Analysis of Carbendazim in Agricultural Samples by Laser Desorption

Juan XXIII-1°, 28040 Madrid, Spain. A method has been developed for the analysis of car- bendazim in agricultural samples. This has been ac- complish...
0 downloads 0 Views 94KB Size
Download PDF
Anal. Chem. 1998, 70, 491-497

Analysis of Carbendazim in Agricultural Samples by Laser Desorption and REMPI Time-of-Flight Mass Spectrometry J. M. Orea, B. Besco´s, C. Montero, and A. Gonzalez Uren˜a*

Unidad de La´ seres y Haces Moleculares, Instituto Pluridisciplinar, Universidad Complutense de Madrid, Juan XXIII-1°, 28040 Madrid, Spain

A method has been developed for the analysis of carbendazim in agricultural samples. This has been accomplished by the combination of laser desorption with resonance-enhanced multi-photon ionization (REMPI) coupled to time-of-flight mass spectrometry detection. After the optimization of the experimental conditions and the location of the resonant wavelength of the substance, several samples of pepper extract enriched with carbendazim were analyzed, finding a detection limit of the same order of magnitude as that of current GC and HPLC techniques, but with higher sensitivity and faster sample preparation Carbendazim (methyl benzimidazol-2-ylcarbamate) is a systemic pesticide of common use on a wide number of agricultural products;1 its maximum residue limits (MRLs) fixed by the European Commission range between 0.10 and 5.00 mg/kg depending on the kind of crop.2 Due to its extensive use and because carbendazim can also be used as indicator for the analysis of other pesticides as benomyl and methyl tiofanate, it is of great interest to have a suitable analytical method to determine its residues in agricultural samples.3 Carbamate pesticides have been mainly analyzed by gas chromatography (GC)4 and high-performance liquid chromatography (HPLC).5-7 Methyl carbamates are labile compounds difficult to analyze by GC,8 even minimizing the effects of thermal instability. Thus, its analysis is carried out in an indirect way by the detection of some thermally stable derivatives.4 Due to this unstability, the technique usually employed for the analysis of carbendazim is reversed-phase HPLC with fluorescence or ultraviolet detection;9,10 (1) Hampely,H.; Lo ¨cher, F. Proc. Br. Insectic. Fungic. Conf. 1973, 1 (301), 127. (2) Community Directive 93/38/CE, Official Journal of the European Community L211/6, Brussels, 1993. (3) Reed, D. V.; Lombardo, P.; Wessel, J. R. J. Assoc. Off. Anal. Chem. 1987, 70, 591. (4) Bagheri, H.; Creaser, S. J. Chromatogr. 1991, 547, 345. (5) Jime´nez, J. J.; Atienza, J.; Bernal, J. L.; Toribio, L. Chromatographia 1994 38, 395. (6) Kiigemagi, U.; Inman, D. D.; Mellenthin, N. M.; Deinzer, M. L. J. Agric. Food. Chem. 1991, 39, 400. (7) Sa´nchez-Brunete, C.; de la Cal, A.; Melgarejo, P.; Tadeo, J. L. Int. J. Environ, Anal. Chem. 1989, 37, 35. (8) De Kok, A.; Hiemstra, M.; Vreeker, C. P. Chromatographia 1987, 24, 469. (9) Liu, C. H.; Mattern, G. C.; Yu, X.; Rosen, J. D. J. Agric. Food. Chem.1990, 38, 167. S0003-2700(97)00443-5 CCC: $15.00 Published on Web 02/01/1998

© 1998 American Chemical Society

this technique requires a long procedure for the separation and extraction of the compound prior to its analysis.5,11,12 The detection limit of the conventional method for the analysis of benzimidazoles adapted to carbendazim is 0.02 mg/kg for 5-mg samples and 0.01 mg/kg for 8-mg samples.13 An easier supercritical fluid extraction (SFE)-HPLC method for the determination of carbendazim in lettuce samples has been described, but with higher detection limit.4 The present work was aimed at developing a method for the analysis of carbendazim in agricultural samples in a direct manner, without the tedious and time-consuming sample preparation necessary for classical techniques. This has been accomplished by the combination of laser desorption (LD) with resonanceenhanced multi-photon ionization (REMPI) coupled to time-offlight mass spectrometry (TOF-MS) detection. The combination of REMPI and TOF-MS is a well-known tool for the selective detection of trace compounds in complex matrixes.14-20 The main advantages of this technique are the selective ionization of minor components in a complex matrix, the great sensitivity and resolution, major ionization efficiency, and the possibility of simultaneous analysis of different components present in a matrix. Those advantages made the REMPI-TOFMS technique one of the most promising analytical methods as reflected by the increasing research carried out recently in this field.21-27 (10) Bicchi, C.; Belliardo, F.; Cantamessa, L.; Gasparini, G.; Icardi, M.; Sesia, E. Pestic. Sci. 1989, 25, 355. (11) Monico Pifarre, A.; Xirau-Vayreda, M. J. Assoc. Off. Anal. Chem. 1990, 73, 553. (12) Monico Pifarre, A.; Xirau-Vayreda, M. J. Assoc. Off. Anal. Chem. 1987, 70, 596. (13) Farrow, J. E. Analyst 1977, 102, 752. (14) Zare, R. N. Science 1984, 226, 298. (15) Zimmerman, F. M.; Who, W. J. Chem. Phys. 1994, 100, 7700. (16) Boels, U.; Zimmermann, R.; Weickhardt, C.; Lenoir, D.; Schramm, K.-W.; Kettrup, A.; Slach, E. W. Chemosphere 1994, 29, 1429. (17) Boesl, U.; Weinkauf, R.; Slach, E. W. Int. J. Mass Spectrom. Ion Processes 1992, 112, 121. (18) Cotter, R. J. Anal. Chim. Acta 1987, 195, 45. (19) Bernstein, E. R.; Law, K.; Schauer, M. J. Chem. Phys. 1984, 80, 207. (20) Mayo, S.; Lucatorto, T. B.; Luther, G. G. Anal. Chem. 1982, 54, 553. (21) Zhang, L.; Pei, L.; Dai, J.; Zhang, T.; Chen, C.; Yu, S.; Ma, X. Chem. Phys. Lett. 1996, 259, 403. (22) Al-Kahali, M. N.; Donovan, R. J.; Ridley, T. Chem. Phys. 1996, 208, 165. (23) Milan, J. B.; Buma, W. J.; Ashfold, M. N. R. Chem. Phys. Lett. 1995, 239, 326. (24) Weickhardt, C.; Boels, U.; Slach, E. W. Anal. Chem. 1994, 66, 1062.

Analytical Chemistry, Vol. 70, No. 3, February 1, 1998 491

Figure 1. Experimental setup. The inset shows the internal parts of the ionization chamber. IC stands for the ionization chamber, TOF for the time of flight tube, V1, V2 for the accelerating voltages, and V3 for the ionic lens voltage.

Laser desorption is a poweful technique to produce both ions and neutrals and has been already used to vaporize all kind of molecules.28-34 Coupling this technique with REMPI-TOF-MS allows the analysis of thermally unstable compounds: the laser pulse communicates a high energy in a very short time (a few nanoseconds), and this favors the vaporization over the thermal fragmentation.30 After desorption, the molecule is cooled by expansion in the vacuum which again minimizes the fragmentation. The other main advantage of LD is the absence of previous sample preparation, which facilitates the analysis by avoiding complex and time-consuming operations that normally imply nonnegligible error sources. After finding the resonant wavelength of the substance and subsequent optimization of the experimental conditions, we have analyzed several samples of pepper extract enriched with carbendazim. The results obtained show that this combination of LD with REMPI-TOF-MS is a suitable method for the determination of carbendazim in agricultural samples. The detection limit obtained for carbendazim using this technique is of the same order of magnitude as published for HPLC. However, the present (25) Numata, Y.; Ishii, Y.; Watahiki, M.; Suzuka, I.; Ito, M. J. Phys. Chem. 1993, 97, 4930. (26) Steenvoorden, R. J. J. M.; Vasconcelos, M. H.; Kistemaker, P. G. J. Mol. Spectrosc. 1993, 161, 17. (27) Kovalenko, L. J.; Maescheling, C. R.; Clemett, S. J.; Philippoz, J. M.; Zare, R. N.; Alexander, C. M. Anal. Chem. 1992, 64, 682. (28) Zhang, J. Y.; Nagra, D. S.; Li, L. Anal. Chem. 1993, 65, 2812. (29) Heise, T. W.; Yeung, E. S. Anal. Chem. 1994, 66, 355. (30) Kinsel, G. R.; Lindner, J.; Grotemeyer, J. J. Phys. Chem. 1992, 96, 3162. (31) Kimbrell, S. M.; Yeung, E. S. Appl. Spectrosc. 1991, 45, 442. (32) Spengler, B.; Cotter, R. J. Anal. Chem. 1990, 62, 793. (33) Li, L.; Lubnam, D. Rev. Sci. Instrum. 1988, 59, 557. (34) Heise, T. W.; Yeung, E. S. Anal. Chem. 1992, 64, 2175.

492

Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

technique shows more sensitivity and it requires less time for sample preparation than the later. EXPERIMENTAL SETUP The experimental setup is composed by two vacuum chambers designed and built in our institute. In the first chamber, called the ionization chamber, the following processes take place: sample desorption, ionization of the desorbed molecules and acceleration of the ions toward the second chamber, which is the time-of-flight tube and where ion detection is performed. Figure 1 shows an schematic view of the experimental setup; the inset shows the internal parts of the ionization chamber, i.e.: accelerating plates, deflectors, and ionic lens. The two chambers, connected through a gate valve, have independent vacuum systems composed of a turbomolecular and a rotary pump each (Varian V250 + Telstar RD 18 and Varian V70 + Telstar 2P-3, respectively). During the measurements, the pressure in the two chambers was always kept below 10-6 mbar, and the second chamber was kept at pressure below 5 × 10-8 mbar when the first chamber was opened for sample manipulation. The system for the acceleration of the ions is of the WhileyMcLaren type, composed by a set of plates, each connected to a high-power supply (Stanford Research System Inc., Models PS325 and PS350); the acceleration is done in two steps: V1 f V2 and V2 f V3 ) 0. The system also includes two deflectors in the directions Y and Z, perpendicular to the TOF trajectory, and an ionic Einzel lens to keep the ions in the detector direction. The sample is prepared dissolving the carbendazim (Aldrich, 98%) in acetone (Panreac, 99%) and then sprayed on a Pyrex disk (75 mm i.d.) by means of an air brush. The disk is placed on a sample holder parallel to the first accelerating plate at 2 mm from

Table 1. Experimental Conditions pressure in the ionization chamber (mbar) pressure in the TOF chamber (mbar) V1 (V) V2 (V) V3 (V) VMCP (V) ionization to desorption distance (mm) laser repetition rate (Hz) laser pulse duration (ns) delay between lasers (µs) λ desorption (nm) E desorption (mJ/pulse) λ ionization (nm) E ionization (µJ/pulse) no. of shots/measurement

1 × 10-6-5 × 10-7 1 × 10-7- 5 × 10-8 1840 1580 230 2000 7,5 10 4-6 25 532 8 281.1 150-550 100

it. The disk is rotated by a step motor during the analysis in order to have a fresh surface for the laser to desorbe. The whole system is placed on a positioner that allows the adjustment of the Z direction perpendicular to the desorption laser; this movement increases the number of runs for a given sample. For the sample desorption, the second harmonic of a Nd:YAG laser (Continuum Surelite) was used, with pulses of 5 ns of duration; the desorption energies ranged from 3 to 15 mJ. The pulsed beam of desorbed molecules expands into the acceleration region and is ionized later by the fourth harmonic of a second Nd:YAG laser (Continuum ND81) or by a frequency-doubled dye laser (Continuum ND60). The ionization laser is perpendicular to the one used for the desorption and intercepts the desorbed molecules at a variable distance from the desorption zone adjustable between 6 and 30 mm. The ions produced by the second laser pulse were accelerated toward the TOF tube by the acceleration system previously described. The ions are detected by a two-microchannel plate detector (Comstock CP-625C/50F) placed at the end of the tube, and the signal is collected by a digital scope (Tektronix 540) averaging 100 laser shoots. For some runs, a Tektronix 11403A digital scope with much higher resolution was used. The TOF spectrum is then transferred to a computer for data analysis and storage. The delay between the desorption and ionization lasers was controlled by a pulse generator (Lyons Instruments PG 75A); the laser frequency was 10 Hz, and the pulse energies were monitored either by a pyroelectric detector (Gentec Ed-100A) for low pulse energies ranging between 0 and 10 mJ or by a calorimetric detector (Photon Control 25) for energies ranging from 10 to 40 mJ. Variable diaphragms were incorporated in the system to control the size of both desorption and ionization areas. For the calibration experiments in which a gas sample was used, the sample holder and motor were replaced by a gas inlet consisting in a 6 mm diameter tube with a 0.5 mm diameter nozzle. The gas pressure was controlled in order to keep the chamber pressure below 10-6 mbar. For these experiments, only the ionization laser was used. Table 1 sumarizes the most relevant experimental conditions used in this work for the desorption and ionization of the carbendazim, as well as other relevant features.

PREPARATION OF THE PEPPER EXTRACT BY SUPERCRITICAL FLUID EXTRACTION Reagents and Apparatus. (a) An Isco SFE system, consisting of one Model 260 D syringe pump and controller, a SFX 2-10 extractor with restrictor heater set at 70 °C, and 10 mL stainless steel extraction cartridges with removable 2 µm frits, was used in this study. An uncoated and deactivated fused-silica capillary column, 30 cm length × 50 µm i.d., was used as restrictor, and a 10 mL graduated test tube, immersed in a 15-20 °C water bath, was used as collection vessel. (b) All the solvents used were Panreac, pesticide residue grade. Carbon dioxide, 99.995% purity, was supplied by SEO (Madrid, Spain). Anhydrous magnesium sulfate, >99% purity, was obtained from Fluka. (c) Fresh peppers were obtained from Campos de Nijar S.A. (Almerı´a, Spain). These pepers were no treated with benzimidazole fungicides and were determined not to contain any detectable pesticide residue by using conventional solvent-based extraction methods and GC and HPLC analysis. Experimental Procedure. The sample preparation and SFE methods used to obtain the pepper extract were those proposed by Valverde-Garcı´a et al.35,36 to analyze pesticide residues in fruits and vegetables by SFE. Specifically, 20 g of blended fresh pepper sample was thoroughly mixed with 28 g of anhydrous magnesium sulfate in a glass baker immersed in an ice/water bath. After 5 min, the mixture was thoroughly pounded in a porcelain mortar until a dry and homogeneus powdered mixture was obtained. Extraction was carried out in 10 mL extraction cartridge filled with 8 g of the pepper/MgSO4 mixture, placing first a 1 g layer of MgSO4 to bind any water that migrated during extraction. Extraction was performed in dynamic mode after a static equilibrium period of 1 min. SFE conditions were as follows: pressure, 300 atm; temperature, 50 °C; pressurized CO2 volume, 15 mL; static modifier, 200 µL of methanol; collection solvent, 3 mL of ethyl acetate. The pressurized CO2 flow rate during extraction was ∼1.3 mL/min. After extraction, the volume of the SFE extract (∼1.1 mL) was adjusted to 2.2 mL with cyclohexane. This final extract contained 1.5 g of pepper sample/mL. CHARACTERIZATION OF THE EXPERIMENTAL SYSTEM Mass Calibration. The new time-of-flight equipment was calibrated with a mixture of vapors of toluene (mass 92 amu) and aniline (mass 93 amu); both substances are liquids with high vapor pressure at room temperature and easily ionized at 266 nm.37 A system with two flasks coupled to independent regulation valves was connected to the ionization chamber through a needle valve; after degasification by several freeze-and-pump cycles, the reactants were mixed by controlling each regulation valve so that a similar signal in the TOF spectrum could be obtained. The vapor was ionized with the fourth harmonic of the Nd:YAG laser (266 nm). Figure 2 shows the TOF spectrum obtained for the simultaneous detection of toluene and aniline, in which one can see the (35) Valverde-Garcı´a, A.; Fernandez-Alba, A.; Agu ¨ era, A.; Contreras, M. J. AOAC Int. 1995, 78, 867-873. (36) Valverde-Garcı´a, A.; Fernandez-Alba, A.; Contreras, M.; Agu ¨ era, A. J. Agric. Food Chem. 1996, 44, 1780-1784. (37) Lubman, D. M.; Kronick, M. N. Anal. Chem. 1982, 54, 660.

Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

493

Figure 2. TOF spectrum of the mixture of toluene (91 amu) and aniline (92 amu) vapors.

Figure 3. Carbendazim TOF mass spectrum. Inset: typical resolution, R ) 834, obtained for carbendazim (191 amu).

ability of our system to separate the two compounds differing by only 1 amu. Resolution. To find the optimal conditions for the analysis, a pure sample of carbendazim was desorbed with the second harmonic of the Nd:YAG laser and postionized with the fourth harmonic of the other Nd:YAG laser. The desorption energy was optimized to obtain the maximum signal avoiding fragmentation; the best results were obtained with 8 mJ/pulse. The ionization energy was changed between 1 and 3 mJ. Both laser beams were introduced in the chamber without focusing. Figure 3 shows a carbendazim TOF mass spectrum obtained by MPI at these conditions. The inset shows a typical ion signal, with a resolution of R ) m/∆m ) t/2(∆t)fwhm ) 834; i.e., our system can unambiguously separate masses up to 800 amu. Variation of the Signal with the Desorption and the Ionization Energies. To study the variation of the carbendazim signal with the desorption energy, we measured several time-offlight spectra at different desorption laser energies, keeping the rest of conditions unchanged for a laser ionization energy fixed at 2 mJ. Figure 4 shows the intensity of the carbendazim ion signal vs the desorption energy. It can be noticed that the signal increases with desorption energy below ∼8 mJ; at higher energies, the signal probably decreases due to fragmentation of the molecule. 494 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

Figure 4. Carbendazim intensity signal as a function of laser desorption energy.

Figure 5. Repeatibility of the carbendazim peak area. The carbendazim TOF signal is displayed for 100 consecutive measurements (see text for comments). Table 2. Carbendazim Ion Signal as a Function of the Ionization Laser Energy ionization energy (mJ)

ion signal (arb units)

ionization energy (mJ)

ion signal (arb units)

1.03 1.01 0.84 0.83

3785 2680 2121 3556

0.54 0.51 0.29 0.26

1443 1390 633 273

a

λ ionization 281.5 nm.

A similar study was carried out on the variation of the carbendazim signal with the ionization energy. As before, the signal increases with the laser energy as listed in Table 2. A loglog plot of the intensity of the carbendazim signal vs the ionization energy gives a straight line with a slope of 1.9 ( 0.1, which indicates a two-photon resonant ionization process for the carbendazim ion yield. Precision. After optimizing the analytical conditions, e.g., energy, laser wavelength (see next section of REMPI studies), acelerating voltages, etc., we studied the precision of our experimental technique by determining the amount of scatter in the

Figure 7. Carbendazim and benzimidazole structural formulas.

Figure 6. Carbendazim ion signal as a function of its concentration in different prepared standards. Solid square, experimental results; solid line, linear regression fit. The inset shows a correlation coefficient, R, of the fit of 0.995.

results obtained from multiple analyzes of a sample. The sample consisting of pure carbendazim was prepared using the same procedure as for the analytical samples and the conditions were the same as that used for the final analysis. Figure 5 shows the repeatibility of the carbendazim peak area over 100 spectra each obtained with 1 s (i.e., 10 laser shots) of acquisition time; during the experiment, the sample was slowly rotated in order to have a fresh surface for each new measurement. From these measurements, a relative standard deviation of 7.4% was found; this value give us the intraassay precision of our method and it mainly reflects the inhomogeneity in the sample deposition, variations in the energy of the lasers, and little variations in the sample position as it rotates. Linearity. In order to check whether the analyte response is proportional to concentration in the range of interest, a linearity study was performed using five standard solutions with concentrations ranging between 0.3 and 6 ppm carbendazim in pure pepper (see further section of Analysis of Carbendazim for more details on the preparation of the solutions). The analysis was carried out using the same optimal analytical conditions as before. Figure 6 shows a plot of the total ion signal vs the concentration of carbendazim. A linear fit to the data gives a correlation coeficient of 0.995. REMPI SPECTRUM OF CARBENDAZIM One of the prerequisites for resonance-enhanced mass spectrometric analysis is the knowledge of the highly resolved REMPI spectrum of the compound to be analyzed, in order to find the optimal wavelength for selective ionization. The carbendazim absorption spectrum shows a wide band between 250 and 300 nm, but we could not find in the literature either its ionization potential or its REMPI spectrum. The more similar substance whose REMPI spectrum is known is that of benzimidazole, which constitutes the basic structure of the carbendazim as can be seen from inspection of Figure 7. The REMPI spectrum of the benzimidazol vapor at room temperature28,31 shows three bands between 277 and 280 nm, with the 0-0 transition at 277.57 nm. To carry out the REMPI study of the carbendazim, the dye laser

Figure 8. REMPI spectrum of carbendazim. Top, vapor at room temperature; bottom, after laser desorption. Notice the less congested bands for the desorbed spectrum.

(Rhodamine 590) output was frequency-doubled by a homemade unit with a KDP crystal whose maximum efficiency was optimized at 560 nm. Varying the ionization wavelength between 250 and 300 nm every 0.1 nm, we were able to determine that the wavelength range in which carbendazim shows resonance lies between 280 and 283 nm. We then measured with higher resolution (0.025 nm) its REMPI spectrum over this interval either for the case of the vapor present in the chamber, i.e., due to the carbendazim vapor pressure, or for that of directly produced by laser desorption of the sample. The result is shown in Figure 8; from this spectrum we could determine that the optimal wavelength for the analysis of carbendazim in complex samples is 281.1 nm, which seems to correspond to the 0-0 transition of this compound. Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

495

Figure 9. TOF mass spectrum of the pepper extract in the region of the carbendazim signal (λd ) 532 nm, λi ) 281.1 nm).

ANALYSIS OF CARBENDAZIM The samples for the analysis of carbendazim were prepared by dissolving the compound in a pepper extract obtained by SFE. The pepper extract was fortified with several amounts of carbendazim in order to determine both the detection limit and the sensitivity of our technique. Between two consecutive analyses, both vacuum chambers were heated overnight, but in fact a 2-h heating period proved to be sufficient to avoid any residue coming from the previous sample. A liquid nitrogen trap, currently under design, will be allocated below the desorption zone to strongly reduce the background and so the heating period between consecutive runs. Prior to the analysis of carbendazim, we studied a pure pepper extract sample under the same conditions of the final analysis to verify whether there is some peak at 191 amu that could interfere with our results. Figure 9 shows the TOF mass spectrum obtained from the blank which was prepared by spraying 20 mL onto the disk. The absence of any significative signal at 191 amu is noticeable. With regard to the question of what the peper extract consists of, no full analysis was carried out for this extract. We shall emphasize that the main objective of the present investigation was focused on the analysis of carbendazim in those samples, regardless of other components present in the analyte. As stated, the combination of selective ionization plus the universality of the time-of-flight spectrometry allows one to clearly identify and analyze one component whithout inferences due to the rest of the substances contained in the sample. This is of course one of the significant advantages of the present technique. We show here the most interesting results that have allowed us to calculate the detection limits of our system both for the relative carbendazim content in pepper extract and for the absolute quantity of product in a sample. Figure 10 shows the TOF mass spectrum obtained for a sample with 0.3 ppm carbendazim (49 ng of carbendazim in 152 mg of pure pepper diluted in 20 mL of acetone). Notice the high level of the carbendazim signal as compared with that of background arising from a pepper extract which contains 152 mg of pure pepper. As mentioned above, this signal corresponds to a sample whose total amount of carbendazim is only 49 ng. Hence, the advantage of using selective ionization for carbendazim analysis is clearly demonstrated. From this signal we can calculate the detection limit of our technique for the 496 Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

Figure 10. Carbendazim TOF mass spectrum obtained from a sample with 48.7 ng of carbendazim in 152 mg of pepper (0.3 ppm). The inset shows an enlarged view of the carbendazim peak region with the noise level used to calculate the detection limit.

analysis of carbendazim; as is well-known, the detection limit of a method is the lowest analyte concentration that produces a response detectable above the noise level of the system; generally this is assumed as three times the noise level.38 In this spectrum, the signal intensity, S, is -2.15 × 10-3 V; if we assume a mean noise level, N, of -2.5 × 10-5 V (as is indicated in the inset of Figure 10), we will then obtain a S/N ) 86 for this sample with 0.3 ppm carbendazim in pepper. From here we can calculate that our detection limit would be better than 0.010 ppm, i.e., 10 ppb carbendazim in pepper. This result also shows the great sensitivity of our technique: the sample was deposited on a disk of 7.5 cm diameter (area 44 cm2), and the area of desorption was 0.25 cm2 (0.5 cm i.d.); even if one assumes that the laser pulse desorbs the carbendazim completely (which is clearly far from reality), the quantity desorbed will be 49 ng × 0.25 cm2/44 cm2 ) 0.28 ng. The peak signal measured in this spectrum corresponds then to a quantity of carbendazim desorbed per laser pulse below 0.3 ng. CONCLUSIONS The combination of LD with REMPI-TOF-MS is a suitable method for the determination of carbendazim in agricultural samples. After the optimization of the experimental conditions and the location of the resonant wavelength of the product, we have analyzed several samples of pepper extract enriched with carbendazim with excellent results. The detection limit obtained for carbendazim using this technique is of the same order of magnitude as the one published for HPLC, and the amount of sample required to reach this limit is far less than in the later; the technique also shows a great sensitivity as it is able to detect less than 0.3 ng of product. One of the most interesting findings of the present work is that the REMPI spectrum of carbendazim showing a two-photon ionization process over the 280-283 nm wavelength range with its 0-0 transition at 281.1 nm. This result is of great value since it will facilitate the analysis of this pesticide by laser spectroscopicbased techniques. (38) Green, J. M. Anal. Chem. 1996, 68, 305.

The only drawback of our results is the moderate repeatibility obtained for the intraprecision assay done. The value obtained for the relative standard deviation includes several parameters that could affect the precision in our measurements, but it is clear to us that the main factor to be considered is the poor homogeneity achieved by spraying the sample with the air brush; we are incorporating an electrodeposition system to our laboratory in order to improve the sample homogeneity. One clear advantage of our technique is the selective ionization of minor components in a complex matrix; the great sensitivity and resolution, a major ionization efficiency, and the possibility of simultaneous analysis of all the components present in a matrix are also significant advantages to be remarked. Nevertheless, the difficulties associated with the presence of such matrix interferences could manifest for some pesticides so that, in principle, they cannot be ruled out and a specific investigation would then be required. Obviously the present work was not intended to provide a critical assessment on the SFE method of extraction but to demonstrate the advantage of using the REMPI coupled to the

TOF-MS detection method. The great advantage of the latter should be considered irrespective of any specific method of extraction. In fact, work is now in progress in our laboratory to perform carbendazim analysis in pepper using other extraction methods. The results, including a comparison among them, will be the subject of a future paper. ACKNOWLEDGMENT The authors acknowledge Prof. Valverde from the University of Almerı´a for providing the SFE pepper extract. This work received financial support from the DGICYT of Spain Grant PB95/ 391 and the Comunidad Auto´noma de Madrid Grant 06M/058/ 96. J.M.O. acknowledges his postdoctoral contract from the Ministerio de Educacio´n y Cultura of Spain. B.B. thanks to the Ministerio de Educacio´n y Cultura of Spain for her fellowship. Received for review April 29, 1997. Accepted October 27, 1997.X AC970443U X

Abstract published in Advance ACS Abstracts, December 15, 1997.

Analytical Chemistry, Vol. 70, No. 3, February 1, 1998

497