemiluminescence intensity was proportional to the concentration of sudan I, giving a calibration graph linear over the concentration from 10 pg mL-1 to 7 ng mL-1 (R 2 = 0.9980) with the detection limit of 3 pg mL-1 (3sigma) and the quantification limit of 7.5 pg mL-1. At a flow rate of 2.0 mL min-1, one analysis cycle, including sampling and washing, could be accomplished in 60 s with a relative standard deviation of <5.0%. The method has been applied successfully to the determination of sudan I in Pixian douban, Golden Mark guilin chilli sauce, and Golden Mark satay sauce, and the recovery was 90.6-110.0%.     

Fast determination of Sudan I by HPLC/APCI-MS in hot chilli, spices, and oven-baked foods

The Commission Decision of EC dated 20 June 2003, on emergency measures concerning hot chilli and hot chilli products coming into any EC member state, required that the consignments of such products should be accompanied by an analytical report showing that they are free of artificial dye Sudan I. The opportunity to set a confirmatory method is evident, and the paper proposes a HPLC/APCI-MS method useful for identification and quantitation of Sudan I, also at very low levels in hot chilli, other spices, and oven-baked foods. Validation data are reported.     

Production of the polyclonal antibody against Sudan 3 and Immunoassay of Sudan dyes in food samples

In this study, 4-aminophenylacetic acid was covalently coupled to aniline to synthesize an intermediate hapten and the intermediate hapten was coupled to β-naphthol to synthesize a tentative hapten of Sudan 3. The hapten was coupled to bovine serum albumin as the immunogen to produce the polyclonal antibody. The obtained antibody was highly specific to Sudan 3, Sudan 1, and Para red, but showed relative low binding ability to Sudan 2, Sudan 4, and Sudan red G. After evaluation of different coating antigens, a heterologous indirect competitive immunoassay was developed to multidetermine the six red dyes in food samples. The cross reactivities to the six analytes were in a range of 21-105%, and the limits of detection were in a range of 0.1-0.8 ng/mL depending on the compound. Intra- and interassay recoveries from the standard fortified blank samples were in a range of 74.5-96.3% with coefficients of variation lower than 15.1%.     

Development of a highly sensitive and specific enzyme-linked immunosorbent assay for detection of Sudan I in food samples

A highly selective and sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) for Sudan I was developed. Two hapten derivatives with different lengths of carboxylic spacer at the azo-bound para-position were synthesized and coupled to carrier proteins. The hapten-bovine serum albumin (BSA) conjugates were used as immunogens, while the hapten-ovalbumin (OA) conjugates were applied as coating antigens. The antisera which were obtained from four immunized rabbits were characterized in terms of sensitivity and specificity. At optimal experimental conditions it was found that IC50 and LOD values of seven pairs based on four antisera and two coating antigens were in the range of 0.3-2 ng/mL and 0.02-0.1 ng/mL, respectively. The most sensitive ELISA could be established with Sudan I-propionic acid-OA coating antigen and the antiserum which was obtained with the corresponding immunogen. The cross-reactivity values of the four antisera with Sudan II, III, and IV was estimated with 0.1-14.3%. No cross-reactivity was found with six edible colorants Sunset yellow, Amarant, Kermes, Indigotin, Bright blue and Lemon yellow, indicating high specificity for Sudan I. Six food samples were fortified with Sudan I and extracted by simple sample preparation. The methanolic extracts after dilution with methanol:water (5:95, v/v) were analyzed by the developed ELISA. Assay precision and accuracy was estimated by determination of three replicates. Acceptable recovery rates of 92.5-114% and intra-assay coefficients of variation of 5.9-24.8% were obtained. The data were validated by conventional HPLC method. As revealed, both methods were highly correlated (r = 0.9851, n = 7), demonstrating the applicability of the developed ELISA for Sudan I analysis in food samples.     

Determination of haloanisols in white wine by immunosorbent solid-phase extraction followed by enzyme-linked immunosorbent assay

A high through-put screening immunochemical method to control the presence of 2,4,6-trichloroanisol (TCA) and 2,4,6-tribromoanisol (TBA), the main agents responsible for the musty odor in wine samples, has been developed. The method involves a selective (antibody-antigen) solid-phase extraction (SPE), followed by enzyme-linked immunosorbent assay (ELISA) analysis. The sample preparation method established uses for immunosorbents (ISs) prepared by covalently coupling antibodies developed for TCA on a sepharose support. At present, about 200-400 ng L-1 of TBA and TCA can be detected in white wine samples by the IS-SPE-ELISA method described here without any preconcentration step. Simultaneous analyses of many samples are possible with this method. Related chloroanisoles (2,3- and 2,6-dichloroanisols and 2,3,4,5-tetrachloroanisol) and chlorophenols (2,3,4,6-tetrachlorophenol and pentachlorophenol) usually present in contaminated wine samples are also effectively retained by the IS, although only 2,4,6-TCA and 2,4,6-TBA are detected by the ELISA used. The immunopurification procedure developed could also be useful as a selective cleanup method prior to chromatographic analysis.     

Determination of 4(5)-methylimidazole in soy sauce and other foods by LC-MS/MS after solid-phase extraction

A method for the determination of 4(5)-methylimidazole (4MeI) in naturally brewed soy sauce was developed for the first time using solid-phase extraction (SPE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). SPE on silica-based reversed-phase cartridges with heptafluorobutyric acid as an ion-pairing reagent was used for the efficient cleanup of 4MeI. A multimode ODS column was employed for the chromatographic separation. To subtract the matrix effect during LC-MS/MS analysis, a standard addition method was used. The levels of 4MeI found in naturally brewed soy sauce were extremely low (ranging from <0.002 to 0.023 μg/g), whereas those in soy sauces containing caramel color were generally high (ranging from 0.43 to 4.8 μg/g). The method proved to be useful for the analysis of 4MeI in other foods such as caramel colors, drinks, and Worcestershire sauce.     

Dummy molecularly imprinted solid-phase extraction for selective determination of five phthalate esters in plastic bottled functional beverages

In this paper, a highly selective sample cleanup procedure combing dummy molecular imprinting and solid-phase extraction (DMI-SPE) was developed for the simultaneous isolation and determination of five phthalate esters in plastic bottled beverages. The new imprinted microspheres were synthesized via precipitation polymerization using diisononyl phthalate as a dummy template that showed high selectivity and affinity to the five kinds of phthalate esters and were successfully applied as selective sorbents of DMI-SPE for the simultaneous determination of the phthalate esters from plastic bottled beverages. Good linearity was obtained in a range of 5.0-750.0 μg/L, and the average recoveries of the five phthalate esters at three spiked levels ranged from 84.3 to 96.2% with the relative standard deviations less than 5.49%. The developed extraction protocol eliminated the effect of template leakage on quantitative analysis and could be applied for the determination of phthalate esters in complicated functional beverages products.     

Use of Tetragenococcus halophilus as a starter culture for flavor improvement in fish sauce fermentation

The potential of Tetragenococcus halophilus as a starter culture for flavor improvement in fish sauce fermentation was elucidated. Four strains of T. halophilus isolated from fish sauce mashes were inoculated to anchovy mixed with 25% NaCl with an approximate cell count of 10(6) CFU/mL. The α-amino content of 6-month-old fish sauce samples inoculated with T. halophilus was 780-784 mM. The addition of T. halophilus MRC10-1-3 and T. halophilus MCD10-5-10 resulted in a reduction of histamine (P < 0.05). Fish sauce inoculated with T. halophilus showed high contents of total amino acids with predominantly high glutamic acid. Major volatile compounds in fish sauce were 2-methylpropanal, 2-methylbutanal, 3-methylbutanal, and benzaldehyde. T. halophilus-inoculated fish sauce samples demonstrated the ability to reduce dimethyl disulfide, a compound contributing to a fecal note. The use of T. halophilus for fish sauce fermentation improves amino acid profiles and volatile compounds as well as reduces biogenic amine content of a fish sauce product.     

Solid-phase extraction combined with high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry analysis of pesticides in water: method performance and application in a reconnaissance survey of residues in drinking water in Greater Cairo, Egypt

Monitoring of water resources for pesticide residues is often needed to ensure that pesticide use does not adversely impact the quality of public water supplies or the environment. In many rural areas and throughout much of the developing world, monitoring is often constrained by lack of testing facilities; thus, collection of samples and shipment to centralized laboratories for analysis is required. The portability, ease of use, and potential to enhance analyte stability make solid-phase extraction (SPE) an attractive technique for handling water samples prior to their shipment. We describe performance of an SPE method targeting a structurally diverse mixture of 25 current-use pesticides and two common degradates in samples of raw and filtered drinking water collected in Greater Cairo, Egypt. SPE was completed in a field laboratory in Egypt, and cartridges were shipped to the United States for elution and high-performance liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry analysis. Quantitative and reproducible recovery of 23 of 27 compounds (average = 96%; percent relative standard deviation = 21%) from matrix spikes (1 microg L-1 per component) prepared in the field and from deionized water fortified similarly in the analytical laboratory was obtained. Concurrent analysis of unspiked samples identified four parent compounds and one degradate in drinking water samples. No significant differences were observed between raw and filtered samples. Residue levels in all cases were below drinking water and "harm to aquatic-life" thresholds, indicating that human and ecological risks of pesticide contamination were relatively small; however, the study was limited in scale and scope. Further monitoring is needed to define spatial and temporal variation in residue concentrations. The study has demonstrated the feasibility of performing studies of this type using SPE to extract and preserve samples in the field. The approach should be broadly applicable in many settings.     

Development of an immunoassay for the pyrethroid insecticide esfenvalerate.

A competitive enzyme-linked immunosorbent assay was developed for the detection of the pyrethroid insecticide esfenvalerate. Two haptens containing amine or propanoic acid groups on the terminal aromatic ring of the fenvalerate molecule were synthesized and coupled to carrier proteins as immunogens. Five antisera were produced and screened against eight different coating antigens. The assay that had the least interference and was the most sensitive for esfenvalerate was optimized and characterized. The I(50) for esfenvalerate was 30 +/- 6.2 microg/L, and the lower detection limit (LDL) was 3.0 +/- 1.8 microg/L. The assay was very selective. Other pyrethroid analogues and esfenvalerate metabolites tested did not cross-react significantly in this assay. To increase the sensitivity of the overall method, a C(18) sorbent-based solid-phase extraction (SPE) was used for water matrix. With this SPE step, the LDL of the overall method for esfenvalerate was 0.1 microg/L in water samples.     

Multiresidue analysis of cotton defoliant, herbicide, and insecticide residues in water by solid-phase extraction and GC-NPD, GC-MS, and HPLC-diode array detection

A multiresidue procedure was developed for analysis of cotton pesticide and harvest-aid chemicals in water using solid-phase extraction and analysis by GC-NPD, GC-MS, and HPLC-DAD. Target compounds included the defoliants tribufos, dimethipin, thidiazuron; the herbicide diuron; and the insecticide methyl parathion. Three solid-phase extraction (SPE) media, octadecylsilyl (ODS), graphitized carbon black (GCB), and a divinylbenzene-N-vinyl pyrollidine copolymer (DVBVP), were evaluated. On GCB and ODS, recoveries varied depending on compound type. Recoveries were quantitative for all compounds on DVBVP, ranging from 87 to 115% in spiked deionized water and surface runoff. The method detection limit was less than 0.1 microg L(-)(1). SPE with DVBVP was applied to post-defoliation samples of surface runoff and tile drainage from a cotton research plot and surface runoff from a commercial field. The research plot was defoliated with a tank mixture of dimethipin and thidiazuron, and the commercial field, with tribufos. Dimethipin was detected (1.9-9.6 microg L(-)(1)) in all research plot samples. In the commercial field samples, tribufos concentration ranged from 0.1 to 135 microg L(-)(1). An exponentially decreasing concentration trend was observed with each successive storm event.     

Characteristics of salt-fermented sauces from shrimp processing byproducts

A salt-fermented sauce from shrimp processing byproducts (heads, shells, and tails) was prepared and characterized. Three types of sauces were prepared; sauce C, with 30 g of salt/100 g of byproduct (high salt); sauce E, with 30 g of salt and 0.2 g of sodium erythorbate (high salt); and sauce L, with 20 g of salt, 0.2 g of sodium erythorbate, 6 g of sorbitol, 0.5 mL of lactic acid, and 5 mL of ethanol (low salt). Sauces C and E showed higher exopeptidase activities than sauce L, whereas sauce L showed the highest endopeptidase activity. After 3 months of fermentation, the amino N content of sauce increased from 150-200 to 500-600 mg/100 g and the nonprotein nitrogen content increased from 300 to 950-1050 mg/100 g. Volatile basic nitrogen content increased significantly from 18 to 60 mg/100 g. The total carotenoids retained in sauces C, E, and L were 26.3, 76.2, and 73%, respectively, thus indicating that the addition of sodium erythorbate to sauces E and L retarded oxidation. Water activities of sauces C, E, and L were 0.753, 0.751, and 0.773, respectively. According to the omission test, the taste of sauces was influenced by the content of free amino acids, mainly glutamic acid and aspartic acid. All three sauces examined showed a 35% higher total amino acid content than commercial salt-fermented shrimp sauces. Therefore, shrimp processing byproducts may lend themselves to the preparation of high-quality salt-fermented sauces.     

Assay of the set of all Sudan azodye (I, II, III, IV, and Para-Red) contaminating agents by liquid chromatography-tandem mass spectrometry and isotope dilution methodology

A high-throughput mass spectrometric method is presented for the simultaneous detection of Sudan I, II, III, IV, and Para-Red azodyes in foodstuff. The method is based on the use of deuterium-labeled internal standards and atmospheric pressure chemical ionization (APCI) in a triple-quadrupole instrument. The gas-phase breakdown pattern of each labeled and unlabeled analogue displays the naphthoic moiety as a common fragment. The search for the parents of this common species (parent ion scans) allows, by flow injection and in a single run, the evaluation of the presence of each polluting species spiked in typical foodstuffs. A detailed assay of each azodye was performed by LC-APCI and isotope dilution method, through the multiple reaction monitoring approach, using deuterium-labeled internal standards. Sudan dyes can be quantified above the threshold of 10 ppb except for Sudan Para-Red, for which the limit of quantification was 20 ppb, likely due to the different ionization efficiency.     

基于电子鼻的鱼露香气品质识别

为了识别鱼露的品质,并为缩短发酵周期的工艺优选提供理论依据.利用电子鼻对7种鱼露样品的挥发性气味进行了分析,并与项空-气质联用( GC-MS)和感官分析结果进行比较.结果表明:鱼露香气成分复杂,工艺改良对气味影响很大,电子鼻18个金属传感器能很好地将不同样品的气味进行识别.以传统发酵原汁鱼露为标样,电子鼻分析结果表明,加曲改良工艺的4号样品与标样香气最为接近,相似系数达87.8％,该结果与GC-MS数据和感官分析结果一致,可为鱼露速酿工艺的优选提供参考.     

Rapid determination of domoic acid in serum and urine by liquid chromatography-electrospray tandem mass spectrometry

A rapid, selective, and sensitive LC-MS/MS method was developed for the quantitative determination of domoic acid in serum and urine samples. Samples were prepared for analysis using an Oasis HLB SPE column. Determination was by a reversed phase HPLC using a mixture of methanol, acetonitrile, and water containing 1% acetic acid and an electrospray ionization (ESI) ion-trap mass spectrometer (Finnigan LCQ). The method was validated by analyzing five replicates each of negative control bovine serum or urine fortified with domoic acid at the 0.005 microg/g method detection limit (MDL) and at the 0.05 microg/g level. Recoveries ranged from 90 to 95% for fortifications at the MDL and from 92 to 98% for fortifications 10 times higher than the MDL. The diagnostic utility of the method was tested by analyzing samples from live animals showing clinical signs suggestive of domoic acid poisoning submitted to the veterinary toxicology laboratory.     

Direct optical biosensor analysis of folate-binding protein in milk

An automated, rapid, sensitive, and label-free biosensor-based assay for folate-binding protein (FBP) in bovine milk utilizing surface plasmon resonance optical detection is described. The active concentration of FBP is estimated from its specific interaction with a pteroyl-l-glutamic (folic) acid derivative immobilized on the sensor surface in a direct binding assay format. Milk, colostrum, and milk powders are prepared for analysis by dilution into buffer. Analysis conditions, including ligand immobilization, flow rate, contact time, and regeneration, have been defined, and nonspecific binding considerations were evaluated. Performance parameters include a working range for FBP in buffer of 0-200 ng/mL, a method detection limit of 0.13 microg/mL in fluid milk, overall instrument response RSD(R) of 0.64%, a mean interassay RSD(R) of 7.3% for skim milk powder, and surface stability over ca. 200 samples. The technique was applied to the measurement of active FBP content of consumer milks, the heat classification of skim milk powders manufactured under a wide range of thermal processing protocols, and change during early bovine lactation.