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Simultaneous Determination of Residues of Approximately 100 Pesticides ,,, and Metabolites in Fruit and Vegetables by LC/MS/MS

Many pesticides, which are difficult or generally not to analyse by GC/MS, can be identified and quantified by LC/MS/MS. After simple solvent extraction and fast cleanup the extracts are analysed using matrix-matched standards. The drawback of MS/MS methods is laborious parameter optimization for each target analyte. This effort can significantly be reduced by the use of standard parameters, which are given in the attached CD ROM.


Actually more than 800 pesticides are used worldwide. For many of these compounds legal action levels (e.g. maximum residue limits or tolerances) in food exist, which have to be controlled. For this type of target analysis multi residue analytical methods are preferred to reduce the workload. Up to now, such methods based on LC/MS/MS had been published for a reduced number of pesticides only. Furthermore, there is no knowledge about the maximum number of compounds, which can be determined simultaneously.


Extraction and cleanup procedures are described in Figure 1. Using the readyfor- use method files on the CD ROM in combination with API 2000, API 3000, and API 4000 LC/MS/MS Systems following pesticides and their metabolites can be identified and quantified: 3,4,5-trimethacarb, 3-hydroxycarbofuron, 5-hydroxyclethodimsulfone, 5-hydroxythiabendazole, acephate, aldicarb, aldicarbsulfoxide, aldoxycarb, amidosulfuron, atrazine, azoxystrobin, bendiocarb, bensulfuronmethyl, butocarboxim, butocarboxim-sulfoxide, butoxycarboxim, carbaryl, carbendazim, carbofuran, chlorsulfuron, cinosulfuron, clethodim, clethodimiminsulfone, clethodimiminsulfoxide, clethodimsulfone, clethodimsulfoxide, cyprodinil, daminozid, demeton-S-methyl, demeton-S-methylsulfone, desmedipham, desmethylformamidopirimicarb, desmethylpirimicarb, dimethoate, diuron, ethiofencarb, ethiofencarbsulfone, ethiofencarbsulfoxide, fenhexamid, fenoxycarb, fenpropimorph, flazasulfuron, florasulam, fluazifoppbutyl, flufenoxuron, fosthiazate, furathiocarb, haloxyfopethoxyethyl, haloxyfopethyl, imazalil, imidacloprid, indoxacarb, iodosulfuronmethyl, iprovalicarb, isoproturon, linuron, metalaxyl, metamitron, methamidophos, methiocarb, methomyl, metolachlor, metsulfuronmethyl, monocrotophos, nicosulfuron, omethoate, oxamyl, oxydemetonmethyl, phenmedipham, pirimicarb, primisulfuronmethyl, promecarb, propamocarb, propoxur, prosulfuron, pymetrozin, pyridate, pyridatmetabolite, pyrimethanil, quinmerac, quizalofopethyl, rimsulfuron, spiroxamine, tebuconazole, tebufenozid, thiabendazol, thiacloprid, thifensulfuronmethyl, thiodicarb, thiofanox, thiofanoxsulfone,

thiofanoxsulfoxide, thiophanatmethyl, triasulfuron, tribenuronmethyl, triflusulfuronmethyl and vamidothion (Figure 2). Liquid chromatography was carried out using a system equipped with a binary pump, degasser and autosampler. For the separation an analytical column Aqua 5 125 A C18 (Phenomenex, Aschaffenburg, Germany) with the dimensions 50 mm x 2 mm was used. The mobile phase A consisted of 80% water and 20% methanol, mobile phase B of 90% methanol and 10% water. Both phases A and B contained 5 mmoL/L ammonium formiate. The flow rate was 0.2mL/min. The mobile phase composition was changed during a run as follows: Starting with 0%, the percentage of B was increased linearly to 100% over 11 min and then kept constant for another 12 min. Equilibration time prior to the next injection was 15 min.

ESI versus APCI

Using the optimized MRM transitions and identical sample amounts only 8 pesticides (propoxur, carbofuran, bendiocarb, carbaryl, tebuconazole, amidosulfuron, atrazine, and promecarb) out of 74 tested gave higher signal intensities with APCI+ under the conditions applied. The other 66 gave a better response with ESI+. Therefore, the TurboIonSpray source was used here.

Number of simultaneously detectable MRM transitions

As demonstrated in Figure 3, a dwell time reduction down to 25 ms is accompanied with only a minor reduction in signal intensity. The signal to noise ratio decreases in the worst two cases to about 40% of the 100 ms value. However, this still compares to a sufficient S/N ratio. Consequently,

with 25 ms dwell time and a pause between mass ranges of 5 ms within the cycle time of 3 seconds the simultaneous measurement of 100 MRM transitions. The signal to noise ratio decreases in the worst two cases to about 40% of the 100 ms value. However, this still compares to a sufficient S/N ratio. Consequently, with 25 ms dwell time and a pause between mass ranges of 5 ms within the cycle time of 3 seconds the simultaneous measurement of 100 MRM transitions is possible. In cases, where one deals with sufficiently high concentrations, even measurements with a minimum dwell time of 10 ms should be possible, allowing the simultaneous detection of about 200 MRM transitions using an LC/MS/MS system with a LINAC collision cell. However, the parallel measurement of positive and negative ions in one run should be avoided because of the necessary stabilization of the changed polarity , which needs approximately 700 ms. This has to be done twice in an ESI+/ESI- sequence and therefore consuming about 50% of the total cycle time of 3 seconds. Figure 2 shows the simultaneous detection of 97 pesticides at the level of 0.025 mg/kg. The figure suffers from the overlay of so many traces. Therefore, a better demonstration of selectivity and sensitivity of MS/MS detection is given in Figure 4, which shows the acquired MRM traces of 12 selected analytes at 0.01 mg/kg level individually. Due to higher selectivity compared to chromatograms obtained with GC/MS in the Selected Ion Monitoring mode (SIM) significant matrix peaks are rare in LC/MS/MS, resulting in a more convenient detection and integration of relevant analyte peaks. Usually, retention time windows (periods) are used to enhance the individual dwell times and consequently the sensitivity of mass spectrometric detection. As shown in Figure 4 (and in the demo files given on the CD), such periods are unnecessary here, avoiding the resetting of switching times with new columns, other pumps etc. An example of a real sample with incurred residues is given in Figure 5, which shows the chromatogram of a lettuce extract with incurred residues.

Dependence of compound specific parameters on the type of MS/MS instrument

As mentioned above, the time consuming part in the development of a MS/MS method is the optimisation of six compound specific parameters for each analyte (totally 600 for 100 pesticides!). Therefore, the transformation of all compound specific parameters originally optimized for an API 2000 system was tested. This was done by the use of a tuning mixture, which contains 8 out of all 97 compounds (aldicarb, a cephate, carbofuran, atrazine, desmedipham, triasulfuron, cyprodinil and flufenoxuron). The criteria of selection of individual pesticides for this tuning mixture were the necessary Declustering Potential (DP) and the Collision Energy (CE). The aim was to take into account the whole range of possible parameter settings. The results of this instrument comparison are summarised in Table 1 (see over page). All parameters optimized for an API 2000 system can be directly used for the API 3000 and API 4000 systems or can be obtained with the help of the test mixture containing 8 compounds only. One parameter (DP) has to be shifted by a constant voltage and two parameters are fixed on API 3000 and API 4000 systems. Consequently, the transformation of optimum conditions from one instrument type to another is possible. At least a satisfactory sensitivity for the pesticides covered by the method will be obtained without individual optimizations of compound specific parameters. Eventually, if distinct pesticides show an unexpected low response, for these compounds only a specific tuning may be needed.

Content of the CD ROM

To assist users of Applied Biosystems/ MDS SCIEX mass spectrometers the CD ROM of this application note contains several files, which work with Analyst software version 1.2 or higher.

Method files (.dam): Method files for API 2000, API 3000, API 4000 LC/MS/MS Systems equipped with Agilent 1100 binary pump, degasser, autosampler, column oven and optional with UV detector, and for API 2000, API 3000, API 4000 LC/MS/MS Systems equipped with Agilent 1100 binary pump, degasser, column oven and HTC/CTC autosampler (PAL) plus optional UV detector are given.

Quantitation file (.qmf ) Quantitation files are given, that contain retention times, appropriate integration parameter and compounds names.

Data files (.wiff ) For test and comparison purposes the data files of a tomato sample spiked at 0.01 mg/kg is given. Additionally, the project contains appropriate matrixmatched standards (0.005, 0.001, 0.025, 0.050 and 0.10 mg/kg) and the data file of the lettuce extract of Figure 4. Supplementary files Q1 spectra of the compounds covered by the method (pdf format). Readme.txt contains information about installation of demo files.


We acknowledge the skilled technical assistance of Volker Happel, Nata?sa Markovic and Marilyn Menden


Jeannette Klein and Lutz Alder1, Andr Schreiber2 1Federal Institute for Risk Assessment P.O.B. 33 00 13, D-14191 Berlin, Germany 2Applied Biosystems, Frankfurter Strasse 129B, D-64293 Darmstadt, Germany



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