Food Chemistry 210 (2016) 26–34

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Food Chemistry

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Analytical Methods

Optimized combination of dilution and refined QuEChERS to overcomematrix effects of six types of tea for determination eight neonicotinoidinsecticides by ultra performance liquid chromatography–electrospraytandem mass spectrometry

http://dx.doi.org/10.1016/j.foodchem.2016.04.0970308-8146/� 2016 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors at: No. 130, Changjiang Road, ShuShan District, HefeiCity, Anhui Province, PR China.

E-mail addresses: hry@ahau.edu.cn (R. Hou), rimaohua@ahau.edu.cn (R. Hua).1 Equal contribution.

Weiting Jiao a,1, Yu Xiao a,1, Xiaosan Qian a, Mengmeng Tong a, Yizheng Hu a, Ruyan Hou a,⇑, Rimao Hua b,⇑a State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, Hefei 230036, PR Chinab School of Resource & Environment of Anhui Agricultural University, Key Lab. of Agri-food Safety of Anhui Province, Hefei 230036, PR China

a r t i c l e i n f o

Article history:Received 29 August 2015Received in revised form 13 February 2016Accepted 20 April 2016Available online 22 April 2016

Keywords:Tea matrixMatrix effectDilutionRefined QuEChERSNeonicotinoidsLC–ESI-MS/MS

a b s t r a c t

Liquid chromatography–electrospray ionization tandem mass spectrometry (LC–ESI-MS/MS) is a primarytool for analysis of low volatility compounds in complex matrices. However, complex matrices, such asdifferent types of tea, complicate analysis through ionization suppression or enhancement. In this study,sample preparation by a refined QuEChERS method combined with a dilution strategy removed almost allmatrix effects caused by six types of tea. Tea samples were soaked with water and extracted withacetonitrile, cleaned up with a combination of PVPP (160 mg) and GCB (20 mg), and dried. Dried extractswere diluted with 20 mL acetonitrile/water (15:85, v/v) before analysis by UPLC–MS/MS. The averagerecoveries of eight neonicotinoid insecticides (dinotefuran, nitenpyram, thiamethoxam, imidacloprid,clothianidin, imidaclothiz, acetamiprid, and thiacloprid) ranged from 66.3 to 108.0% from tea samplesspiked at 0.01–0.5 mg kg�1. Relative standard deviations were below 16% for all recovery tests. The limitof quantification ranged from 0.01 to 0.05 mg kg�1.

� 2016 Elsevier Ltd. All rights reserved.

1. Introduction

Tea (Camellia sinensis L.) is classified into 6 types, according toits processing and chemical composition (Oellig & Schwack,2012; Sadowska-Rociek, Surma, & Cieslik, 2014): green tea (unfer-mented tea), black tea (fermented tea), oolong tea (semi-fermented tea), dark tea (fermented tea), white tea (slightly fer-mented tea) and yellow tea (slightly fermented tea) (Chen, Yin,Wang, Jiang, & Liu, 2014; Luo et al., 2012), which are the mosthighly consumed traditionally non-alcoholic beverage around theworld. Tea cultivation is hindered by diseases and pests, leadingto pesticide use throughout tea planting and growth (Chen et al.,2014; Zhao et al., 2013; Kopjar, Tadic, & Pilizota, 2015; Zhang &Xu, 2014). Neonicotinoid insecticides are one of the fastest growingclasses of pesticides in the past three decades, due to their broadspectrum efficacy and systemic action for crop protection againstnumerous sucking and biting insects, including aphids, whiteflies,

beetles and some lepidopteran species (Muccio et al., 2006; Hou,Jiao, et al., 2013; Zhang et al., 2013). There are several commercial-ized neonicotinoids: imidacloprid, acetamiprid, nitenpyram,thiacloprid, thiamethoxam, clothianidin, dinotefuran and imida-clothiz [1-(5-chloro-thiazolemethyl)-4,5-dihydro-N-ni-tro-1H-imidazole-2-amine], the newest synthetic neonicotinoid producedin China. Imidaclothiz has been increasingly used for controllinggreen leafhoppers, whiteflies and thrips in tea cultivation becauseof its greater systemic activity and lower acute mammalian toxic-ity (Hou, Jiao, et al., 2013; Wu, Cai, Yao, Dai, & Lu, 2010; Fang et al.,2011). As polar compounds, it is possible that neonicotinoid insec-ticides may reside on the surface of treated plants and penetrateinto the plant, increasing the chance of being found in the con-sumer product (Hou, Jiao, et al., 2013; Zhang et al., 2013; Yánez,Bernal, Nozal, Martín, & Bernal, 2013; Gbylik-Sikorska, Sniegocki,& Posyniak, 2015; Kamel, 2010;Wang et al., 2012). Thus, the devel-opment of a method capable of analyzing multiple neonicotinoidinsecticides is of great significance.

Liquid chromatography coupled to tandem mass spectrometrywith electrospray ionization (LC–ESI-MS/MS) has proven to bethe most powerful tool available for identification and quantitationof polar pesticide residues in complex mixtures, due to its

W. Jiao et al. / Food Chemistry 210 (2016) 26–34 27

precision, robustness, high sensitivity and selectivity (Ferrer,Lozano, Aguera, Giron, & Fernandez-Alba, 2011; Benijts, Dams,Lambert, & Leenheer, 2004). Despite all the advantages, the matrixeffect, which was first described by Kebarle and Tang (Kruve,Künnapas, Herodes, & Leito, 2008; Kebarle & Tang, 1993), in elec-trospray ionization (ESI) is a severely compromising phenomenon,especially for dirty or difficult matrices such as tea leaf extract(Oellig & Schwack, 2012). How the matrix influences LC–ESI-MS/MS is still not fully understood (Taylor, 2005); it has been sug-gested that there is a competition between matrix componentsand target analytes for access to the droplet surface prior to gasphase emission (Benijts et al., 2004). Some strategies to minimizematrix interference include improvement of chromatographicselectivity to avoid interference of co-extracted matrix compo-nents and modification of sample preparation with the aim toremove interfering components. When the matrix effect cannotbe reduced, some methods can compensate for matrix effects, suchas using isotopically labeled standards (ILS), matrix-matched cali-bration curves (MMCC), the ECHO peak technique, and post-column infusion (PCI). Lehotay, Mastovska, Lightfield, and Gates(2010) and Stahnke, Kittlaus, Kempe, and Alder (2012) have exten-sively discussed the compensation methods, including their disad-vantages: isotopically labeled standards are expensive and oflimited availability; MMCC requires many blank extracts; theECHO peak technique can not completely compensate matrixeffects; and PCI cannot adequately compensate in all matrices.Investigation of the relationship between the matrix concentrationand the matrix effect for four matrices (avocado, black tea, orangeand arugula) showed that an appropriate dilution factor couldreduce the matrix effects to less than 20% (Stahnke et al., 2012).Since only one type of tea (black tea) was investigated, it remainsunclear whether this method could be applied to other types of tea.

Methods combining different solid phase extraction (SPE) andd-SPE (QuEChERS) and HPLC-MS methods have been publishedfor determination of several neonicotinoid insecticide residues intea (Oellig & Schwack, 2012; Chen, Cao, & Liu, 2011; Wu et al.,2013; Xie et al., 2009; Liu et al., 2013; Hou et al., 2015; GB/T23205-2008). Both MMCC and ILS have been used to compensatefor the tea matrix effect (Chen et al., 2011; Xie et al., 2009; Houet al., 2015). Our previous study showed that different types oftea had very different matrix effects (Hou et al., 2015). AlthoughMMCC from green, black and oolong tea were separately used tocompensate for the matrix effects, the use of MMCC could notresolve the problem of the diversity of tea types. Furthermore, itis difficult to obtain tea samples for each of the different types ofteas that are truly blank for all target residues.

The objective of this work was to develop a simple, rapid andeffective method based on a combination of a refined QuEChERSand dilution method by LC–MS/MS to diminish the matrix effectsfrom six different types of tea during the determination of eightneonicotinoid insecticide residues. This method could serve as amodel procedure for the analysis of other pesticide residues in dif-ferent types of tea.

2. Materials and methods

2.1. Chemicals and reagents

All certified neonicotinoid insecticide standards, namely dinote-furan (98.6%), nitenpyram (98.6%) thiamethoxam (98.5%), clothian-idin (99%), imidacloprid (98.0%), acetamiprid (98.1%), thiacloprid(98%), as well as the internal standards thiamethoxam D4, imida-cloprid D4 and acetamiprid D3, were obtained from Dr. Ehrenstor-fer (Augsburg, Germany). Imidaclothiz (100 lg mL�1 inacetonitrile, ACN), was purchased from Agro-Environmental Pro-

tection Institute, Ministry of Agriculture (Tianjin, China). Standardstock solutions of seven insecticides (except imidaclothiz) andthree internal standards were prepared by weighing about 5 mgof each analyte and dissolving in 10 ml of acetonitrile (ACN). Work-ing standard solutions were prepared by diluting the standardstock solution with ACN:water (1:9, v/v). The solvent solutionsrequired for preparing a standard curve (0.025, 0.5, 1.25, 2.5,12.5, 25 lg L�1) were obtained from the working standard solu-tions by serial dilution. All solutions were stored at �20 �C. ACN,hypergrade (99.9%) for LC–MS, was purchased from Tedia Com-pany, OH, USA. Water used for LC–MS/MS was produced directlyin the laboratory with a Milli-Q water purification system(Millipore, Bedford, MA). Polyvinylpolypyrrolidone (PVPP) waspurchased from Solarbio Science & Technology Co., Ltd. (Beijing,China). Graphitized Carbon Black (GCB, 120/400 Mesh) wasobtained from Supelco Company (Bellefonte, PA, USA). Primarysecondary amine (PSA, 230–400 Mesh) and (C18, 230–400 Mesh,60 Å; SiliCycle, Canada) absorbents were obtained from ShanghaiANPEL Scientific Instrument Co., Ltd. Anhydrous Na2SO4 andMgSO4 were dried at 550 �C for 5 h and stored in desiccators.Ammonium formate was obtained from Anaqua Chemicals Supply,USA. A nitrogen evaporator (N-EVAP, Organomation, USA), vortexand pulverizer (MS 3 digital, and A11, respectively, both fromIKA, Germany) were used.

Six types of tea samples (Green, black, oolong, dark, white andyellow tea) were checked to be free of the neonicotinoid insecti-cides in order to provide blanks or post-spiked samples for analysisof matrix effects. Tea samples used for application of the methodcompared to the recommended national standard method (GB/T23205-2008) were purchased from local supermarkets in Hefei,Anhui province, China.

2.2. Sample preparation

Samples of green, black, oolong, dark, white and yellow teawere ground with a pulverizer and sized by 50 mesh sieve. In brief,a 1.0 g aliquot of each sieved sample (six types of tea) was mixedwith 2 mL of water, allowed to soak for 30 min, then mixed with20 mL of ACN and homogenized for 2 min. The supernatant wasobtained by filtration (through Whatman No. 1 paper) into a35 mL centrifuge tube containing 2 g anhydrous sodium sulfate.After mixing by vortex for 1 min, the samples were allowed to restin order to partition the ACN and water phases (Hou et al., 2015).

An aliquot of 2 mL of supernatant was cleaned up by a refinedQuEChERS method with 160 mg of PVPP and 20 mg of GCB in thepresence of 150 mg of anhydrous MgSO4. The extracts were shakenby vortex for 2 min and then centrifuged at 10,000 rpm for 8 min.1 mL aliquot of the supernatant was evaporated to near-drynesswith a nitrogen evaporator at 40 �C. The residue was dissolved in20 mL ACN:water (15:85, v/v), being passed through a 0.22 lmpore size filter membrane (Millipore, Billerica, MA), and finally,analyzed by LC–MS/MS.

2.3. LC–MS/MS analysis

A 6460 triple quadrupole mass spectrometer (QQQ; AgilentTechnologies, Palo Alto, CA, USA) was used. This mass spectrometerwas coupled to an Agilent Series 1290 ultra-performance liquidchromatography system (UPLC), equipped with a quaternarypump, a vacuum solvent degasser, a column oven, and an autosam-pler and controlled by MassHunter software qualitative analysisB.01.04 software.

The employed chromatographic separation and MS parametersof the quantification (primary) and qualification ion transitions(secondary) of the respective insecticides were the same as inour previous report (Hou et al., 2015). The only difference was

28 W. Jiao et al. / Food Chemistry 210 (2016) 26–34

further comparison of different mobile phase additives: formic acidand acetic acid (both at 0.02%, v/v), 5 mM ammonium formate,5 mM ammonium acetate and 5 mM ammonium acetate mixedwith 0.1% formic acid.

2.4. Dilution chart and matrix effect

The spiked level corresponds to the residue amount of0.05 mg kg�1 in all tea samples, so post-spiked solutions(2.5 lg L�1) of each of the eight neonicotinoid insecticides andthree internal standards were prepared. The residue (Section 2.2)was dissolved in 1 mL standard solution (2.5 lg L�1) prepared withACN:water (15:85, v/v) before being passed through a 0.22 lmpore size filter membrane. These spiked extracts, indicated as ‘‘di-lution factor 20” due to the sample matrix concentration(0.05 g mL�1), were ready for the next dilution step before injec-tion into LC–MS/MS.

The dilution factors ranged from 20 to 10,000 for all tea sam-ples. The 40, 100, and 200-fold diluted extracts were obtained froma volume of 500, 200 or 100 lL of the dilution factor 20 sample (seeabove) into a final volume of 1000 lL ACN:water (15:85, v/v). Thespiked extracts with dilution factors 400, 1000 and 2000 resultedfrom 1:10 dilution of these three solutions (100 lL filled to1000 lL). An analogous 1:10 dilution resulted in extracts with dilu-tion factors of 4000 and 10,000, respectively. The correspondingstandards in solvent were prepared in an identical way using1000 lL of ACN:water (15:85, v/v) instead of 1000 lL of blankextracts.

The matrix effect (ME) was described as the change of ioniza-tion efficiency in the presence of other compounds, expressed asthe response of the insecticide in matrix compared to the signalin solvent, calculated by the following equation (Stahnke et al.,2012):

ME ¼ peak area ðspiked extractÞpeak area ðsolvent standardÞ � 1

� �� 100% ð1Þ

An ME value equal to 0% means that no matrix effect existed.Positive and negative values stand for enhancements and suppres-sions, respectively, of the analyte signal by matrix compounds.

Matrix effects were classified into different categories based onthe value of this percentage. No matrix effect occurred when thevalues were between �20% and 20%, a medium matrix effect wasbetween �50% and �20% or 20% and 50%, and a strong matrixeffect was below �50% or above 50% (Ferrer et al., 2011).

2.5. Real sample analysis

Real samples were used to compare the two sample preparationmethods, the dilution method described above and the Recom-mended Chinese National Standard method (GB/T 23205-2008),as follows:

2.5.1. Sample extractionTea samples (six types of tea) were ground into powder. Tea

powder (10 g) was weighed into a 50 mL centrifuge tube, extractedwith 30 mL of acetonitrile using a homogenizer at 15,000 rpm for1 min, then centrifuged at 4200 rpm for 5 min. The entire upperlayer was transferred to a spin steaming bottle and re-extractedtwice with 30 ml and 20 mL of acetonitrile, respectively. All theupper layers were mixed into one spin steaming bottle, whichwas evaporated at 40 �C until dryness using a rotary evaporatorand a nitrogen stream. The remaining dry residue was dissolvedin 5 mL acetonitrile. Aliquots of 1 mL were used for clean-up.

2.5.2. SPE cleanupThe concentrated extract in acetonitrile (1 mL) was passed

through a Cleanet TPT SPE that had been preconditioned with5 mL acetonitrile:methylbenzene (3:1, v/v), adding 2 cm anhy-drous sodium sulfate. The sample was eluted from the TPT-SPEwith 25 mL acetonitrile:methylbenzene (3:1, v/v). The effluentwas concentrated to 0.5 mL by a rotary evaporator at 45 �C, andthen evaporated at 35 �C until dryness using a nitrogen stream.The residue was dissolved with 1 mL acetonitrile:water (3:2, v/v)and filtered with a 0.2 lm pore size filter membrane. The samplewas now ready for injection into LC–ESI-MS/MS.

3. Results and discussion

3.1. Mobile-phase additives

In LC–MS, the composition of the mobile-phase has a greatinfluence on ionization efficiency (Benijts et al., 2004; Chen et al.,2011; Hajšlová & Zrostlıkova, 2003; Pascoe, Foley, & Gusev,2001). Our previous study on neonicotinoid analysis compared fivedifferent mobile-phase additives, namely 0.1% and 0.3% formicacid, 5 mM ammonium formate, 5 mM ammonium formate with0.1% formic acid, and no additives, and showed that 5 mM ammo-nium formate performed the best (Hou et al., 2015). In this study,lower concentrations of formic acid and acetic acid (both at 0.02%,v/v, and pH 3.65 and 4.22, respectively), 5 mM ammonium acetate(pH 7.23) and 5 mM ammonium acetate mixed with 0.1% formicacid (pH 2.76) were used to investigated and supplemented toour previous study further (Hou et al., 2015; Chen et al., 2011)for determination of neonicotinoid insecticides, and 5 mmol/Lammonium formate was compared again. As shown in Fig. 1, thehighest signals were achieved for all insecticides in the buffer. Add-ing 5 mM ammonium formate (pH 6.98) in the phase A resulted ina 5 to 32-fold higher response than using formic acid or acetic acidin the mobile phase. The signals of all the insecticides were alsohigher (2–6 times) in 5 mM ammonium formate than in 5 mMammonium acetate or 5 mM ammonium acetate mixed with 0.1%formic acid (Fig. 1). These results verified our previous study(Hou et al., 2015) and also showed that a relative higher pH andammonium benefits the ionization of neonicotinoid insecticides(Mayer-Helm, 2009; Cech & Enke, 2001). Additionally, threeisotopically labeled standards (thiamethoxam D4, imidaclopridD4 and acetamiprid D3) also showed the same highest responseas their individual unlabeled standards when 5 mM ammoniumformate was added to mobile phase A (Fig. 1).

The higher neonicotinoid signals when the mobile phase con-tained ammonium formate, rather than ammonium acetate, maybe due to different capabilities of the different ammonium saltsto change the spray condition and/or ion-molecule reaction, ideaswhich need further research (Benijts et al., 2004). Based on thesefindings, 5 mM ammonium formate was selected as the optimizedmobile-phase in this study.

3.2. Dilution of QuEChERS extracts to reduce matrix effects

Matrix effects may differ for six types of tea samples, whichnaturally contain different quantities of polyphenols, phenolic acid,pigment, flavonals, etc (Lu et al., 2010), due to the various pro-cesses of manufacture. Compared to black and oolong tea, greentea has a higher content of catechins, which are broken down dur-ing fermentation. On the other hand, fermentation significantlyincreases the gallic acid levels in black tea (Cabrera, Giménez, &López, 2003). Fully fermented and half-fermented teas (black andoolong) contain less L-theanine than unfermented teas, whilewhite tea contains more than green tea (Keenan, Finnie, Jones,

Fig. 1. Peak response of eight neonicotinoid standards and three internal standards (0.05 mg kg�1) isolated using five different mobile phases.

W. Jiao et al. / Food Chemistry 210 (2016) 26–34 29

Rogers, & Priestley, 2011). Yellow tea has higher phenolic contentthan black and green tea (Kopjar et al., 2015). Matrix interferencefrom tea can especially affect the determination of multiple neon-icotinoid insecticide residues (Hou, Jiao, et al., 2013; Oellig &Schwack, 2012; Chen et al., 2011; Wu et al., 2013; Xie et al.,2009; Liu et al., 2013; Hou et al., 2015).

In our previous study, a QuEChERS preparation method wasestablish using matrix-matched calibration (MMCC) to compen-sate for the matrix from three types of tea (Hou et al., 2015). Inorder to establish a more quick and convenient method and tobe able to analyze all six types of tea, a dilution-based methodwas investigated with the aim of diminishing the matrix effects.The matrix effects of six types of tea on eight neonicotinoids weresystematically obtained across a dilution factor range of 20–2000(Fig. 2).

For any one matrix, the magnitude of the effect varied for eachof the eight neonicotinoid insecticides at different dilution factors.For most of the neonicotinoids, dilution tended to diminish thematrix effect. The 6 initial tea sample extracts (dilution factor 20,matrix concentration of 0.05 g/ml) all showed suppressed insecti-cide signals. Yellow tea had the greatest matrix suppression effectson dinotefuran, nitenpyram, thiamethoxam, clothianidin, andimidaclothiz quantitation of �64.8%, �59.0%, �43.8%, �49.1% and�57.8%, respectively. The least suppressive matrix for these 5 resi-dues was green tea (�20.6%, �36.7%, �4.0%, �26.7% and �35.2%,respectively). Moreover, there was an interfering peak in theyellow tea sample between nitenpyram and thiamethoxam(Fig. S-1, between peaks 2 and 3, and Table S-1) that was higherthan in the other five types of tea. Together these results indicatedthat yellow tea was the most interfering tea matrix for the analysisof neonicotinoids.

There were some surprising responses of the matrix effect todilution (Fig. 2). For imidaclothiz, the magnitude of the matrixeffect in the initial tea sample extracts (20-fold DF) was below�50% in five types of tea, but not in green tea, and this matrix sup-pression was ameliorated by moderate dilution (about 200-fold).The imidacloprid signal in the initial green tea sample (20-foldDF) was suppressed by less than 20%, was enhanced to a very highlevel (>50%) at a dilution factor of 40, but was unaffected at a dilu-tion factor of 400. This imidacloprid response pattern was similarin all 6 tea samples. For most other insecticides in the six types

of tea, a 400-fold dilution of the initial QuEChERS extracts reducedthe matrix effects to acceptable levels (�20 � + 20%), with theexception for nitenpyram (28.9, 24.3, 31.3%) in black, oolong andyellow teas (Table S-1).

As the odd case, the matrix effect on nitenpyram was enhancedas the dilution factor increased. For instance, a 1000-fold dilutionof dark or white tea samples resulted in matrix effects of 55.8%or 54.7%, respectively, and a 2000-fold dilution of oolong tearesulted in 64.76% effect. The matrix effects of all the tea typeson the eight neonicotinoid insecticides at 4000 and 10,000-folddilution could not be evaluated since that level of dilution resultedin an insufficient signal-to-noise ratio (S/N < 10). Therefore, a 400-fold dilution of the QuEChERS extracts was considered to be themost appropriate dilution factor for the analysis of neonicotinoidsacross the six types of tea.

3.3. Adsorbing material optimization

In order to decrease the cost and time for sample preparation,we hoped to reduce the matrix effect through dilution of the initialextract without use of any adsorbent. Therefore, the initial extractfrom yellow tea (the dirtiest matrix) was processed in one of twoways before dilution by 400-fold: either untreated or treated withan adsorbent mix consisting of 400 mg PVPP, 25 mg PSA, 100 mgGCB, 50 mg C18 (Hou et al., 2015). The yellow tea matrix effectcould not be eliminated completely through dilution without priorclean-up by an adsorbent. For example, the dinotefuran signal wassuppressed by more than 50% (�64.9%) without the adsorptionstep (Fig. 3). Addition of a clean-up step for yellow tea extractbefore dilution resulted in a reduction of the matrix effect to arange of �6.4–11.7% for most of the insecticides, except niten-pyram (31.3%).

Although we now deemed the clean-up procedure necessary,we aimed to reduce the amount of adsorbents required in orderto develop a quicker and cheaper method. Since our previousexperiments verified that PVPP and PSA yielded nearly identicalreduction of polar interfering compounds in tea matrix (Houet al., 2015), an adsorbent composed of only PVPP and GCB wasproposed for cleanup of the raw extracts. Since GCB is the morecostly of the two and probably adsorbs some polar pesticides(Oellig & Schwack, 2012; Chen et al., 2011; Liu et al., 2013;

Fig. 2. Matrix effects of six types of tea on determination of eight neonicotinoid insecticides across 7 dilution factors over the range of 20–2000.

Fig. 3. The matrix effect on the determination of eight neonicotinoid insecticideresidues from yellow tea without further clean-up by sorbent or with treatment bysorbent before a 400-fold dilution (sorbent mixture composed of PVPP: 400 mg,PSA: 25 mg, GCB: 100 mg, C18: 50 mg).

30 W. Jiao et al. / Food Chemistry 210 (2016) 26–34

Castillo, González, & Miralles, 2011; Fernández-Alba & García-Reyes, 2008), different doses of GCB (0, 10, 20, 50, 100 mg) weretested with a constant amount of 160 mg PVPP for clean-up of

raw yellow tea extracts. The magnitude of the matrix effects forall insecticides reached a satisfactory level (�0.51% to 10.4%;Fig. 4A) when 20 mg GCB and 160 mg PVPP was used, althoughthe extract still appeared slightly yellow. The yellow tea extractwas colorless when treated with 50 mg GCB (Fig. 4B). Interestingly,the matrix effect on nitenpyram was higher (�31.3%) at highersorbent levels (Fig. 3) and was lower (10.4%) when the dosage ofsorbent materials was reduced (Fig. 4A). The reason behind thisphenomenon requires further detailed study. Based on theseresults, a sorbent composed of 160 mg PVPP and 20 mg GCB wasused for cleanup in the final method.

3.4. Internal standard

Although the use of internal standards has been a highly effec-tive tool for addressing quantitative issues associated with signalsuppression, the use of one internal standard may not be a goodchoice for simultaneous detection of multiple neonicotinoid resi-dues in tea (Choi, Hercules, & Gusev, 2001). The commerciallyavailable internal standards thiamethoxam D4, imidacloprid D4and acetamiprid D3 were used to compensate for the matrix effectof 7 different dilutions of yellow tea (ranging from 20 to 2000-fold)on the measurement of eight neonicotinoid insecticides (Fig. 5).The signals of the three internal standards reflected the samematrix effects as their corresponding compounds across the 7dilution factors, except for in the 2000-fold dilution where thematrix effect on imidacloprid D4 was seven times higher than onimidacloprid (Table S-1).

However, thematrix effects on the three internal standardsweredifferent from those on the other neonicotinoid insecticides. At a

Fig. 4. (A) The matrix effect at dilution factor 20 on signals of eight neonicotinoid insecticides in yellow tea treated with 20 mg GCB and 160 mg PVPP. (B) Yellow tea extractcolor after absorbance using different dosages of GCB (0, 10, 20, 50, 100 mg) with 160 mg PVPP.

W. Jiao et al. / Food Chemistry 210 (2016) 26–34 31

dilution factor of 20, the matrix suppressed the signals of all theinsecticides. The matrix effects were improved to a certain degreeif imidacloprid D4 was used as an internal calibration standard,however the improvementwas not equal across the tested residues,as the matrix effects on dinotefuran, nitenpyram and imidaclothizwere still below �50% (�63.1, �59.4 and �56.5%). Similar patternswere seen when the other two internal standards were used forthe calibration. At dilution factor 40, imidacloprid and imidaclopridD4 showed signal enhancement, while the signals of other insecti-cides were still suppressed. If an insecticide analysis protocol wereto rely on imidacloprid D4 for calibration of tea matrix effects, itcould result in serious underestimation of residue levels. Use of thi-amethoxamD4or acetamipridD3 for calibrationdidbetter diminishthe teamatrix effects. At the dilution factors between 100 and 1000-fold, the matrix effects of yellow tea could be negligible, except foron dinotefuran and nitenpyram, whose values of matrix effect wereout of the range of no matrix effects. At the dilution factor of 2000,the matrix effects on three kinds of insecticides (nitenpyram, cloth-ianidin and imidacloprid D4)were beyond the negligible scope, per-haps due to the low concentration of insecticides. The above resultsverified that it is not ideal to use one internal standard for compen-sation of matrix effects during multi-residue insecticide analysis.Furthermore, only systematical research can provide the data intohow the various matrices affect analysis of different insecticides.Since this developed method used dilution to mitigate the matrixeffect on the insecticide, there was no need to include calibrationby isotopic internal standard(s), which means that the standardscould be used for quality control in daily analysis. Acetamiprid D3showed the lowest variation in matrix effects compensation, eventhough imidacloprid D4 has often been used in the publishedmeth-ods. These results indicated that acetamiprid D3 better compen-sated for the matrix effect and is thus the best internal standardfor method evaluation.

3.5. Validation of the dilution method

For quantitative validation of the method, standard solutioncurves ranging over six concentrations of each insecticide wereanalyzed (0.025–25 lg L�1). The linearities and correlation coeffi-cients (R2) for neonicotinoid insecticides were all higher than0.997 (Table 1).

The recoveries with relative standard deviations (RSDs) for eachneonicotinoid insecticide were measured by spiking blank yellow

tea samples in six replicates at three different spiked levels (0.01,0.05 and 0.5 mg kg�1). The recoveries ranged from 66.3 to108.0%, with RSDs of 0.5–15.2% (Table 1).

The Limit of Quantitation (LOQ) was calculated as a signal-to-noise ratio of 10 (S/N = 10), using the lowest concentrationresponse for each insecticide at the primary ion transition (quanti-tation ion transition) obtained from the MS/MS mode and as asignal-to-noise ratio of 3 (S/N = 3) from the secondary ion transi-tion (confirmation ion transition). For dinotefuran, imidaclothizand thiacloprid, the LOQs were 0.05 mg kg�1, due to their recover-ies being below 70%. Additionally, the signal-to-noise ratio of theimidaclothiz confirmation ion was below 3. For the other 5 insec-ticides, the LOQs were 0.01 mg kg�1. All LOQs for the 8 neonicoti-noids were below the MRLs for tea set by both the EuropeanUnion (EU) and Japan.

3.6. Comparison to the Recommended National Standard in realsample analysis

The validated method was compared to the Recommended Chi-nese National Standard method (GB/T 23205-2008) for the analysisof pesticide residues in commercial samples of six types of tea. Threerepresentative samples obtained from themarketplace for each typeof teawere analyzed in triplicate. Using themethod developed here,imidacloprid was detected in black, oolong, dark and white tea(0.029, 0.013, 0.022 and 0.005 mg kg�1); acetamiprid was detectedin black, oolong and dark tea (at 0.046, 0.004 and 0.063 mg kg�1;Table S-2). The insecticide levels detected in the six types of teawerebelow the MRLs established by the EU (0.05–20 mg kg�1), with theexception of acetamiprid in one dark tea sample, which was a littlehigher than 0.05 mg kg�1 at 0.063 mg kg�1. No neonicotinoid insec-ticide residues were detected when the recommended ChineseNational Standard method was used, which may be due to lowerextraction rates of theses insecticides by this protocol, which doesnot include soaking the samples in water before solvent extraction(Hou, Hu, et al., 2013; Hou, Jiao, et al., 2013). As discussed therein,pre-soaking the dry tea samples with water apparently results in ahigher extractionefficiencyof theneonicotinoids anda lowermatrixeffect,which together result in a higher sensitivity. Significantly, ourapproach is not onlymore efficient and accurate in extraction of theinsecticides, but also saves time, reagents and money, when com-pared to the current Recommended Chinese National Standardmethod.

Fig. 5. The matrix effect on determination of eight neonicotinoid insecticides and three internal standards across 7 dilution factors ranging from 20 to 2000 in yellow tea.

Table 1Method validation using yellow tea: correlation coefficient, recoveries, Relative Standard Deviations and LOQs.

Pesticide R2 Recoveries,% (RSD,%) n = 6 Spiked level (mg kg�1) LOQ (mg kg�1)

0.01 0.05 0.5

Dinotefuran 0.9998 68.9 (3.9) 94.5 (4.6) 81.6 (4.6) 0.05Nitenpyram 0.9999 108.0 (3.9) 93.3 (15.2) 79.7 (3.2) 0.01Thiamethoxam 0.9998 79.4 (2.7) 91.9 (1.1) 93.8 (1.7) 0.01Clothianidin 0.9989 69.8 (6.4) 80.3 (2.2) 78.4 (1.4) 0.01Imidacloprid 0.9997 96.9 (4.6) 97.3 (6.1) 100.8 (0.5) 0.01Imidaclothiz 0.9996 67.4 (5.8) 88.0 (5.1) 88.3 (1.8) 0.05Acetamiprid 0.9999 69.6 (9.5) 96.9 (3.6) 92.5 (0.5) 0.01Acetamiprid d3 0.9993 70.7 (3.9) 97.9 (3.6) 93.0 (1.2) 0.01Thiacloprid 0.9972 66.3 (7.2) 98.6 (1.6) 93.7 (1.1) 0.05

32 W. Jiao et al. / Food Chemistry 210 (2016) 26–34

W. Jiao et al. / Food Chemistry 210 (2016) 26–34 33

4. Conclusions

For the determination of multiple pesticide residues withLC–MS/MS, this is the first report combining a QuEChERS methodusing PVPP and GCB clean-up with a dilution method in order todiminish the complex and varied matrix interference due toco-extracted from six different types of tea. This method is quick,effective and accurate, for there is no need to use other compensat-ing methods.

In this work, different dilutions of six tea matrices were inves-tigated systematically in order to diminish the matrix effect onthe determination of eight neonicotinoid insecticides. A dilutionfactor of 400 was demonstrated to sufficiently eliminate the matrixeffect in most cases. The matrix effect compensation capabilities ofthree isotopically labeled were also compared and discussed. Theinternal standard method was verified as a poor choice for com-pensating an analytical target other than the insecticide corre-sponding to the standard. Furthermore, we recommend testingthis extraction, clean-up and dilution protocol for determinationof other kinds of pesticides in all types of tea by LC–MS/MS.

Acknowledgements

This work was supported by the National Natural ScientificFoundation of China (No. 31270728), the project of ‘‘Nutritionand Quality & Safety of Agricultural Products, Universities LeadingTalent Team of Anhui Province”, and Anhui Major DemonstrationProject for Leading Talent Team on Tea Chemistry and Health,National Modern Agriculture Technology System (CARS-23). Wesincerely thank Mrs. Huarong Tan and Mr. Jingwei Hu (of theCenter of Biology Technology at Anhui Agricultural University)for their assistance in using LC–MS/MS.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.foodchem.2016.04.097.

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