Development of Aflatoxin B1 Certified Reference Material in Peanut Oil
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摘要: 根据我国食品安全监测工作的需求,研制了花生油中黄曲霉毒素B1成分分析标准物质。该标准物质由自然污染的阳性样本,经混匀、充氮、分装等多种加工工艺制备。使用高效液相色谱法(荧光检测器,HPLC-FLD)对标准物质候选物的均匀性进行评估,高效液相色谱-稳定同位素稀释质谱法(HPLC-ID-MS/MS)对稳定性进行考察,高效液相色谱法(荧光检测器)和高效液相色谱-稳定同位素稀释质谱法两种方法进行多家实验室合作定值。研究表明:该标准物质满足技术规范要求,均匀性和稳定性良好,可在−18 ℃储藏下保存16个月,黄曲霉毒素B1的标准值为15.6 μg/kg,扩展不确定度为1.6 μg/kg (k = 2)。标准物质的不确定度评估综合考虑了均匀性、稳定性和定值过程引入的不确定度。研制的花生油中黄曲霉毒素B1成分分析标准物质,已获得国家市场监督管理总局批准的有证标准物质证书,编号为GBW(E) 100604。该标准物质能够满足食用油中黄曲霉毒素B1测量急需溯源标准的应用需求,为保证准确可靠的测量结果提供了有效支撑。Abstract: To fulfill the requirements of food safety monitoring in China, a certified reference material (CRM) for aflatoxin B1 (AFB1) analysis in peanut oil was developed. This CRM was derived from naturally contaminated positive samples, undergoing processes such as blending, nitrogen purging, and packaging. Homogeneity of the CRM candidate was assessed using High-Performance Liquid Chromatography with Fluorescence Detection (HPLC-FLD), while its stability was evaluated using High-Performance Liquid Chromatography - Isotope Dilution Mass Spectrometry (HPLC-ID-MS/MS). Value assignment was performed through collaborative laboratory efforts utilizing both HPLC-FLD and HPLC-ID-MS/MS. The results indicated that the CRM meets technical specifications, displaying excellent homogeneity and stability for 16 months at −18 ℃. The certified value of AFB1 is 15.6 μg/kg with an expanded uncertainty of 1.6 μg/kg (k = 2). The uncertainty assessment encompassed factors from homogeneity, stability, and value assignment. The developed CRM for AFB1 in peanut oil, certified as GBW(E) 100604, fulfills the urgent need for traceable standards in edible oil AFB1 measurement and ensures accurate and reliable results.
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Key words:
- metrology /
- peanut oil /
- aflatoxin B1 /
- certified reference material /
- homogeneity /
- stability /
- quantification
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表 1 两种不同原理方法结果比较(μg/kg)
Table 1. Comparative results from two distinct analytical methods for AFB1 (μg/kg)
序号 液相法 质谱法 1 15.2 15.1 2 15.4 15.4 3 15.6 15.2 4 15.1 15.6 5 15.8 15.1 6 15.3 15.7 7 15.7 15.2 8 15.8 15.0 9 15.0 15.6 10 15.9 15.3 平均值 15.48 15.32 SD 0.32 0.24 RSD 2.08% 1.59% 表 2 方差分析结果
Table 2. Statistical analysis of variance results
差异源 差方和 自由度 方差 F统计量 F临界值 组间 10.984 24 0.458 1.226 1.737 组内 18.660 50 0.373 总计 29.644 74 表 3 稳定性研究线性回归分析
Table 3. Linear regression analysis of long-term stability
类型 时间/月 平均测定值
(μg/kg)类型 时间/天 平均测定值
(μg/kg)长期稳定性 0 15.8 短期稳定性 0 16.2 0.5 14.9 1 16.9 1 16.1 2 16.8 2 15.2 3 16.2 4 15.8 5 16.2 8 15.5 6 16.1 12 15.3 7 15.9 16 15.2 − − 平均值 15.5 平均值 16.3 β1 −0.021 β1 0.0932 β0 15.590 β0 16.648 s2 0.168 s 0.0945 s(β1) 0.0260 s(β1) 0.0476 t 0.95, n-2 2.45 t 0.95, n-2 2.45 表 4 花生油中AFB1 标准物质定值结果
Table 4. Certified value assignment results for aflatoxin B1 in peanut oil
实验室 测定值(μg/kg) 平均值(μg/kg) 标准偏差 定值方法 A 15.6, 14.9, 15.7, 16.9, 15.2, 16.2 15.8 0.72 HPLC-ID-MS/MS B 14.9, 15.3, 14.6, 15.0, 16.6, 15.3 15.3 0.70 HPLC-ID-MS/MS C 15.9, 15.4, 15.4, 16.0, 15.3, 15.7 15.6 0.29 HPLC-ID-MS/MS D 15.6, 16.3, 15.4, 15.2, 15.4, 15.6 15.6 0.38 HPLC-FLD E 15.6, 15.7, 15.9, 15.5, 16.6, 16.0 15.9 0.40 HPLC-FLD F 14.9, 15.7, 15.5, 15.5, 15.5, 15.8 15.5 0.31 HPLC-FLD G 15.8, 15.9, 16.1, 15.7, 15.5, 16.0 15.8 0.22 HPLC-FLD 总平均值 15.6 0.20 表 5 花生油中AFB1标准物质研制过程的不确定度来源
Table 5. Uncertainty sources identified during the development of aflatoxin B1 CRM in peanut oil
不确定度来源 计算公式 相对不确定度 % 均匀性引入 $ {u}_{\mathrm{H}}=\dfrac{{S}_{\mathrm{H}}}{\overline{X}} $ 1.06 稳定性引入 $ {u}_{\text{S}}=\sqrt{{{u}_{\mathrm{l}\mathrm{S}}}^{2}+{{u}_{\mathrm{S}\mathrm{S}}}^{2}} $ 3.42 定值引入 $ {u}_{\mathrm{A}}=\dfrac{{S}_{\overline{X}}}{\overline{X}}=\dfrac{\sqrt{{\displaystyle\sum }_{i=1}^{m}(\overline{{X}_{i}}-\overline{X}{)}^{2}/m(m-1)}}{\overline{X}} $ 1.11 $ {u}_{\mathrm{B}} $ 3.32 合成相对不确定度 5.1 k = 2(95%置信区间),相对扩展不确定度 10.2 -
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