曹小云，陳樹干，曾楚瑩，黎秀娟，王 露

(廣東省深圳市鹽田區(qū)疾病預(yù)防控制中心，廣東 深圳 518081)

農(nóng)產(chǎn)品中農(nóng)藥多殘留是食品安全中最令人關(guān)注的問題之一[1]，由于其對人體的長期健康風(fēng)險(xiǎn)[2-3]，國際食品法典委員會(huì)(CAC)和多數(shù)國家均制定了嚴(yán)格的農(nóng)產(chǎn)品中農(nóng)藥最高殘留限量(MRLs)[4]，日本、歐盟等發(fā)達(dá)國家對未設(shè)MRLs的農(nóng)藥，一般均要求其含量小于0.01 mg/kg。我國《GB2763-2016食品中農(nóng)藥最大殘留限量》[5]已對433種農(nóng)藥制定了4 140項(xiàng)標(biāo)準(zhǔn)，但仍與發(fā)達(dá)國家有差距[4]，蔬果中農(nóng)藥殘留情況依然嚴(yán)峻[6]。因此，亟待建立新鮮蔬果中高靈敏度、高準(zhǔn)確性和高通量的檢測農(nóng)藥殘留的分析方法。

由于新鮮蔬果的基質(zhì)非常復(fù)雜，對其進(jìn)行農(nóng)殘檢測時(shí)需繁瑣的樣品前處理以減少干擾。隨著科學(xué)技術(shù)的發(fā)展，特別是氣相色譜-質(zhì)譜法(GC-MS)[7-8]、氣相色譜-串聯(lián)質(zhì)譜法(GC-MS/MS)[9-10]、超高效液相色譜-串聯(lián)質(zhì)譜法(LC-MS/MS)[11]等聯(lián)用技術(shù)的發(fā)展，將色譜高效的分離性能與質(zhì)譜優(yōu)越的抗干擾、高靈敏度、高通量離子監(jiān)測能力相結(jié)合，能夠在復(fù)雜基質(zhì)背景下實(shí)現(xiàn)目標(biāo)物的準(zhǔn)確測定，從而可以摒棄繁雜、污染大、不經(jīng)濟(jì)的傳統(tǒng)前處理方法，而采用分散固相萃取(DSPE)、QuEChERS法、液相微萃取(LPME或LLME)等[12-13]新技術(shù)。如GB 23200.113-2018[14]采用QuEChERS法，使用特定吸附填料吸附除去雜質(zhì)，凈化萃取液[9]；Rezaee等[15]發(fā)展的分散液液微萃取法(DLLME)，用微量有機(jī)溶劑即可實(shí)現(xiàn)目標(biāo)物的提取和富集[13,16]，操作簡便、成本低、快速、環(huán)保、效果好。然而每種方法都有各自的優(yōu)缺點(diǎn)，如QuEChERS法在濃縮痕量殘留農(nóng)藥的同時(shí)也濃縮了雜質(zhì)，需根據(jù)不同樣品優(yōu)選凈化吸附劑及用量[12,14]；DLLME法則只適用于水樣等基質(zhì)簡單的樣品，難以直接應(yīng)用于基質(zhì)復(fù)雜的食品中[7,13]。雖然通過方法聯(lián)用則可發(fā)揮各自優(yōu)勢互補(bǔ)不足，但將QuEChERS和DLLME及GC-MS/MS三者聯(lián)合應(yīng)用的報(bào)道較少[8,13]，且涉及的農(nóng)藥種類較少。本研究將QuEChERS和DLLME及GC-MS/MS 3種技術(shù)聯(lián)用，并通過實(shí)驗(yàn)條件優(yōu)化，建立了同時(shí)檢測新鮮蔬果中152種(含異構(gòu)體)農(nóng)藥殘留的分析方法，以滿足新鮮蔬菜水果中農(nóng)藥多殘留的高通量快速篩查及應(yīng)急檢測需要。

1 實(shí)驗(yàn)部分

1.1 儀器與試劑

Trace1300 TSQ8000 Evo氣相色譜-質(zhì)譜儀(美國ThermoFisher公司)；Allegra 64R Centrifuge高速冷凍離心機(jī)(美國BeckmanCoulter公司)；GENIUS 3 VORTEX漩渦混合器、A11分析研磨機(jī)(德國IKA公司)。

乙腈、丙酮、二氯甲烷、乙酸乙酯(優(yōu)級純，德國Merck公司);三氯甲烷和氯化鈉(分析純，上海凌峰化學(xué)試劑有限公司)：QuEChERS提取鹽由2 g 無水MgSO4、0.5 g NaCl、0.5 g檸檬酸三鈉二水合物、0.25 g檸檬酸氫二鈉組成，凈化劑由300 mg無水MgSO4、50 mg乙二胺-N-丙基硅烷(PSA)和5 mg石墨化碳黑(GCB)組成，均購于美國ThermoFisher公司。

農(nóng)藥標(biāo)準(zhǔn)物質(zhì)：有機(jī)磷類、有機(jī)氯類、氨基甲酸酯類、菊酯類、除草劑、殺菌劑等農(nóng)藥及其異構(gòu)體共152種(日本林純藥、美國O2si smart solutions和德國Dr.Ehrenstorfer)。標(biāo)準(zhǔn)工作溶液：將各農(nóng)藥標(biāo)準(zhǔn)品用丙酮稀釋成質(zhì)量濃度10.0 mg/L的標(biāo)準(zhǔn)儲(chǔ)備液，臨用前再用乙腈混合逐級稀釋成低濃度混合標(biāo)準(zhǔn)工作溶液；用空白基質(zhì)溶液配制用于繪制校準(zhǔn)曲線的標(biāo)準(zhǔn)溶液。

1.2 樣品前處理

取蔬果新鮮樣品100 g用研磨機(jī)研磨成勻漿，攪拌均勻后稱取5.0 g于50 mL離心管中(剩余勻漿于-20 ℃冰箱中儲(chǔ)存?zhèn)溆?，加入5.0 mL乙腈，于-20 ℃冷凍10 min，再加入提取鹽，渦旋1 min后以9 000 r/min離心5 min，吸取2.0 mL上清液于15 mL離心管(預(yù)先加入凈化劑)中；再渦旋離心1次，取1.0 mL上清液于15 mL離心管，準(zhǔn)確加入50 μL三氯甲烷萃取劑，再加4 mL純水，渦旋離心后用微量注射器取管底部液滴至含有內(nèi)襯管的進(jìn)樣瓶中，GC-MS/MS自動(dòng)進(jìn)樣測定。

1.3 色譜-質(zhì)譜條件

色譜條件：色譜柱TG-5MS(30 m×0.25 μm×0.25 mm)；進(jìn)樣口溫度280 ℃；柱箱升溫程序：初始柱溫60 ℃，保持2 min，以25 ℃/min升至180 ℃，再以5 ℃/min升至280 ℃，最后以10 ℃/min升溫至300 ℃，保持5 min；不分流進(jìn)樣，進(jìn)樣量1.0 μL；氦氣柱流量1.20 mL/min。

質(zhì)譜條件：電子轟擊電離源(EI)溫度300 ℃；傳輸線溫度280 ℃；選擇反應(yīng)監(jiān)測掃描(SRM)模式；根據(jù)農(nóng)藥各組分保留時(shí)間分段掃描，窗帶寬(Window)為1.2 min，循環(huán)時(shí)間0.3 s，其余參數(shù)用儀器默認(rèn)值；152種農(nóng)藥的保留時(shí)間、離子對信息和碰撞能見表1。

表1 152種農(nóng)藥的保留時(shí)間、離子對信息和碰撞能Table 1 Retention times,ion pairs information and collision energy of 152 pesticides

(續(xù)表1)

No.CompoundRetentiontime/minIonpairs(m/z)Collisionenergy(eV)60Cyprodinil(嘧菌環(huán)胺)14.79224.1>196.9?,224.1>208.0,225.1>209.720,18,1661Chlorfenvinphos-E-trans(E-反式毒蟲畏)14.87266.9>159.0?,268.8>161.0,322.9>267.014,12,1262Dimethametryn(異戊乙凈)14.97212.1>68.1?,212.1>94.1,212.1>122.130,20,1263Heptachlorepoxide(環(huán)氧七氯)15.06262.9>192.9?,352.8>262.9,354.7>264.930,16,1264Oxychlordane(氧化氯丹)15.08115.0>50.9?,184.9>84.9,184.9>121.022,26,1265Trichlorfon(敵百蟲)15.2292.9>65.0?,109.0>63.0,109.0>81.06,10,666Isofenphos(異丙胺磷)15.22185.0>121.0?,213.0>121.0,213.0>185.010,14,667Chlorfenvinphos-Z-cis(Z-順式毒蟲畏)15.22266.9>159.0?,294.9>267.0,323.0>267.014,8,1268Isocarbophos(水胺硫磷)15.22121.1>65.0?,136.0>69.0,136.0>108.014,30,1269Isofenphos-methyl(甲基異丙胺磷)15.23199.0>65.0?,199.0>121.0,241.1>121.134,10,2070Esprocarb(禾草畏)15.3291.0>65.0?,119.0>65.0,119.0>91.016,26,1071Dimepiperate(哌草丹)15.32119.0>65.0?,119.0>91.1,145.0>112.126,10,872Phoxim(辛硫磷)15.33135.2>77.1?,135.2>94.0,135.2>134.514,8,673Quinalphos(喹硫磷)15.33146.0>118.1?,157.1>102.0,157.1>129.010,22,1474Phenthoate(殺蟲殺螨劑)15.33121.0>77.0?,246.0>121.0,274.0>121.022,8,1075Methidathion(殺撲磷)15.76145.0>58.0?,145.0>85.0,302.6>284.914,6,1476Propaphos(丙蟲磷)15.77139.9>125.0?,220.0>125.0,220.0>140.012,24,1077α-cis-Chlordane(α-順式氯丹)15.77372.8>265.8?,374.7>265.8,376.6>268.020,20,2078o,p′-DDE(o,p′-滴滴伊)15.90246.0>176.1?,317.8>246.0,317.8>248.028,20,1879Fenothiocarb(苯硫威)15.9172.1>56.0?,105.7>72.0,160.1>72.112,10,1080Paclobutrazol(多效唑)15.95125.0>89.0?,236.0>125.0,236.0>167.018,12,1081Pentachlorobiphenyl1(PCB101,五氯聯(lián)苯峰1)15.95253.9>184.0?,323.8>253.9,325.8>255.930,25,2582Tetrachlorvinphos(殺蟲畏)16.07109.0>79.0?,328.9>109.0,330.8>109.06,18,1883Endosulfan1(硫丹峰1)16.15194.7>125.0?,194.7>159.4,240.6>205.922,8,1484γ-trans-Chlordane(γ-反式-氯丹)16.23271.7>236.8?,372.7>263.7,374.7>265.912,20,2285Fenamiphos(克線磷)16.42154.0>139.0?,216.9>202.0,303.1>195.210,12,886Butamifos(丁胺磷)16.46200.0>65.1?,286.0>185.0,286.0>202.020,24,1487Napropamide(敵草胺)16.48100.1>72.1?,128.2>72.1,271.1>72.16,6,1488Prothiofos(丙硫磷)16.65266.7>220.9?,266.7>238.9,308.9>239.018,8,1489Metominostrobin-E(E-苯氧菌胺)16.73191.0>119.0?,191.0>132.6,191.0>160.120,18,1090Profenofos(丙溴磷)16.75296.7>268.9?,336.9>266.9,336.9>308.910,12,891Pretilachlor(丙草胺)16.83202.1>145.5?,202.1>174.2,238.1>146.114,8,1092Butachlor(丁草胺)16.84160.0>131.7?,176.1>146.9,188.1>160.112,12,1093p,p′-DDE(p,p′-滴滴伊)16.88246.0>176.1?,317.8>246.0,317.8>248.028,20,1894Dieldrin(狄氏劑)16.95262.8>190.9?,262.8>192.9,262.8>227.830,30,1695p,p′-DDD(p,p′-滴滴滴)17.16235.0>165.1?,235.0>199.0,236.8>165.020,14,2096Flusilazole(氟硅唑)17.22206.0>151.3?,233.0>151.9,233.0>164.914,14,1697Cyproconazole(環(huán)唑醇)17.62222.0>82.1?,222.0>89.3,222.0>125.010,38,2098Endrin(異狄氏劑)17.65245.0>173.0?,262.8>192.9,280.8>245.322,30,899Fenoxanil(稻瘟酰胺)17.76189.0>125.1?,293.1>155.0,293.1>198.012,20,12100Chlorobenzilate(殺螨酯)17.93111.0>75.1?,139.0>74.9,139.0>111.014,26,12101Endosulfan2(硫丹峰2)17.96158.9>123.0?,194.7>159.0,240.6>205.812,8,12102Fensulfothion(芬硫磷)18.04307.9>96.9?,307.9>153.1,307.9>293.026,12,8103o,p′-DDD(o,p′-滴滴滴)18.25235.0>165.1?,235.0>199.0,236.8>165.020,14,20104o,p′-DDT(o,p′-滴滴涕)18.33235.0>165.1?,235.0>199.5,236.8>165.022,10,22105Ethion(乙硫磷)18.36153.0>97.0?,230.9>128.9,230.9>174.910,22,12106Nonachlor-trans(反式九氯)18.38236.8>142.9?,262.8>192.9,408.7>300.024,28,18107Endrinaldehyde(乙醛異狄氏劑)18.57173.0>138.1?,249.8>214.9,344.9>281.016,24,8108Hexachlorobiphenyl1(PCB138,六氯聯(lián)苯峰1)18.64289.9>218.0?,357.9>287.8,359.8>289.830,25,25109Mepronil(擔(dān)菌寧)18.66119.0>65.0?,119.0>91.0,269.1>119.122,12,14110Triazophos(三唑磷)18.80161.0>105.7?,161.0>134.1,172.1>77.112,8,25111Benalaxyl(苯霜靈)19.1491.0>65.0?,148.1>77.0,148.1>79.012,30,22112Carfentrazon-ethyl(唑草酮)19.18290.0>99.9?,311.9>150.7,340.1>312.136,18,10113Edifenphos(克瘟散)19.20172.9>65.1?,172.9>109.0,310.0>109.030,8,26114Propiconazole1(丙環(huán)唑峰1)19.31172.9>74.0?,172.9>109.0,172.9>145.038,26,16115Endosulfansulfate(硫丹硫酸鹽)19.34238.7>203.9?,271.7>234.9,271.7>236.812,12,12116p,p′-DDT(p,p′-滴滴涕)19.46235.0>165.1?,235.0>199.5,236.8>165.022,10,22117Propiconazole2(丙環(huán)唑峰2)19.53172.9>74.0?,172.9>109.0,172.9>145.038,24,14118Hexachlorobiphenyl(PCB153,六氯聯(lián)苯)19.53287.8>217.9?,357.7>287.8,359.7>287.930,35,35119Thenylchlor(甲氧噻草胺)19.90127.0>53.0?,127.0>59.0,127.0>97.018,10,12

(續(xù)表1)

No.CompoundRetentiontime/minIonpairs(m/z)Collisionenergy(eV)120Tebuconazole(戊唑醇)19.92125.0>89.0?,125.0>99.0,250.0>125.016,16,20121Bioresmethrin(生物芐呋菊酯)20.29123.1>81.1?,143.0>128.1,171.0>128.08,10,14122Endrin-ketone(異狄氏劑酮)20.86209.2>138.4?,316.8>208.9,316.8>281.030,28,10123Pyridaphenthion(噠嗪硫磷)20.95199.0>77.1?,199.0>92.1,340.0>199.124,14,8124EPN(苯硫磷)21.17157.0>77.0?,169.0>77.0,169.0>141.022,22,8125Bifenthrin(聯(lián)苯菊酯)21.24165.1>163.6?,181.0>165.9,181.0>179.024,10,12126Piperophos(哌草磷)21.32122.1>53.0?,122.1>79.7,320.1>122.120,12,10127Methoxychlor(甲氧滴滴涕)21.39227.1>141.1?,227.1>169.1,227.1>212.132,22,12128Fenpropathrin(甲氰菊酯)21.4797.1>55.1?,181.0>126.8,181.0>151.96,28,22129Etoxazole(乙螨唑)21.55140.9>62.9?,140.9>113.0,204.0>176.126,14,10130Tebufenpyrad(吡螨胺)21.60276.1>171.0?,318.1>131.1,318.1>145.110,14,14131Anilofos(莎稗磷)21.82183.7>157.0?,225.9>157.0,225.9>184.08,14,6132Heptachlorobiphenyl1(PCB180,七氯聯(lián)苯峰1)21.84323.8>253.9?,391.8>321.7,393.7>323.735,25,25133Azinphos-methyl(谷硫磷)22.38132.0>77.0?,160.0>50.9,160.0>77.012,34,16134Phosalone(伏殺硫磷)22.38121.1>65.0?,182.0>74.8,182.0>111.010,30,14135Mirex(滅蟻靈)22.70236.8>142.9?,272.0>236.8,273.8>238.826,14,14136Iambdacyhalothrin(高效氯氟氰菊酯)23.15180.9>152.0?,197.1>141.1,207.9>180.922,10,8137Pyraclofos(吡唑硫磷)23.79360.0>96.9?,360.0>139.0,360.0>194.120,14,10138Etobenzanid(乙氧苯草胺)25.32149.1>121.0?,179.1>59.1,179.1>121.08,10,10139Butafenacil(氟丙嘧草酯)25.32180.0>124.0?,331.0>124.0,331.0>180.016,38,16140Cyfluthrin1(氟氯氰菊酯峰1)25.64163.0>127.1?,163.0>91.1,163.0>65.16,12,26141Cyfluthrin2(氟氯氰菊酯峰2)25.83163.0>127?,163.0>91.1,206.0>151.16,12,18142Cyfluthrin3(氟氯氰菊酯峰3)25.97163.0>127.0?,163.0>91.1,226.0>206.16,12,12143Cyfluthrin4(氟氯氰菊酯峰4)26.05163.0>127.0?,163.0>91.1,226.0>206.16,12,10144Cypermethrin1(氯氰菊酯峰1)26.22163.0>91.1?,163.0>127.1,180.9>152.16,12,20145Cypermethrin2(氯氰菊酯峰2)26.42163.0>127.0?,163.0>91.1,180.9>151.96,12,18146Cypermethrin3(氯氰菊酯峰3)26.56163.0>127.0?,163.0>91.0,180.9>152.26,12,20147Cypermethrin4(氯氰菊酯峰4)26.63163.0>127.1?,163.0>91.1,180.9>152.26,12,20148Etofenprox(醚菊酯)26.76163.1>77.1?,163.1>107.1,163.1>135.132,16,10149Fenvalerate(氰戊菊酯)27.90125.0>89.0?,125.0>89.0,167.0>89.0,167.0>125.018,18,10,32150Flumioxazin(丙炔氟草胺)27.92354.1>176.1?,354.1>312.0,354.1>326.116,8,8151Indoxacarb(二唑蟲)29.02133.9>106.0?,203.0>106.1,203.0>134.08,22,12152Deltamethrin(溴氰菊酯)29.07181.0>152.1?,252.8>92.9,252.8>172.022,16,8

*quantitative ion pairs(定量離子對)

圖1 不同提取條件下152種農(nóng)藥回收率(10 μg/kg)Fig.1 152 Pesticides recoveries under different extraction conditions(10 μg/kg)

2 結(jié)果與討論

2.1 QuEChERS法優(yōu)化

2.1.1 提取與凈化本研究采用蔬果中農(nóng)殘檢測常用溶劑乙腈[13]為提取劑，考察了樣品與乙腈用量不同比例(料液比)對結(jié)果的影響，發(fā)現(xiàn)料液比為1∶1(即5.0 g樣品加5.0 mL乙腈)時(shí)的提取效果最好，可滿足樣品均勻性、代表性及后續(xù)步驟所需。另外，實(shí)驗(yàn)還考察了樣品萃取時(shí)不加鹽、加入氯化鈉或緩沖鹽(檸檬酸緩沖鹽)、凈化劑用量(1倍量)(2.0 mL萃取液)對結(jié)果的影響(見圖1)。由圖可見，檸檬酸緩沖鹽會(huì)使溶液酸堿平衡，比只用NaCl萃取液鹽析的效果更好，且農(nóng)藥的回收率更高；凈化劑用量對測定結(jié)果也有較大影響，隨著凈化劑用量的增大，色素類雜質(zhì)去除明顯，干擾峰減少，但農(nóng)藥的回收率降低，上清液量減少，綜合考慮，選擇1倍量的凈化試劑較適宜。

2.1.2 QuEChERS法驗(yàn)證實(shí)驗(yàn)取3組空白菜心樣品各6份，每份5.0 g，152種農(nóng)藥的加標(biāo)濃度為20、50、100 μg/kg，在優(yōu)化條件下測定。結(jié)果顯示，152種農(nóng)藥(包括異構(gòu)體)在3個(gè)濃度水平下的加標(biāo)回收率為68.6%～106%，相對標(biāo)準(zhǔn)偏差(RSD)為1.3%～14%(見表2)，表明經(jīng)QuEChERS方法提取凈化后的效果較好，滿足實(shí)驗(yàn)要求。

表2 QuEChERS法凈化條件下菜心中152種農(nóng)藥在3個(gè)添加水平下的回收率統(tǒng)計(jì)結(jié)果Table 2 The statistical results of recoveries of 152 pesticides in Chinese cabbages at 3 levels by QuEChERS

2.2 DLLME法優(yōu)化

2.2.1 萃取劑與分散劑選擇實(shí)驗(yàn)選取輕質(zhì)溶劑(正己烷、環(huán)己烷、甲苯)、三氯甲烷(CHCl3)、四氯化碳、二硫化碳、正辛醇、氯苯為萃取劑進(jìn)行DLLME實(shí)驗(yàn)，比較了萃取體系中萃取劑聚集成的液滴狀態(tài)、體積穩(wěn)定性、取出便利性和加標(biāo)回收率，結(jié)果發(fā)現(xiàn)以CHCl3為萃取劑時(shí)的效果最好。另外，實(shí)驗(yàn)還比較了丙酮、乙腈、二氯甲烷、乙酸乙酯為分散劑時(shí)的分離效果，發(fā)現(xiàn)采用乙腈時(shí)效果最佳。因此，本研究中乙腈既是QuEChERS法萃取溶劑，又是DLLME分散劑。

圖2 1.0 mL萃取液在不同水體積中不同萃取劑量DLLME的152種農(nóng)藥回收率(10 μg/kg)Fig.2 The recoveries of 152 pesticides of DLLME from 1.0 mL extract with different extraction doses in different water volumes(10 μg/kg)

2.2.2 DLLME萃取體系組成比例DLLME體系中分散劑、萃取劑和水的比例不同將影響農(nóng)藥組分萃取效率。分別在2～6 mL純水和1.0 mL加標(biāo)乙腈萃取液(分散劑)的不同比例體系中加入50 μL 或100 μL萃取劑CHCl3進(jìn)行DLLME實(shí)驗(yàn)，結(jié)果如圖2。實(shí)驗(yàn)顯示4 mL水中添加1.0 mL萃取液和50 μL CHCl3于DLLME效果最佳。

2.3 GC-MS/MS條件的優(yōu)化

2.3.1 質(zhì)譜條件的優(yōu)化以1.00 mg/L的152種農(nóng)藥標(biāo)準(zhǔn)液進(jìn)行GC-MS/MS檢測，以各組分實(shí)際出峰時(shí)間校準(zhǔn)和調(diào)整儀器方法參數(shù)，使各組分盡可能分離良好；利用儀器全自動(dòng)SRM離子對優(yōu)化軟件(AutoSRM)功能，優(yōu)化組分的特征母離子和子離子對及其碰撞能量，提高農(nóng)藥組分定性和定量準(zhǔn)確性。經(jīng)優(yōu)化調(diào)整質(zhì)譜參數(shù)后，152種農(nóng)藥組分(100 μg/L)的SRM譜圖見圖3。

圖3 152種農(nóng)藥標(biāo)準(zhǔn)的SRM(選擇反應(yīng)監(jiān)測)色譜圖(100 μg/L)Fig.3 SRM chromatogram of 152 pesticide standards(100 μg/L)

2.3.2 基質(zhì)效應(yīng)基質(zhì)效應(yīng)(Matrix effects，ME)[17]是指樣品中非待測物在分析過程中對待測物檢測有顯著干擾并影響結(jié)果準(zhǔn)確性的現(xiàn)象。減少M(fèi)E的方法有基質(zhì)凈化法、同位素內(nèi)標(biāo)法、掩蔽劑補(bǔ)償法[10,17]、空白基質(zhì)配制標(biāo)準(zhǔn)溶液等[17]。用基質(zhì)匹配的校準(zhǔn)曲線斜率(A)與其純?nèi)軇┑臉?biāo)準(zhǔn)曲線斜率(B)進(jìn)行比較，若A/B值在0.8～1.2之間為弱ME。實(shí)驗(yàn)結(jié)果發(fā)現(xiàn)，不采用基質(zhì)匹配時(shí)不足30%農(nóng)藥的A/B值在上述范圍內(nèi)，而經(jīng)基質(zhì)匹配標(biāo)準(zhǔn)溶液校準(zhǔn)后，A/B值在0.8～1.2范圍內(nèi)的農(nóng)藥達(dá)92.0%，表明基質(zhì)效應(yīng)被有效控制，故本方法采用空白基質(zhì)匹配標(biāo)準(zhǔn)溶液繪制標(biāo)準(zhǔn)曲線。

2.4 方法學(xué)考察

2.4.1 方法校準(zhǔn)曲線及定量下限用空白樣品基質(zhì)溶液配制152種農(nóng)藥(包括異構(gòu)體)的質(zhì)量濃度分別為5.0、10、20、50、100、150、200 μg/L的標(biāo)準(zhǔn)溶液，在優(yōu)化條件下測定。結(jié)果顯示，各組分峰面積(y)與其質(zhì)量濃度(x,μg/L)在5.0～200 μg/L范圍內(nèi)呈良好的線性關(guān)系，相關(guān)系數(shù)均在0.995以上，計(jì)算得方法的檢出限(LOD，S/N=3)和定量下限(LOQ，S/N=10)分別為0.14～2.3 μg/kg和0.45～7.7 μg/kg，結(jié)果見表3。

2.4.2 準(zhǔn)確度與精密度取空白樣品(梨)勻漿3組每組6份，每份樣品5.0 g，152種農(nóng)藥的加標(biāo)濃度為5.0、10.0、20.0 μg/kg，在優(yōu)化條件下測定(表3)。結(jié)果顯示，152種農(nóng)藥在3個(gè)加標(biāo)水平下的回收率為60.6%～119%，RSD為1.5%～20%，其中樣品平均加標(biāo)回收率在70%～120%范圍內(nèi)的農(nóng)藥種數(shù)達(dá)133種，占總組分?jǐn)?shù)87.5%。

表3 152種農(nóng)藥的線性方程、相關(guān)系數(shù)、檢出限、定量下限及梨樣品在3個(gè)添加水平的回收率和相對標(biāo)準(zhǔn)偏差(RSDs)Table 3 Linear equations,correlation coefficients(r2),limits of detections(LODs),limits of quantification(LOQs),recoveries and relative standard deviations(RSDs) of 152 pesticides in pears at three levels

(續(xù)表3)

No.CompoundLinearequationr2LOD(μg/kg)LOQ(μg/kg)Recoveries(RSD,n=6)/%5.0μg/kg10.0μg/kg20.0μg/kg33Dimethenamidy=4.36×104x+2.56×1040.99850.301.076.4(5.7)84.5(5.7)88.1(9.9)34Chlorpyrifos-methyly=4.87×104x-8.82×1040.99930.411.468.6(7.3)80.9(12)86.2(13)35Parathion-methyly=4.85×104x-1.10×1050.99880.331.168.0(7.2)79.6(12)85.0(11)36Tolclofos-methyly=1.87×104x+2.59×1030.99840.511.767.0(12)75.5(6.8)69.5(9.9)37Pirimicarby=1.92×102x-1.27×1020.99741.55.072.9(16)81.4(3.9)87.8(5.7)38Prometryny=2.15×104x+6.11×1030.99840.742.567.6(5.7)70.9(6.5)69.9(5.7)39Metalaxyly=2.58×104x+2.14×1040.99950.571.968.0(7.5)77.8(17)91.2(10)40Terbutryny=2.82×104x+4.96×1030.99910.722.463.2(10)70.3(7.7)73.2(2.9)41Fenchlorfosy=4.38×104x-6.31×1040.99940.301.071.0(10)81.1(9.2)87.3(12)42Fenitrothiony=3.45×104x-7.84×1040.99910.291.071.6(9.3)78.4(6.4)89.5(6.2)43Pirimiphos-methyly=1.79×104x+5.78×1030.99960.431.478.3(12)88.5(6.8)89.6(6.9)44Carbofuran-3-hydroxyy=9.67×102x-2.81×1020.99981.55.064.3(10)79.3(6.9)75.6(13)45Tetrachlorobiphenyl1(PCB52)y=7.16×104x+7.24×1040.99940.200.6665.8(17)72.6(1.6)78.7(12)46Dimethylvinphosy=2.49×104x-6.79×1040.99860.391.389.2(6.2)97.2(6.3)100(16)47Malathiony=5.92×104x-2.20×1050.99770.291.071.4(14)79.9(6.2)88.9(9.0)48Diethofencarby=3.66×104x-2.83×1040.99920.341.166.2(15)72.8(7.2)83.3(12)49Acetochlory=9.39×103x-4.37×1030.99900.481.668.7(5.4)75.7(12)97.7(14)50Metolachlory=1.53×105x+9.90×1020.99830.291.067.7(14)76.4(6.5)82.1(9.7)51Carbaryly=2.20×103x+4.97×1030.99911.65.571.9(15)72.3(6.9)68.5(11)52Fenthiony=2.91×104x-3.30×1040.99860.321.174.5(6.9)84.4(10)84.1(6.0)53Methiocarby=8.56×103x+3.67×1030.99951.75.881.4(15)80.0(6.2)75.3(18)54Chlorpyrifos-ethyly=3.81×104x+4.75×1020.99880.230.7576.0(10)83.4(18)83.2(9.2)55Parathiony=2.87×104x-5.77×1030.99991.13.792.0(11)96.8(2.9)104(14)56Tetraconazoley=1.07×104x-1.49×1040.99931.96.583.6(2.0)90.3(5.2)98.3(12)57Fosthiazatey=1.01×103x-2.93×1030.99871.55.174.7(16)75.9(10)69.5(7.0)58Diphenamidy=1.91×104x+1.79×1040.99950.481.664.2(8.5)71.6(7.0)70.7(17)59Aldriny=8.87×103x+1.66×1040.99930.642.163.2(10)73.6(9.3)72.6(5.6)60Cyprodinily=5.91×104x+7.36×1040.99910.341.162.0(17)78.3(13)69.9(18)61Chlorfenvinphos-E-transy=3.68×104x-4.15×1040.99940.421.480.7(5.0)87.7(4.9)89.7(10)62Dimethametryny=5.00×104x+3.50×1040.99920.411.464.9(14)76.4(10)68.2(7.6)63Heptachlorepoxidey=1.52×104x+2.34×1040.99890.672.261.6(5.0)72.0(16)68.7(19)64Oxychlordaney=2.70×104x+3.61×1040.99930.531.861.7(6.0)72.8(15)65.0(18)65Trichlorfony=6.89×103x-1.49×1030.99971.24.086.0(19)91.5(4.5)91.8(7.8)66Isofenphosy=6.95×104x-1.70×1040.99790.270.8967.9(11)78.9(12)72.2(19)67Chlorfenvinphos-Z-cisy=3.47×104x-4.07×1040.99970.461.578.0(7.6)84.0(4.0)79.2(12)68Isocarbophosy=1.15×104x+1.45×1040.99941.24.068.3(11)78.7(9.0)86.8(15)69Isofenphos-methyly=1.36×103x+2.86×1020.99982.37.768.8(12)80.1(9.3)80.4(17)70Esprocarey=1.90×105x+4.13×1020.99930.371.274.6(19)91.9(5.1)86.6(17)71Dimepiperatey=1.83×105x+5.69×1040.99970.220.7469.9(14)79.3(8.9)64.8(16)72Phoximy=1.09×104x+6.93×1030.99951.03.397.0(6.4)111(15)89.3(12)73Quinalphosy=1.73×105x-1.52×1050.99940.190.6379.8(16)84.4(4.6)92.9(11)74Phenthoatey=1.54×104x-3.86×1040.99890.501.776.0(7.9)82.7(7.8)85.7(14)75Methidathiony=1.10×105x-3.37×1050.99860.301.068.0(11)79.5(16)83.7(12)76Propaphosy=6.32×104x-3.87×1040.99980.702.372.1(12)79.7(7.5)78.1(14)77α-cis-Chlordaney=2.96×104x+5.16×1040.99890.732.460.6(3.5)71.5(14)75.5(8.1)78o,p′-DDEy=1.04×105x+1.26×1050.99910.190.6465.1(5.2)66.3(5.0)66.1(14)79Fenothiocarby=6.04×104x+5.29×1040.99940.461.577.6(10)82.2(7.8)78.0(19)80Paclobutrazoly=4.44×104x-9.09×1040.99850.642.168.0(18)75.8(4.0)78.4(15)81Pentachlorobiphenyl1(PCB101)y=4.76×104x+3.59×1030.99990.260.8661.0(10)75.5(14)76.6(16)82Tetrachlorvinphosy=1.22×104x-6.38×1040.99550.662.282.8(4.5)91.8(14)93.4(6.5)83Endosulfan1y=1.38×104x+1.39×1040.99920.451.563.8(8.4)73.1(11)71.2(7.8)84γ-trans-Chlordaney=2.08×104x+1.64×1040.99981.13.665.5(7.6)76.4(10)69.7(11)85Fenamiphosy=1.90×104x-3.71×1040.99930.431.472.1(15)81.8(4.2)80.5(19)86Butamifosy=2.61×104x-1.68×1040.99960.431.481.4(17)88.2(1.9)93.9(17)87Napropamidey=5.97×104x+4.40×1040.99910.230.7565.3(19)77.1(6.6)78.2(18)88Prothiofosy=3.31×104x+1.28×1040.99940.692.371.6(7.1)72.5(16)78.0(15)89Metominostrobin-Ey=5.28×104x+6.36×1040.99930.571.972.7(18)77.3(6.9)81.8(11)

(續(xù)表3)

No.CompoundLinearequationr2LOD(μg/kg)LOQ(μg/kg)Recoveries(RSD,n=6)/%5.0μg/kg10.0μg/kg20.0μg/kg90Profenofosy=1.68×104x-5.06×1040.99860.451.570.7(13)83.8(7.5)94.9(14)91Pretilachloy=1.02×104x+6.24×1030.99950.882.964.8(4.8)71.3(10)75.6(9.1)92Butachlory=1.91×104x+5.24×1030.99980.441.577.7(16)89.2(1.6)82.5(13)93p,p′-DDEy=1.82×105x+2.66×1050.99840.250.8565.9(6.3)65.7(14)69.3(7.4)94Dieldriny=1.20×104x+7.84×1030.99940.742.563.5(15)69.3(8.2)73.3(9.1)95p,p′-DDDy=1.75×105x+2.79×1050.99940.250.8275.0(16)61.5(13)75.8(15)96Flusilazoley=1.74×104x-1.67×1040.99961.13.664.3(15)75.2(2.8)76.0(18)97Cyproconazoley=5.17×104x-9.44×1040.99880.481.662.7(19)70.4(7.1)79.1(16)98Endriny=8.28×103x+4.06×1030.99960.491.671.1(3.7)75.2(7.3)79.7(6.4)99Fenoxanily=7.96×103x-1.71×1040.99910.361.270.5(17)81.5(4.6)84.1(18)100Chlorobenzilatey=1.47×105x-4.09×1030.99960.260.8874.4(11)82.8(10)79.9(16)101Endosulfan2y=1.87×104x+7.08×1030.99950.521.766.2(12)76.1(6.9)74.2(16)102Fensulfothiony=3.50×103x-1.52×1030.99910.752.595.3(12)92.3(13)90.1(3.9)103o,p′-DDDy=3.26×105x+2.34×1050.99980.170.5869.8(17)72.0(14)82.8(18)104o,p′-DDTy=1.23×105x-8.32×1040.99980.541.866.0(6.9)76.4(7.5)63.6(10)105Ethiony=5.21×104x-2.18×1030.99980.672.273.8(2.4)80.6(4.4)89.8(13)106Nonachlor-transy=9.52×103x+1.33×1040.99900.782.663.5(5.0)74.8(11)75.9(5.7)107EndrinAldehydey=5.06×103x+5.56×1030.99950.581.969.7(10)79.7(6.3)79.7(8.4)108Hexachlorobiphenyl1(PCB138)y=5.95×104x+5.58×1040.99970.220.7471.2(20)73.6(8.2)84.0(10)109Mepronily=3.18×105x+9.51×1040.99960.612.078.9(14)85.2(2.1)93.3(9.6)110Triazophosy=4.31×104x-1.12×1050.99860.491.687.5(14)93.5(4.2)100(14)111Benalaxyly=5.59×104x+5.22×1040.99950.301.093.7(15)91.8(7.1)93.4(17)112Carfentrazon-ethyly=4.22×103x-2.74×1030.99990.521.785.1(9.1)98.6(9.3)101(14)113Edifenphosy=2.23×104x-1.05×1050.99680.481.685.6(7.9)92.4(6.6)90.9(16)114Propiconazole1y=2.55×104x+1.28×1040.99960.682.380.6(6.9)87.0(11)89.3(8.2)115Endosulfansulfatey=1.57×104x-4.20×1040.99880.371.263.6(4.7)76.0(19)71.8(18)116p,p′-DDTy=7.00×104x-2.30×1050.99820.712.462.5(8.9)68.5(5.7)62.4(4.5)117Propiconazole2y=3.32×104x+3.21×1030.99980.672.269.3(12)76.2(17)77.9(14)118Hexachlorobiphenyl(PCB153)y=3.68×104x+3.22×1040.99970.150.5067.6(16)78.6(13)89.3(11)119Thenylchlory=4.30×104x+7.42×1030.99960.411.481.0(10)89.4(6.9)91.0(14)120Tebuconazoley=3.59×104x-6.94×1040.99860.401.370.0(9.7)79.5(7.1)85.8(11)121Bioresmethriny=5.26×104x+5.12×1040.99950.441.564.7(8.6)67.3(15)61.8(11)122Endrin-ketoney=6.14×103x-1.06×1030.99960.401.362.5(4.1)73.1(19)62.7(5.6)123Pyridaphenthiony=1.51×104x-3.67×1040.99900.541.895.8(2.6)96.6(9.1)115(16)124EPNy=4.78×104x-8.87×1040.99950.301.077.7(16)84.5(4.8)87.2(4.4)125Bifenthriny=2.22×105x+2.90×1050.99910.210.7170.8(17)85.4(8.0)85.4(14)126Piperophosy=2.96×104x-6.05×1040.99930.411.491.7(17)82.7(17)91.3(12)127Methoxychlory=4.11×104x-1.15×1050.99880.501.769.8(4.2)90.7(17)75.4(7.1)128Fenpropathriny=5.36×104x-1.64×1040.99930.571.965.2(3.7)80.4(17)79.7(13)129Etoxazoley=2.69×104x+5.09×1030.99930.521.771.4(11)81.7(9.4)84.0(17)130Tebufenpyrady=4.21×104x+3.75×1040.99950.331.184.8(9.0)92.5(2.1)101(15)131Anilofosy=1.37×104x-5.49×1040.99750.461.584.3(5.5)99.1(9.0)93.6(9.0)132Heptachlorobiphenyl1(PCB180)y=3.36×104x+2.58×1040.99990.150.5067.6(17)72.3(9.7)81.0(11)133Azinphos-methyly=3.88×103x-4.85×1030.99911.55.194.4(16)118(15)103(14)134Phosaloney=6.39×104x-2.32×1050.99760.361.276.8(4.7)91.7(13)83.7(15)135Mirexy=1.58×104x+1.58×1050.99960.220.7263.3(12)75.5(18)65.9(17)136Iambdacyhalothriny=3.82×104x-7.01×1040.99950.180.5976.1(15)76.8(11)76.7(13)137Pyraclofosy=1.67×103x-7.54×1030.99711.75.795.9(14)99.4(5.7)104(11)138Etobenzanidy=3.23×104x-3.75×1040.99971.44.6113(10)119(5.1)114(9.0)139Butafenacily=1.22×105x+7.31×1040.99990.150.5085.1(2.8)89.1(2.9)95.8(9.6)140Cyfluthrin1y=8.37×103x-9.03×1030.99970.431.471.2(5.0)82.6(9.6)66.8(10)141Cyfluthrin2y=1.08×104x-1.46×1040.99950.341.174.0(4.6)83.8(8.2)77.1(15)142Cyfluthrin3y=7.14×103x-4.12×1030.99970.301.074.0(3.9)82.9(5.2)76.4(16)143Cyfluthrin4y=8.84×103x-1.60×1040.99940.361.269.3(11)76.3(15)79.3(8.8)144Cypermethrin1y=9.47×103x-1.42×1030.99950.531.877.9(12)74.9(13)85.1(7.3)145Cypermethrin2y=9.81×103x-8.73×1030.99930.762.571.1(11)72.4(12)70.2(10)

(續(xù)表3)

No.CompoundLinearequationr2LOD(μg/kg)LOQ(μg/kg)Recoveries(RSD,n=6)/%5.0μg/kg10.0μg/kg20.0μg/kg146Cypermethrin3y=9.29×103x-8.19×1030.99970.461.574.0(14)71.3(12)79.4(7.4)147Cypermethrin4y=8.20×103x-1.80×1030.99990.491.668.9(8.6)75.3(7.9)81.4(15)148Etofenproxy=7.61×104x+1.37×1040.99990.381.376.9(4.8)84.9(7.5)86.1(12)149Fenvaleratey=1.93×104x-2.84×1040.99950.290.9870.7(1.5)81.2(4.9)80.1(13)150Flumioxaziny=4.34×103x-4.69×1030.99980.602.077.1(8.8)83.4(5.5)85.6(15)151Indoxacarby=6.62×103x-8.45×1030.99961.24.192.5(13)93.4(5.1)95.4(11)152Deltamethriny=1.36×104x-1.35×1040.99970.421.470.1(7.2)79.7(4.7)79.1(16)

the linear range of the standard curve is 5.0-200 μg/L(標(biāo)準(zhǔn)曲線線性范圍是5.0～200 μg/L)

2.5 實(shí)際樣品檢測

應(yīng)用本文建立的方法對超市和農(nóng)貿(mào)市場的蔬果樣品進(jìn)行檢測，包括26種蔬菜(油麥菜、西藍(lán)花、苦瓜、小白菜、番茄、四季豆、胡蘿卜、紅薯、通菜、卷心菜、茄瓜、圣女果、南瓜、芋頭、洋蔥、韭黃、菠菜、花椰菜、甜椒、韭菜花、豇豆、芡實(shí)、山藥、蒜薹、菜心、芥藍(lán))和14種水果(蘋果、梨、桃、藍(lán)莓、葡萄、獼猴桃、香蕉、櫻桃、柑橘、李子、橙子、哈密瓜、柿子、火龍果)，各30份樣品。結(jié)果顯示，有24份蔬菜樣品共檢出31種農(nóng)藥殘留，占總152種農(nóng)藥的20.4%；檢出含量為1.5～2 649.8 μg/kg，其中含量低于50 μg/kg的占70.7%；有3份蔬菜樣品中的農(nóng)殘超標(biāo)：菠菜中氟硅唑含量為1.23 mg/kg；苦瓜中氯氰菊酯(異構(gòu)體之和)為0.93 mg/kg；油麥菜中戊唑醇為2.65 mg/kg，此外還檢出氟硅唑、丙環(huán)唑等農(nóng)藥(見圖4)。有25種水果樣品檢出農(nóng)殘，涉及20種農(nóng)藥組分，占農(nóng)藥總數(shù)13.2%；沒有水果樣品農(nóng)藥超標(biāo)，檢出范圍是1.7～325.5 μg/kg，含量低于50 μg/kg的占91.2%。

圖4 油麥菜SRM色譜圖Fig.4 SRM chromatogram of leaf lettuce

3 結(jié) 論

采用優(yōu)化QuEChERS法和DLLME法聯(lián)合處理樣品，使樣品農(nóng)殘的萃取效率、液滴穩(wěn)定性和易操作性好，目標(biāo)物濃縮倍數(shù)高，檢測更靈敏準(zhǔn)確；這兩種前處理方法與優(yōu)化的GC-MS/MS方法聯(lián)用構(gòu)建的高通量農(nóng)藥多殘留檢測方法，更經(jīng)濟(jì)、高效且操作簡單,能較好地滿足新鮮蔬果中農(nóng)藥殘留的快速篩查和應(yīng)急檢測的需要。