以FaPEx搭配同位素稀釋極致效能液相層析串聯質譜法同時測定即食生菜沙拉中新菸鹼類農藥及對羥基苯甲酸酯類

Abstract

近年隨著消費者健康意識提升及飲食西化，市場發展出生鮮蔬果等即食產品，例如便利超商販售之即食生菜沙拉，因利於消費者攝取營養素而日漸受到青睞。然而這類產品除種植過程中可能產生農藥如新菸鹼類農藥(neonicotinoids, NEOs)殘留問題外，也可能因部分業者為延長產品保存期限，選擇添加防腐劑如對羥基苯甲酸酯類(esters of para-hydroxybenzoic acid, parabens)，亦或是在加工製造過程環境污染導致物質殘留，進而對人體健康造成潛在風險。 NEOs為目前廣泛使用的系統性殺蟲劑之一，於農藥噴灑使用後可透過植株吸收並擴散至所有組織，在環境中穩定存在且不易降解。近年研究指出NEOs對哺乳動物可能影響腦部發展及生殖發育，且因NEOs屬系統性殺蟲劑，無法透過去皮或洗淨的方式去除殘留，可能經由食物鏈對生物多樣性及生態環境造成影響；而Parabens為一類可從植物中萃取的天然防腐劑，亦可由人工化學方式合成，因具低成本、高水溶性和廣效性抗菌之優勢，常應用於食品及個人護理產品，但文獻顯示Parabens可能對人體健康產生不良影響。為了解國人經食用即食生菜沙拉同時暴露NEOs及Parabens之可能性及劑量，本研究擬建立一套同時測定即食生菜沙拉中NEOs及Parabens之定量分析方法，並實際應用於市售即食生菜沙拉檢測。 本研究參考食品藥物管理署公告標準方法之農藥檢驗快速萃取前處理技術 (Fast Pesticide Extraction, FaPEx)並改良之，搭配同位素稀釋極致液相層析串聯質譜法 (isotope dilution ultra-performance tandem mass spectrometry, ID-UPLC-MS/MS)，針對即食生菜沙拉中7種NEOs及9種parabens進行同時定量分析方法開發。實驗方法參數最佳化包含樣品前處理、管柱選擇、管柱及游離源溫度，以得到最佳分析結果。最終優化後方法之確效結果顯示，16種目標分析物之檢量線線性範圍為0.1  250.0 ng/g，呈現良好線性關係 (R2 > 0.990)，其定量極限為0.003  0.968 ng/g；同日間(intra-day)與異日間(inter-day)實驗中，16 種分析物均有良好之準確度(以回收率表示)與精密度(以相對標準偏差relative standard deviation, %RSD表示)，回收率範圍分別為94.58%  111.90%與 96.27%  109.70%，%RSD分別為 0.89%  10.63%與 2.49%  7.81%，符合國際食品法典委員會(Codex Alimentarius Commission)之確效規範。實際應用於2020年12月至2021年7月間採集之111件市售即食生菜沙拉分析，結果顯示，NEOs均檢出微量濃度，其範圍為未檢出(not detected, N.D.)  439.958 ng/g；parabens部分，僅PePB皆低於偵測極限外，其餘物質以MePB檢出濃度最高，介於N.D. – 8.546 ng/g。整體而言，16種代測物檢出濃度皆低於我國法規訂定之最大殘留容許量標準(maximum residue limits)，故雖有檢出但仍在可接受範圍內。本研究建立之FaPEx搭配ID-UPLC-MS/MS食品微量分析方法除可定期檢測即食生菜沙拉中NEOs及parabens之濃度，亦可結合國家攝食資料庫及相關毒理數據，應用於食品風險評估中，以更全面性的方式了解消費者飲食上可能存在的危害及風險。

In recent years, as consumers’ health awareness and diet westernization have increased, the market has developed ready-to-eat (RTE) products such as RTE salads sold by convenience stores, which have become increasingly popular due to the convenience of consumers' intake of nutrients. However, the residual problems of pesticides such as neonicotinoids (NEOs) during the planting process, some suppliers may also add preservatives like esters of para-hydroxybenzoic acid (parabens) in order to extend the shelf life of the products, which may increase the potential risks to human health. Therefore, the study hopes to establish a set of methods to simultaneously analyze Neonicotinoids and parabens. This study developed and validated an isotope dilution-liquid chromatography-tandem mass spectrometry (ID-UPLC-MS/MS) followed by the Fast Pesticide Extraction (FaPEx) pretreatment method announced by the Taiwan Food and Drug Administration with minor modification to simultaneously determine seven NEOs and nine parabens in ready-to-eat (RTE) salads. The optimization of experimental parameters included the condition of the sample pretreatment, temperatures of column oven and the ionization source, and the flow rate of UPLC to obtain the optimal analysis results. The validation results of the optimized analytical method indicated that the linear ranges of 16 target analytes were 0.1  250.0 ng/g, showing a good linearity (R2 > 0.990), and their limits of quantification (LOQ) were 0.003  0.968 ng/g. Regarding the intra-day and inter-day assays, good accuracy (expressed by recovery) and precision (expressed by relative standard deviation, %RSD) of 16 target analytes were observed by using FaPEx coupled with ID-UPLC-MS/MS, and were in line with the acceptable criteria of Codex Alimentarius Commission. Their recoveries were 94.58%  111.90% and 96.27%  109.70%, respectively, in the intra-day and inter-day assays; the %RSD were 0.89%  10.63% and 2.49%  7.81%, respectively, in the intra-day and inter-day assays. After the method was validated, it was applied to the analysis of 111 RTE collected from December 2020 to July 2021. The results showed that the detection rate of acetamiprid, one of NEOs, was as high as 87%, and pentyl paraben were all below its limit of detection. Moreover, the detection concentration of neonicotinoid ranged from not detected (N.D.) to 439.958 ng/g, and parabens ranged from N.D. to 8.546 ng/g. Both of them were lower than the maximum residue limits of pesticides and parabens in the regulation of Taiwan. This study developed and validated an ID-UPLC-MS/MS followed by FaPEx to quantitatively analyze 111 real samples to realize the distribution and occurrence of seven NEOs and nice parabens in RTE salads. In addition, this method and its application data can also apply to assess the risk of exposure to NEOs and parabens via intake of RTE salads, which can provide a more comprehensive understanding of the relevant risks that consumers might be exposed.