direct analysis of dithiocarbamate fungicides in fruit by...

Food Additives and ContaminantsVol. 28, No. 10, October 2011, 1372–1382

Direct analysis of dithiocarbamate fungicides in fruit by ambient mass spectrometry

Tomas Cajkaa, Katerina Riddellovaa, Paul Zomerb, Hans Molb and Jana Hajslovaa*

aDepartment of Food Chemistry and Analysis, Faculty of Food and Biochemical Technology, Institute of Chemical TechnologyPrague, Technicka 3, 166 28 Prague 6, Czech Republic; bRIKILT Institute of Food Safety, Wageningen University and ResearchCentre, Akkermaalsbos 2, 6708 WB Wageningen, The Netherlands

(Received 2 March 2011; final version received 19 May 2011)

Dithiocarbamates (DTCs) are fungicides that require a specific single-residue method for detection andverification of compliance with maximum residue limits (MRLs) as established for fruit and vegetables in the EU.In this study, the use of ambient mass spectrometry was investigated for specific determination of individualDTCs (thiram, ziram) in fruit. Two complementary approaches have been investigated for their rapid analysis:(i) direct analysis in real time (DART) combined with medium-high resolution/accurate mass time-of-flight massspectrometry (TOFMS) and high-resolution/accurate mass Orbitrap MS, and (ii) desorption electrosprayionization (DESI) combined with tandem-in-time mass spectrometry (MS2). With both techniques, thiramdeposited on a glass surface (DART) or Teflon (DESI) could be directly detected. With DART, this was alsopossible for ziram. Before the instrumental analysis of fruit matrix, an extract had to be prepared followinga straightforward procedure. The raw extracts were deposited on a slide (DESI), or rods were dipped into theextracts (DART), after which thiram and ziram could be rapidly detected (typically 10 samples in a few minutes).In the case of thiram, the lowest calibration levels were 1mg kg�1 (DART–TOFMS, DESI–MS2) and 0.1mg kg�1

(DART–Orbitrap MS). For ziram, the achieved lowest calibration levels were 0.5mg kg�1 (DART–TOFMS) and1mgkg�1 (DART–Orbitrap MS). In all cases, this was sufficiently low to test samples against EU-MRLs for anumber of fruit crops. Using an internal standard, (semi)quantitative results could be obtained.

Keywords: method validation; pesticide residues; fruit

Introduction

Introduced 40–70 years ago, dithiocarbamate fungi-cides (DTCs) still represent an important class of plantprotection product widely used in agriculture. They arecharacterized by a broad spectrum of activity againstvarious plant pathogens, by low acute mammaliantoxicity, and low production costs. In combinationwith modern systemic fungicides, they are also used tomanage resistances and to broaden the spectrum ofactivity. It is therefore not surprising that the so-called‘‘maneb group’’ (zineb, maneb, mancozeb, propineb,metiram) was not only one of the most frequentlydetected pesticides reported in the document onMonitoring of Pesticide Residues in Products ofPlant Origin in the European Union, Norway,Iceland, and Liechtenstein, but this group also hadthe highest frequency in exceeding maximum residuelimits (MRLs) (European Commission (EC), SEC(2007) 1411).

DTCs are non-systemic fungicides, the residuesof which mostly remain on the surface of thesprayed crop. DTCs can be categorized into three

sub-classes depending upon their carbon skeleton –dimethyldithiocarbamates (DMDs), ethylenebis(dithiocarbamates) (EBDs), and propylenebis(dithiocarbamates) (PBDs). Without the presenceof sodium salts, EBDs and PBDs forming polymericchelates are almost insoluble in both, water and organicsolvents. However, DMDs (e.g. thiram, ziram, ferbam)are slightly soluble in water and some polar organicsolvents. Due to the insolubility in common extractionsolvents and also poor stability, they are not amenableto multi-residue methods. On this account, a singleresidue method which involves their conversion intocarbon disulfide (CS2) is commonly used. CS2 is thendetected spectrophotometrically or by gas chromatog-raphy with various detection options (Crnogorac andSchwack 2009). Inherent to this approach is that noinformation is obtained on the origin or the sourceof CS2, i.e. which DTC had been applied in the field.Although current EU legislation for routineenforcement is still based on the determination ofDTCs as CS2, specific maximum residue limits (MRLs)have been established for thiram, ziram and propineb,

*Corresponding author. Email: [email protected]

ISSN 1944–0049 print/ISSN 1944–0057 online

� 2011 Taylor & Francis

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these therefore should be determined as such whenrequired (European Community, CommissionDirective 2007/57/EC).

Over the few recent years, a large number of novelambient desorption ionization techniques, such asdesorption electrospray ionization (DESI), atmo-spheric-pressure solids analysis probe (ASAP), directanalysis in real time (DART), and many others, havebecome available. Their main advantages, comparedto conventional techniques, involve the possibility ofdirect sample examination in the open atmosphere,minimal or no sample preparation requirements, andremarkably high sample throughput (Takats et al.2004; Cody et al. 2005; McEwen et al. 2005).

DART represents one of APCI-related techniquesemploying a glow discharge for ionization. Metastablehelium atoms, originated in the plasma, react withambient water, oxygen, or other atmospheric compo-nents to produce the reactive ionizing species. DARTion source was shown to be efficient for soft ionizationof a wide range of both polar and non-polarcompounds. DART produces relatively simple massspectra characterized in most cases by [MþH]þ inpositive-ion mode, and [M�H]� in negative-ion mode.Coupling the DART ion source with a mass spectro-meter (MS) with a high mass resolving power (e.g.time-of-flight MS, Orbitrap MS) enabling accuratemass measurements allows confirmation of the targetanalyte identity and the calculation of elementalcomposition of ‘‘unknowns’’ (Cody et al. 2005;Hajslova et al. 2011).

In the case of DESI, an electrospray (ESI) needle iskept at high voltage and the spray is pneumaticallyassisted with nitrogen gas. DESI solvents are usuallyaqueous mixtures of methanol or acetonitrile withformic acid or acetic acid, and are sprayed at a few mlper min. The solvent spray is directed onto the surfaceto be analyzed. In the case of liquids, sample aliquotscan be pipetted onto a surface, usually a glass orpolytetrafluoroethylene (PTFE) slide. The pneumati-cally assisted electrospray is used to produce chargedsolvent droplets and gas phase solvent ions that collidewith analytes on the sample surface. The ionizationprocess closely resembles conventional ESI–MS; how-ever, the sample is not present in the solvent or ionizedduring the electrospray process, and might be lessvulnerable to ionization suppression caused by thepresence of salts and other interfering matrix compo-nents. Similarly to ESI, the resulting mass spectra showmainly singly or multiply charged molecular ion of theanalyte (Takats et al. 2004; Nielen et al. 2011).

In this study, we have addressed the challenge todevelop a rapid method for the specific determinationof individual DTCs based on the use of ambient massspectrometry. Two complementary approaches havebeen investigated for rapid analysis of DTC fungicidesin fruits by: (i) direct analysis in real time (DART)

combined with medium-high resolution/accurate masstime-of-flight mass spectrometry (TOFMS) and high-resolution/accurate mass Orbitrap MS, and (ii) desorp-tion electrospray ionization (DESI) combined withtandem-in-time mass spectrometry (MS2).

Materials and methods

Chemicals and reagents

Standards of target DTCs thiram and ziram togetherwith triphenyl phosphate (TPP) used as an internalstandard (purity 99.0, 97.0 and 99.0%, respectively)were obtained from Dr. Ehrenstorfer (Augsburg,Germany) and Sigma-Aldrich (Taufkirchen,Germany). Working standard solutions were preparedin acetonitrile (0.5mgml�1). In the case of ziram, thedissolving process was supported by ultrasonicationfor 5 s. Acetonitrile (HPLC grade) was provided byMerck (Darmstadt, Germany). Magnesium sulfate andsodium chloride were delivered from Sigma-Aldrichand Lach-ner (Neratovice, Czech Republic), respec-tively. Poly(ethylene glycol), PEG 600, obtained fromSigma-Aldrich was used as a mass calibrant. Ultra-pure water was produced by Milli-Q system (MilliporeCorporation, Bedford, MA, USA).

For the DESI experiments acetonitrile and meth-anol from Biosolve (Valkenswaard, The Netherlands)were used. Formic acid, ammonium formate, sodiumacetate, sodium hydrogen carbonate, DL-penicillamineand a sodium hydroxide solution were from Sigma-Aldrich; magnesium sulfate, acetic acid, and ammoniafrom Merck. Primary secondary amine from AgilentTechnologies (Santa Clara, CA, USA) was used to testthe effect of clean up of the extract by dispersive solidphase extraction (dSPE). TMTD, a commercial plantprotection product containing thiram, was purchasedfrom a local agrochemicals supplier. Standards ofziram and propineb were purchased from Dr.Ehrenstorfer.

Instrumentation

DART–TOFMS

For DART–TOFMS analyses, the system consistedof a DART ion source (model DART-100; IonSense,Danvers, MA, USA), an AccuTOF LP medium-highresolution TOF mass spectrometer (JEOL (Europe)SAS, Croissy sur Seine, France), and an HTC PALautosampler AutoDART-96 (Leap Technologies,Carrboro, NC, USA).

The DART ion source was operated in a positiveion mode with helium as the ionizing medium at a flowrate of 3.5 lmin�1. The gas beam was heated to 300�C.The discharge needle voltage of the DART source wasset to �3000V and the discharge/grid electrode volt-ages were þ150/þ250V, respectively. The desorption

Food Additives and Contaminants 1373

time was 5 s. Accurate mass profiles were acquiredwithin the range m/z 100–500, the spectra recordinginterval was 1 s (1 spectrum s�1), and the peak voltagevalue was 1000V. A solution of PEG 600 in methanol(200 mg l�1) was used for mass axis calibration of theDART–TOFMS instrument.

MassCenter (JEOL) software (v. 1.3.0) was used forinstrument control, data acquisition and data process-ing. Mass spectral data were obtained by averaging ofthe mass spectra recorded during the exposure of thesample to the DART gas beam; background ions weresubtracted and a mass drift was corrected.

DART–Orbitrap MS

The DART–Orbitrap MS system consisted of a DARTion source (model DART-SVP) with a 12 Dip-It tipscanner autosampler (IonSense, Saugus, MA, USA)coupled to an Exactive high-resolution mass spectro-meter (Thermo Fisher Scientific, San Jose, CA, USA).A Vapur interface (IonSense, Saugus, MA, USA) wasemployed to hyphenate the ion source and the massspectrometer. Low vacuum in the interface chamberwas maintained by a membrane pump (Vacuubrand,Wertheim, Germany).

The DART ion source was operated in a positiveion mode with helium as the ionizing medium at apressure of 4 bar. The gas beam was heated to 400�C.The discharge needle voltage of the DART source wasset to �5 kV and the grid electrode voltage wasþ350V. The parameters of the mass spectrometerwere as follows: capillary voltage: þ30V; tube lensvoltage: þ110V; capillary temperature: 250�C. Theacquisition rate was set to 4 spectra s�1, correspondingto a mass resolving power of 25,000 FWHM (m/z 200).A constant speed of 0.5mm s�1 was used for the Dip-Ittip scanner autosampler.

XCALIBUR software (v. 2.1) was used for instru-ment control, data acquisition and data processing.Mass spectral data were obtained by averaging ofthe mass spectra recorded during the exposure of thesample to the DART gas beam followed by thesubtraction of background ions.

DESI–MS2

An Omnispray DESI source from Prosolia Inc.(Indianapolis, IN, USA) was coupled to a LXQlinear ion trap from Thermo Fisher Scientific. Aglass microscope slide with PTFE spots (Prosolia)was used as a sample substrate. The final spraysolution was MeOH :H2O (1 : 1, v/v) with 0.1%formic acid and 10mM ammonium formate at a flowof 5 ml min�1. The spray voltage was 5 kV, the iontransfer tube was held at 50�C. The distance betweenspray tip and sample was 3mm, and the distancebetween sample and the MS inlet was 0.5mm.

The nitrogen pressure was 120 psi. A modified iontransfer tube (Thermo Fisher Scientific) was used tominimize the distance between MS inlet and thesample. The angle between spray tip and sample was55�. The sample slide moved at a speed of 250 mms�1.

Sample extraction

Procedure I for DART–MS analyses

Fruit samples (pear) were weight as whole (100–250 g)in 1-l polyethylene bags. After addition of acetonitrile(1ml per 1 g of the sample) and internal standardsolution (2mg kg�1), the bag was clipped shut and theextraction was conducted for 15min on a shaker (HS260 basic, IKA, Staufen, Germany) operating at180min�1. A volume of 0.7ml of sample extract wasput into the sampling hole of a deep well micro-platefor direct analysis. The final concentration of matrixwas 1 gml�1.

For the validation experiments, appropriate vol-umes of thiram, ziram and TPP from the stocksolutions (all at 0.5mgml�1 in acetonitrile) wereadded in a polyethylene bag containing the wholefruit and the extraction solvent (1 : 1, w/v) to achieveconcentrations of 5, 1, and 2mgkg�1, respectively.

Procedure II for DART–MS analyses

The QuEChERS-based procedure (Anastassiades et al.2003) was applied: 5 g of roughly homogenized samplewas mixed with 5ml of water and extracted using 10mlof acetonitrile in a 50-ml PTFE vessel. The mixture wasvortexed for 1min. Subsequently, 4 g of MgSO4 and1 g NaCl were added, and the mixture was shakenfor another minute. The mixture was then fortified byinternal standard solution (2mg kg�1). To provide awell-defined phase separation, centrifugation at11,000 rpm for 5min was used. A volume of 0.7ml ofsample extract was put into the sampling hole of a deepwell micro-plate for direct analysis. The final concen-tration of matrix was 0.5 gml�1 (Note: Roughly,medium, and finely homogenized samples wereobtained by homogenization of �400 g of pears for15, 30, and 60 s using a homogenizer (2094Homogenizer, Foss Tecator, Hoganas, Sweden)).

For the optimization experiments, appropriatevolumes of thiram and ziram from the stock solutions(both at 0.5mgml�1 in acetonitrile) were added toeither homogenized sample or the whole fruits toachieve the concentrations of 10 and 5mgkg�1,respectively. In the case of spikes added to homoge-nized fruit, the standard solutions were added to (i) thehomogenized sample in a PTFE vessel, followed by animmediate extraction step, or (ii) the homogenizedsample in a PTFE vessel, followed by an extractionstep after 10min. For spikes carried out before sample

1374 T. Cajka et al.

homogenization, the standard solutions were spread on

the whole fruits (400 g) placed in a homogenizer. After

sample homogenization, the QuEChERS extractionprocedure followed. The internal standard (TPP) was

added (2mg kg�1) before the centrifugation step.For the validation experiments, appropriate vol-

umes of thiram and ziram from the stock solutions(both at 0.5mgml�1 in acetonitrile) were spread on the

whole fruits (400 g) present in a homogenizer to

achieve the concentrations of 5 and 1mgkg�1, respec-tively (MRLs). After sample homogenization, the

QuEChERS extraction procedure followed. The inter-

nal standard (TPP) was added (2mg kg�1) before thecentrifugation step.

Note: Matrix-matched standards of thiram and

ziram containing also an internal standard (TPP) were

used for the quantification purposes (determination ofrecoveries). Calibration graphs were constructed by

plotting the analyte concentrations (x-axis) against the

ratio of the target DTCs and internal standard TPPresponse (y-axis).

Procedure I for DESI–MS2 analyses

Two types of QuEChERS extraction were tested, a

non-buffered version, the same as described in thesection ‘‘Procedure II for DART–MS analyses’’ and an

acetate-buffered version (Lehotay, de Kok et al. 2005).

In short: 10 g of homogenized sample was extractedwith 10ml acetonitrile. A phase partitioning was

induced by addition of salts. After vigorous shaking

and centrifugation, the acetonitrile phase was mea-sured. An optional clean up by dispersive SPE was

done by transferring 0.5ml to an Eppendorf cup

containing 25mg PSA and 150mg magnesium sulfate.The cup was vortexed and centrifuged. For DESI–MS2

analysis, 1.5 ml aliquots of the extracts were pipetted

onto 1.5-mm ID PTFE spots printed on a microscopeslide. After evaporation of the solvent under ambient

conditions, the slide was put on a moving stage and the

spots could automatically be positioned in the spraybeam of the DESI source for detection of the analyte.

Procedure II for DESI–MS2 analyses

As an alternative to the procedure involving extraction

of a homogenized product, the rinsing procedure asdescribed in the section ‘‘Procedure I for DART–MS

analyses’’ was also performed. Again, 1.5 ml of extractwere pipetted onto PTFE spots on the microscope slidefor DESI–MS2 measurement.

For both extraction procedures, azoxystrobin

(arbitrarily chosen for method development purposes)

was used as an internal standard, and was added to theextracts just before the DESI measurements.

Results and discussion

DART–MS experiments

Optimization of DART parameters

In the first part of our experiments, the relationshipbetween the setting of various DART operatingparameters and features of mass spectra generatedunder particular conditions was investigated. Twomodels of DART ion source (i.e. DART-100,DART-SVP) were tested within our experiments,each requiring optimization of particular settings. Ingeneral, helium beam temperature, flow rate anddesorption time were the major parameters affectingDART ion formation and effectiveness of their trans-mission into MS.

Protonated molecules [MþH]þ were obtainedunder conditions of positive DART ionizationwhen analyzing standards of thiram and ziramdissolved in acetonitrile. In the case of thiram, the[MþH]þ ion corresponded to an elemental composi-tion of [C6H12N2S4þH]þ, while that of ziram to[C6H12N2S4ZnþH]þ, each characterized by the char-acteristic isotope pattern.

The impact of gas beam temperature wasmonitored for temperatures 250, 300, 350, 400, and450�C. For the model DART-100, the temperature of300�C provided the highest responses for both ana-lytes tested, while a temperature of 400�C wasoptimal for the model DART-SVP. Helium flowrates were also observed to have an influence on theDART–TOFMS responses of target analytes. Thisparameter was tested for 3.0, 3.5, 4.0 and 4.5 lmin�1

(DART-100). A helium flow of 3.5 lmin�1 gave thehighest responses for both analytes. In the case ofDART-SVP, a constant pressure of 4 bar is recom-mended; thus, this value was kept during the allexperiments.

Another important factor optimized was(thermo)desorption time. It is worth to mention thattwo different commercial autosampler devices wereused to improve the quantitative DART measurementsand also the throughput of analyses. The AutoDARTautosampler (HTC PAL autosampler AutoDART-96)uses the robotic arm to deliver the sample from a deep-well reservoir into the sampling region where desorp-tion at fixed position occurs. On the other hand, a 12Dip-It tip scanner autosampler scans the glass-rodsurfaces with samples through the DART gas streamat a constant speed. In the case of an AutoDARTautosampler, the tested values of (thermo)desorptiontime included 1, 2, 5, 10 and 30 s. It was observed that5 s provided sufficient intensity of ions. Longer desorp-tion time led only to increased detection of variousmatrix co-extracts present in the sample extracts. Fora 12 Dip-It tip scanner autosampler, a constant speedof 0.5mm s�1 was optimal providing the MS peakwidth of 5 s.


The last parameter considered was the use of aVapur interface (DART-100), which is designed toimprove collection of ions desorbed from the sampleby collimating them for transfer into the MS. However,using this ‘‘ion concentrator’’, only a relatively slightincrease of signal intensity was observed (�20%) forthiram and ziram. On the other hand, a much highersignal intensity of the chemical background wasobserved. Therefore, a Vapur interface was not usedin subsequent experiments (DART–TOFMS).However, the use of a Vapur gas ion separatorduring DART ionization was essential to maintainstable vacuum within the operating pressure limits ofthe Exactive instrument (Orbitrap MS).

Optimization of sample preparation for DART–MS

Once the DART parameters were optimized, thedetection of thiram and ziram in real-world samples(fruits) was investigated. The recovery studies wereperformed with pears at a level of 10mgkg�1 forthiram and 5mgkg�1 for ziram. After that, theoptimized method was tested at the levels of5mgkg�1 for thiram and 1mgkg�1 for ziram, whichrepresent the MRLs for this type of matrix.

In the initial phase of the experiment, we used amethod developed by Crnogorac and Schwack (2007),which was modified in terms of using acetonitrile as anextraction solvent instead of water containingsodium hydrogen carbonate and DL-penicillamine(Procedure I). Employing this sample preparationmethod, recoveries of 94 and 102% and RSDs of 8and 17%, were achieved for thiram and ziram, respec-tively.However, the drawback of thismethodwas a highconsumption of the solvent (1ml per 1 g of sample) sincethe whole fruit (�100–250 g) needed to be analyzed.

To overcome this limitation, we decided to developan alternative strategy for sample preparation.Choosing QuEChERS-based procedure as a conceiv-able option, we were concerned about recoveries,which were reported low when using this protocol forthiram analysis in matrices such as oranges and lettuce(Lehotay, Mastovska et al. 2005). This problem seemsto be related to poor stability of DTCs in acidic mediaas well as to the action of various enzymes releasedduring the extensive sample homogenization of bioticmatrices (Roberts and Hutson 1999).

To learn more about DTCs breakdown, in the firstpart of experiments dealing with the QuEChERS-based procedure (Procedure II), we analyzed spikes ofthiram and ziram added to differently homogenizedpears (roughly, medium, finely). The best recoverieswere obtained for finely homogenized samples(�100%) supposing immediate extraction followed.However, as far as it started 10min after addition ofthe spike, the degradation of both analytes wasobserved (�40% decrease), thus, demonstrating

their lower stability in the presence of pear matrix(Figure 1A).

The lower recoveries of roughly and mediumhomogenized samples can be explained by lowamount of water released from the sample.Therefore, we also investigated the addition of waterto the sample to increase the ‘‘active’’ water contentbefore the extraction (10 g of sample versus 5 g ofsample plus 5ml of water). As a consequence, therecoveries of target analytes improved also for the

Figure 1. Dependence of thiram and ziram recovery onsample preparation conditions. (A) The pear samples werespiked by thiram and ziram at 10 and 5mgkg�1, respectively,after completing homogenization of the matrix. For theextraction QuEChERS-based procedure with 10 g of samplesper 10ml of acetonitrile was used with subsequent immediateanalysis by DART–TOFMS. (B) The pear samples werespiked by thiram and ziram at 10 and 5mgkg�1, respectively,before homogenization of the matrix. For the extractionQuEChERS-based procedure with 5 g of samples plus 5ml ofwater per 10ml of acetonitrile was used with subsequentimmediate analysis by DART–TOFMS. For the quantifica-tion (A)þ (B) matrix-matched standards were used with TPPas an internal standard. The error bars represent standarddeviations (n¼ 3).


roughly and medium homogenized samples as far aswater was added to the sample before the extraction.

In the follow-up part of the experiments, weinvestigated the recoveries of thiram and ziram whenboth analytes were added to the sample beforehomogenization (�400 g of pears were used for eachexperiment). Again, roughly, medium, and finelyhomogenized samples were analyzed. Under theseconditions, the best recoveries were obtained forroughly homogenized samples, probably due to lessintensive interaction of target analytes with the matrixcomponents (Figure 1B). Further improvement ofrecoveries was observed as far as the pear sampleswere cryogenically homogenized (the samples were putinto the freezer (�18�C) overnight and were commi-nuted in frozen condition). Using this approach, theenzymatic activity reduces, thus, an improvement ofrecoveries of both target analytes was achieved.

To conclude, we recommend cryogenic homogeni-zation with subsequent extraction of 5 g sample plus5ml of water per 10ml of acetonitrile. Under theseconditions, the recoveries 68 and 75%, and RSDs of11 and 10%, were achieved for thiram and ziram,respectively, spiked at 10 and 5mgkg�1, respectively.The recoveries of thiram and ziram spiked at theirMRLs of 5 and 1mgkg�1, respectively, were slightlylower: 65 and 73% with RSDs of 12 and 13%,respectively. It should be noted, that the use ofisotopically labeled internal standards (currently avail-able as d12-thiram and d12-ziram) can be an effectiveway to compensate for the losses of both analytesduring the homogenization/extraction step.Unfortunately, the cost of isotopically labeled versusnative DTCs is currently prohibitively high (25Eversus 0.1E, respectively, per 1mg); moreover, isotopicexchange (D–H) may occur, thus biasing the quanti-tative data. For the quantification purposes, thematrix-matched standards containing an internal stan-dard (TPP) should be prepared shortly before the

analysis of the extracts prepared by the QuEChERSmethod. When checking the stability of the matrix-matched standards, a decrease of responses of DTCs ashigh as 20% was observed after 2 h.

Comparison of detection: TOFMS versusOrbitrap MS

A limitation of the TOFMS used in this work was therelatively low mass resolving power (typically around5000 FWHM). Consequently, a high risk of interfer-ence of the target ion with matrix components withsimilar masses can be expected, especially since inambient MS there is no LC separation. Therefore, analternative high resolution MS system equipped withan orbitrap mass analyzer offering enhanced massresolving power was also used. The DART–OrbitrapMS system showed a high ability to eliminate matrixinterferences. Figure 2 clearly illustrates the benefit ofhigh mass resolving power of this system – using the25,000 FWHM resolving power allowed completespectral separation of thiram from matrix interferenceseven if their intensities were higher compared to thisanalyte.

As illustrated in Figure 3, for quantitative analysisusing a DART ion source, it is necessary to use aninternal standard to compensate relatively high varia-tion of the ion intensities of analytes. Triphenylphosphate (TPP), yielding [MþH]þ at m/z 327.079,was used for this purpose (spiked at a level of2mgkg�1). Calibration curves obtained by analysesof matrix-matched standards were constructed byplotting the ratio of analyte/internal standard ionintensity versus concentration of the particular analyte.Acceptable linearity was obtained for the testedconcentration range; regression coefficients of calibra-tion curves were 40.97.

Using DART–TOFMS, the limits of quantification(expressed as the lowest calibration levels, LCLs) were

Figure 2. Thiram (m/z 240.996) in pear extract analyzed by DART–Orbitrap MS (0.1mg kg�1) and DART–TOFMS(1mg kg�1). The mass resolving power (R) is also indicated.


1 and 0.5mg kg�1 for thiram and ziram, respectively,and 0.1 and 1mgkg�1 for thiram and ziram, respec-tively, when employing DART–Orbitrap MS. The highvalue of LCL of thiram in the case of TOFMS wasattributed to the low mass resolving power of theTOFMS, resulting in insufficient resolution of thiramfrom matrix coextractants. However, even with thisdifference of sensitivity/selectivity, the achieved LCLfor thiram by both instruments are acceptable for thecontrol of the MRLs of fruit commodities such aspears, apples, plums, and strawberries (EU-MRLs of 5,5, 2 and 10mgkg�1, respectively). In the case of ziram,the obtained LCLs would be suitable to control theMRL of fruit commodities such as pears and plums(1 and 2mgkg�1, respectively). Figure 4 shows MSrecords of both analytes at their MRLs in pear extractusing DART–TOFMS and DART–Orbitrap MS. Therecords of blank samples are also presented to illustratethe selectivity of detection.

DESI–MS2 experiments

Optimization of DESI–MS2 parameters

As the first step, the MS response of different DTCswas tested by dilution of standard solutions into the

spray solvent of the DESI source. Thiram wasdissolved in acetonitrile. Ziram and probineb weredissolved in a mixture of DL-penicillamine and sodiumhydrogen carbonate in water (at pH 12) as describedby Crnogorac and Schwack (2007), which resulted inthe disintegration of the polymer and formation ofclass-specific anions. Several spray solvent composi-tions were tested: aqueous methanol or acetonitrile,with and without addition of acids (acetic acid, formicacid, ammonia, and/or ammonium formate). In addi-tion, the ion transfer tube temperature was varied. Theziram (transition m/z 120 ! m/z 76 with a collisionenergy of 10 eV) and propineb (transition m/z 225 !m/z 191 with a collision energy of 20 eV) anions couldbe detected but only with relatively low sensitivity. Dueto limitations of the linear ion trap in the differencebetween precursor and product ions that can bedetected, some of the precursor/product ion combina-tions described by Crnogorac and Schwack (2007)could not be measured. Thiram was sensitivelydetected when using a water–methanol mixture (1 : 1,v/v) with 0.1% formic acid as spray solvent. An[MþNa]þ adduct (m/z 263) was obtained but frag-mentation of this ion did not give sensitive productions. After adding 10mM of ammonium formate tothe spray solution, the protonated molecules [MþH]þ

Figure 3. DART–Orbitrap MS and DART–TOFMS analysis of matrix-matched standards. Concentration of thiram (m/z240.996) was in the range of 0.1–10mg kg�1. To illustrate the fluctuation of signal intensity, the internal standard TPP (m/z327.079) is also shown (concentration 2mgkg�1).


of thiram (m/z 241) were the most intense signal. Thision could be fragmented to m/z 196 and m/z 88 byapplying a collision energy of 25 eV.

Next, the standards were deposited on PTFE spotsprinted on a glass slide and the different geometricparameters of the DESI source were optimized, mea-suring the most intense DTC transition.Unfortunately, only thiram could be detected byDESI–MS2 (optimum conditions provided in theexperimental section). Therefore, further work withDESI–MS2 was limited to this DTC.

Attempts to directly detect thiram from crop surfaces

One of the attractive features of DESI–MS is directdetection of analytes from a sample surface. The directdetection of a pesticide from a crop surface has beendemonstrated by Garcia-Reyes et al. (2009). Althoughdetection of thiram from a (small) surface area ofa crop sample is not easily related to an MRL set inmg kg�1 whole crop, such direct detection might bevery useful for fast qualitative screening purposes.Therefore, attempts were made to directly detectthiram deposited on pear leaves taped onto the

microscope slide, but unfortunately without success.The signal was much lower compared to that of solventstandard deposits, and, in addition, the signal wasinterfered by matrix. Possible explanations for thesephenomena include interference of the matrix on theionization process and the fact that the leaves are adifferent material than the slides, which can influencewetting of the surface and the formation of secondarydroplets which are thought to play an important role inthe ion formation during DESI (Gao et al. 2010).

Indirect analysis through an intermediateextraction step

As an alternative to the direct surface analysis, differentextraction methods, which are widely used in pesticideresidue analysis, were studied. Since thiram dissolveswell in acetonitrile, three variations of the QuEChERSextraction procedure were examined, a non-bufferedversion and an acetate-buffered version with andwithout dSPE clean up. Here, extraction was doneusing a spiked homogenized pear sample. Anotherapproach involving a surface extraction of the intactproduct with acetonitrile was also carried out.

Figure 4. DART–Orbitrap MS and DART–TOFMS analysis of spikes of thiram (m/z 240.996) and ziram (m/z 304.925) in thepear extracts at the MRLs of 5 and 1mgkg�1, respectively.


Aliquots of the different extracts were pipetted ontothe PTFE spots of the slide and then measured byDESI–MS2.

Figure 5 shows a comparison between the differentextraction methods (note that the signals for samplesfrom the QuEChERS extraction are enhanced 15times). As is clear from Figure 5, the surface extractionprovided the least suppression and highest response.Therefore, this method was chosen for further work.The method was additionally tested on apple, pear,strawberries and lettuce leaves spiked with thiram atthe respective MRLs of the different matrices (2–10mgkg�1). Immediately after surface extraction ofthe samples, 1.5 ml aliquots were deposited on thePTFE spots of the glass slide. The solvent evaporatesquickly under ambient conditions leaving a stablethiram deposit. Thiram could be successfully detectedfrom all crops tested.

Semi-quantitative analysis of samples

In solvent standards, thiram could be detected down toconcentrations of 0.1mg l�1 (corresponding to 0.15 ngabsolute or �0.1 ngmm�2). No spot-to-spot carry-overoccurred. The response increased with the amount ofthiram deposited but the absolute response was oflimited reproducibility. By addition of an internalstandard (in this case, rather arbitrary, azoxystrobinwas used) for normalization of the response, semi-quantitative data could be obtained. Using solventstandards (0–1mg l�1) and averaging three analyses of

the same spots, a five-point calibration curve wasconstructed. The linearity was considered sufficient(R2¼ 0.98) to allow at least semi-quantitative analysis.The applicability of the method to real-world sampleswas tested by spiking six pear samples at 10mg kg�1

and measuring them against a calibration solution inmatrix at the same level. The average recovery was85% with an RSD of 26%.

Detection of thiram applied as TMTD

In the field, thiram is not applied as the pure substance.Instead the formulated plant protection products suchas TMTD, which contains 80% of thiram, is suspendedin water at 2 g l�1 and applied onto the crops. To test ifthe DESI–MS2 method was also able to detect thiramwhen applied as TMTD, strawberries were spiked atthe MRL of 10mgkg�1 using both the TMTDsuspension and a solution of thiram in acetonitrile.After spraying, the fruits were left for 30min to allowdrying and interaction with the surface.

Figure 6 shows that thiram could be easily detectedboth when deposited as TMTD and neat thiram.During MS measurement, product ion spectra areobtained which facilitate identification.

Figure 7 shows the product ion spectra of m/z 241for a solvent standard and a strawberry spiked at10mgkg�1. Both the quantifier at m/z 196 and thequalifier at m/z 88 can be seen in the correct ion ratioaccording to the applicable EU guideline (SANCO/10684/2009).

Figure 5. DESI–MS2 analysis of thiram and azoxystrobin (internal standard) in various pear extracts deposited on PTFE spots.


The results obtained demonstrate that theDESI–MS2 method is suited for detection of thiramin real-world samples and to provide at least asemi-quantitative concentration.

Conclusions

Ambient MS was investigated for rapid detection of

DTCs in fruits. Two complementary ionization tech-

niques were investigated: DART and DESI. With both

Figure 7. Scan of product ions of m/z 241 at a collision energy of 25 eV. (A) Solvent standard of thiram at a concentration of10 mg ml�1. (B) Spiked strawberry sample by thiram at 10mg kg�1.

Figure 6. DESI–MS2 analysis of (i) blank strawberry extract, (ii) extract of strawberry spiked with a solvent standard solution ofthiram (equivalent to 10mgkg�1), (iii) extract of strawberry sprayed with commercial crop protection product TMTD(equivalent to 10mg thiramkg�1). Each deposit in duplicate. To illustrate the fluctuation of signal intensity, internal standard(azoxystrobin) is also shown.


approaches, an extraction step was found to benecessary to facilitate detection.

With DESI–MS2, the best results were obtainedusing a surface extraction of the intact product. Forextracts of homogenized product, severe suppressioneffects were observed. Thiram could be rapidlydetected (typically 10 samples in few minutes) downto 0.1mg l�1 in standard solutions and at MRL levelin extracts from various fruits. Using an internalstandard, semi-quantitative results could be obtained.With the current instrumentation and interface design,it was not possible to detect the other DTCs tested.

In the case of DART, also here an extraction stepwas employed. For detection, a glass rod was dippedinto the raw extract and simply positioned between theDART source and MS detector. Using this strategy,thiram was easily detected. In addition, ziram couldalso be detected as an intact molecule after dissolutionin acetonitrile. The detectability of both DTCs wasdemonstrated at MRL level in pears. Using an internalstandard, quantitative analysis was possible.

Overall, despite a high solvent consumption, thesurface extraction method combined with DARTanalysis would be the preferred method due to higherrecovery and the applicability to two of the three DTCsfor which the individual MRLs have been established.

Disclaimer

The information reported reflects the authors’ views;the European Commission is not liable for any useof the information contained therein. Mention ofbrand or firm names in this publication is solely forthe purpose of providing specific information and doesnot imply recommendation or endorsement by theInstitute of Chemical Technology, Prague or theRIKILT Institute of Food Safety.

Acknowledgements

This project is part of the EU-project ‘‘Contaminants in foodand feed: Inexpensive detection for control of exposure’’(acronym Conffidence) and is financially supported by theEuropean Commission, contract 211326-CP CollaborativeProject. A financial support of the Ministry of Education,Youth and Sports of the Czech Republic (project MSM6046137305) is also acknowledged.

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