Screening of pesticides residues by the time of flight analyzer (ToF) : Myth or reality? Laure Joly & Vincent Hanot WIV-ISP, Juliette Wytsmanstraat 14, 15 Brussel The passengers pesticides are requested...... at the airport desk Over 8 pesticides are currently listed by the European Union. At present, the triple quadrupole Multiple Reaction Monitoring (MRM) coupled to a liquid or gas chromatography is the most commonly used analyzer for detecting several hundreds of molecules simultaneously. With this instrument, every potential pesticide is observed by two MRM transitions. However, the amount of pesticides to be analyzed at the same time is constantly increasing and unfortunately the amount of MRM transitions is not unlimited. Nevertheless, alternative solutions for triple quadrupole analyses are now under development. Among these, the most endorsed solution is undoubtedly the Time of Flight analyzer (ToF) used for screening prior to pesticides quantification. Moreover, it seems that this type of mass spectrometer perfectly meets the new SANCO 12495/211 criteria [1]. Finally, with regard to the latest developments made by the engineers, it seems that it is now high time for our pesticides to...... go to the gate in order to embark The time of flight analyzer (ToF) makes it possible to know the mass-to-charge ratio (m/z) of an ion based on the time (t) it needs to pass through the flight tube and reach the detector. Before entering this tube, ions are accelerated by a potential difference (U) of known strength. The potential energy charged in this manner corresponds to Ep = zeu, e being the elementary charge. Once the ions are in the tube (of length L), they evolve in an ultra vacuum condition, their potential energy is entirely converted into kinetic energy : Ec = 1/2mv 2, v being the velocity of the ion. Considering Ep = Ec and v = L/t, it appears that : m/z = t 2. Cste In real terms this means that an ion with an m/z ratio equal to 1 needs twice the time to pass through the flight tube of an ion with an m/z ratio equal to 25. Now that we have acquired the theory, it is time... 28
... to breach the sound barrier Successive technological improvements have radically changed the ToF method. First of all, a reflectron (mirror or electrostatic reflector) allowed to compensate the differences in potential energy that may occur between ions of the same m/z ratio by a difference in path length within this reflectron, so as to allow them to arrive at the detector simultaneously. Secondly, the installation of the pusher. The electrospray source which continuously generates ions makes it hazardous to determine an initial reference time (t = ) that is typical of every ion and marks the introduction of it in the tube. In order to solve this tricky problem, a pusher has now been added. Using an orthogonal ion acceleration, this device allows the ions to be packed together when entering the ToF. Moreover, this ingenious technique also allows neutrons to be kept off. Finally, an extremely low pressure (~5.1-11 bar) has to be maintained in the ToF in order to prevent all fragmentation that may occur when accelerated ions and the environment collide. Reflectron Detector Pusher Source Figure 1 : Ion path in a flight tube, the reflectron compensates the differences in energy which has been acquired before entering the flight tube : the blue and red ions having the same mass arrive at the same time on the detector. 29
Before the plane takes off, it is time to... contact the control tower The identification of pesticides is partly based, in the case of a ToF, on the molecular mass of the parent ion and not on the MRM transitions parent fragment as would have been the case with a triple quadrupole. So it is crucial to measure the masses accurately when using a ToF analyzer. In order to ensure that the measured mass is as close as possible to the theoretical value, sensitivity, accuracy and resolution are optimized as follows : At first, the working parameters of the source, the transfer parameters and the ToF parameters are optimized by a component with a mass similar to that of the pesticides to be analyzed. Secondly, an external calibration has to be realized to obtain a mass measurement that is close to the exact mass at all points of the spectrum. Such calibrations are executed daily or even weekly according to the target accuracy level. Finally, a continuous internal calibration will be executed : a reference component or lock mass and analytes are introduced in the source and this during the whole analysis process. This last phase makes it possible to break variations that might occur in time (e.g. due to temperature changes). Flight conditions seem to be optimal and we can... draw up our flight plan First of all, considering the fact that there has been no mass selection in advance by the ToF analyser, all ions generated in the source are simultaneously detected : most of the detected ions are matrix ions. The obtained chromatogram for a mandarin orange matrix containing no pesticide at all is shown in figure 2. The same ions are generated in the source with a triple quadrupole, but the analyzer serves as a filter and these ions are not detected. Co-pilot s journal Resolution is the capacity of the analyzer to separate two related m/z components and is specified by R=m/δm. Resolution measurement for an isolated mass ion (m) consists of measuring its mass defect (δm) at half peak height. The accuracy of the m/z measurement of the analyzed molecule represents the possibility to measure a mass as closely as possible to the theoretical mass. This measurement has to be true and accurate. In mass spectrometry, the measurement error (defining the accuracy) is expressed - either in dalton (absolute value), error (Da) = M exp - M th or in parts-per-million (ppm) (relative value) = M exp M th / M th * 1 6 3
2 4 6 8 1 12 14 Time (min) Figure 2 : Chromatogram of a mandarin orange matrix containing no pesticide at all Finally pesticides are identified by high resolution and high mass accuracy of the ToF analyzer. The monoisotopic mass corresponding to the first peak of the isotopic profile is the mass we were looking for. This peak only takes into account the masses of the most stable isotopes (figure 3). In comparison, the medium ion masses are the masses we are looking for with a triple quadrupole instrument. Figure 3 : in black the theoretical medium mass of cyprodinil and in violet its theoretical monoisotopic mass Monoisotopic mass 226.1344 Average mass 226.2969 22 225 23 m/z 31
After these considerations, it is time to...... take a test flight When using a ToF instrument for analyses, a software program compares theoretical monoisotopic masses (M th ) of all pesticides with the different masses of the mass spectrum (M exp ). A 5 to 1 ppm tolerance is generally accepted. Starting from a total chromatogram of a mandarin orange sample (figure 4a) the presence of cyprodinil is confirmed with a chromatographic retention time of 8.38 min by extracting the chromatogram which corresponds with its theoretical monoisotopic mass (226.1344) and a 1 ppm tolerance (figure 4b). 1 a) 1: TOF MS ES+ BPI 1.2e5 1 2. 4. 6. 8. 1. 12. 14. 8.38 1: TOF MS ES+ 226.134 1PPM 1.42e3 b) 2. 4. 6. 8. 1. 12. 14. Time (min) Figure 4 : (a) chromatogram of a mandarin orange sample, (b) chromatogram of the same sample filtered for the mass 226.1344 (theoretical mass of cyprodinil) and a 1 ppm tolerance. The mass spectrum linked to the retention time of 8.38 min is shown in figure 5a. Processing software programs subduing background noise are very useful to identify a pesticide among a very large amount of masses (figure 5b). 32
SG211126-9 2184 (8.421) Cm (2153:2191) 1 1 4.78e4 1.19e4 a) 217.1115 SG211126-9 226.135 2184 (8.421) Cm (2152:2191-(2128:2151+2191:2217)) 3.284 b) m/z 2 4 6 8 1 m/z 2 4 6 8 1 Figure 5 : mass spectra linked to a retention time of 8.38 min without (a) and with (b) subtraction of the background noise. After this successful maiden flight, it is time to... go for a long-distance flight Several hundreds of pesticides are examined when screening : the combination of the known retention time/ theoretical monoisotopic mass of every pesticide will be assessed. Previous phases are automatically or semiautomatically repeated several hundreds of times. However, different source voltages (capillary, conical) and ToF transmission voltages cannot be individualized. As it is, contrarily to what happens when using a triple quadrupole, mean generic requirements are applied to all pesticides using a ToF. Every non optimal voltage for a certain pesticide consequently reduces its chance to reach the detector undamaged. An inadequate voltage is enough for the pesticide to be badly ionized or fragmented or even for not being transmitted. A slight calibration shift is also enough for the pesticide to be tilted out of the 1 ppm tolerance zone and not to be recovered by the software program. Such shifts occur when the intensity of the signal is too weak - the form of the peak is not well defined and the mass accuracy diminishes - but also when the signal is too intense - saturation of the detector causes a peak distortion and a mass shift occurs. A calibration loss in the course of time or a momentarily blinding of the detector caused by an extremely intense matrix peak can also result in mass accuracy problems. There is a large variety of matrices in which pesticides are detected and each of them has a specific impact on ionization, transmission and detection of pesticides : a drastic loss of sensitivity can be observed when using a specific matrix for a certain pesticide without suggesting such problems in previous analyses in other matrices. Scan velocity also has an impact on sensitivity : the lower the velocity, the higher the sensitivity of the instrument, but on the contrary the amount of points per peak decreases. This affects the deconvolution software and consequently mass accuracy. Manufacturers make every effort to improve the sensitivity, the resolution as well as the accuracy of the new generation ToF instruments. But the critical eye of the analyst will always be necessary in order to avoid false positive and false negative results when using a ToF analyzer. False positives are not a problem as such because confirmation can annul the result, on the other hand false negatives do not have a second chance. This is the end of the journey, so it is time... 33
... for a soft landing Finally the ToF analyzer is an extremely high performing instrument based on a simple principle : measuring the trajectory time of an ion so as to know its mass. High resolution, high accuracy and high sensitivity of these instruments allow us to measure the monoisotopic mass of the pesticide so that it can be identified. On the other hand, results always have to be scrutinized by a critical mind as regards any risk involving pesticides in extraction and purification phases, in chromatography and detection, as well as during the processing of results by computer software. Fortunately, the most important sources of errors (false positives and false negatives) are blocked because of the various warnings in the SANCO criteria. The analyst has to prove that he can detect the detection limit (SDL, screening detection limit) of the pesticide at least in 95 of the samples. Today, at European level, several laboratories are using the ToF technology for routine screening analyses. Logbook [1] SANCO 12495/211 Method validation and quality control procedures for pesticide residues analysis in food and feed Laure.Joly@wiv-isp.be 34