针对国家标准方法GB/T 23210-2008测定乳粉中二苯胺残留量时假阳性率高等现象,提出了题示方法,并进行气相色谱-串联质谱法(GC-MS/MS)确证、回收试验以及基质效应试验,比较了十八烷基键合硅胶固相萃取柱(C₁₈小柱)、弗罗里硅土固相萃取柱(Florisil小柱)和石墨化碳黑-氨基复合固相萃取柱(Carb/NH₂小柱)对实际样品的净化效果。在3 g乳粉样品中加入20 mL乙腈和5 g氯化钠,均质2 min,离心5 min,重复提取一次。合并上清液于50 mL鸡心瓶中,于40℃旋蒸至1 mL,过预先活化好的Carb/NH₂小柱,收集流出液。用体积比3∶1乙腈-甲苯混合溶液5 mL洗涤上述鸡心瓶,洗涤液过柱,再加入体积比3∶1乙腈-甲苯混合溶液20 mL洗脱柱子。净化过程控制流出速率为1 mL·min^-1,合并全部流出液,于40℃旋蒸至近干,氮气吹干,用1 mL乙酸乙酯溶解后用气相色谱-质谱法测定。结果显示:以C₁₈小柱净化10种市售乳粉样品时,二苯胺的阳性率为100%,二级质谱结果显示,质荷比(m/z)169,167,168(二苯胺的定量及定性离子)处对应的物质为样品溶液中的干扰物质,说明国家标准方法检测结果呈假阳性;Florisil小柱所得样品溶液中干扰物较多,二苯胺回收率偏高,且显示出强基质效应;Carb/NH₂小柱净化效果好,且不需要基质匹配法定量,所得二苯胺的质量浓度在0.01~1.00 mg·L^-1内与其对应的峰面积呈线性关系,检出限为3.4 μg·kg^-1。对空白乳粉样品进行3个浓度水平的加标回收试验,回收率为84.6%~93.1%,测定值的相对标准偏差(n=6)为4.2%~7.5%。方法用于以上10种市售乳粉样品的分析,均未检出二苯胺的残留。

The method shown in the title was proposed based on the fact of high false positive rate of diphenylamine residue in milk powder by the national standard method of GB/T 23210-2008. Tests for confirmation with gas chromatography-tandem mass spectrometry (GC-MS/MS), recovery and matrix effect were made, to compare purification effects of C₁₈ cartridge, Florisil cartridge and Carb/NH₂ cartridge on the actual samples. Milk powder sample (3 g) was homogenized with 20 mL of acetonitrile and 5 g of sodium chloride for 2 min, and centrifuged for 5 min, and the extraction was repeated once again. The supernatants obtained were combined to a heart-shaped pendant (50 mL), and evaporated by rotation to 1 mL at 40 ℃. The above solution was passed through the Carb/NH₂ cartridge activated beforehand, and the effluent was collected. Acetonitrile-toluene mixed solution (15 mL) at volume ratio of 3∶1 was used to wash the heart-shaped pendant, and the rinsing solution was loaded on the Carb/NH₂ cartridge. Acetonitrile-toluene mixed solution (20 mL) at volume ratio of 3∶1 was taken, and passed through the Carb/NH₂ cartridge for elution. Throughout the purification process, the effluence flow was controlled at 1 mL·min^-1. The collected eluate was combined, evaporated by rotation to nearly dry at 40 ℃, and dried by nitrogen. The residue was dissolved with 1 mL of ethyl acetate, and the solution was analyzed by gas chromatography-mass spectrometry. It was shown that the positive rate of diphenylamine was 100% when the 10 commercial milk powder samples were purified with C₁₈ cartridge, and the corresponding substances at m/z 169, 167, 168 (quantitative and qualitative ions of diphenylamine) were interfering substances in the sample solution which was confirmed by MS/MS, and it was concluded that the results provided by the national standard method were false positive. When purified with Florisil cartridge, many interferents in the sample solution were detected, with high recovery and strong matrix effect. When purified with Carb/NH₂ cartridge, the purification effect was better, and matrix matching method was not needed for quantification. Linear relationship between values of mass concentration and peak area of diphenylamine was kept in the range of 0.01-1.00 mg·L^-1, with detection limit of 3.4 μg·kg^-1. Test for the spiked recovery was made at the 3 concentration levels with negative milk powder sample as matrix, giving recoveries in the range of 84.6%-93.1%, and RSDs (n=6) of the determined values ranged from 4.2% to 7.5%. This method was applied to the analysis of the 10 milk powder samples described above, and the residues of diphenylamine were not detected.