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International Journal of Environmental AnalyticalChemistry

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Multiresidue GC-MS/MS pesticide analysis forevaluation of tea and herbal infusion safety

Antonija Beneta, Dragana Mutavdžić Pavlović, Ivan Periša & Mira Petrović

To cite this article: Antonija Beneta, Dragana Mutavdžić Pavlović, Ivan Periša & Mira Petrović(2018) Multiresidue GC-MS/MS pesticide analysis for evaluation of tea and herbal infusionsafety, International Journal of Environmental Analytical Chemistry, 98:11, 987-1004, DOI:10.1080/03067319.2018.1518439

To link to this article: https://doi.org/10.1080/03067319.2018.1518439

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ARTICLE

Multiresidue GC-MS/MS pesticide analysis for evaluation oftea and herbal infusion safetyAntonija Benetaa, Dragana Mutavdžić Pavlović b, Ivan Perišac and Mira Petrovićd

aAnalytical Instruments Department, Shimadzu d.o.o., Zagreb, Croatia; bFaculty of Chemical Engineeringand Technology, Department of Analytical Chemistry, University of Zagreb, Zagreb, Croatia; cLaboratorijQuanta, Quanta d.o.o, Rakov Potok, Croatia; dCatalan Institute for Water Research (ICRA), H2O Building,Scientific and Technological Park of the University of Girona, Girona, Spain

ABSTRACTA fast, simple, low-cost and high-throughput multiresidue pesticideanalysis method was developed and validated for 300 pesticides inherbal and fruit infusion samples based onmodified QuEChERS (quick,easy, cheap, effective, rugged and safe) procedure combined with gaschromatography coupled with tandem mass spectrometry method(GC-MS/MS). The objectives were to develop low cost GC-MS/MSmethod, validate the method in accordance to SANTE/11,813/2017guidance document and application in routine. The results obtainedusing different GC and MS/MS parameters were evaluated in order todevelop quick, robust, accurate and effective multiresidue method.Total analysis time was 28 min with 0.6 µL injection volume. Foraccurate quantification, matrix-matched calibration (MMC) curves (inrange of 10 µg/kg – 250 µg/kg) were applied to compensate matrixeffect. The limits of quantification (LOQ)were rangedbetween 0.06 µg/kg and 135 µg/kg, and for the majority of the pesticides the LOQ werebelow the regulatory maximum residue limits. Most recoveries at10 µg/kg and 100 µg/kg were in the range 70%–120% indicatingsatisfactory accuracy. The validatedmethodwas applied to commercialherbal and fruit infusion products detecting chlorpyriphos, DEET, tebu-conazole, terbuthylazine, piperonyl butoxide, biphenyl, pendimethalin,pirimiphos-methyl and p,p’-DDE in more than 100 samples from 1,466so risk assessment on human health was calculated specially for thosepesticides.

ARTICLE HISTORYReceived 3 May 2018Accepted 18 August 2018

KEYWORDSMultiresidue method;pesticides; tea and herbalinfusions; gaschromatography - tandemmass spectrometry; riskassessment

1. Introduction

Tea (Camellia sinensis L.) is consumed by over two-thirds of the world’s population due toits medicinal, refreshing and mild stimulant effects. There are mainly four types of tea:black or red, oolong, green and white which are used for tea infusion (water extract fromfermented tea leaf) worldwide [1]. According to European Tea Committee (ETC) andEuropean Herbal Infusions Association (EHIA) definition, herbal and fruit infusion (HFI)materials are plants or parts of plants that do not originate from the tea plant (Cameliasinensis L.) and are intended for food use by brewing with freshly boiling water. They alsoinclude blends of HFI with tea as a minor component [2]. Tea together with HFI is amongst

CONTACT Dragana Mutavdžić Pavlović dmutavdz@fkit.hrSupplemental data for this article can be accessed here

INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY2018, VOL. 98, NO. 11, 987–1004https://doi.org/10.1080/03067319.2018.1518439

© 2018 Informa UK Limited, trading as Taylor & Francis Group

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the world’s most popular and widely enjoyed beverages, thanks to their almost unlimitedvariety and their convenience [3]. Cultivation of herbs and tea is quite sensitive becausemites, winged insects, caterpillars, variety of diseases and weeds can cause huge pro-blems. In order to minimise all the above problems, the most common practise incultivation and production is use of pesticides [4]. Determination of pesticide residues isvery important, especially for prevention in the field of public health safety [5] but it is abig challenge due to the complex matrix of herbs, consisting of polyphenol compounds,pigments, sugars and alkaloids [6]. Those components, by their nature, can influence thephysical and chemical properties of the pesticide, and thus directly on the insulatingcavity. With all these difficulties, there are also potential international trade barriers due tothe maximum residue limit (MRL) in tea and HFI established by most countries and someinternational organisations. The current MRLs for some pesticides in tea occasionallychanging and becoming more stringent [4]. Regarding to this, sample preparation andmultiresidue method for pesticide residues are key steps for identification and quantifica-tion. In nowadays, there are a lot of different pesticides available and it is very hard tocover all of them by a single analytical method due to the differences in physical/chemicalproperties. From these reasons there is tremendous need of tea and HFI producers andother quality control laboratories worldwide for low-cost multiresidue method for a largenumber of pesticides in one method, reliable identification and quantification with theshortest possible analysis time [7]. Very important feature of analytical method must berobustness with the shortest possible downtime which impacts a lot on TCO (total cost ofownership) for analytical technique and at the end competitive analysis price.

Thus, the aims of this study were as follows:

(1) Optimisation of extraction and cleanup step for the GC-MS/MS analysis of 300pesticide residues in tea and HFI

(2) Development of low cost, sensitive, selective and robust analytical method(3) Validation according to SANTE/11,813/2017 [8](4) Application in routine for real samples(5) Risk assessment on human health for most presented pesticides in tea and HFI

from region Balkans.

Sample preparation is one of the most important parts of analytical procedure and itis preferred that it would be fast, accurate, precise and economic. QuEChERS (quick,easy, cheap, effective, rugged and safe) method has already proved successful for thedetermination of pesticide residues in food samples [9–12]. For multiresidue method oflarge number of pesticides, a generic procedure of sample preparation is required.During the sample preparation, other presented organic matter and impurities may beextracted along with the targeted analyte so tea and HFI sample is considered to be adifficult task to extract and clean.

Initial investment in hardware and TCO including maintenance, spare parts andconsumables are extremely high and most of laboratories have tendency for reductionof total costs in order to be more competitive on the market. To date, there have beenreports on the multiresidue analysis of pesticide residues in tea using GC-MS/MS or LC-MS/MS techniques. Even if there are an increasing number of publications using LC-MS/MS for pesticide residues, the main goal of this work is low cost analysis. LC-MS/MS is

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more than twice hardware, software and maintenance price comparing to GC-MS/MS.QuEChERS procedure in combination with GC-MS/MS is one of the preferred approachesat present for residue determination of GC suitable pesticides [13].

Most of analysts in laboratories worldwide are using standard procedures and applica-tions given from instrument suppliers without any adjustment and optimisation. Suchkind of approach can be fast in implementation but it is always better to do the optimisa-tion on site regarding the instrument and matrix in order to have higher sensitivity androbustness of the analytical method. Analyst is able to achieve much better performanceof the instrument with customised approach because all instrument types in the marketare a bit different and need to be considered in individual way. So, different injection portparameters, columns and MS/MS parameters were compared in experimental part inorder to create rapid, accurate multiresiduemethod for the analysis in tea and HFI sampleswith improved sensitivity, analysis time, TCO and instrument downtime.

Pesticides were selected on the basis of information on the frequent detection of theirresidues since 2010 in unknown samples by Quanta Company. Most of them wereavailable in 203 compounds mix and all other were added in the mix as single standards.Final analytical procedure has been applied to 1,466 (Balkans) tea and HFI samples ofdifferent varieties in order to provide insight into pesticide residue levels, application anddistribution in region Balkans. Risk assessment on human health has been calculatedspecially for the most common pesticides. For that purpose, the acute/short-term (aHI)and the chronic/long-term (hazard quotient, HQ) consumer health risk was calculated [14].

2. Experimental

2.1. Reagents and chemicals

Pesticide certified reference standards (purity higher than 99%): comprehensive 203-compound GC Multiresidue Pesticide Kit was obtained from Restek Corporation(Bellefonte, Pennsylvania, USA) and individual certified standards from Accustandard(New Haven, CT, USA). QuEChERS extraction salt packets (EN and AOAC method),dispersive solid phase extraction and SPE step were purchased from RestekCorporation (Bellefonte, Pennsylvania, USA). Stock standard solutions of the targetedcompounds were prepared at default concentration 100 ng/mL in acetonitrile. Thesesolutions were stored in a freezer at −20ºC. Working solutions containing in total 300compounds were prepared by appropriate dilution of the stock solutions with acetoni-trile at concentration levels of 2, 5, 10, 20, 50 ng/mL and stored under refrigeration at4ºC and renewed monthly. Acetonitrile is pesticide residue grade and purchased fromJ.T. Baker.

2.2. Samples

The blank samples used for MMC and recovery experiments were uncontaminatedsamples verified to present residue below LOD. Around 137 different tea and HFIsorts and in total 1,466 samples were obtained from tea and HFI growers in regionBalkans.

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2.3. Sample preparation

Samples were extracted following by modified QuEChERS-based approach in two steps:extraction with salting-out and sample clean-up. Briefly, 10 mL of acetonitrile was addedto 2 g of homogenised sample (diluted with 10 mL of water for matrix hydration)contained in a polypropylene centrifuge tube and the sample was shaken. Salting-outwas performed with salt mixture 4 g MgSO4, 1 g NaCl, 1 g TSCD (Trisodium citratedihydrate) and 0.5 g DHS (Disodium hydrogen citrate sesquihydrate). Afterward clean-upstep was performed with a mixture of salts; 250 mg of MgSO4 (magnesium sulphate),150 mg primary and secondary amine (PSA) and 45 mg graphitised carbon black (GCB).

2.4. Instrument and apparatus

GC-MS/MS analysis was performed using a Shimadzu GC-2010Plus gas chromatographwith SPL-2010Plus (Split/splitless injector), OCI/PTV-2010 (On column/ProgrammableTemperature Vaporization Injector) and Optic-4 (Multi mode Injector). GC is coupledwith a AOC-20i+ s autoinjector and autosampler, a GCMS-TQ8050 triple-quadrupole anda computer with GCMS solution software together with Insight software for dataacquisition, processing and statistics (Shimadzu Corporation, Kyoto, Japan). Analyteswere separated on the Rxi-5Sil MS capillary column from Restek (0.25 mm i.d., x 30 m,0.25 µm film thickness), which is selected as most optimum column comparing to Rxi-5SilMS with precolumn, Rxi-5MS and InertCap-5. The column was set at a linear velocityof 47.2 mL/min using helium as carrier gas. The column temperature was programmedas follows: the initial temperature was 40ºC (for 1 min) and increased to 125ºC at 40ºC/min, ramped to 300ºC at 8ºC/min, then was held for 7.00 min. The total run time was28 min. The injection volume was 0.6 µL in splitless mode at constant injector tempera-ture of 250ºC. The three transitions were determined for each compound and thecollision energy was optimised for each pesticide. Quantitation by GC-MS/MS wasbased on MMC using the GCMS solution software. Identification of pesticides in fortifiedsamples by GC-MS/MS was determined by comparing expected retention time and theratio of the two qualifier transition results to MMC.

2.5. Method validation

In the validation, the main parameters including limit of detection (LOD), limit ofquantification (LOQ), linearity, accuracy and precision were evaluated. The methodvalidation followed the EU guidelines on analytical quality control and validation pro-cedures for pesticide residue analysis in food and feed described in SANTE [8]. Theselectivity of the method was evaluated by injecting extracted blank samples. MMC wasused in order to minimise the matrix effect because matrix constituents may increase ordecrease the analytical signal. Matrix effects were assessed by comparison of the slopesof five-point MMC with the slopes of the calibration curves in solvent.

Recovery study was carried out to determine the method accuracy and precision. Inorder to avoid quantitative errors, MMC standards were used to calculate the analyterecoveries. Solvent-based standards were also analysed to assess the matrix effects.Linearity of the MS/MS system was evaluated by assessing the signal responses of the

990 A. BENETA ET AL.

target analytes from MMC solutions prepared by spiking blank extracts at 5 sampleconcentrations, from 2 to 50 ng/mL, which correspond in the sample to 10 to 250 µg/kgbecause of correction factor which is 5.

2.6. Risk assessment

Risk assessment on human health was calculated specially for the most commonpesticides. For that purpose, the acute/short-term (aHI) and the chronic/long-term(hazard quotient, HQ) consumer health risk was calculated and impact on human healthwas estimated.

3. Results and discussion

3.1. Optimisation of GC-MS/MS conditions

Identification of pesticides in fortified samples by GC-MS/MS was determined by com-paring expected retention time and the ratio of the two qualifier transition results. MS/MS detector parameters (transitions and collision cell energies) were optimised togetherwith fine adjustment of MRM transitions in order to get the best intensity and optimumparameters for all pesticides. Transitions with the highest intensities with related colli-sion energies were selected for quantification analysis as confirmation together withretention times for all the pesticides.

Different approaches were obtained in order to achieve low cost, robust and highsensitivity method. On column injection (OCI) [15], programmed-temperature-vapor-isation (PTV) injection [16] and high pressure splitless injection with split-splitess(SPL) [17] were tested. OCI is the simplest and most reliable of these techniques,however, contamination of the column inlet with non-volatile sample materials isfrequent and it is not suitable for fast elution components. With programmedtemperature sample introduction, the negative effect of column contamination canbe more or less avoided, because nonvolatile products are retained in a vaporisationchamber without reaching the analytical column. Large volume injection (LVI) tech-niques have gained wide attention for lowering system detection limits to meetnewer and more stringent regulations. By introducing more samples into the system,the mass of analyte reaching the detector will be proportionately increased. If thebaseline noise stays constant, larger peak height means greater signal-to-noise ratiosand lower system detection limits. Another advantage of LVI is the decrease insolvent that reaches the detector. In LVI, the solvent is carefully evaporated andvented from the inlet before the analytes are transferred to the analytical column.Disadvantages of the LVI technique are that sample-to-sample cycle time increasesbecause the GC oven and inlet need to be cooled down to a lower initial tempera-ture, technique requires clean samples (not suitable for dirty and complex matrices),it is less flexible technique and lower intensities have been noticed for late elutingpeaks. Also, LVI causes that downtime of the instrument because of necessarymaintenance is more frequent which directly increase total cost of ownership forthe analytical instrumentation and decrease efficiency throughput of samples.

INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 991

In Supplementary information, in Fig. S1, comparation of peak height for differentinjection techniques and liners for pesticides with the biggest difference in height ispresented. Different injection techniques and temperature programmes used in optimi-sation are listed below:

● SPL injector with inert ‘Sky’ liner with improved deactivation for trace-level analysisat constant temperature of 250, 200 and 170°C

● SPL injector with single tapper with wool liner at constant 250°C temperature● SPL injector with focused liner used for focusing a high pressure splitless injection

at constant 250°C temperature● OCI/PTV with standard PTV liner and two different temperature programmes

(Programme 1 = 60°C–10°C/min–250°C and Programme 2 = 60°C–50°C/min–250°C)● Multi-mode injector for LVI with standard LVI liner and temperature pro-

gramme = 60°C–800°C/min–250°C

The highest sensitivity was obtained with high pressure injection with focused liner in highpressure splitless injection mode-SPL injector (Fig. S1). Result of obtained data showed thathigh pressure splitless injection is the most suitable for larger number of pesticides.

After optimisation of injection port conditions and techniques, high pressure splitlessinjection with focused liner using Shimadzu SPL-2010Plus injection port is taken for furthermethod development and selection of suitable capillary column. Analytes were separatedon the Rxi-5Sil MS capillary column fromRestek (0.25mm i.d., x 30m, 0.25 µm film thickness)which is selected asmost optimum column comparingwith Rxi-5SilMSwith precolumn, Rtx-5MS and InertCap-5 (Fig. S2) for pesticides with biggest difference between differentcapillary columns. MS/MS detector with scan rate of 20,000 units/second is fast enough sothere is no need for perfect separation. MS/MS detector parameters (collision cell energies,dwell time) were optimised together with fine adjustment ofMRM transitions in order to getthe best intensity and optimum parameters for all pesticides.

Final GC-MS/MS parameters are listed below:

● SPL injector with focused liner used for focusing a high pressure splitless injectionat constant 250°C temperature

● Linear Velocity mode with 100 kPa pressure and high pressure injection mode with250 kPa in first minute

● Column oven programme: initial temperature of 50°C _25°C/minute up to 125°C _10°C/minute up to 300°C with hold time of 11 min (Rxi-5Sil MS capillary column from Restekwith 0.25 mm i.d., x 30 m, 0.25 µm film thickness)

● Column flow: 1.7 mL/minute● Interface temperature: 250°C/Ion source temperature: 230°C● Transitions with highest intensities with related collision energies are given in

Table S1.

Experimental part for GC-MS/MS parameters optimisation was carried out with 1 µLinjection volume. Around 0.6 µL injection volume is used for method validation mea-surements because peak areas were higher than 10,000 for most of the analytes whichhelps in accurate and precise peak integration.

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Splitless high pressure injection, high temperature isothermal programme of injectionport together with inert liner have the biggest influence in better peak shape and higherresponse time for all pesticides. This could be a main reason for satisfying methodvalidation results for most of target pesticides.

3.2. Sample preparation

The QuEChERS method includes simple extraction step with acetonitrile, salting-out andclean-up step. For products with water content lower than 25% and high content organicacids, fatty acids, pigments, caffeine and sugars such as tea and HFI samples which areconsidered as dry commodity, the QuEChERS method has to be modified [13]. Importantkey elements in QuEChERS theory is salting out effect with magnesium sulphate andadjusting the polarity of organic phase [18,19]. Based on recoveries alone MgSO4 is thebest choice, but selectivity of the extraction process has to be considered.

Like for other food matrices, a widely used sorbent (purification material) for theremoval of free fatty acids, sugars and other polar compounds present in extracts offood samples is PSA. As a polar sorbent, PSA can form hydrogen bonds with polarcompounds from the matrix, but retention of more polar analytes can also occur and,therefore, the amount of this sorbent was tested in the method development.

Tea and HFI samples besides PSA and MgSO4 required GCB [4,20] in order to removehigh levels of pigments from the matrix. GCB can remove sterols and pigments such aschlorophyll [19] and strongly retains planar pesticides [4].

In order to achieve better recoveries, a clean-up step was tested using mentionedsalting-out mixtures so aliquot of the supernatant was transferred to a tube containingmixture of PSA, MgSO4 and GCB. Three different producers of mixtures were tested inorder to choose one with the best recovery (Figure 1). These mixtures were selectedbecause of different amount of PSA and GCB content: Mixture 1 tube containing mixtureof salts; 900 mg MgSO4, 150 mg PSA and 15 mg GCB, Mixture 2 tube containing mixtureof salts; 900 mg MgSO4, 150 mg PSA and 45 mg GCB and Mixture 3 is handmade mix ofadsorbents and salts in quantities: 900 mg MgSO4, 300 mg PSA and 150 mg GCB. Fromthe Figure 1 it is obvious that Mixture 2 (Restek salt mix) provides better recoveries for85 pesticides comparing to results with Mixture 1 (VWR handmade mix) and 3 (Supelcobulk adsorbents and salts). Differences in recoveries were presented only for selected 85pesticides which showed the biggest difference and influence of mixtures.

On the basis of the obtained results it is clear that the different mixture contentaffects the efficiency of removing pigments from the tea and HFI matrix and improvingthe recoveries of the investigated pesticides.

Final extraction, salting-out and clean-up process:

● Add 10 mL water in 2 g of sample and shake 1 min● Add 10 mL of acetonitrile in hydrated sample and shake 1 min● Add salt mixture 4 g MgSO4,1 g NaCl, 1g TSCD and 0.5 g DHS, shake for 1 min and

centrifuge at 4,000 rpm for 5 min● Transfer 6 mL of supernatant to 15 mL sample tube add 900 mg MgSO4, 150 mg

PSA and 45 mg GCB, shake for 2 min and centrifuge at 4,000 rpm for 5 min● Filter through 0.45µm syringe filter and inject to GC-MS/MS.

INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 993

The purification materials are used are more than traditional QuEChERS method, whichis usually added to 1 mL of supernatant [4,20]. However, there are also papers in whichthis supernatant volume is considerably greater than 1 mL (3 mL [12] or even 8 mL [13])as in this work. Otherwise, there is a difference in comparison between the EN and AOACstandard method. This difference is based on the corresponding changes in the stepwith salt mixture and the volume of supernatant and purification salts [13]. So, 6 mL ofsupernatant was sellected according to EN 15,662 standard method and differentcombinations of amounts for MgSO4/PSA/GCB were tested for best recoveries.

3.3. Matrix effect

To evaluate the matrix effect for all analytes, the calibration curves of pure solventstandards and matrix-matched standards were measured in this work, and the calibra-tion curve slopes of matrix-matched standards and pure solvent standards were com-pared subsequently. It is well known that matrix effects are one of the main drawbacksof MS/MS methods, making quantification in samples problematic in some cases [4,21].In gas chromatography matrix-effect is attributed to the presence of active sites in theinjector, which causes the differences in the observed response for the given analyte insolvent compared to response in sample matrix (signal suppression or enhancement).The presence of matrix in the injected sample extract can block active sites in theinjector, reduce thermal stress for labile compounds, prevent thermal degradation/adsorption of analytes delivering more analytes to the column and can have significanteffect on recovery values [22]. Matrix effect can be very variable, dependent on specificcombination of an analytes in a particular matrix and concentration level. Because of itsrandom nature, it cannot be predicted for a specific analyte-matrix combination, soavailable tools should be applied to minimise its effect on the results. Soft matrix effects

Figure 1. Recoveries for pesticides (10 µg/kg) with Salt 1, 2 and 3.

994 A. BENETA ET AL.

(suppression or enhancement of 0–20%) are negligible. However, if the pesticides suffermedium (suppression or enhancement of 20–50%) or strong (suppression or enhance-ment of ˃50%) matrix effects, it is necessary to use certain methods to overcome theinfluence of matrix. The matrix effect was calculated by the equation:

ME %ð Þ ¼ slope of calibration curve in matrixslope of calibration curve in solvent

� 1� �

x100 (1)

In view of the above equation, the negative and positive values of the ME signify matrix-induced suppression and enhancement, respectively.

Values of ME are presented in Figure 2 and as can be seen most of the pesticides intea and HFI samples suffer strong matrix effect.

Knowing that the nature of matrix effect is pretty varying, the percentage is just a relativeindicator of the degree of suppression and enhancement. For most of the investigatedpesticides, matrix affects the analyte signal in terms of enhancement, but it was not a generalpattern because matrix effect is different for each analyte. Hexachlorobenzene and oxy-Chlordane were not significantly affected by the matrix because change in signal is lessthan 20%. Pesticides like p,p’-DDE, trans-chlordane, cis-chlordane, heptachlor, o,p’-DDE,dichlobenil, beta-HCH, beta-endosulfan, biphenyl, chlorthal-dimethyl, alpha-endosulfan, pen-tachloroanisole and mirex show some degree (20%-50%) of medium signal enhancement.Eighty-nine per cent of the compounds like: bendiocarb, dichlofluanid, pyrethrin, fenoxycarb,famoxadone, folpet, etoxazole, bromuconazole-1, carbaryl, bifenox, fenvalerate-2 (esfenvale-rate) and fluridone showed the highest signal enhancement. Only dichlorvos show signalsuppression. This fact about signal enhancement goes in favour to lower detection limits forsuch small injection volume (0.6 µL).

The results proved that MMC is indispensable for accurate quantification by GC-MS/MS.Besides all mentioned results, more important is the fact that matrix effects for a givenpesticide were similar in all matrices [12]. Since in this article the validated method will beapplied to different types of tea and HFI samples, this fact greatly simplifies the procedures.

3.4. Method validation

The optimised methodology was validated in order to verify its applicability to theroutine analysis of samples and to ensure the reliability of the results. Validation of

Figure 2. Matrix-effect in tea.

INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 995

the method was based on the European Union guidelines [8]. The selectivity of themethod was evaluated by injecting blank sample extracts. The absence of signal above asignal-to-noise ratio of three at the retention times of the target compounds showedthat the method is free of interferences. The MMC curves for each compound were builtusing blank samples. For this, five concentrations levels were selected. The criteriaadopted for the selection of the analytical curve levels were the signal to noise ratioand also the results of recovery studies. From this evaluation we selected the followingconcentration levels for the MMC curves: 2, 5, 10, 20 and 50 ng/mL. The LOD and LOQfor all pesticides, which were defined at a signal-to-noise (S/N) ratio of 3 and 10, wereobtained, ranging from 0.018 to 40 µg/kg and 0.06 µg/kg and 135 µg/kg, respectivelylike it is shown in Figure 3.

Recovery study, inter-day and intra-day precision were carried out to determine themethod accuracy and precision Table S2. In order to avoid quantitative errors, MMCstandards were used to calculate the analyte recoveries. Solvent-based standards werealso analysed to assess the matrix effects.

Method selectivity was assessed based on the presence of specific ion transitions(quantifier ion and two transitions for compound confirmation) at the correspondingretention time (Table S1), as well as the observed ion ratio values corresponding tothose of the standards.

Method trueness was assessed by recovery studies using blank matrices spiked atconcentration level 2 ng/mL which is equivalent to lowest possible maximum residuelevel (MRL) concentration and it is injected in five individually prepared replicates inFigure 4. Found concentrations, recovery and relative standard deviation (% RSD) werecalculated. According to SANTE requirements recovery values are deemed acceptable ifbetween 70% and 120% and relative standard deviation within ± 20%.

Figure 3. LOQ results for 300 pesticides.

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Two hundred and sixty-three pesticides in concentration level of 10 µg/kg are withinSANTE [8] recovery requirement range and only 1.3% of pesticides: difenoconazole-2,cypermethrin-2, terbacil and monocrotophos have recovery value higher than 120%.Pesticides like omethoate, thiabendazole, fluvalinate-2, boscalid, fenvalerate, nicotine,propargite, pyrethrin and cyproconazole-2 have lower recovery in range between 28%and 58% and it can be compared to publication [22] where those pesticides weremeasured with liquid chromatography which is probably preferred technique for it.Only boscalide was measured in mentioned article with both, liquid and gas chromato-graphy techniques and recovery of 75.6% was obtained (for a high concentration of200 µg/kg) with gas chromatography which also proves together with our result thatliquid chromatography would be better choice for boscalide. Based on obtained results,this GC-MS/MS multi residue method has proven to be very suitable for 87% ofinvestigated pesticides, including pesticides like chlozolinate and fenobucarb whichshowed better recoveries comparing to other scientific publications. The triazole basedpesticides (bitertanol, cyproconazole-1, myclobutanil, penconazole and triadimenol)showed nice recoveries comparing to very low recoveries in [23].

If there is a situation for the identification of pesticides in unknown sample whoserecovery or RSD is outside the target range, accurate quantification will continue withstandard addition method that is an alternative approach MMC method.

3.5. Comparison of the proposed method with other works

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and GC-MS/MS methods in combination with QuEChERS sample preparation have been widelyused for pesticide residue analysis. LC-MS/MS instrument cost and its TCO are almostdouble price comparing to GC-MS/MS technique so that is the main reason why GC-MS/MS has been chosen as target technique for method development. There are no manyarticles with GC-MS/MS determination of pesticides in tea and HFI samples because ofmatrix complexity and difficulties with extraction. In this study, a very simple and robustGC-MS/MS method was developed for the determination of 300 pesticides in tea and HFIsamples taking special care of costs, a potential competitiveness of laboratory on the

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Figure 4. 2D plot representing the recovery percentages for all pesticides over the entire analysis time.

INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 997

market. Compared to other works involving pesticide residue analysis with GC-MS/MS intea samples and cereals, the proposed method has some superiorities in respect to thenumber of target pesticides in only one method, number of real samples, analysisruntime, injection volume and method validation parameters such as linearity andLOQ, demonstrating the suitability of the method for multiresidue analysis in tea andHFI samples for regulatory and routine residue qualification and quantification (Table 1).Total analysis time of proposed method is shorter even up to 30% comparing to othermethods which means reduction in consumption of carrier/collision cell gases andlonger lifetime for filaments. Injection volume of proposed method is smaller from40% up to 88% comparing to proposed methods so this also leads to reduction intotal cost of ownership because much longer lifetime of liner, capillary column, quadru-pole and electron multiplier detector.

Although it may seem that large amounts of relatively expensive purification salts(MgSO4 is quite cheap; PSA price is 7–8 times higher than MgSO4; GCB price is 2–4 timeshigher than MgSO4) are used for 6 mL of supernatant, the resulting method is compro-mise between standard method, number of pesticides analysed in one method, totalanalysis time, good recovery and the lowest possible amount of GCB/PSA, and as such isstill, despite all above-mentioned, quite economical.

3.6. Sample analysis

The validated method was applied to detect and quantify residues of the pesticides indifferent sorts of tea and HFI samples acquired from region Balkans. In order to ensure thequality of the results and evaluate the stability of the method proposed, an internal qualitycontrol was carried out on every batch of samples. In order to test the feasibility of theproposed approach for routine identification and quantification of pesticide residues in realsamples, 1,466 samples were analysed for the target compounds and 21 pesticides weredetected in more than 3% of total analysed samples as shown in Figure 5.

Chlorpyriphos, DEET, tebuconazole, terbuthylazine, piperonyl butoxide, biphenyl,pendimethalin, pirimiphos-methyl and p,p’-DDE were detected more than 100 timesso those pesticides are considered further. It is interesting to point out that in 1,466analysed samples included 137 different tea and HFI sorts and only 4 of them arecontaminated the most with mentioned 9 pesticides: 10.0% of total biphenyl and19.9% of tertbuthylazine positive samples are chamomile samples; 10.9% of chlorpyrifos,13.1% of pirymiphos-methyl, 16.4% of tebuconazole and 36.0 % of pendimethalinpositive samples are nettle herb samples; 14.9% of DEET and 16.1% of piperonyl-butoxidpositive samples are blue mallow samples and 16.0 % of tertbuthylazine positivesamples are shavegrass samples as shown in Figure 6. This finding is confirmed by thefact that tea matrices have the highest percentage of the European Union MaximumResidual Limit and contain more than five pesticides per sample [30], which is the reasonfor constant need for a suitable, versatile and reliable extraction method that can bedetermined.

In addition to the sorts of tea and HFI, it is important to emphasise the geographic originof plants which are contaminated the most with these pesticides. More than 90% ofmeasured nettle samples grew in Bulgaria, more than 50% of chamomile and shavegrasssamples grew in Croatia and 100% of blue mallow flower samples grew in Croatia.

998 A. BENETA ET AL.

Table1.

Comparison

oftheprop

osed

workwith

anotherworks.

No.

ofpesticides

Metho

dMatrix

Linearity

Concentration;

Recovery

(RSD

)LO

QAn

alysis

time

Injection

volume

Num

berof

realsamples

Reference

300pesticides

inon

emetho

dfile

GC-MS/MS

137differen

tdriedtea

sorts

≥0.99

910

μg/kg

;70

%-110

%(≤20

%)

≤50

µg/kg(for

90%

ofpe

sticides)

28min

0.6µL

1466

samples

Prop

osed

metho

d

200pesticides

GC-MS/MS

3different

cereals

>0.99

100μg/kg;

70%–120%

(<20%)

5–50

µg/kg

38min

1µL

10samples

[13]

8pesticides

GC-MS

Green

tea

>0.995

100–500μg/kg;7

7–114%

(≤16%)

15–30µg

/kg

40min

1µL

N/A

[24]

8pesticides

GC-MS

5differenttea

sorts

N/A

60μg

/kg;

92.1

–99.6%

(<6%

)N/A

40min

1µL

10samples

[25]

78pesticides

GC-MS/MS

N/A

>0.99

50–100

μg/kg;7

0%-120%

(<20%)

N/A

60min

5µL

55samples

[26]

33pesticides

GC-MS

1tea

N/A

50μg

/kg;

70%–120%

(<20%)

N/A

18min

1µL

1teasample

[27]

490pesticides

insixmetho

ds(app

roximately90

compo

unds

ineach

grou

p)

GC-MS

4differenttea

sorts

N/A

10–1000μg/kg;6

0%-

120%

(for94%

pesticides)

N/A

40min

1µL

N/A

[28]

101pesticides

GC-MS/MS

Freshtealeaves

>0.99

50and1000

μg/kg;

70%–120%

(<20%)

1.1–25.3

µg/kg

40min

5µL

N/A

[29]

INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 999

3.7. Risk assessment on human health for most presented pesticides

Whereas the aforementioned pesticides (chlorpyriphos, DEET, tebuconazole, terbuthyla-zine, piperonyl butoxide, biphenyl, pendimethalin, pirimiphos-methyl and p,p’-DDE)mostly detected in 1,466 real samples, we decided to calculate risk assessment onhuman health especially for those pesticides. Exposure to certain harmful substancesis a function of the share of the food consumed and the concentration of harmfulsubstances and can be chronic or acute. The obtained data are compared with thetoxicological reference values. In terms of food safety, certain types of foods consideredsafe for consumers if the estimated intake of pesticide residues does not exceed the ADI(Acceptable Daily Intake) or ARfD (Acute Reference Dose) [31] (Table S3).

To assess the acute or short-term consumer health risk (aHI) it is important to knowestimated short-term intake (ESTI) and ARfD value, while estimation of the chronic orlong-term consumer health risk (health hazard quotient, HQ) was based on the esti-mated daily intake (EDI) and ADI. The relevant formulas are following [32]:

313

269

208

157 155

131

123

115108

95

84

74

66 64 62

52 52 5046 46 44

0

50

100

150

200

250

300

350

0.000

2.000

4.000

6.000

8.000

10.000

12.000C

ON

CE

NT

RA

TIO

N

PESTICIDES

Highest residue level in ug/kg Average of residue level in ug/kg Count of positive analysis

Figure 5. Maximum residue level, average level of all measured real samples and count of positivemeasured samples for pesticides presented in more than 3% of analysed samples.

1000 A. BENETA ET AL.

ESTI ¼ the highest residue level � food consumptionbody weight

(2)

aHI ¼ ESTIArfD

� 100% (3)

EDImeanresiduelevel� foodconsumption

bodyweight(4)

HQ ¼ EDIADI

� 100% (5)

For the precise evaluation, the ARfD and ADI are expressed as a percentage of dailyintakes for a 60 kg person. HQ was used to assess the non-carcinogenic health risk tohazard materials in food consumption. The HQ level lower than 100% is acceptable riskfor human health unlike the case when it is higher than 100% [33]. For risk assessment ofconsumer’s exposure to pesticide residues, the estimated intakes on the daily basis areexpressed as percentages of the ArfD and ADI values for the tested pesticides [34] andevaluations [35]. Food consumption of tea in Croatia is 0.18 g/day [36].

The dietary exposure to pesticides (mgkg−1bw−1day−1) was calculated based onconsumption data and individual body weights, and residue monitoring data (thehighest residue and mean residue). The results are shown in Table 2. For short-termrisk assessment, all the ESTI values were much less than the ARfD values. Furthermore,the highest risk was from chlorpyrifos with aHI 0.6598%. All the aHIs were little, whichmeant there was a negligible short-term or acute risk with the exposure to the tested

0.00

5.00

10.00

15.00

20.00

25.00

30.00

35.00

40.00

Chamomile Shavegrass Blue Mallow flower Nettle herb

% o

f p

osit

ive

sa

mp

les

Most presented pesticides in different tea samples

Biphenyl Chlorpyrifos DEET

p,p'-DDE Pendimethalin Piperonyl butoxide

Pirimiphos-methyl Tebuconazole Terbuthylazine

Figure 6. Distribution of most presented pesticides in positive real samples.

INTERNATIONAL JOURNAL OF ENVIRONMENTAL ANALYTICAL CHEMISTRY 1001

pesticides via tea consumption. However, in the long-term risk assessment, the riskindexes (HQs) were notable lower than aHIs, which indicated the chronic risk frompesticide exposure via tea consumption do not need to be considered further.

Water solubility is one of the crucial physical and chemical parameters that influencesthe transfer rates of pesticides from herb to infusion because water is the mostimportant carryover of pesticides during brewing. In this article worst case scenariocalculated althought it will be more precise or more preferable to determine the transferrate, especially for more water soluble pesticides. Chlorpyrifos, detected in real samplesin highest residue level, has solubility in water < 1 mg/l and octanol/water partitioncoefficient (log Kow) = 4.7 [37]. Less water soluble and high Kow pesticides do not infuseinto tea brew from prepared tea [24] so this is additional indicator of negligible chronicrisk from detected pesticide exposure via tea consumption.

4. Conclusions

A low cost, fast, simple and high-throughput multiresidue pesticide analysis method wasdeveloped and validated for 300 pesticides in tea and HFI samples based on QuEChERSprocedure combined with GC-MS/MS. Method with total analysis time of 28 min with only0.6 µL injection volume has been successfully applied to the analysis of commercial tea andHFI samples, such novelty of analytical method without the need for continual, high-costand time-consuming maintenance influence a lot on TCO in terms of reduction of main-tenance costs especially regarding to sample preparation, injection port, columns, filaments,quadrupole and detector. Accurate retention time information combined with fragmenta-tion was used for accurate analyte detection and quantification and MMC was applied tocompensate matrix effect. The overall performance of the method was satisfactory for mostof targeted compounds. The coefficient of determination (R2) for 91% pesticides were> 0.999 within the calibration linearity range of 2 µg/kg–50 µg/kg and limits of quantifica-tion (LOQ) were ranged between 0.05 µg/kg and 135 µg/kg. Recoveries for 88% of pesticidesat 10 µg/kg were in the range of 60%–120% indicating satisfactory accuracy. Around 174pesticides were detected in 1,466 real tea and HFI samples but chlorpyrifos, DEET, tebuco-nazole, terbuthylazine, piperonyl butoxide, biphenyl, pendimethalin, pirimiphos-methyl andp,p’-DDE were most presented pesticides. Regardless of the large number of pesticides

Table 2. The short-term and long-term risks due to average daily intake of pesticides through teaconsumption in Croatia.

Pesticide

Short-term risk Long-term risk

ESTI(mg kg−1

day−1)ARfD (mg/kg

bw)aHI(%)

EDI(mg kg−1

day−1)ADI (mg/kg bw per

day)HQ(%)

Chlorpyrifos 3.2988E-05 0.005 0.6598 7.7288E-07 0.001 0.0773DEET 0.2697E-05 - - 0.7390E-07 - -Tebuconazole 0.3638E-05 0.030 0.0121 1.2872E-07 0.030 0.0004Terbuthylazine 0.1241E-05 0.080 0.0016 0.5862E-07 0.040 0.0001Piperonylbutoxide

0.0971E-05 - - 0.4660E-07 0.200 0.0000

Biphenyl 0.0281E-05 - - 0.1905E-07 0.038 0.0000Pendimethalin 0.0620E-05 1.000 0.0001 0.3781E-07 0.125 0.0000Pirimiphos-methyl 0.1878E-05 0.150 0.0013 1.2066E-07 0.040 0.0003p.p’-DDE 0.0734E-05 - - 0.5080E-07 0.010 0.0005

1002 A. BENETA ET AL.

detected and especially for the most presented pesticides, the calculation of short-term andlong-term estimates of exposure to human health does not give worrisome information. Allthe aHIs were tiny, which meant there was a negligible short-term or acute risk with theexposure to the tested pesticides via tea and HFI consumption. In the long-term riskassessment, the HQs were notabely lower than aHIs, which indicated the chronic risk frompesticide exposure via tea and HFI consumption do not need to be considered further.

Concentrations of pesticides found in tea and HFI samples confirmed the globalconcern regarding HFI and tea contamination with pesticide residues. Large numberof pesticides, high precision of the method and simple sample preparation together withcertificate of accreditation makes proposed method a valid candidate for economicquality control of tea and HFI samples.

Acknowledgments

The authors gratefully acknowledge use of the instruments, standards, real samples and overallsupport services of Shimadzu Ltd and Quanta Ltd.

Disclosure statement

No potential conflict of interest was reported by the authors.

ORCID

Dragana Mutavdžić Pavlović http://orcid.org/0000-0001-7689-7078

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Abstract1. Introduction2. Experimental2.1. Reagents and chemicals2.2. Samples2.3. Sample preparation2.4. Instrument and apparatus2.5. Method validation2.6. Risk assessment

3. Results and discussion3.1. Optimisation of GC-MS/MS conditions3.2. Sample preparation3.3. Matrix effect3.4. Method validation3.5. Comparison of the proposed method with other works3.6. Sample analysis3.7. Risk assessment on human health for most presented pesticides

4. ConclusionsAcknowledgmentsDisclosure statementReferences