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        果蔬中農(nóng)藥殘留檢測分析研究進展

        2021-05-06 03:16:56李俊霞馬麗雅林河通
        江蘇農(nóng)業(yè)科學(xué) 2021年6期
        關(guān)鍵詞:前處理檢測

        李俊霞 馬麗雅 林河通

        摘要:果蔬中殘留農(nóng)藥對人類的健康造成威脅,因此檢測果蔬中農(nóng)藥殘留具有重要意義。本文綜述了果蔬中農(nóng)藥殘留檢測分析方法的原理及應(yīng)用,包括前處理方法和檢測技術(shù)??偨Y(jié)了應(yīng)用較廣的前處理技術(shù):液液萃取、固相萃取、QuEChERS、分析比較色譜法、色譜-質(zhì)譜聯(lián)用法、酶聯(lián)免疫法及生物傳感器等檢測技術(shù)。另外,介紹了納米材料在果蔬農(nóng)藥殘留分析中的應(yīng)用。

        關(guān)鍵詞:果蔬;農(nóng)藥殘留;前處理;檢測

        水果和蔬菜中含有豐富的維生素、礦物質(zhì)和膳食纖維,合理攝入果蔬不僅能夠減少中風(fēng)和缺血性心臟病的風(fēng)險,而且還可以降低腸胃的患癌率[1],是現(xiàn)代生活中不可或缺的部分。但是果蔬生長周期長,存在極大的病蟲害隱患,而農(nóng)藥或復(fù)合農(nóng)藥的施用在確保果蔬產(chǎn)量和質(zhì)量的過程中發(fā)揮著重要作用[2-4]。同時,農(nóng)藥種類和劑量的不合理施用,會造成果蔬中農(nóng)藥殘留甚至超標(biāo)現(xiàn)象,影響果蔬的出口貿(mào)易和人類健康[5-8]。近年來,食品安全問題已經(jīng)成為全球熱點,加強果蔬中農(nóng)藥殘留檢測具有重大意義。

        由圖1可知,果蔬中農(nóng)藥殘留檢測所涉及的主要步驟包括樣品前處理(提取、凈化及富集),檢測(測定目標(biāo)物)及數(shù)據(jù)分析(評估可靠性)[9]。農(nóng)藥殘留種類多、樣品基質(zhì)復(fù)雜、干擾物多等是目前果蔬中農(nóng)藥殘留檢測面臨的挑戰(zhàn)[10-12],因此,須要選擇合適的前處理和檢測技術(shù),以提高果蔬中農(nóng)藥殘留檢測的精確度和靈敏度[13]。本文綜述了近年來國內(nèi)外在果蔬中農(nóng)藥殘留的前處理及檢測技術(shù),并分析比較優(yōu)劣,以期為相關(guān)領(lǐng)域工作者提供借鑒。

        1 農(nóng)藥殘留前處理技術(shù)

        目前多種前處理技術(shù)已經(jīng)在果蔬中農(nóng)藥殘留檢測方面得以應(yīng)用,樣品前處理是農(nóng)藥殘留檢測過程中至關(guān)重要的步驟。針對基質(zhì)的差異性和目標(biāo)物的多樣性,果蔬中農(nóng)藥殘留前處理技術(shù)應(yīng)具有差異性,應(yīng)結(jié)合果蔬種類、數(shù)據(jù)分析要求、檢測儀器等確定合適的前處理技術(shù)[14]。

        1.1 液液萃取法

        液液萃?。╨iquid-liquid extraction,簡稱LLE)也稱溶劑萃取和分離,它主要根據(jù)化合物在特殊不混溶液體中的相對溶解度來分離化合物[15]。LLE要使用不同的單一或者混合提取溶劑,例如乙腈[16]、二氯甲烷/丙酮[17]及氯仿/二氯甲烷[18]等溶劑。該技術(shù)適應(yīng)性強并且與大多數(shù)儀器兼容,過去10年來,LLE方法已作為常規(guī)技術(shù)在果蔬農(nóng)藥殘留檢測中廣泛應(yīng)用[19-20]。但LLE技術(shù)耗時,難以自動化,毒性溶劑如氯仿等消耗大,大量有毒的有機溶劑可能對人類和環(huán)境構(gòu)成潛在威脅。同時,該技術(shù)對極性農(nóng)藥的提取效果較差[15]。

        針對液液萃取的弊端,目前該技術(shù)已經(jīng)衍生出大量的改進技術(shù),如分散液-液微萃?。╠ispersive liquid-liquid micro-extraction,簡稱DLLME)、空氣輔助液-液微萃?。╝ir-assisted liquid-liquid microextraction,簡稱AALLME)、加糖液液萃?。╯ugaring-out liquid-liquid extraction,簡稱SULLE)、鹽析輔助均相液液萃?。╯alting-out homogenous liquid-liquid extraction,簡稱SHLLE)等,這些技術(shù)在果蔬農(nóng)藥殘留檢測中的應(yīng)用見表1。

        1.2 固相萃取法

        固相萃?。╯olid-phase extraction,簡稱SPE)是一種柱色譜分離過程,以固體吸附劑作為固定相,將樣品中目標(biāo)化合物選擇性吸附,分離樣品的基體和干擾物,然后再通過合適的洗脫液進行洗脫,達到分離和富集目標(biāo)化合物的效果[25]。與LLE相比,SPE消耗有機溶劑更少,分析時間更短,方法回收率更高,同時還能更有效地去除干擾化合物,在樣品預(yù)處理中起著越來越重要的作用[26]。但基于SPE技術(shù)是將分析物吸附到固體吸附劑上的特質(zhì),因此選擇合適的吸附劑非常重要,SPE應(yīng)用于農(nóng)藥殘留的幾種常見商業(yè)吸附劑的類型及適用范圍見圖2,另外氨基固相吸附劑(—NH2)、復(fù)合吸附劑等也常用于果蔬樣品前處理[25,27-29]。

        在固相萃取的原理上,SPE技術(shù)不斷發(fā)展,主要包括以下6種SPE:固相萃取、固相分散萃?。╠ispersive solid-phase extraction,簡稱DSPE)、固相微萃取(solid-phase microextraction,簡稱SPME)、磁固相萃取(magnetic solid-phase extraction,簡稱MSPE)、基質(zhì)固相分散萃取(matrix solid-phase dispersion,簡稱MSPD)、攪拌棒吸附萃取(stir bar sorptive extraction,簡稱SBSE),這6種主要SPE模式的原理及優(yōu)缺點見表2。

        近年來,大量的學(xué)者在果蔬農(nóng)藥殘留檢測過程中展開基于固相萃取技術(shù)的研究。Guan等利用基質(zhì)固相分散萃取快速濃縮,然后結(jié)合液相色譜-串聯(lián)質(zhì)譜同時分離檢測8種不同果蔬中的9種有機磷農(nóng)藥[30]。Zuin等比較了攪拌棒吸附萃取和膜輔助溶劑萃?。∕ASE)前處理技術(shù)在測定甘蔗汁中農(nóng)藥殘留的效果,2種前處理技術(shù)結(jié)合GC-MS,檢出限均可達到1 μg/L,但SBSE前處理具有更高的靈敏度和重復(fù)性[31]。Kin等在氣相色譜儀(電子捕獲檢測器,ECD)檢測前,選用固相微萃取法進行前處理,評估了草莓和黃瓜中有機磷和有機氯農(nóng)藥殘留水平,具有較低的檢出限(0.01~1.00 μg/L)[32]。

        1.3 QuEChERS法

        QuEChERS(quick,easy,cheap,effective,rugged,safe)技術(shù)分為提取和凈化2個部分,第1個部分是用乙腈和鹽的混合物通過分配進行萃取,第2個部分是通過包含1種或幾種吸附劑的分散固相萃取(d-SPE)進行凈化,去除潛在的干擾化合物,包括有機酸、色素、糖等,具體操作見圖3[33]。該方法因省時、安全、操作簡單、成本低、可去除多種雜質(zhì)對分析物的干擾而在果蔬農(nóng)藥殘留分析中得以廣泛應(yīng)用[34-36]。盡管原始的QuEChERS法在大多數(shù)果蔬基質(zhì)中農(nóng)藥殘留提取十分有效,但針對特殊基質(zhì)或特殊農(nóng)藥,須對QuEChERS方法不斷完善發(fā)展。近年來,大量的學(xué)者針對QuEChERS方法的提取和凈化部分進行不同程度的改進,主要包括pH值、提取溶劑、凈化的優(yōu)化。

        QuEChERS方法最初使用無緩沖液條件下進行,在應(yīng)用過程中發(fā)現(xiàn)在高或低pH值下降解的敏感化合物的回收率差。為了克服局限性,歐洲標(biāo)準(zhǔn)委員會(CEN)[37]和美國分析化學(xué)家協(xié)會(AOAC)[38]制定了官方方法: 在提取過程中引入檸檬酸鹽緩沖液(相對較低的緩沖能力)或乙酸鹽緩沖液(較強的緩沖能力)。通過添加緩沖溶液,2種方法均出現(xiàn)pH值為5左右的萃取溶劑,有利于萃取pH值依賴性農(nóng)藥。Lehotay等在果蔬農(nóng)藥殘留檢測中添加醋酸鈉形成緩沖萃取劑,測定了32種農(nóng)藥殘留,回收率為 (95±10)%,包括百菌清等pH值敏感農(nóng)藥[39]。

        目前大量的有機溶劑,如丙酮、乙腈、乙酸乙酯等,廣泛用于果蔬中農(nóng)藥殘留分析[40],而乙腈(醋酸或甲酸酸化的乙腈)是最常見的萃取溶劑[37,40],不僅在水果和蔬菜等含水量高的基質(zhì)中提取農(nóng)藥回收率高[41-42],同時可以穿透樣品基體的水相,添加鹽后可以實現(xiàn)兩相分離[43]。隨著基質(zhì)的復(fù)雜化以及農(nóng)藥的多樣化,混合萃取溶劑在QuEChERS提取過程中不斷發(fā)展。Sivaperumal等用乙腈、乙酸乙酯(體積比為25 ∶ 75)混合溶液萃取,經(jīng)d-SPE凈化后采用超高效液相色譜串聯(lián)飛行質(zhì)譜(UHPLC-Q-TOF/MS)技術(shù)對芒果中68種殘留農(nóng)藥進行測定,3種濃度水平(10、50、100 μg/kg)的回收率都在70%~122%之間,檢出限和定量限范圍分別為 0.5~7.0 μg/kg、2~25 μg/kg[44]。

        凈化是QuEChERS法的關(guān)鍵步驟,可以極大程度地影響農(nóng)藥殘留檢測的定量限和檢出限,其中最常見的凈化劑為MgSO4、石墨化碳黑(GCB)、十八烷基硅烷(C18)等。根據(jù)這些傳統(tǒng)凈化劑的優(yōu)缺點[45],可將果蔬分為3類,一般的果蔬、高色素的果蔬和高色素及脂肪的果蔬。在QuEChERS法中選擇合適的吸附劑組合很大程度上取決于果蔬的類別。一般的果蔬采用N-丙基乙二胺(PSA)+MgSO4進行去除有機酸、部分糖[44,46],高色素的果蔬則一般以PSA、石墨化碳(GCB)+MgSO4的組合去除有機酸、部分糖以及色素[47-48],而對于高色素及脂肪的果蔬,會利用C18可以消除脂肪等非極性雜質(zhì)的優(yōu)勢,在此基礎(chǔ)上添加C18凈化劑進行除雜[49]。

        1.4 其他前處理技術(shù)

        除以上幾種比較常用的前處理技術(shù)外,凝膠滲透色譜法(gel permeation chromatography,簡稱GPC),超聲波輔助萃?。╱ltrasound assisted extraction,簡稱UAE),濁點萃取法(cloud point extraction,簡稱CPE)等在果蔬農(nóng)藥殘留前處理過程中也有所應(yīng)用。Ramos等開發(fā)了超聲輔助基質(zhì)固相分散法,用于提取和凈化水果中的15種有機磷農(nóng)藥和9種三嗪類農(nóng)藥,超聲反應(yīng)器在50%振幅下進行 1 min 的超聲處理前處理效果最佳,基本沒有基質(zhì)效應(yīng)[50]。周璐等建立了濁點萃取-正己烷反萃取氣相色譜(FPD)聯(lián)用法對蘋果汁中5種有機磷農(nóng)藥的殘留進行測定[51]。5種目標(biāo)物在0.05~2.00 mg/L范圍內(nèi)線性相關(guān)系數(shù)范圍為0.998 6~0.999 6,方法的檢出限為0.13~1.50 μg/kg。

        2 農(nóng)藥殘留檢測技術(shù)

        在過去的幾十年中,已經(jīng)開發(fā)出許多檢測技術(shù)來測定果蔬中的農(nóng)藥殘留,其中色譜法和色譜質(zhì)譜聯(lián)用法是檢測的主要手段?;谄潇`敏度、分離和鑒定能力,氣相色譜和液相色譜通常是農(nóng)藥殘留檢測的首選。但是色譜法對復(fù)雜樣品的農(nóng)藥殘留檢測有一定的限制,針對這一局限性,色譜-質(zhì)譜聯(lián)用法(氣相色譜-質(zhì)譜聯(lián)用技術(shù)、液相色譜-質(zhì)譜聯(lián)用技術(shù))得到了廣泛應(yīng)用。近年來,為了滿足快速、簡單及選擇性高的農(nóng)藥檢測需求,酶聯(lián)免疫分析技術(shù)、生物傳感器等檢測技術(shù)不斷發(fā)展。表3總結(jié)了幾種檢測技術(shù)在果蔬農(nóng)藥殘留中的應(yīng)用。

        2.1 色譜法

        氣相色譜法(gas chromatography,GC)適用于以氣體和可揮發(fā)物質(zhì)作為分析對象,是一種經(jīng)典分析方法。其原理是將前處理后的樣品注入氣相色譜柱,升溫汽化固相分離檢測,通過物質(zhì)的保留時間進行定性,峰高和標(biāo)準(zhǔn)曲線進行定量。通過GC進行農(nóng)藥殘留分析通常與特定的檢測器結(jié)合使用,例如電子捕獲檢測器(ECD)[52]、火焰光度檢測器(FPD)[8,53]、氮磷檢測器(NPD)[55]和火焰電離檢測器(FID)[22-23]。然而,隨著持久性和毒性較低的極性農(nóng)藥的使用增加,由于其熱穩(wěn)定性差和高沸點的特質(zhì),GC檢測方法的弊端顯現(xiàn),使用有所減少[15]。

        液相色譜法(liquid chromatography,簡稱LC)廣泛應(yīng)用于農(nóng)藥殘留分析,絕大部分采用了光電二極管陳列檢測器(PDA)、紫外檢測器(UV)、二極管陣列檢測器(DAD)[26]。高效液相色譜法(HPLC)是液相色譜法中最常用的方法,適用于相對分子量較大、極性較強、沸點較高及熱穩(wěn)定性較差的農(nóng)藥的分離檢測,彌補了氣相色譜不能分離熱穩(wěn)定性和揮發(fā)性差的農(nóng)藥的局限[67]。同時,HPLC因其快速、高效、準(zhǔn)確性高等優(yōu)勢在果蔬農(nóng)藥殘留檢測中廣泛應(yīng)用(表3)。

        2.2 色譜-質(zhì)譜聯(lián)用法

        色譜-質(zhì)譜聯(lián)用技術(shù)是結(jié)合色譜法和質(zhì)譜(MS)的新檢測技術(shù),常用在果蔬農(nóng)藥多殘留分析領(lǐng)域。質(zhì)譜的引入可以克服結(jié)構(gòu)干擾,有效分離復(fù)雜樣品中的農(nóng)藥,檢測多殘留農(nóng)藥及其代謝物,還可同時對其進行定量、定性分析,并提供來自精準(zhǔn)分子質(zhì)量和裂解模式的結(jié)構(gòu)信息。質(zhì)譜分析器種類很多,其中四級桿分析器(quadrupole,簡稱Q)、離子阱分析器(ion trap,簡稱IT)和飛行時間分析器(time of flight mass,簡稱TOF)最為常用。為了達到增加結(jié)構(gòu)信息的目的,大多數(shù)情況下選用具有串聯(lián)質(zhì)譜功能的質(zhì)量分析器,如Q-TOF、Q-Q-Q[15]。在色譜-質(zhì)譜聯(lián)用系統(tǒng)中,被分析的樣品先在色譜系統(tǒng)中分離,然后從色譜柱中洗脫出來的餾分進行電離并進入質(zhì)量分析器進行測定。農(nóng)藥覆蓋范圍廣、樣品制備簡便、無需衍生化、靈敏度高、選擇性強等優(yōu)點[26,68-70]是色譜-質(zhì)譜聯(lián)用技術(shù)廣泛應(yīng)用于農(nóng)藥檢測、鑒定和定量分析的重要原因。表3總結(jié)了色譜-質(zhì)譜聯(lián)用在果蔬農(nóng)藥殘留檢測的研究。

        2.3 其他檢測方法

        近年來,酶聯(lián)免疫分析技術(shù)及生物傳感器法在果蔬農(nóng)藥殘留檢測中的應(yīng)用頻頻被報道。酶聯(lián)免疫法(ELISA)在免疫分析中使用最為廣泛,是根據(jù)抗原與抗體相互作用原理來確定農(nóng)藥的含量[71]。該技術(shù)的缺點是抗體不穩(wěn)定,會導(dǎo)致實驗結(jié)果有偏差,且不能同時準(zhǔn)確分析多種農(nóng)藥成分,只能作為輔助方法進行監(jiān)測[15],但基于具有簡易快捷的特點以及較高的靈敏度和選擇性,應(yīng)用在果蔬農(nóng)藥殘留快速檢測中具有很大的發(fā)展?jié)摿Αavarro等使用雙酶聯(lián)免疫吸附法檢測了柑橘汁中氯吡硫磷和倍硫磷的殘留量,該方法測得氯吡硫磷的檢出限為(0.20±0.04) μg/L,倍硫磷檢出限為(0.50±006) μg/L,且二者的回收率均為95%~106%[62]。Sun等建立了一種多酶示蹤劑形式的ElISA法測定蔬菜和果汁中西維因和速滅威含量,2種農(nóng)藥回收率均超過70%,檢出限為0.15 μg/L(西維因)和1.2 μg/L(速滅威)[63]。

        生物傳感器通過生物功能物質(zhì)與合適的轉(zhuǎn)換元件充分結(jié)合,對特定類別的化合物、生物活性物質(zhì)進行選擇性分析。與傳統(tǒng)檢測技術(shù)相比,生物傳感器檢測法具有檢樣微量、成本低、靈敏度高、分析速度快等優(yōu)點[72],其中壓電生物傳感器、光學(xué)生物傳感器、電化學(xué)生物傳感器等是果蔬農(nóng)藥殘留檢測的主要生物傳感器類型[15]。Caetano等構(gòu)建了基于抑制乙酰膽堿酯酶(AchE)活性的電化學(xué)生物傳感器,用于測定番茄中西維因的殘留量,該方法檢出限為3.2 μg/L[64]。

        3 納米材料在果蔬農(nóng)藥殘留檢測的應(yīng)用

        隨著納米材料的不斷發(fā)展,研究者們不斷開發(fā)基于納米材料的農(nóng)藥前處理技術(shù)和快速檢測方法。納米材料是一種三維空間中至少有一維在納米尺度范圍內(nèi)(1~100 nm)的材料。近年來,碳納米材料(碳納米管、石墨烯)、半導(dǎo)體納米材料(量子點)及納米氧化物(二氧化鈦、四氧化三鐵)等在果蔬農(nóng)藥殘留檢測中成為不可或缺的一部分[73-74]。

        納米級別的材料具有塊狀材料所不具備的表面效應(yīng)及強吸附能力[75]。為實現(xiàn)檢出限低、分離和富集一體化的凈化效果,納米材料在固相微萃取、磁固相萃取、基質(zhì)固相萃取和QuEChERS等果蔬農(nóng)藥殘留前處理技術(shù)中應(yīng)用廣泛。Chatzimitakos等在基質(zhì)固相萃取時使用磁性氧化石墨烯進行凈化后,利用GC-MS分離檢測了從蔬菜(白菜、韭菜、菊苣)提取的45種多類農(nóng)藥[73]。磁性氧化石墨烯具有親水性和強吸附性,可以與高含水量蔬菜有效混合,在3種蔬菜中檢出率均為89%~106%,定量限更是達到0.4~4.0 μg/kg。多壁碳納米管(MWCNT)結(jié)合其大表面積和獨特結(jié)構(gòu),具有強吸附性,是一種固相微萃取的可替代凈化劑。Han等建立了QuEChERS-HPLC-MS/MS法測定韭菜、萵苣和花環(huán)菊花中70種農(nóng)藥殘留,前處理分別采用MWCNT、GCB、PSA作為凈化劑,結(jié)果表明,MWCNT的凈化性能優(yōu)于GCB和PSA,回收率較高,范圍為74%~119%,同時70種農(nóng)藥的檢出限(0.1~2.4 μg/kg)和定量限(0.3~7.9 μg/kg)都較低[76]。

        金屬半導(dǎo)晶體納米材料量子點(quantum dot,簡稱QD)擁有獨特的光學(xué)性質(zhì),量子點與目標(biāo)分析物發(fā)生物理或化學(xué)反應(yīng),能夠?qū)е掳l(fā)光增強或猝滅,以此來測定目標(biāo)物的濃度[77]。量子點表面易進行功能修飾的特點以及光學(xué)性質(zhì),促進了其在農(nóng)藥殘留檢測中的應(yīng)用。Luan等以建立了CdTe量子點為信號傳感器、乙酰膽堿酯酶(AchE)為識別分子的生物傳感器,已經(jīng)應(yīng)用于蘋果中有機磷農(nóng)藥的測定[78]。有機磷農(nóng)藥抑制了AchE活力,從而改變CdTe/AchE的熒光強度,可以衡量有機磷農(nóng)藥含量。在最佳條件下,對硫磷和對氧磷的線性范圍為5~100 μg/L,檢測限為10 μg/L。為實現(xiàn)特異性檢測果蔬中農(nóng)藥殘留,Huang等用O,O-二甲基-(2,2-二氯乙烯基)磷酸酯的分子印跡聚合物(MIPs)包覆混合量子點,選擇性吸附測定敵敵畏,而量子點的加入大大提高了測定敵敵畏的靈敏度,檢出限達到1.27 μg/L,并成功應(yīng)用于白菜中敵敵畏的測定,回收率為87.4%~101.0%[79]。

        4 總結(jié)與展望

        隨著人們食品安全意識的不斷增強,果蔬的農(nóng)藥殘留問題越來越受重視。果蔬基質(zhì)的復(fù)雜性、農(nóng)藥的多樣性,對果蔬農(nóng)藥殘留分析技術(shù)的發(fā)展起了推動作用。近年來,樣品前處理過程已進行了很大改進,這些改進技術(shù)具有提高靈敏度,減少樣品量、有機試劑、分析時間、基質(zhì)干擾的發(fā)展趨勢。而檢測技術(shù)也逐漸從色譜技術(shù)向色譜-質(zhì)譜聯(lián)用技術(shù)轉(zhuǎn)移,同時,生物傳感器法和免疫技術(shù)近年來在果蔬農(nóng)藥殘留檢測中不斷開發(fā)應(yīng)用。不同的前處理和檢測技術(shù)都具有各自的適用范圍和優(yōu)缺點,在實際檢測中,需要結(jié)合果蔬的種類、農(nóng)藥的種類和限度,選擇適當(dāng)?shù)那疤幚砗蜋z測技術(shù),來提高果蔬中農(nóng)藥殘留檢測的準(zhǔn)確度。

        最近幾年,農(nóng)藥產(chǎn)業(yè)迅速發(fā)展,出現(xiàn)了不少新型農(nóng)藥,新型農(nóng)藥正朝著復(fù)合農(nóng)藥的方向發(fā)展,農(nóng)藥殘留檢測也逐漸向多種組分同時檢測分析的趨勢發(fā)展。農(nóng)藥殘留檢測需要生物技術(shù)與多種現(xiàn)代儀器分析技術(shù)相結(jié)合來提高檢測的準(zhǔn)確性和靈敏度。未來的農(nóng)藥殘留分析將與新材料結(jié)合,朝著安全化、微型化和自動化分析的方向發(fā)展。研發(fā)高效快捷、高靈敏、高通量及自動化的新型農(nóng)藥殘留檢測技術(shù)并將其應(yīng)用到實踐,將是未來研究熱門方向之一,將為我國農(nóng)藥殘留檢測打開一個多元化局面。

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