交联聚乙烯基吡咯烷酮快速前处理方法结合液-...（三）

2020.5.18

在低浓度下,PVPP 法同样能达到较高回收率。如表2 所示, 在0. 1 mg/ L 酪蛋白(100 μg/ L)添加量下,特征性肽段的回收率均可达到75%以上,肽段ALNEINQFYQK 由于响应太低,无法准确计算回收率。

考察了0. 1 mg/ L 酪蛋白在4 种不同品牌红酒中的回收率。如表3 所示,特征性肽段的回收率均保持在64% ~ 150% 之间,进一步证明了PVPP用于红酒酪蛋白快速前处理方法高效、可行。

表3 不同品牌红酒经PVPP 处理后的回收率考察(n =4)

Table 3Efficiency of PVPP method for casein in different red wines (n =4)

3. 4 方法的线性、灵敏度考察

为了考察PVPP 法的线性、检测限和定量限,添加量为50 mg/ L 的红酒经PVPP 处理后,用空白基质稀释为0. 01 ~50 mg/ L 的12 个浓度点,分别质谱分析并绘制标准曲线。图7 展示了 β-酪蛋白VLPVPQK肽段的标准曲线,在3 ~4 个数量级范围内,该肽段具有良好的线性(R2 =0. 9996)和重现性(RSD<6%)。

图6红酒酪蛋白前处理方法的回收率比较:上图为 β-酪蛋白VLPVPQK 肽段不同处理方法的LC-MS/ MS 色谱峰对比;下图为6 条肽段的回收率对比(n =6)

Fig. 6 Recovery comparison of different pre-treatment methods for casein in red wine. Comparison of LC-MS/ MS chromatographic peaks of β-casein peptide VLPVPQK (up figures); Comparison of recovery of 6 peptides (lower figures) (n =6)

图7 红酒中 β-酪蛋白VLPVPQK 肽段的线性范围(PVPP法)(n =4)

Fig. 7 Linear response range of peptide VLPVPQK from β-casein in red wine (PVPP method) (n =4)

表4 总结了3 种亚型酪蛋白共6 条肽段的线性范围,在0. 01 ~50 mg/ L 浓度范围内,相关系数R2 >0. 99,显示出良好的线性。

表4 PVPP 法的线性范围与定量限

Table 4Linear response ranges and limits of quantification (LOQs) of PVPP method

根据α-酪蛋白中α-S1 和α-S2 所占比例计算,最终确定6 条肽段的定量限在10 ~100 μg/ L 之间, 对应到 α-S1, α-S2 和β-酪蛋白的定量限分别为40, 40 和10 μg/ L(表4),比文献[3] 报道方法提高约一个数量级。此外,在10 μg/ L 浓度下,所有肽段均有响应(信噪比>3), 因此 α-S1, α-S2, β-酪蛋白的检测限均在10 μg/ L 以下,同样比文献[3]报道方法提高约一个数量级。

4 结论

通过PVPP 快速前处理,使红酒中酪蛋白回收率提高到80%以上,使整个前处理时间缩短到2 h 之内,有效解决了红酒中大量单宁对蛋白提取与酶解的抑制。将PVPP 法与三重四极杆液相色谱-质谱结合,分析了红酒中3 种亚型的酪蛋白 α-S1, α-S2, β的过敏原,检测限均低至10 μg/ L,定量限达10 ~40 μg/ L,同时结果具有良好的重现性。PVPP 前处理方法简单有效、重现性好,适用于高通量的红葡萄酒过敏原检测。

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Rapid Polyvinylpolypyrrolidone Pretreatment Approach for Liquid Chromatography Tandem Mass Spectrometric Analysis of Casein Allergen in Red Wine

ZHANG Wei1 , HE Yan2 , LIU Wei2 , WU Zhi-Yuan1 , JIANG Zheng1 , XU Hong2 ,

WANG Yong-Wei1 , ZHANG Yu-Jun2 , ZHENG Wen-Jie*2

1(ThermoFisher Scientific China, Shanghai 201206, China)

2(Animal & Plant & Foodstuffs Inspection Center, Tianjin Entry-Exit Inspection and Quarantine Bureau, Tianjin 300461, China)

Abstract A MS method for casein allergen analysis was developed to reduce the restriction of protein extraction and digestion from tannins in red wine. Unique selective reaction monitoring (SRM) transitions were selected and confirmed for qualitative and quantitative detection of α-S1, α-S2 and β-caseins. Effective extraction and fast digestion of red wine protein were realized by utilizing polyvinylpolypyrrolidone (PVPP),

which also reduced the pre-treatment time within 2 h. Results showed that the PVPP method increased red wine Casein recovery toat least 80%. Simultaneously, the limits of detection decreased to 10 μg/ L, and the limits of quantification to 10-100 μg/ L, which lowered at least one order of magnitude compared to previous reports.

Keywords Red wine; Casein; Allergen; Pretreatment; Liquid chromatography tandem mass spectrometry

(Received 14 May 2013; accepted 29 June 2013)

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