| Flour and flour products are one of the most important staple foods in China, of which the quality and security has raised great concern. Azodicarbonamide (ADC) used as a flour gluten fortifier has been proved to easily produce biurea (HDC) and semicarbazide (SEM) by transformation and degradation under critical temperature and humidity conditions during handling process. SEM has been widely recognized as a teratogenic and carcinogenic substance. However, there is still lack of standardized analytical methods for determination of ADC, HDC and SEM in flour and flour products in domestic regulations. Therefore, the aim of this duty is to develop appropriate analytical methods for determination of ADC and its transformation products, i.e. HDC and SEM in flour and flour products. Sample pretreatment procedure including extraction solvent, time, temperature and clean-up methods (liquid-liquid extraction, enzymatic hydrolysis, solid phase extraction, etc.) were optimized to effectively remove matrix-introduced interfering materials, while instrumental analysis was accomplished using high performance liquid chromatography (HPLC) and high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). The proposed methods showed high sensitivity, good selectivity and were successfully applied to analyze real samples collected from local markets in Shenzhen. Finally, the occurrence level of ADC, HDC and SEM in flour and flour products was stermined and preliminary assessment of their hazardous risk to human health through dietary intake exposure was conducted. The detailed information is listed as below:1. A high-performance liquid chromatographic (HPLC) method was established for the determination of ADC in flour and flour productsADC in certain matrices was extracted by acetone/dimethylformamide (DMF) mixture (95:5, v/v), defatted with n-hexane when needed, and then analyzed by HPLC. LC separation was performed on a Waters XBridge BEH Amide column (3.5 ?m,4.6 mm×150 mm) by isocratic elution using acetonitrile and 20 mmol/L ammonium acetate, both containing 5% DMF (v/v). Quantification was achieved by external standard calibration. The calibration curve of ADC showed good linearity in the range of 1-100 mg/L with correlation coefficient of 0.9999. The average recovery of three spiking levels (six aliquots for each level) in flour, steamed bun, bread, deep-fried dough stick and noodle matrices ranged from 91.13% to 101.41%, with RSDs of 2.82%-11.5%. The LOQ of the method was 0.5 mg/kg.2. An isotope diluted high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method was established for the determination of HDC in flour and flour products.The HDC in certain matrices was ultrasonic extracted by ultrapure water, defatted with n-hexane when needed, and then analyzed by HPLC-MS/MS. LC separation was performed on a Waters XBridge BEH Amide column (3.5 ?m,4.6 mm×150 mm) by gradient elution using ultrapure water and acetonitrile. Identification was achieved by electrospray ionization (ESI+) in positive mode using multiple reaction monitoring. Quantification was performed by isotope labeled 13C2,15N2-HDC internal standard calibration. The calibration curve of HDC showed good linearity in the range of 0.2-100 ?g/L with correlation coefficient of 0.9999. The average recovery of three spiking levels (six aliquots for each level) in flour, steamed bun, bread, deep-fried dough stick and noodle matrices ranged from 82.0% to 113.6%, with RSDs of 2.1%-9.3%. The LOQ of the method was 20 ?g/kg. This method is applicable for the determination of HDC in flour and different flour products due to its operation convenience, high sensitivity and good accuracy.3. A high performance liquid chromatography-tandem mass spectrometric (HPLC-MS/MS) method was established for the determination of SEM in flour and flour products.The SEM was extracted by 0.2 mol/L hydrochloric acid and derivatized with 2-nitrobenzaldehyde (2-NBA), kept for 16 h (overnight) in a waterbath at 37 ?. After this period of time, samples were removed and allowed to cool to room temperature. Aqueous sodium phosphate was added to terminate the derivatization reaction, and then a pH value about 7.4 was adjusted by addition of aqueous sodium hydroxide. The 2-NBA derivatives were extracted by liquid-liquid extraction with ethyl acetate. The eluate was evaporated to dryness under a gentle stream of nitrogen gas, and the residue was redissolved in 1 mL of initial mobile phase. After defatting with 2 mL acetonitrile saturated n-hexane, the resulting solution was filtered through a 0.22 ?m membrane filter and collected into an LC autosampler vial, which was subjected to HPLC-MS/MS analysis. The analytes were spearated on a Waters Atlantis dC18 column (150 mm×2.1 mm,5 ?m) by gradient elution using ultrapure water and methanol. Identification was achieved by electrospray ionization (ESI+) in positive mode using multiple reaction monitoring, and quantification was performed by internal standard calibration. The calibration curve of SEM showed good linearity in the range of 0.5-50 ?g/L with correlation coefficient of 0.9999. The average recovery of three spiking levels (six aliquots for each level) in flour, steamed bun, bread, deep-fried dough stick and noodle matrices ranged from 91.8% to 113%, with RSDs of 2.08%-9.11%. The LOQ of the method was 0.5 ?g/kg.4. Market survey and data analysis of the occurence level for ADC, HDC and SEM in local flour and flour products of ShenzhenA total amount of 375 samples including flour, noodles, biscuits, Chinese pastry and western-style snacks were randomly collected from 10 sites through the monitoring project of food contamination network in Shenzhen during 2015 to 2016, considering resident and geographical distribution. The collected samples were prepared and analyzed using cerfain methods mentioned above, and consequently the content and contamination level of ADC, HDC and SEM in local flour and flour products of Shenzhen was obtained. Among those tested samples, ADC was found in 28 (6.67%) samples at 1.08?190.6 mg/kg, while 4 of them beyond the maximum residue level (45 mg/kg) of China. SEM was found in 117 (31.2%) samples at 0.509?1210 ?g/kg, HDC was found in 115 (30.67%) samples at 20.2?10780 ?g/kg. The market survey results indicate that there are still abuse of ADC in flour, which would lead to the occurrence of HDC and SEM in flour products due to the transformation and degradation of ADC in handling process. Since SEM has been widely recognized as a teratogenic, carcinogenic and mutagenic compound, which will cause heavy damage to human health through dietary intake, the useage of ADC as flour additives should be strictly regulated and supervised in the future. |
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