Trace Level Determination of Organophosphorus Pesticides in Water

May 24, 2002 - Direct-EI is a new device that, in a very simple fashion, couples a nano-HPLC system with a mass spectrometer equipped with electron io...
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Anal. Chem. 2002, 74, 3547-3554

Trace Level Determination of Organophosphorus Pesticides in Water with the New Direct-Electron Ionization LC/MS Interface Achille Cappiello,* Giorgio Famiglini, Pierangela Palma, and Filippo Mangani

Istituto di Scienze Chimiche “F. Bruner”, Universita` di Urbino, Urbino, Italy

A new LC/MS method for the determination of organophosphorus pesticides in water, based on the use of direct-electron ionization (EI) interface, is presented. Direct-EI is a new device that, in a very simple fashion, couples a nano-HPLC system with a mass spectrometer equipped with electron ionization capability. The nanoscale liquid flow allows for a direct introduction of eluate into the ion source and, after nebulization, for its ionization under typical EI conditions. Library-matchable EI spectra are generated for a choice of full scan or SIM detection of the analytes. In our case, a selection of organophosphorus pesticides, commonly distributed in local sugar beet cultivation, were considered. The new interface permits a very sensitive detection of the analytes in a wide range of linear response (0.09-9 ng). When applied to a real sample, the method allowed detecting four different pesticides at a concentration level of approximately 3 ng‚L-1. Direct-electron ionization is a new interfacing device designed to couple a nano-HPLC column with a mass spectrometer equipped with electron ionization (EI) capability. It is designed to work between 0.3 and 1.5 µL‚min-1 of mobile phase flow rate, and it does not rely on any special interfacing apparatus, but optimizes the direct introduction of a liquid effluent into an EI ion source. Differently from the old direct liquid introduction1-8 (DLI) interface, direct-EI is not affected by problems such as frequent clogging of the transport capillary, unstable source pressure, and sample sputtering. In addition, the ionization is not chemical unless when specifically required. It is noteworthy that unlike 20 years ago, direct-EI can take advantage of excellent micro- and nano-HPLC columns capable of performing at their best at a very low flow rate. A thorough description of a direct-EI interfacing mechanism is given in reference.9,10 * Corresponding author. Tel.: +39-07224164. Fax: +39-07222754. E-mail: [email protected]. (1) Vestal M.; Blakley C.; Ryan P.; Futrell J. H.; Adv. Mass Spectrom. 1974, 6, 781. (2) Arpino P. J.; Krien P.; Vajta S.; Devant G. J. Chromatogr. 1981, 203, 117. (3) Tijssen R:; Bleumer J. P. A.; Smit A. L. C.; Van Kreveld M. E. J. Chromatogr. 1981, 218,137. (4) Sha¨fer K. H.; Levsen K. J. Chromatogr. 1981, 206, 245. (5) Mauchamp B.; Krien P. J. Chromatogr. 1982, 236, 17. (6) Arpino P. J.; Beaugrand D. Int. J. Mass Spectrom. Ion Proc. 1985, 64, 275. (7) De Wit, J. S. M.; Tomer K. B.; Jorgenson J. W. J. Chromatogr. 1989, 462, 365. 10.1021/ac015685f CCC: $22.00 Published on Web 05/24/2002

© 2002 American Chemical Society

Direct-EI can be ideally located at the end of an evolutionary process that has the particle beam interface11-13 as a progenitor. It generates high-quality electron ionization spectra using a very simple approach as easily as in a typical gas chromatography/ mass spectrometry (GC/MS) interface. The same benefits obtained with the simplicity of a GC/MS system are transferred into the HPLC world, of course, only for those compounds amenable by electron ionization. Unlike atmospheric pressure ionization (API), the EI spectra are readily interpretable and easily obtainable from almost any compound under 600 u. The typical EI spectrum has plenty of structural information that can be usually traced back to a specific compound. The main reasons for the limited success of electron ionization in the LC/MS arena are the lack of sensitivity due to a low ionization efficiency and the necessity to convert the analytes in vapor phase. The latter is mainly due to sample transport difficulties and scarce ionization efficiency. An API interface performs well on both sides, but because of a poor fragmentation, it lacks structural information. On the other hand, ion fragmentation, an essential step for any analyte characterization, can be induced in an API interface under collision ion dissociation (CID) conditions either in the interface (source CID) or in the analyzer region (MS/MS), with an obvious detriment of the instrument simplicity, even though and unlike EI, in an API interface, the entire ionization process is heavily influenced by the experimental conditions and mobile phase composition and, although informative, it can be used only as a confirmatory technique. EI spectra, thanks to their high reproducibility, have been recorded in libraries and used as references and are considered to be the “fingerprint” of a specific molecule. In a direct-EI interface, sensitivity is improved, as compared to previous attempts, and picogram-level detection is often achieved in selected ion monitoring (SIM) mode. In scan mode, the typical EI spectrum is recorded allowing the comparison with thousands of spectra from commercially available sources (such as NIST or Wiley). In addition, EI detection can benefit from recent sophisticated procedures, (8) Bruins, A. P.; Drenth, B. F. H. J. Chromatogr. 1983, 271, 71. (9) Cappiello, A.; Famiglini, G; Mangani, F.; Palma, P. J. Am. Soc. Mass Spectrom. 2002, 13, 265-273. (10) Cappiello, A.; Famiglini, G.; Mangani, F.; Palma, P. Mass Spectrom. Rev. 2001, 20, 88-104. (11) Willoughby, R. C.; Browner, R. F. Anal. Chem. 1984, 56, 2626. (12) Cappiello, A.; Bruner, F. Anal. Chem. 1993, 65, 1281-1287. (13) Cappiello, A.; Balogh, M.; Famiglini, G.; Mangani, F.; Palma, P. Anal. Chem. 2000, 72, 3841-3846.

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such as the one developed by the National Institute of Standards and Technology (NIST) called AMDIS (Automated MS Deconvolution and Identification System), which extracts the analytes mass spectra in complex chromatographic mixtures, even in those with overlapping peaks. These aspects turn out to be essential in all cases when unknown peaks might be encountered or legal defensibility is a necessity, such as in pharmaceutical studies, drug abuse testing in forensic applications, or environmental monitoring. An interesting application in which the role of the direct-EI interface can be particularly emphasized is the determination of organophosphorus pesticides in water samples at a concentration level of a few parts per billion (w/v) in the environment. Organophosphorus pesticides are widely used in large quantities all over the world to combat harvest-threatening insects and can persist in the environment for some days depending, basically, on their intrinsic properties as well as weather conditions. Because of their persistence, they can be easily transported in surface or groundwater, resulting in a possible contamination of water resources intended for human consumption. Trace-level determination is normally required for such compounds and, because of their low concentration as well as their chemical nature, the analytical methods normally employ solid-phase extraction and LC/MS analysis. Electrospray ionization has a solid reputation for the analysis of trace-level contaminants, but the very low polarity does not allow a quantitative detection of organophosphorus pesticides. Several papers have been published on the use of particle beam (PB)14-18 and thermospray (TSP)19-29 for the analysis of pesticides, and some of these methods were validated by the Environmental Protection Agency (EPA) as recommended protocols for environmental monitoring. In some cases, GC with flame photometric (FPD), nitrogen-phosphorus (NPD) or MS detection can be advantageously used in the determination of organophosphorus pesticides, as demonstrated by many papers in the literature.30-38 HPLC methods coupled with MS represent (14) Cappiello, A.; Famiglini, G. Anal. Chem. 1994, 66, 3970-3976. (15) Behymer, T. D.; Bellar, T. A.; Budde W. L. Anal. Chem. 1990, 62, 16861690. (16) Jones, T. L.; Betowski L. D.; Lesnik, B.; Chiang, T. C.; Teberg, J. E. Environ. Sci. Technol. 1991, 25, 1880-1884. (17) Kim, I. S.; Sasinos, F. I.; Stephens, R. D.; Wang, J.; Brown, M. A. Anal. Chem. 1991, 63, 819-823. (18) Cappiello, A.; Famiglini, G.; Mangani, F.; Angelino, S.; Gennaro, M. C. Environ. Sci. Technol. 1999, 33, 3905-3910. (19) Bellar, T. A.; Budde, W. L. Anal. Chem. 1988, 60, 2076-2083. (20) Volmer, D.; Preiss, A; Levsen, K.; Wu ¨ nsch, G. J. Chromatogr. 1993, 647, 235-259. (21) Volmer, D.; Levsen, K. J. Am. Soc. Mass Spectrom. 1994, 5, 655-675. (22) Volmer, D.; Levsen, K.; Engewald, W. Wasser, 1994, 82, 335-364. (23) Sennert, S.; Volmer, D.; Levsen, K.; Wu ¨ nsch, G. Fresenius’ J. Anal. Chem. 1995, 67, 642-649. (24) Volmer, D.; Wilkes, J. G.; Levsen, K. Rapid Commun. Mass Spectrom. 1995, 9, 767-771. (25) Shalaby, L.; Bramble, F. Q., Jr.; Lee, P. W. J. Agric. Food Chem. 1992, 40, 513-517. (26) Chiron, S. Dupas, S. Scribe, P. Barcelo´, D. J. Chromatogr. 1994, 665, 295305. (27) Lacorte, S.; Ehresmann, N.; Barcelo´, D. Environ. Sci. Technol. 1996, 502, 917-923. (28) Durand, G.; Barcelo´, D. J. Chromatogr. 1990, 502, 275-286. (29) Volmer, D.; Levsen, K.; Honing, M.; Barcelo´, D. J. Am. Soc. Mass Spectrom. 1995, 6, 656-667. (30) Organophosphorus Compounds by Gas Chromatography: Capillary Column Technique; EPA Method 8141A; U.S. Environmental Protection Agency, U.S. Governmental Printing Office: Washington, DC, 1994.

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a valid alternative to GC/MS, since some of such compounds are thermally labile or chemically reactive and may decompose during analysis, leading to poor recoveries.39 Nowadays, atmospheric pressure chemical ionization interfaces (APCI) are widely used for the determination of nonpolar or scarcely polar analytes, such as organophosphorus pesticides, and several methods have been proposed.40-44 APCI is characterized by high ionization efficiency and provides mainly molecular-weight information. Structural information can be obtained by means of cone-voltage induced fragmentation before the analyzer region or by relying on a CID approach to induce fragmentation into a MS/MS apparatus. In this context, direct-EI may offer a simpler approach with comparable results either in terms of sensitivity or specificity. In the present study, the performance of direct-EI interface in the determination and identification of several organophosphorus pesticides in water samples below 0.5 µg‚L-1 level was evaluated. The lowest limit of detection (LOD) was obtained in SIM using a selection of 1-3 ions/analyte. Solid-phase extraction was incorporated into the method for a suitable sample preparation and preconcentration. EXPERIMENTAL SECTION Reagents. All solvents were HPLC grade from Baker (Deventer, Holland) and were filtered and degassed before use. Ten pesticides (Table 1) were selected for this work on the basis of their presence in the local environment and their particular amenability to HPLC. They were purchased from Riedel-De Hae¨n (Hannover, Germany). TFA was purchased from Sigma Scientific (St. Louis, MO). Reagent water was obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA). Direct-EI Interface. The new interface accomplishes the following objectives: 1. Maximum simplicity: no complex mechanisms are involved. 2. High sensitivity: no sample loss due to transport process, since the whole effluent is transferred into the ion source by means of capillary tubing. 3. Broad spectrum of application for low-to-medium-weight molecules. The first two objectives were accomplished by directly introducing the eluate into the ion source. The full accomplishment of the third objective is limited only by the possibility of obtaining an EI spectrum for a specific substance. Thermal decomposition (31) Aguera, A.; Pietra, L.; Hernando, M. D.; Fernandez-Alba, A. R.; Contreras, M. Analyst 2000, 125 (8), 1397-1402. (32) Lambropoulou, D.; Sakellarides, T.; Albanis, T. Fresenius’ J. Anal. Chem. 2000, 368 (6), 616-623. (33) Gamon, M.; Lleo, C.; Ten, A.; Mocholi, F. J. AOAC Int. 2001, 84 (4) 12091216 (34) Schachterle, S.; Feigel, C. J. Chromatogr. A 1996, 754 (1-2) 411-422. (35) Lartiges, S. B.; Garrigues, P. Analusis, 1995, 23 (8) 418-421. (36) Okumura, T.; Nishikawa, Y. J. Chromatogr. A 1995, 709 (2), 319-331. (37) Lartiges, S. B.; Garrigues, P. Analusis, 1993, 21 (3), 157-165. (38) Aguilar, C.; Borrull, F.; Marce, R. M. LC-GC, 1996, 14 (12), 1048-1054. (39) Betowsky, L. D.; Jones, T. L. Anal. Chem. 1988, 22, 1430-1434. (40) Kawasaki, S.; Ueda, H.; Itoh, H.; Tadano, J. J. Chromatogr. 1992, 595, 193202. (41) Itoh, H.; Kawasaki, S.; Tadano, J. J. Chromatogr. 1996, 754, 61-76. (42) Slobodnı´k, J.; Hogenboom, A. C.; Vreuls, J. J.; Rontree J. A.; van Baar, B. L. M.; Niessen, W. M. A.; Brinkman, U. A. Th. J. Chromatogr. 1996, 741, 59-74. (43) Lacorte, S.; Jeanty, G.; Marty, J.; Barcelo´, D. J. Chromatogr. 1997, 777, 99-114. (44) Lacorte, S.; Molina, C.; Barcelo´, D. J. Chromatogr. 1998, 795, 13-26.

Table 1. List of the Selected Pesticides compd

solubility in H2O (mg‚L-1)

CAS RN

MW

m/z

LOD, pg (no. of ions)

dimethoate paraoxon azinphos-methyl parathion-methyl malathion azinphos-ethyl parathion-ethyl diazinon phorate phoxim

25 000 2.4 28.0 55.0 145.0 4.0 11.0 40.0 50.0 1.5

60-51-5 311-45-5 86-50-0 298-00-0 121-75-5 2642-71-9 56-38-2 333-41-5 298-02-2 14816-18-3

229 275 317 263 330 345 291 304 260 298

87-125 109-149-275 132-160 109-125-263 125-127-173 132-160 109-125-291 137-179-304 121-231-260 129-157

90 (2) 90 (3), 60 (2) 30 (2) 20 (3), 20 (2) 20 (3), 20 (2) 10 (2) 30 (3), 30 (2) 20 (3), 20 (2) 90 (3), 30 (2) 50 (2)

Figure 1. Schematic view of the direct-EI interface.

or insufficient vaporization may not extend the use of direct-EI to high-molecular-weight analytes, but it offers a good solution for thousands of compounds that cannot be determined by GC/MS. The major drawbacks encountered when a liquid effluent is introduced into the high vacuum of a mass spectrometer in a hightemperature environment are essentially two: 1. The first one appears to be an unstable loss of vacuum in the ion chamber and follows the immediate vaporization of the eluate. In addition, solvent vapors create the ideal conditions for unwanted chemical ionization processes. 2. The second one is related to the quick solvent evaporation that generally leads to solute buildup in the connecting capillary tubing or the freezing of solvents at the capillary tip. The prevalence of one phenomenon or the other depends on many factors, such as mobile phase composition and flow rate, source temperature and pressure, tubing size, etc. Both drawbacks are responsible for signal instability and poor detection capability. Our research group has gained good experience in aerosol formation at the microscale flow rate, and we used it for developing this interface. The interfacing mechanism is based on the formation of aerosol in high vacuum conditions, followed by a quick droplet desolvation and final vaporization of the solute prior to ionization. The completion of the process requires less than 8-mm in space, all inside the ion chamber, with only a slight modification of the original EI hardware (Figure 1). The sequence of the eluate’s physical state modifications was proved by direct observations of the process (see ref 9). Fused-silica capillary tubing connects the nebulizer with the capillary column and regulates

the critical passage from atmospheric pressure to high vacuum. To avoid in-tube evaporation of the mobile phase as a result of the radiating source heat, the capillary tubing is thermally insulated. The aerosol is formed at the tubing end where a coneshaped tip, slightly bent sideways (∼10°), is placed. In this way, the aerosol jet is directed to a specific target surface inside the ion source. Eventually, the hot, smooth, stainless steel surface converts the remaining solute into the gas phase for the final ionization.10 The nebulizer tip protrudes 2 mm into the ion source and has an orifice of ∼5 µm. In this way, it creates a backpressure that limits any premature, in-tube solvent evaporation. The emerging liquid phase is rapidly converted into concentrated aerosol droplets by the shape of the nebulizer tip, and they are easily desolvated during the 8-mm-long journey between the nebulizer and the surface. The partial pressure of the solvent vapors is kept low by the reduced flow rate (0.3-1.5 µL‚min-1) and by an additional opening (6-mm diameter) in the ion volume. A mobile phase flow rate higher than 1.5 µL‚min-1 is larger than the exhausting capacity of the system, and it may induce chemical ionization. The aerosol formation is the key to the interfacing process, whereas the high temperature of the ion source, between 150 and 300 °C, has a double function: it compensates for the latent heat of vaporization during the droplet desolvation, and it converts the solute into the gas phase upon contact on the hot target surface. Temperature also prevents the liquid from freezing right off the tip. The extreme compactness and simplicity and the lack of any particular transport mechanism reduce sample loss and enhance Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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Figure 2. Effect of the loop shape on the chromatographic profile, separation of dimethoate, malathion, and phorate using (a) a 500-nL external loop, and (b) a 500-nL internal loop. The separation was achieved in gradient elution from 0 to 100% acetonitrile in water in 30 min at a flow rate of 500 nL‚min-1.

sensitivity. The commercial availability of reliable capillary HPLC columns has brought the liquid-phase separations into a new dimension where complex mixtures can be resolved at a microliterper-minute flow rate or less. Mass Spectrometer. The new interface was realized using a Hewlett-Packard 5989A quadrupole mass spectrometer. The operating pressure was ∼8-10‚10-5 Torr measured in the manifold of the ion source at the highest operating flow rate (1.5 µL‚min-1) and using a 1:1 mixture of water and acetonitrile. Mass spectrometer tuning and calibration were performed automatically using perfluorotributylamine (PFTBA) as a reference compound and monitoring m/z 69, 219, and 502. Considering the average molecular weight of our test compounds, the repeller potential was adjusted manually and optimized for the m/z 219 signal. No mobile phase was allowed into the ion source during calibration. The dwell times during SIM and scan times for full spectrum acquisition were adjusted to obtain 0.5 cycles‚s-1 and a mean of 10 acquisition samples for each HPLC peak. One-to-three-ion detection was used in SIM mode. The final transfer tube, prior to the ion source, was shifted to a fully retracted position after the tuning procedure, allowing an additional pumping stage for more efficient solvent vapor removal. Source temperature is, of course, an important issue in the mechanism of the direct-EI interface, and its effect must be evaluated carefully. The temperature must be high enough to vaporize the solute without inducing thermal decomposition of the most delicate analytes. As reported elsewhere,45 the peak (45) Cappiello, A.; Famiglini G. Anal. Chem. 1995, 67, 412.

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temperature can be significantly reduced if the solute can be quickly vaporized by an efficient contact with a hot surface. The right combination of solute mass and temperature represents the recipe for a correct analysis and has to be evaluated case by case. For our compound selection, we found that 240 °C was the best temperature to obtain the highest signal and the best quality spectrum. Nanoscale Liquid Chromatography. Liquid chromatography was carried out using a Kontron Instrument 420 dual-pump, binarygradient, conventional HPLC system (Kontron Instrument, Milano, Italy). Microliter and nanoliter flow rates were obtained with a laboratory-made splitter that was placed between the pumping system and the injector.46 This device allows the conversion of flow rates as high as 200µL‚min-1, generated by a conventional HPLC binary system, into capillary-scale flow rates. A two-step splitting of the main stream of solvents generates, for instance, a 0.5 µL‚min-1 mobile phase flow rate with a splitting ratio of 400: 1. The splitting device accurately reproduces at lower scale any solvent concentration gradient generated at higher flow rate in the pumping system. Acetonitrile was used as the organic solvent in the mobile phase because of its lower viscosity, a parameter that is crucial in micro-HPLC. Large-volume injections were performed to optimize sample quantitation and to lower the limit of detection of real samples. For this purpose, a Valco injector, equipped with a 60-nL, 100-nL, or 500-nL internal loop, was employed (Valco, Houston, TX). For flow injection analysis (FIA) experiments, two different injection valves were used: an Upchurch Scientific M-435 microinjection valve (Upchurch Scientific, Oak Harbor, WA) and a Rheodyne 7520 (Rheodyne L. P., Rohnert Park, CA). The latter can deliver as low as 2 nL of sample into the system. A LC Packings 75-µm-i.d. column (LC Packings, Amsterdam, The Netherlands), 15 cm long, packed with C18, 3-µm particles, was used for the chromatographic separations. A 75µm bore column has an ideal flow rate of between 200 and 300 nL‚min-1 and an ideal injection volume of a few nanoliters. To enhance sensitivity in real world applications, larger injection volumes are in great demand. For instance, a 500-nL injection represents a 100-fold decrease in the detection limit for any analyzed substance. When it is used as the sample solvent during the displacement of a large volume into the column from a long capillary injection loop water plays the role of a weak, noneluting mobile phase, promoting focusing of the solute at the column’s head. In this case, it is important to use 100% water both in the mobile phase and in the sample matrix. The combined action of sample and mobile phase water helps to ensure a correct solute focusing at the head of the column. Of course, organic solvent gradients must follow for a suitable separation of complex mixtures. This method has proven its validity for the analysis of several basic-neutral pesticides with a good solubility in water.47 With the exception of dimethoate, the selected organophosphorus pesticides are scarcely soluble in water, and samples cannot be prepared in pure water. On the other hand, the injection of methanolic sample extracts, obtained after the SPE procedure, cannot be performed in large injection loops. To overcome this limitation, wide-bore injection loops were adopted. A short Valco 500-nL internal loop contains the same volume as a 20-cm-long, (46) Berloni, A.; Cappiello, A.; Famiglini, G.; Palma P. Chromatographia 1994, 39, 279. (47) Rezai, M. A.; Famiglini, G.; Cappiello, A. J. Chromatogr. 1996, 742, 69.

Figure 3. Mass spectra of parathion-ethyl, (a) 60-ng injected; (b) 6-ng injected; and (c) Wiley reference spectrum.

50-µm-wide external loop. Its particular broad shape mixes the incoming mobile phase with the sample solution and, in our case, quickly dilutes the methanol in a large amount of water. The solute is pushed to the column head in a watery environment and, thus, retained by the stationary phase. The subsequent steady increase in acetonitrile, during the gradient slope, will mobilize the analytes through the column for a suitable separation. Figure 2 shows the effect of loop shape on the chromatographic profile. For this example, three pesticides of our group, dimethoate, malathion, and phorate, with different solubilities in water, were chosen, and a UV detector at 230 nm was employed. It is evident that at this flow rate, a change in the loop shape can promote an appropriate sample solvent replacement with a positive effect in the solute focusing. At the moment, the largest volume that can be injected under these conditions is 500 nL. Larger injected volumes can be achieved only with aqueous samples and external loops. Sample Preparation. A solid-phase extraction (SPE) procedure for the isolation and preconcentration of pesticides from water was implemented. All of the labware, except the LSE

cartridge, was made of Teflon, including the water samples container and the connection tubings and related hardware. Although its cost is higher, a Teflon surface is less prone to adsorption of analyte molecules at very low concentration, and it avoids losses of important aliquots of pesticides during the extraction process. The cartridge was filled with graphitized carbon black (Carbograph 1) and was capable of sampling up to 2 L of water. The extraction cartridge, made of polypropylene and measuring 6.5 × 1.4 cm i.d., was packed with 250 mg of Carbograph 1, 120-400-mesh (Alltech, Deerfield, IL) with a specific surface area of 100 m2‚g-1. Polyethylene frits, 20-µm pore size, were located above and below the sorbent bed. The cartridge was washed with 5 mL of methylene chloride/methanol (80:20 by volume) followed by 2 mL of methanol and 15 mL of 10 g‚L-1 ascorbic acid in HCl-acidified water (pH 2) before the water sample extraction. Water samples were forced through the trap at a flow rate of ∼150 mL‚min-1 by using a vacuum apparatus placed below the cartridge. The trap was washed with distilled water (7 mL) after the sample passage. A stream of nitrogen was used for 30 Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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Figure 4. Reconstructed ion chromatogram relative to a SIM acquisition of 90 pg of standard solution of the selected pesticides. Injection volume, 500-nL; flow rate, 500-nL‚min-1. (1) dimethoate, (2) paraoxon, (3) azinphos-methyl, (4) parathion-methyl, (5) malathion, (6) azinphosethy, (7) parathion-ethyl, (8) diazinon, (9) phorate, and (10) phoxim.

min to dry out the residues of water from the cartridge. The extraction solution was prepared with methylene chloride/ methanol (60:40 by volume).A 6-mL portion of this solution was passed through the cartridge and collected in a Teflon vial. The sample was heated to 40 °C, and a gentle stream of nitrogen over the liquid surface rapidly evaporated the methylene chloride fraction. At the end of the evaporation process, ∼100 µL of a methanolic solution of the extracted pesticides was found at the bottom of the vial. Methanol was added, and the extract was brought to 1 mL, reaching a preconcentration factor of 2000. Very good recovery can be obtained with this method, even at a parts-per-billion concentration. As reported elsewhere,48 recovery varied from 92.6% for phoxim to 100.3% for azimphosethyl, with a relative standard deviation always lower than (4%. RESULTS AND DISCUSSION From a chemical point of view, the selected compounds are characterized by low polarity and reduced thermal stability; both characteristics make them very suitable for liquid chromatography/mass spectrometry detection. In our case, electron ionization generates very informative spectra with abundant ion signals for selective detection. It is important to consider that a full EI spectrum acquisition represents a point of strength for any EIbased interface, since no other interfacing mechanism can compete with it in terms of specificity and simplicity. Spectra were recorded in flow injection analysis (FIA) using a large loop volume (60 nL) in isocratic conditions (water/acetonitrile 1:1) and injecting 10 ng for each pesticide. The spectra were acquired at low mobile phase flow rate (500 nL‚min-1), the same used in other steps of the present method. As reported elsewhere (ref 9), the design of this interface prototype cannot tolerate flow rates higher than 1-1.5 µL‚min-1, depending on the analyte. Over this limit, (48) Di Corcia, A.; Marchetti, M. Anal. Chem. 1991, 63, 580-585.

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chemical ionization ions can prevail in the spectrum and may impair EI library-matching quality. The 10 selected organophosphorus pesticides showed different responses to chemical ionization between 0.8 and 1.2 µL‚min-1 so that, for safety reasons, the flow rate was set below 600 nL‚min-1 for all experiments. To maximize sensitivity and ignore the chemical noise generated in the ion source, the mass window for full spectrum acquisition was set between 80 and 400 u, wide enough to cover the main analyte fragmentation. The spectra of the 10 selected pesticides were electronically compared to those in the Wiley reference library to evaluate the quality of those generated by the direct-EI interface. All of them are very much comparable to the reference spectra, as confirmed by the matching quality values provided by the library software: these values span from 68% for parathion to 98% for diazinon, with most of them >90%. Lowering sample concentration does not affect fragmentation so that the identification capability of direct-EI is accessible even at trace levels. To note the good reliability of direct-EI, Figure 3 shows the comparison among three mass spectra obtained from two different standard solutions of parathion-ethyl (6 and 60 ng) and the Wiley reference spectra. Although some CI interference is noticeable in these spectra, it does not appreciably alter their matching quality, and it does not compromise their utilization in real sample applications. Quantitative detection was performed in SIM using two or three characteristic ions chosen from among the highest signals and mass-to-charge ratios found in the spectrum. The acquisition program was subdivided into two parts and for each one was assigned only those m/z values relative to the group of pesticides eluted during the corresponding time window. In this way, dwell times increased approximately by a factor of 2 and sensitivity could be maximized as well. Switching time is found at 12 min after the start. Because of its retention time, only dimethoate is included in the first program. SIM conditions are reported in Table 1.

Figure 5. Reconstructed ion chromatogram relative to the SIM acquisition of the extract of 2 L of river water: (a) azinphos-methyl and azinphosethyl, (b) parathion-methyl and parathion-ethyl. Injection volume, 500-nL; flow rate, 500 nL‚min-1.

Corrected analyte identification in the sample is confirmed only when retention times, fragment ions, and their relative abundance match the standard profile. Two different chromatographic conditions were used for the separation of 90 pg of the selected pesticides. In both cases, the injected volume is 500 nL, and the flow rate is 500 nL‚min-1. The first one inverts the solvent composition in 35 min, starting from 100% water to promote solute focusing at the column head. Almost complete separation of the 10 organophosphorus pesticides is achieved under these conditions, and the combination with the selective SIM acquisition ensured full detection of the entire set of the selected pesticides (Figure 4). For instance, phoxim and phorate are completely overlapped in the column, thanks to a different ion selection (m/z 129 and 157 for phoxim, m/z 121, 231 and 260 for phorate) that can be easily distinguished in the final

ion profile. The conditions described above were used for every step in the method validation. A second, faster separation was performed for routine detection of a smaller group of pesticides found in a local set of samples. The SIM program was simplified to maximize sensitivity for the target analytes. Solvent composition change was faster, inverting water and acetonitrile percentage in only 15 min. The combination of a faster solvent program and a specific SIM program was adequate for the four-analyte detection. Instrument limits of detection (LODs) were obtained in actual chromatographic and mass spectrometric conditions, injecting diluted solutions of the initial standard mixture containing all of the analytes. Each LOD was assigned in SIM mode when the least intense ion signal of a specific group exceeded the noise intensity of 6σ. For quantitative data, a 10σ difference was required. Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

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Table 2. Concentration Calibration Statistic Data compd dimethoate paraoxon azinphos-methyl parathion-methyl malathion azinphos-ethyl parathion-ethyl diazinon phorate phoxim

linear regression equation y ) 770.2x + 71.5 y ) 1300.0x + 367.9 y ) 398.8x + 322.0 y ) 1296.2x - 197.1 y ) 1879.3x + 26.7 y ) 822.6x + 198.1 y ) 712.1x + 354.7 y ) 1047.8x - 2.1 y ) 768.1x + 496.2 y ) 291.1x + 152.3

corr coeff

RSD

0.99951 0.99975 0.99974 0.99791 0.99866 0.99994 0.99955 0.99686 0.99961 0.99928

7.6 7.5 7.3 6.6 6.4 3.6 6.0 4.9 6.6 8.1

Instrument detection limits of the analytes are reported in Table 1. Generally, LODs improve when fewer, intense ions are selected. To ensure higher specificity, a larger number of ions for each analyte must be selected. A balance between the two aspects of sample detection has to be evaluated case by case. Concentration calibration experiments were performed for all pesticides in 2 orders of magnitude ranging from 0.09 to 9 nanograms. The lowest value corresponds to the detection limit for several analytes. The experiment was carried out in FIA using eluents A and B in equal proportion. The flow rate was set at 0.5 µL‚min-1, and mass detection was obtained in SIM (see Table 1) using only the most intense signal. Linear regression equations and mean standard deviation data were calculated on the basis of five replicates for each concentration. Precision data were obtained by averaging the relative standard deviation values collected from each concentration calibration experiments (five replicates). The results are reported in Table 2. Excellent linearity and repeatability were demonstrated along the range of concentrations considered. Access to field measurements of organophosphorus pesticides was found in a local river near Rimini, Italy, where a mixture of four compounds, azinphos-methyl and -ethyl, parathion-methyl and -ethyl, were extensively used over the surrounding sugar beet cultivation. A precise knowledge of the products used by farmers

3554 Analytical Chemistry, Vol. 74, No. 14, July 15, 2002

in the area allowed us to restrict the search to the four pesticides alone and use the faster chromatographic program. A 2-L portion of river water was extracted according to the procedure described in the Sample Preparation section. The results obtained are shown in Figure 5a and b. Identification was based on a combination of retention times and ion abundance matching between standard and sample ion profiles. A single ion profile for each compound was used for quantitation. The concentrations calculated using the linear regression plots were azinphos-methyl, 2.9 mg‚L-1; parathion-methyl, 3.2 mg‚L-1; azinphos-ethyl, 2.9 mg‚L-1; and parathion-ethyl 2.0 mg‚L-1. CONCLUSIONS Direct-EI interface generates classical, well-characterized electron ionization spectra, and unlike other LC/MS approaches, it represents a very simple and straightforward LC detector for a variety of applications at trace-level concentration. In the determination of organophosphorus pesticides, direct-EI provides necessary linearity, ruggedness, reproducibility of response, and readily interpretable spectra from commercially available sources (such as NIST or Wiley), for positive identification of the target analytes. An evolved apparatus for electron ionization of compounds amenable by LC will certainly find a strategic role in the field of analytical chemistry. ACKNOWLEDGMENT The authors thank the following companies for their technical and financial support: Rheodyne L.P., Rohnert Park, CA, for providing the 7520 micro-injector; Upchurch Scientific, Oak Harbor, WA, for providing the M-435 microinjection valve; and LC Packings, Amsterdam, The Netherlands, for providing the microcolumn. Received for review November 13, 2001. Accepted April 17, 2002. AC015685F