Application Research Of Online HPLC-MS Technich For Food Safety Analysis

The main goal of this study was to establish the key technology and validated methods characterized with effective separation, high selective and high sensitive detection for food safety analysis. A series of novel methods for the analysis of dyes and drugs residues in foods were established by application of advanced HPLC-MS technique. These substances were all currently with popular concerned for food safety, including 10 banned azo dyes,8 banned nitroimidazole drugs and two classes of drugs whose MRL were established. A practical template for application of the HPLC-MS technique to food satety analysis was brought forward, which can afford some theory guidance and practical example for the method development in this field. Meanwhile the cleavage pattern and mechanism of ions in MS spectrum were also investigated and attemped to give a reasonable explaination.A HPLC method for simultaneous determination of Sudan I, Sudan II, Sudan III and Sudan IV in foodstuffs was proposed. The Sudan dye in foodstuffs was extracted with hexane and cleaned up by neutral Aluminum oxide SPE column. Quantitative determination was carried out with HPLC-DAD and qualified with the similar index of their VIS spectrometry. Four kinds of sudan dyes(i.e. Sudan I, II, III, IV) in foodstuffs were characterized and determined simultaneously by the hyphenation of HPLC and ion-trap MS/MS. The powdered sample was extracted for 15min with methanol in ultrasonic bath. After standing, the mixture was filtered and introduced directly into the hyphenated analytical instrument. The Agilent Zorbax XB-C18 column was used for the separation of the four dyes. A mixture containing 0.1% acetic acid water solution and acetonitrile in the portion of 15 to 85 was used as the mobile phase. ESI ion trap MS/MS was used for the MS analysis. The smart mode of seting the condition of the Agigent 1100 ion trap mass spectrometer was adopted and the collision energy was optimized. The pattern and mechanism of the molecules were studied based on which a mistake of the structure of Sudan IV and corresponded explaination of its mas spectrum reported in an authorized literature has been corrected. The limit of detection (LOD) was lng/ml, and linear responding range was from 5ng/ml to 100 ng/ml. Fortified recovery was in the range of 85～97%. An accurate method based on combination of gel permeation chromatography (GPC)-HPLC-ESI-MS/MS was devised for simultaneous determination of Sudan (I-IV), Sudan Orange G, Sudan Red B, Sudan Red G, Sudan Red 7B, Butter Yellow and Para Red in hot chili products. A GPC clean-up procedure was developed for simultaneous quantification of 10 dyes in hot chili and its products. A HPLC was performed on an Inertsil C18 column using a multistep gradient elution with 0.1% formic acid and methanol as the mobile phase. MS/MS acquisition was done in positive ion mode. Linearity of around three orders in the magnitude of concentration was generally obtained, and the limit of detection (LOD) and limit of quantification (LOQ) for the investigated dyes were in the ranges of 0.1～1.8 and 0.4～5.0μg/kg depending on matrices, respectively. The recoveries of the 10 synthetic dyes in five matrices were in the range of 81.7 to 92.9%. The intra- and inter-day precisions (RSDs) were between 2.9～7.8% and 3.9～8.1%, respectively.A sensitive and validated method based on solid-phase extraction(SPE)-HP LC-ESI-MS/MS was devised for the simultaneous determination of the eight nitroimidazole(NOZ) drugs including metronidazole(MNZ), ronidazole(RNZ), dimetridazole (DMZ), tinidazole (TNZ), ornidazole(ONZ), secnidazole(SNZ), metronidazole-OH (MNZOH, the metabolite of MNZ) and 2-hydroxymethyl-l-methyl-5-nitroim- idazole (HMMNI, the metabolite of RNZ and DMZ) in natural animal casings. After extraction with ethyl acetate and evaporation, the nitroimidazoles were redissolved in ethyl acetate and purified using strong cation exchange (SCX) SPE column, and then HPLC-MS/MS analysis was performed by positive electrospray ionisation (ESI) applying multiple reaction monitoring of three transition reactions for each compound. The whole method has been validated, according to the EU Commission Decision 2002/657/EC. Specificity, linearity, decision limit (CCa), detection capability (CCβ), accuracy and precision were determined. Average recoveries of the eight NOZs from natural animal casing fortified at three levels of 0.1,0.5 and 1μg/kg were in the range from 87.3 to 116.5%. Relative standard deviations (RSD) of recoveries at all fortification levels were less than 10.3% for the eight NOZs. The calculated CCa for NOZs was in the range from 0.029 to 0.049μg/kg, and CCβwas in the range from 0.049 to 0.083μg/kg.A new SPE-HPLC-ESI-MS/MS method was developed and validated for determination of closantel residues in bovine tissues and milk. An acetonitrile-acetone mixture (80:20, v/v) was used for one-stage extraction of closantel residues from the samples, and the extract was cleaned by SPE with Oasis MAX cartridges. The MS/MS was operated in MRM mode with negative electrospray interface.Extemal standard method was used to quantitation. The linearity is satisfactory with r2 from 0.9899 to 0.9987 at both concentration ranges of 0.02～100μg/kg and 500～5000μg/kg. The overall recoveries for bovine muscle, liver, kidney and milk samples spiked at different levels including MRL were in the range of 73.0～95.7%. The overall RSDs were in the range of 3.29～9.87%. The LOD and LOQ were 0.008μg/kg and 0.02μg/kg respectively. The method has been listed as a trade standard for inspection and quarantine in China.A novel GPC-HPLC-ESI-MS/MS method was developed for determination of diclazuril, toltrazuril and its two metabolites (toltrazuril sulfone, toltrazuril sulfoxide) residues in animal tissues and eggs were established. The extraction of the samples with ethyle acetate was clean up with GPC, then analysed by HPLC-ESI-MS/MS with isotope internal standard dilution technique. The results show that the linearity is satisfactory for the four compounds were above 0.9921 in the concentration ranges of 1-1000μg/kg. The overall recoveries spiked at three levels were in the range of 84.6-103%. The overall relative standard deviations were in the range of 2.64-8.82%. The LODs were 10μg/kg for livers and kidneys, and 3μg/kg for tissues and eggs. The LOQs were 20μg/kg for livers and kidneys, and 10μg/kg for tissues and eggs.Through above-mentioned study, a practical template for application of the HPLC-MS technique to food safety analysis was brought forward as follows:a). Choose suitable solvents and manner such as ultrasonic to extract the residues from the samples.b). Remove the co-extractions such as protein, fat, natural pigments etc which have higher molecular weight with GPC technique to avoid the interference and possible damage of the columns. If the last results show that the interference still remains, use SPE for further cleanup; c). Separate the analytes, also the possible impurities having similar molecular size with HPLC; d). Optimize the MS/MS parameters for the molecular weight of the analytes in the corresponding time segments around its retention time. Compounds with different molecular weights can be detected individually through selection its molecular ions as monitor ions. Compound with same molecular weight such as isomer can be further distinguished in MS/MS with different daughter ions derived from different fragmentation.e). The matrix effect can be checked through comparation between the solvent calibration curve and the matrix matched calibration curve. Whenever the significant difference exists, a matrix match calibration curve is needed for quantitation. The isotope dilution internal standard.can be used to eliminate the effect of matrix and other errors caused during the sample treatment process.f). Specificity, linearity, matrix calibration, accuracy, precision, CCα,CCβetc. of the method should be and must be validated in according to IUPAC and 2002/657/EC as well as the methodology demands.