The toxic protein of ricin has drawn wide attention in recent years as a potential bioterrorism agent due to its high toxicity and wide availability. For the verification of the potential anti-terrorism activities, it is urgent for the quantification of ricin in food-related matrices. Here, a novel strategy of trypsin/Glu-C tandem digestion was introduced for quantitative detection of ricin marker peptides in several beverage matrices using isotope-labeled internal standard (IS)-mass spectrometry. The ricin in beverages was captured and enriched by biotinylated anti-ricin polyclonal antibodies conjugated to streptavidin magnetic beads. The purified ricin was cleaved using the developed trypsin/Glu-C tandem digestion method and then quantitatively detected by ultra-high-pressure liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) with isotope-labeled T7A and TG11B selected as IS. The use of trypsin/Glu-C digestion allows shorter peptides, which are more suitable for MS detection, to be obtained than the use of single trypsin digestion. Under the optimized tandem digestion condition, except for T7A in the A-chain, two resulting specific peptides of TG13A, TG28A from the A-chain and two of TG11B, TG33B from the B-chain were chosen as novel marker peptides with high MS response. The uniqueness of the selected marker peptides allows for unambiguous identification of ricin among its homologous proteins in a single run. The MS response of the four novel marker peptides is increased by more than 10 times compared with that of individual corresponding tryptic peptides. Both the marker peptides of A-chain T7A and B-chain TG11B were selected as quantitative peptides based on the highest MS response among the marker peptides from their individual chains. The limit of detection (LOD) of ricin is 0.1 ng/mL in PBS and 0.5 ng/mL in either milk or orange juice. The linear range of calibration curves for ricin were 0.5–300 ng/mL in PBS, 1.0–400 ng/mL in milk, and 1.0–250 ng/mL in orange juice. The method accuracy ranged between 82.6 and 101.8% for PBS, 88.9–105.2% for milk, and 95.3–118.7% for orange juice. The intra-day and inter-day precision had relative standard deviations (%RSD) of 0.3–9.4%, 0.7–8.9%, and 0.2–6.9% in the three matrices respectively. Furthermore, whether T7A or TG11B is used as a quantitative peptide, the quantitative results of ricin are consistent. This study provides not only a practical method for the absolute quantification of ricin in beverage matrices but also a new strategy for the investigation of illegal use of ricin in chemical weapon verification tasks such as OPCW biotoxin sample analysis exercises.

近年来，蓖麻毒素的毒性蛋白作为一种潜在的生物恐怖剂，由于其高毒性和广泛的可用性而受到广泛关注。为了验证潜在的反恐活动，迫切需要对食品相关基质中的蓖麻毒素进行定量。在这里，引入了一种胰蛋白酶/Glu-C 串联消化的新策略，用于使用同位素标记的内标 (IS) 质谱法定量检测几种饮料基质中的蓖麻毒素标记肽。通过与链霉亲和素磁珠结合的生物素化抗蓖麻毒蛋白多克隆抗体来捕获和富集饮料中的蓖麻毒蛋白。使用开发的胰蛋白酶/Glu-C 串联消化法裂解纯化的蓖麻毒蛋白，然后通过超高压液相色谱-串联质谱 (UHPLC-MS/MS) 以同位素标记的 T7A 和 TG11B 作为 IS 进行定量检测。与使用单一胰蛋白酶消化相比，使用胰蛋白酶/Glu-C 消化可以获得更适合 MS 检测的较短肽。在优化的串联消化条件下，除 A 链中的 T7A 外，A 链中的 TG13A、TG28A 和 B 链中的 TG11B、TG33B 中的两个特异性肽被选为具有高 MS 响应的新型标记肽. 所选标记肽的独特性允许在单次运行中明确识别其同源蛋白质中的蓖麻毒蛋白。与单个相应胰蛋白酶肽相比，四种新型标记肽的 MS 响应提高了 10 倍以上。A 链 T7A 和 B 链 TG11B 的标记肽都被选为定量肽，这是基于来自它们各自链的标记肽中最高的 MS 响应。蓖麻毒素的检测限 (LOD) 在 PBS 中为 0.1 ng/mL，在牛奶或橙汁中为 0.5 ng/mL。蓖麻毒素校准曲线的线性范围为 PBS 中 0.5–300 ng/mL、牛奶中 1.0–400 ng/mL 和橙汁中 1.0–250 ng/mL。PBS 的方法准确度介于 82.6% 和 101.8% 之间，牛奶为 88.9-105.2%，橙汁为 95.3-118.7%。三个矩阵的日内和日间精密度的相对标准偏差 (%RSD) 分别为 0.3-9.4%、0.7-8.9% 和 0.2-6.9%。此外，无论是用T7A还是TG11B作为定量肽，蓖麻毒素的定量结果都是一致的。该研究不仅为饮料基质中蓖麻毒素的绝对定量提供了一种实用方法，而且为在禁化武组织生物毒素样品分析练习等化学武器验证任务中调查非法使用蓖麻毒素提供了一种新策略。