以非目標胺類指紋鑑別臺灣烏魚卵：化學同位素標誌法之應用

Abstract

烏魚子是臺灣高價水產加工品，其價格與原物料烏魚卵之產地來源、生產方式來源與物種來源有關。本研究目的是以化學同位素標誌結合高解析質譜法分析烏魚子之胺指紋（amine fingerprints），以及利用胺指紋鑑別不同來源之烏魚卵。 本研究蒐集70個不同來源的烏魚卵樣本，分成：不同產地（臺灣50個與進口20個）、生產方式（野生35個與養殖15個）及物種（NWP1種26個與NWP2種24個）。樣本（200 mg）經蛋白質沉澱與液－液萃取後，以12C2－丹磺醯氯（dansyl chloride, DnsCl）進行個別樣本胺類輕標誌衍生化；另以13C2－丹磺醯氯（13C2-DnsCl）進行混合樣本重標誌胺類衍生化。衍生後的個別樣本與混合樣本以等體積均勻混合後，以極致液相層析－四極桿飛行時間質譜儀在資料相依擷取模式下收集所有小分子化合物（m/z 50-1200）高解析質譜資訊。正離子波峰經提取後，挑選同時標誌了12C2/13C2之潛在胺類化合物，並以12C2/13C2波峰面積比值為相對濃度。來源鑑別是以胺類化合物的相對濃度作為變量，建立偏最小平方判別分析模型。候選指標化合物的篩選條件是variable importance in the projection（VIP）值 > 1以及Wilcoxon rank sum test檢定結果有顯著差異（p < 0.05）。最後透過搜尋資料庫、比對滯留時間、分子離子與碎片離子，進行指標胺的鑑定。 本研究成功應用化學同位素標誌法結合非目標胺分析法建立烏魚卵胺指紋。前處理方法最適化之結果顯示，相較於僅使用蛋白質沉澱，加上液－液萃取使前處理效率提升了1.6倍。以酸性甲醇進行蛋白質沉澱，再利用氯仿及超純水進行液－液萃取，使胺類化合物的前處理效率高於80%、基質干擾低於50%，確保良好的生物胺分析回收率。以501個正離子衍生化分子建立的偏最小平方判別分析模型能穩健鑑別不同產地、生產方式及物種的烏魚卵（|" R2Y - Q2 " | < 0.3，Q2 > 0.5，permutation test Q2斜率 > 0），顯示不同來源組別的烏魚卵之胺類化合物組成有顯著差異。本研究共篩選118個具有來源特徵之候選指標化合物（產地：61個；生產方式：52個；物種：31個），並發現組胺酸在進口烏魚卵中之相對濃度高於臺灣烏魚卵（1.39至8.31倍），可能與烏魚的生長水體鹽度有關。本研究未發現與人體健康危害的胺類化合物隨烏魚卵的來源有所差異。 本研究完成烏魚卵胺指紋分析，且證實此胺指紋具有鑑別來源之應用價值。本研究所發現的候選指標胺可供後續研究進行進一步鑑定，以做為未來烏魚子溯源檢驗及品質精進之參考。

Wuyutsu (烏魚子) is a high-value processed aquatic product in Taiwan, and its price depends on the mullet roe origins, including geographic origins, production methods, and species. This study aimed to apply chemical isotope labeling (CIL) combined with high-resolution mass spectrometry to untargeted amine fingerprinting in mullet roe origins. The discrimination of mullet roe origins was performed using the amine fingerprints. A total of 70 mullet roe samples from varied sources, including the geographic origins (Taiwan: n = 50, import: n = 20), production methods (wild: n = 35, culture: n = 15), and species (NWP1: n = 26, NWP2: n = 24) were collected. The samples (200 mg) were processed through protein precipitation and liquid-liquid extraction. Each individual sample was derivatized using 12C2-dansyl chloride (DnsCl) as a light-labeled reagent, while a pooled sample was derivatized using 13C2-DnsCl as a heavy-labeled reagent. Each labeled individual sample was mixed with the labeled pooled sample in equal volumes. Ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry was employed to acquire high-resolution mass spectrometry information of small-molecule compounds (m/z 50-1200) in data-dependent acquisition mode. Molecular features in positive mode were extracted followed by peak pair picking for the amines labeled with 12C2-DnsCl and 13C2-DnsCl. The relative concentration of an amine was the peak area ratio of its 12C2- to 13C2-labeled species. The relative concentrations of all amines were employed in discrimination modeling using partial least squares-discriminant analysis. Candidate markers were selected based on the variable importance in the projection (VIP > 1) and Wilcoxon rank sum test (p < 0.05). Candidate amine markers were identified by searching in databases and comparing the retention time, molecular ions, and fragment ions. This study successfully applied CIL combined with untargeted amine analysis to the amine fingerprints in mullet roe. The results of sample preparation method optimization revealed that including liquid-liquid extraction enhanced the sample preparation efficiency by 1.6 times compared to solely conducting protein precipitation. Adequate amine recoveries were achieved by performing protein using acidic methanol and subsequent liquid-liquid extraction by adding chloroform and water, which resulted in high sample preparation efficiencies (> 80%) and low matrix interference (< 50%). The partial least squares-discriminant analysis models based on 501 derivatized positive ions robustly discriminated mullet roe from different geographic origins, production methods, and species (|" R2Y - Q2 " | < 0.3, Q2 > 0.5 and permutation test Q2 slope > 0), indicating that the amine composition in mullet roe differed by origins. A total of 118 candidate markers (61 for geographic origins, 52 for production methods, and 31 for species) were selected. Histidine was found to be more abundant (1.39 to 8.31 times) in the imported than in Taiwan mullet roe. None of the amines relevant to adverse health effects were found to be different between origins. This study completed mullet roe amine fingerprinting and demonstrated that the amine fingerprint could be applied to origin discrimination. The candidate amine markers found in this study could be identified by further studies and applied to Wuyutsu traceability inspection and quality improvement in the future.