以超高效液相層析搭配飛行時間串聯式質譜術篩查保健食品中之有機污染物

Suspect Screening of Organic Contaminants in Nutraceuticals Using Ultra-high Performance Liquid Chromatography/Quadrupole Time-of-flight Mass Spectrometry

施宏儒(Hung-Ju Shih)

指導教授 ： 陳家揚

國立臺灣大學/公共衛生學院/食品安全與健康研究所/碩士(2022年)

https://doi.org/10.6342/NTU202200641

摘要

「保健食品」泛指具有保健功效，可調節生理機能的食品。近年來，伴隨人口老年化及健康意識的提升，於已開發國家當中保健食品的消費量逐年上升。然而各國在保健食品規範程度不一，且於生產過程中，原料中若含有天然毒素、農藥及環境污染物的殘留，經萃取及濃縮後可能會攝入造成風險；在過往研究中，曾於保健食品中檢出非法的西藥攙雜以及黴菌毒素。本研究藉由對多種保健食品進行篩查，初步辨別出具潛在風險的化學物。
本研究針對保健食品中常見的26種植物、真菌、動物及海洋來源的原料，購入53種市售保健食品進行篩查，抽樣時間為2020年12月至2021年7月，樣本購自網路商城、量販店及藥妝店。樣本經由QuEChERS萃取及Oasis PRiME HLB萃取匣淨化，萃取液經濃縮後，以超高效能液相層析搭配四極柱飛行時間串聯式質譜儀(ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometer)進行分析，以全離子MS/MS模式採集質譜資料。應用MassHunter Qualitative Analysis Software (version B.07.00)軟體及六個資料庫合計含有3376種化合物的二次質譜圖譜進行比對，包含Agilent廠商建立之藥物與毒物資料庫(Forensic and Toxicology)、黴菌毒素資料庫(Mycotoxins)、農藥資料庫(Pesticides)、畜產用藥資料庫(Veterinary)及水體環境新興污染物資料庫(Water)，及本研究團隊的自建高度關注物質資料庫(Concerned Chemicals)。
所有樣本檢出合計552種化合物；檢出頻率較高的化學物質多為原料自身的內生性化學物質或常見的新興環境污染物。杜鵑花酸(azelaic acid，58%)及塑化劑鄰苯二甲酸二(2-乙基己基)酯(DEHP，57%)具有最高的檢出頻率。杜鵑花酸為天然存在於植物中的有機酸類，亦常作為外用藥用於痤瘡的治療；DEHP則為常用於聚氯乙烯之中的塑化劑。於植物類保健食品當中較常檢出有機酸類，包含杜鵑花酸、肉桂酸(cinnamic acid)、龍膽酸(gentisic acid)等。阿糖腺苷(vidarabine)為抗皰疹病毒之藥物，在真菌及動物/海洋性的保健食品中具有較高的檢出頻率，分別為64%及56%。在含有魚油的保健食品當中皆可發現樅酸(abietic acid)，其常作為船隻修補材料及紙漿和油漆的黏著劑。對羥基苯甲酸及對羥基苯甲酯類化合物同時可作為食品、藥品及化妝品的防腐劑，並於64%的全體樣品中檢出。
在列出潛在關注化學物質清單之後，針對篩出率高於10%的化合物進行優先化排序，使用美國環保署開發的ToxPi軟體結合檢出頻率、訊號強度及ToxCast資料庫中的生物活性、暴露評估及每日容許攝取量(acceptable daily intake, ADI)進行評分排序。需要高度關注的化合物多為內分泌干擾物質(endocrine disrupting chemicals, EDCs)，包含類固醇、鄰苯二甲酯及對羥基苯甲酯類化合物，可能源自個人醫藥、護理產品排入環境當中。於植物保健食品中，黴菌毒素altenusin (產自鏈隔孢菌屬)、DEHP及咖啡因具有較高的優先度。咖啡因常見於標榜瘦身及壯陽提神的保健食品，然而部分樣品難以從品名辨識含咖啡因，如田七瑪卡飲中含有的咖啡因源自瓜拿納萃取物而易被忽略；真菌保健食品中排序前三皆為類固醇，分別為1,4-雄二烯-3,17-二酮(boldione)、11-酮睪酮(11-ketotestosterone)及可體松(cortisone)；在動物及海洋來源保健食品中則以樅酸(abietic acid)、阿糖腺苷、甲基木皮素(methylequisetin)及去氧孕烯(desogestrel，口服避孕藥)具有高優先度。
本研究使用優化後的前處理方式對保健食品進行前處理並分析，篩查市售保健食品當中常見具潛在風險的化學物，並對其進行排序，列出優先關注化學物質。當中以鄰苯二甲酯及對羥基苯甲酯類具有較高優先度而需要從源頭管控，其可能的來源包含工廠生產的廢水、放流水及地表水；而保健食品上易讓人混淆的標示也容易使人在非預期的情況下攝入部分化合物，如咖啡因。此排序結果可供日後對保健食品中關注化學物質進行定量及風險評估作為優先化的參考，並為保健食品的安全進行把關。

關鍵字

新興污染物 ； 保健食品 ； 高解析質譜術 ； 非目標物篩查 ； 優先化

並列摘要

The term “nutraceuticals” refers to foods with health benefits and health care effects. In recent years, market values of nutraceuticals have increased year by year because of population aging problems and the desire of health promotion. However, regulations about the safety of nutraceuticals are insufficient in many countries; if there were contaminants or natural toxins in the raw materials, they can be concentrated into the nutraceuticals during manufacturing process. Previous studies have identified pharmaceutical adulterants and mycotoxins in nutraceuticals; thus, this study aims to screen various nutraceuticals for figuring out potential hazardous compounds.
Fifty-three products of nutraceuticals containing 26 different ingredients from plants, fungus, animals were collected from pharmacies and online stores in Taiwan during December 2020 to July 2021. The samples were extracted with QuEChERS and were cleaned up with Oasis PRiME HLB cartridges. After concentrations, the extracts were analyzed with ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometer (UHPLC-QTOF MS) in All Ion MS/MS mode. Peaks were identified with MassHunter Qualitative Analysis Software and the spectra were matched with five libraries from Agilent (Forensic and Toxicology, Mycotoxins, Pesticides, Veterinary, and Water) plus an in-house library.
There were 552 identified chemicals from all samples, and most chemicals in high detection frequencies are either endogenous chemicals from raw materials or emerging contaminants. Azelaic acid (58%) and di(2-ethylhexyl)phthalate (DEHP, 57%) were the most detected compounds. Azelaic acid is used in the treatment of acne and exists in various plants. DEHP is a plasticizer used in polyvinylchloride (PVC). In plant-originated nutraceuticals, organic acids were the most identified chemicals, including azelaic acid, cinnamic acid, gentisic acid, and ferulic acid. Vidarabine is an antivirus drug with high positive rates in fungus-originated (64%) and animal/marine-originated nutraceuticals (56%). Abietic acid, an adhesive in paper and paints, was found in all fish-oil-containing nutraceuticals. Parabens and 4-hydroxybenzoic acid were found in 64% of all samples; they are preservatives in foods, cosmetics, and pharmaceuticals.
The identified chemicals with detection frequencies higher than 10% were prioritized with the US EPA-developed ToxPi (Toxicological Priority Index) software by considering their empirical data (detection frequency and peak abundance) and external information (estimated exposure, bioactivity and acceptable daily intake (ADI)). Altenusin (produced by Alternaria sp.), DEHP, and caffeine were in high priority in plant-originated nutraceuticals. Caffeine was common in nutraceuticals for slim products and aphrodisiacs, such as Tian Qi Maca Essence Drink. Steroids, including boldione, 11-ketotestosterone, and cortisone, were at the top three positions in fungus-originated nutraceuticals. Abietic acid, vidarabine, methylequisetin and desogestrel (an oral contraceptive) were in high priority in animal and marine originated nutraceuticals. Most chemicals in high priority were endocrine disrupting chemicals such as hormone-related steroids, phthalates, and parabens, which were commonly used in pharmaceuticals and personal care products and could come from the environment.
The optimized suspect screening method was applied on identifying contaminants in 26 types of nutraceuticals to clarify potential chemical hazards that consumers may ingest unconsciously and the identified chemicals were prioritized with risk-based approaches. Parabens and DEHP were in high priority and the probable sources include manufacturing processes, wastewater. Fancy product names of nutraceuticals can confuse consumers and make them expose to some chemicals unconsciously, such as caffeine. The list of concerned chemicals can be used for further quantitative analysis and risk assessment in the future, and facilitates the regulations of nutraceuticals.

並列關鍵字

emerging contaminants ； dietary supplements ； high-resolution mass spectrometry ； non-target screening ； chemical prioritization

參考文獻

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