在低濃度下,PVPP 法同樣能達到較高回收率。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達到75%以上,肽段ALNEINQFYQK 由于響應太低,無法準確計算回收率。
考察了0. 1 mg/ L 酪蛋白在4 種不同品牌紅酒中的回收率。如表3 所示,特征性肽段的回收率均保持在64% ~ 150% 之間,進一步證明了PVPP用于紅酒酪蛋白快速前處理方法高效、可行。
表3 不同品牌紅酒經PVPP 處理后的回收率考察(n =4)
Table 3Efficiency of PVPP method for casein in different red wines (n =4)
3. 4 方法的線性、靈敏度考察
為了考察PVPP 法的線性、檢測限和定量限,添加量為50 mg/ L 的紅酒經PVPP 處理后,用空白基質稀釋為0. 01 ~50 mg/ L 的12 個濃度點,分別質譜分析并繪制標準曲線。圖7 展示了 β-酪蛋白VLPVPQK肽段的標準曲線,在3 ~4 個數量級范圍內,該肽段具有良好的線性(R2 =0. 9996)和重現性(RSD<6%)。
圖6紅酒酪蛋白前處理方法的回收率比較:上圖為 β-酪蛋白VLPVPQK 肽段不同處理方法的LC-MS/ MS 色譜峰對比;下圖為6 條肽段的回收率對比(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)
圖7 紅酒中 β-酪蛋白VLPVPQK 肽段的線性范圍(PVPP法)(n =4)
Fig. 7 Linear response range of peptide VLPVPQK from β-casein in red wine (PVPP method) (n =4)
表4 總結了3 種亞型酪蛋白共6 條肽段的線性范圍,在0. 01 ~50 mg/ L 濃度范圍內,相關系數R2 >0. 99,顯示出良好的線性。
表4 PVPP 法的線性范圍與定量限
Table 4Linear response ranges and limits of quantification (LOQs) of PVPP method
根據α-酪蛋白中α-S1 和α-S2 所占比例計算,最終確定6 條肽段的定量限在10 ~100 μg/ L 之間, 對應到 α-S1, α-S2 和β-酪蛋白的定量限分別為40, 40 和10 μg/ L(表4),比文獻[3] 報道方法提高約一個數量級。此外,在10 μg/ L 濃度下,所有肽段均有響應(信噪比>3), 因此 α-S1, α-S2, β-酪蛋白的檢測限均在10 μg/ L 以下,同樣比文獻[3]報道方法提高約一個數量級。
4 結 論
通過PVPP 快速前處理,使紅酒中酪蛋白回收率提高到80%以上,使整個前處理時間縮短到2 h 之內,有效解決了紅酒中大量單寧對蛋白提取與酶解的抑制。將PVPP 法與三重四極桿液相色譜-質譜結合,分析了紅酒中3 種亞型的酪蛋白 α-S1, α-S2, β的過敏原,檢測限均低至10 μg/ L,定量限達10 ~40 μg/ L,同時結果具有良好的重現性。PVPP 前處理方法簡單有效、重現性好,適用于高通量的紅葡萄酒過敏原檢測。
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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)