Zhen LlN，Jian CHEN，Jie Ll，Meixing HUl，Jinchang LAN，Jinbin JlANG，F(xiàn)ang YANG*

1.Fujian Vocational College of Bioengineering，F(xiàn)uzhou 350002，China；

2.Fujian Entry-exit Inspection and Quarantine Bureau，F(xiàn)uzhou 350003，China；

3.Fujian Agriculture and Forestry University，F(xiàn)uzhou 350002，China

Simultaneous Determination of 8 Pesticide Residues in Green Tea Based on Gas Chromatography-mass Spectrometry

Zhen LlN1，Jian CHEN2，Jie Ll2，Meixing HUl3，Jinchang LAN2，Jinbin JlANG2，F(xiàn)ang YANG2*

1.Fujian Vocational College of Bioengineering，F(xiàn)uzhou 350002，China；

2.Fujian Entry-exit Inspection and Quarantine Bureau，F(xiàn)uzhou 350003，China；

3.Fujian Agriculture and Forestry University，F(xiàn)uzhou 350002，China

To improve pesticide residues detection efficiency in tea，simultaneous determination of eight pesticide residues in green tea was developed based on gas chromatography-mass spectrometry （GC-MS）.The results showed that，detected by the GC-MS under following conditions:acetonitrile as extraction and elution solvent，activated carbon and PSA tandem column as the stationary phase，elution volume of 12 ml the relationship between peak area and concentration of each residue（ametryn in the range of 0-100 μg/L，napropamide in the range of 0-10 000 μg/L，other pesticides in the range of 0-1 000 μg/L）were linearly related，and their correlation coefficients were all greater than 0.999.The recovery rate the pesticides added to tea sample at legal concentration limits ranged from 73.6%to 116.8%，and the relative standard deviation from 1.47%to 15.58%.The new method we developed is sensitive，specific and anti-interference，and thus provides test basis for the development of pesticide matrix reference materials in tea.

Tea；Pesticide residues；Gas chromatography-mass spectrometry

T ea is one of the most important agricultural products and a traditional major export commodity in China.At present，Chinese tea export faces severe challenges，because new laws and regulations were published in European Union and Japan and other countries and regions，which formulated the most detection terms and strictest standard for the maximum residue limits（MRT）in pesticides［1-2］.To meet the world tea market demands，rapid method for detecting multi-pesticide residues should be developed in addition to decreasing pesticide residues and modifying the current industry standards［3］. Currently，simultaneous detection of multi-pesticide residues in tea has become a hot issue［4-5］.Previous papers were most about the simultaneous detection of pesticide residues with similar structure or those used to the same pest，less on the simultaneous detection of common pesticides，fungicides and herbicides［6-7］.Therefore，in this study，eight agrochemicals including two amide herbicides（acetochlor and napropamide），one triazine herbicide（ametryn），one pyrethroid pesticide（bifenthrin），one aniline fungicide（metalaxyl），two triazole fungicides（triadimefon，diniconazole）and strobilurin fungicide（trifloxystrobin）were selected as detection objectives based on our earlier work.Then，solid phase extraction technology was adopted to purify tea extract，before simultaneous detection of the pesticides from tea using GC-MS-MS detector.The results proved that the method is highly sensitive，specific and anti-interference［8-14］.

Materials and Methods

Time and place

The study was conducted in Fujian Entry-Exit Inspection and Quarantine from May，2013 to March，2014.

Materials

Activated carbon column was purchased from CNW Technologies，Germany.C18，PSA，silica gel and Floish were purchased from SUPELCO，USA.Chromatographically pure acetonitrile was purchased from Merck KCaA，Germany.Ethyl acetate，acetone，n-hexane，all of which were analytical reagents and aluminum oxide（100-200 mesh）were all bought from Shanghai Shi Yi Chemical Reagent Co.Ltd.，China.

Eight pesticide standards including acetochlor，napropamide，ametryn，bifenthrin，metalaxyl，triadimefon，diniconazole and trifloxystrobin were purchased from Dr.Ehrenstorfer GmbH，Germany.

The main instruments used in this studyincluded Agilent7890A GC 5975C Inert XL MSD triple axis detector，equipped with electron bombardment ion source （EI），PHILIPS highspeed grinder and HGC-12A nitrogen blowing instrument（Tianjin Heng Ao Technology Development Co.，Ltd）.

Methods

Experimental design By screening appropriate extraction solvents，stationary phase，eluents and elution volume，a method for simultaneously detecting eight stable and representative pesticides:acetochlor，napropamide，ametryn，bifenthrin，metalaxyl，triadimefon，diniconazole and trifloxystrobin in green tea can be established based on gas chromatography-mass spectrometry.Meanwhile，the linear regression，detection limit，recovery rate and precision ofthis system should also be proven.

Chromatographic conditions Agilent DB-5 capillary column（0.25 mm× 30 m，0.25 μm）was used.Column temperature was maintained at 80℃for 1 min，and was raised to 200℃at a rate of 10℃/min，then to 280℃at a rate of 25℃/min，and maintained at 280℃for 14 min.Samples were totally eluted out within 30.2 min.The carrier gas（He）flowed through the column at a rate of 1.0 ml/min.The injection volume was 1 μl（splitless）.External-standard high-performance liquid chromatographic method was adopted for quantitative determination.

Mass spectrometry parameters

課程結(jié)束后，我為媽媽拍了幾張照片，媽媽很滿意，還表?yè)P(yáng)我很會(huì)抓拍，人物的表情十分自然。我喜歡攝影，因?yàn)?，它可以把平凡的事物變得更美麗、更溫馨。（指?dǎo)老師：張莉敏）

Mass spectrometry parameters were set as follows:electron impact（EI）ionization at 70 eV；transfer line temperature at 270℃；ion source temperature at 260℃；full scan mode from 50 to 300 m/z；data acquisition mode:selected ion monitoring （SIM）.The detection parameters were described in Table 1.

Pretreatment Tea sample（1 g）was precisely weighed，fine milled and transferred to a 50 ml centrifuge tube，mixed with 1 ml of saturated NaCl，added to 10 ml extract，shaken for 30 min，before being centrifuged to collect the supernatant.Above steps were repeated.The two extracts were bulked，and dried by N2blowing.Then，about 2 ml of eluent was added before being oscillated on an oscillator.The solution sample was transferred to a purification column and eluted.Finally，12 ml of eluent was collected，dried by N2blowing，and diluted to 1 ml with acetonitrile，filtered through organic filter film，and detected with a mass spectrometer.

Statistical analysis Data processing and analysis were finished using Agilent GC-MS system and Excel.

Table 1 Detection parameters for eight pesticides in mass spectrometry

Table 2 The recovery rates of target pesticides extracted using different solvents %

Results and Analysis

Selection of extraction solvents

It was not necessary to consider the purification process to remove lipid，as there is no lipid in tea sample. Therefore，one suitable solvent that can be used to effectively extract the pesticides is enough.Acetonitrile，ethyl acetate，n-hexane and acetone were selected as the solvents to extract the eight pesticides，all of which were weakly polar，by referring to the references［15-22］.

In detail，tea samples added with 100 μg/kg pesticide mixture were extracted with acetonitrile，mixture of ethyl acetate+n-hexane （1∶1），and mixture of acetone+n-hexane （1∶2），separately.The detection results were shown in Table 2.

As shown in Table 2，the recovery rate of the eight pesticides using the mixture of ethyl acetate and n-hexane ranged from about 54%to 103%，among which that of acetochlor and diniconazole was lower，and the extraction of metalaxyl was interfered. The recovery rates of acetochlor，napropamide and ametryn using the mixture of acetone and n-hexane were all below 73%，and that of diniconazole was very low，only about 8%，the extraction of metalaxyl using the mixture of acetone and n-hexane was also in-terfered.Acetonitrile had a relative high recovery rate to metalaxyl，and recovery rates ranging from 88%to 108%to the other seven pesticides.In summary，acetonitrile was the optimal solvent to extract the eight pesticides.

Selection of the stationary phase

Activated carbon，C18，PSA，silicone，F(xiàn)loish and C18+Al2O3（acidic Al2O3powder added to the C18 column，about 1 cm in height）were compared for their recovery and purification efficiency as stationary phase.The results revealed that the activated carbon had the best efficiency in pesticides recovery and tea pigment removal［23］.In addition，PSA could effectively remove organic acids，sugars，metal ions and part of polar pigments［24］.So，activated carbon and PSA were equipped in tandem in column to purify the pesticides in tea in this study.The combined use of activated carbon and PSA exhibited a better effect in pesticide purification. The eluent was colorless or pale yellow，so that the qualitative and quantitative determination was more accurate.

Selection of elution solvents Acetonitrile，the mixture of ethyl acetate+ n-hexane 1∶1），and the mixture of acetone+n-hexane（1∶2）were compared for their elution efficiency.The results showed that the elution of metalaxyl was interfered by the mixture of ethyl acetate+hexane and the mixture of acetone+n-hexane，while the recovery rate of the eight pesticides reached a range of 90%-130%using acetonitrile，so acetonitrile was finally selected as the eluent.

Elution volume Teat samples were purified using PSA+activated carbon as the stationary phase and acetonitrile as the elution solvent.Eluent was finally collected into 10 tubes，3 ml in each，and detected with a mass spectrometer.The elution curves of the eight pesticides are shown in Fig.1.

Acetonitrile had the best elution efficiency in acetochlor，metalaxyl and triadimefon.50%or more of the three pesticides were eluted into the first 3 ml of acetonitrile，and more than 99% of acetochlor，napropamide，ametryn，metalaxyl and triadimefon，more than 98% of bifenthrin and trifloxystrobin but only about 49%of diniconazole were eluted into the first 6 ml of eluent. 94%of diniconazole was recovered using 12 ml of eluent.Therefore，12 ml was considered as the optimal volume of eluent.

In summary，acetonitrile as the extraction and elution solvent，tandem column of activated carbon and PSA as the stationary phase and eluent volume of 12 ml were selected as the best conditions for simultaneous determination of the eight pesticides in tea using GC-MS-MS assay.

Linear relationship and detection limits

Tea samples supplemented with the eight pesticides at eight different concentrations were detected under the optimal GC-MS-MS conditions determined above.Then linear regression analysis was performed using peak area versus pesticide concentration.The resulting regression equations and detection limits are listed in Table 3.The detection limits were determined at signal to noise ratio 3:1.As shown in Table 3，the peak area and mass concentration of all the eight pesticides were linearly related，and their correlation coefficients were all above 0.999.

Table 3 Regression equations and detection limits of the eight pesticides

Table 4 Recovery rate and relative standard deviation of the eight pesticides using GCMS-MS method（n=6）

Recovery and precision

Green tea samples supplemented with the eight pesticides at the concentrations as listed in Table 4 were detected using the GC-MS-MS method we developed to detect the recovery rate and precision.The measurement was repeated for 6 times.As shown in Table 4，the average recovery rates of the eight pesticides rangedfrom 73.6%to 116.8%，all were acceptable except the recovery rate of trifloxystrobin.The relative standard deviations（RSD）of bifenthrin and triadimefon were below 2%，that of ametryn and metalaxyl around 4%，and that of the other four pesticides ranged from 13%to 16%.Such differences may be resulted from their concentration in the samples，because the detection for a large concentration was stable and the RSD was small.On the contrary，the RSD for a small concentration was large.

Conclusion and Discussion

A system for simultaneous determination of eight pesticides of six types using GC-MS-MS was determined:acetonitrile as the extraction and elution solvent，tandem column of activated carbon and PSA as the stationary phase and eluent volume of 12 ml in this study.It was proven that the peak area and mass concentration at a certain range of all the eight pesticides were linearly related，and their correlation coefficients were all above 0.999；the recovery rates of the pesticides reached 73.6%-116.8%；and the relative standard deviation ranged from 1.47%to 15.58%.

The system we developed is sensitive，specific，anti-interference and accurate，and thus can be used to screen and detect green tea samples in bulk.But it still has some disadvantages.First of all，the method was developed based on green tea，whether it is can be used to detect other types such as black tea and oolong tea remains unclear.Second，the traditional method for tea sample pretreatment is time-consuming and labor-intensive. Therefore，we plan to introduce matrix solid-phase dispersion into the system，so that extraction，filtration and purification can be done in one step to shorten the detection time and increase the speed of analysis［23］.

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Responsible editor：Qingqing YlN

Responsible proofreader：Xiaoyan WU

基于氣相色譜-質(zhì)譜聯(lián)用法的綠茶8種農(nóng)藥殘留的同時(shí)檢測(cè)

林真1，陳健2，李捷2，惠美星3，藍(lán)錦昌2，江錦彬2，楊方2*（1.福建生物工程職業(yè)技術(shù)學(xué)院，福建福州 350002；2.福建出入境檢驗(yàn)檢疫局，福建福州 350003；3.福建農(nóng)林大學(xué)，福建福州350002）

為了提高茶葉農(nóng)藥殘留的檢測(cè)效率，該研究從提取溶劑的選擇、凈化條件、線性回歸、回收率及精密度等方面著手，開展了基于氣相色譜-質(zhì)譜聯(lián)用法的綠茶中8種農(nóng)藥殘留的同時(shí)檢測(cè)研究。結(jié)果表明，在以乙腈為提取溶劑、以活性炭和PSA串聯(lián)柱為固定相、以乙腈為洗脫劑、洗脫體積為12 ml的檢測(cè)條件下，莠滅凈在0～100滋g/L范圍，敵草胺在0～10 000滋g/L范圍，其余農(nóng)藥在0～1 000滋g/L范圍內(nèi)，峰面積與樣品質(zhì)量濃度呈線性關(guān)系，相關(guān)系數(shù)均大于0.999。以茶葉農(nóng)藥殘留量限值為添加量，重復(fù)測(cè)定6次，各農(nóng)藥平均加標(biāo)回收率在73.6%～116.8%，相對(duì)標(biāo)準(zhǔn)偏差范圍在1.47%～15.58%。說明該研究所建立的檢測(cè)方法靈敏度高、選擇性好、抗干擾能力強(qiáng)，可在茶葉多農(nóng)殘檢測(cè)中推廣應(yīng)用。

茶葉；農(nóng)藥殘留；氣相色譜-質(zhì)譜聯(lián)用

福建省科技項(xiàng)目“食品中有害殘留測(cè)量基體標(biāo)準(zhǔn)物質(zhì)研制及應(yīng)用”（2012Y6001）；福建省基金項(xiàng)目“基于藥物代謝的茶葉中多種農(nóng)藥殘留基體標(biāo)準(zhǔn)物質(zhì)研制”（2012J01060）。

林真（1980-），女，福建連江人，博士，從事食品安全、保健食品研發(fā)，E-mail：linzhen402@126.com。*通訊作者，E-mail：yffjciq@gmail.com。

2015-07-30

Supported by Science and Technology Program of Fujian Province-Development and Application of Matrix Reference Materials of Harmful Residues in Food（2012Y6001）；Development of Matrix Reference Materials of Multi-pesticide Residues in Tea based on Drug Metabolism（2012J01060）.

*Corresponding author.E-mail:yffjciq@gmail.com

Received:July 30，2015 Accepted:October 6，2015

修回日期 2015-10-06