Simultaneous Determination of Acidic and Basic-Neutral Pesticides in

neutral pesticides in the same chromatographic run is represented by the ion-interaction reagent (hexylammonium phosphate), which is a nonvolatile sal...
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Environ. Sci. Technol. 1999, 33, 3905-3910

Simultaneous Determination of Acidic and Basic-Neutral Pesticides in Water at ppt Concentration Level by Ion-Interaction Micro-HPLC/MS A. CAPPIELLO,* G. FAMIGLINI, AND F. MANGANI Istituto di Scienze Chimiche “F. Bruner”, Universita` di Urbino, Piazza Rinascimento 6, 61029 Urbino, Italy S. ANGELINO AND M. C. GENNARO Dipartimento di Chimica Analitica, Universita` di Torino, Via P. Giuria 5, 10125 Torino, Italy

A new ion-interaction micro-HPLC method, for the simultaneous separation of acidic and base-neutral pesticides, was coupled to a capillary-scale particle beam LC/MS interface for EI mass spectrometric detection of the analytes. The method was employed for analysis of tap water in which a large number of functionally different pesticides can be determined in a single run. Capillaryscale technology of the LC/MS apparatus yields full compatibility with hexylammonium phosphate, a nonvolatile salt used as ion-interaction reagent in the HPLC mobile phase, and provides an excellent sensitivity with instrument detection limits (IDLs) ranging between 0.07 and 0.7 ng in SIM mode. Method sensitivity lower than 0.1 ppb in water was achieved using a large-volume injection technique specifically developed for capillary columns typical flow rates.

Introduction It has been demonstrated (1) that the use of an ion-interaction reagent (IIR) in the mobile phase allows the simultaneous separation of hydrophilic and hydrophobic herbicides with a common reversed-phase HPLC column. IIR-HPLC is based on the dynamic modification of a reversed stationary phase, usually in isocratic conditions, induced by a suitable ioninteraction reagent added to the mobile phase (2). The IIR is a salt of a cation with a lipophilic chain that can be adsorbed onto the surface of the stationary phase. The cation and its counterion give rise to an electrical double layer able to retain and separate cationic and anionic species. Since it is assumed that not the whole original stationary phase is modified, it is also possible to retain, by reverse mode, more lipophilic compounds. A careful tuning of different parameters such as pH, concentration of the ion-interaction reagent, concentration of the organic modifier, etc., can balance the effect of contrasting retention mechanisms thus spacing the peaks in the most convenient chromatographic separation. Due to the high versatility of the technique, new methods have been developed for separations of interest in food, clinical, and environmental chemistry (3-7). The advantage offered by the simultaneous analysis of chemically different pesticides is clearly evident and may * Corresponding author phone: 07224164; fax: 07222754; e-mail: [email protected]. 10.1021/es990340o CCC: $18.00 Published on Web 09/23/1999

 1999 American Chemical Society

positively influence several aspects of the entire analytical process. At trace and ultratrace level, the screening of water samples for pesticide contamination may take advantage of a simplified extraction and chromatographic procedure. In fact, a stepwise extraction for the isolation of functional different pesticides (8, 9) is no longer needed when the IIR method is implemented and the entire organic fraction can be recovered at once. Consequently, only a single instrumental analysis would be required to disclose the global pesticide content of the extract. It is well-known that the coupling between liquid chromatography and mass spectrometry is playing the lion’s share in the analysis of labile substances, and it is almost irreplaceable in any serious application of environmental concern. Several LC/MS methods have been proposed for the analysis of chemically sensitive pesticides in various matrices (10-13) confirming that mass spectrometric detection is unbeatable when specificity and sensitivity come into play. A crucial aspect of the interfacing mechanism, aside from any specific interfacing technique, is a certain degree of coordination between LC and MS requirements. In our case, the key for the appropriate retention of acidic and baseneutral pesticides in the same chromatographic run is represented by the ion-interaction reagent (hexylammonium phosphate), which is a nonvolatile salt added to the mobile phase. On the other hand, mass spectrometry cannot usually tolerate the presence of salts because of their continuous deposition with a rapid fouling of the ion source. The presence of such modifiers in the mobile phase often interferes with the operation of electrospray ion sources by clogging the skimmers and by obscuring or suppressing ionization. A straightforward solution, at least for those analytes amenable by electron ionization, is offered by the capillary-scale particle beam interfacing (14, 15). In the recent past, our group has largely contributed to the analysis of pesticides proposing a certain number of new methods and techniques (13, 16, 17) based on the use of a modified particle beam interface, the only commercially available interface which is still offering an electron ionization alternative. In a world of rampant, soft ionization techniques, the old fashion EI still generates reproducible, high informative, library matchable spectra for a sensitive detection of many analytes. In a particle beam interface, any nonvolatile component present in the mobile phase is not removed from the system, thus joining the solute in the formation of a beam of particles addressed to the mass spectrometer ion source. While the solute is vaporized upon contact on hot source surface and then removed as a mixture of ions and gas phase molecules, crystals of salts remain confined into the ion source. Differently from a conventional apparatus, a capillary-scale interface operates at a mobile phase flow rate of only 1 µL per min, resulting in a salt deposition equal approximately to 0.5 µg per min for a 5-mM reagent concentration in the mobile phase. As demonstrated in a recent paper (18), the very slow reagent deposition, ensured by the microflow rate, is mainly dispersed inside the ion source and remains completely unnoticed in terms of instrument performance and only barely visible during a normally scheduled source cleaning procedure. Differently from other devices, traces of salts trapped inside the system do not interfere with the ionization of the analytes and do not affect their mass spectra or their response. The purpose of the present work is to demonstrate the feasibility of coupling ion-interaction liquid chromatography to mass spectrometry and use it to detect 10 HPLC amenable VOL. 33, NO. 21, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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herbicides ranging from acidic species such as phenoxy acids to weak basic species such as phenol ureic herbicides in a single LC/MS analysis. Besides that, another innovation reported in this paper is the use of a packed capillary HPLC column (0.25 mm i.d.) (19) to perform the pesticide separation under ion-interaction reversed-phase conditions and with a linear variation of the organic modifier concentration in the mobile phase. A method detection limit below 0.1 ppb in water was achieved using a large volume injection technique (20), which allows an injection volume up to 50 µL at 1 µL/min flow rate.

Experimental Section Ion-Interaction Reagent Micro-HPLC. Liquid chromatography was carried out with a Kontron Instrument 420 dualpump, binary-gradient, conventional HPLC system (Kontron Instrument, Milano, Italy). Microliter flow rates were obtained with a laboratory made splitter that was placed between the pumping system and the injector (21). This device allows conversion of almost conventional flow rates (200-µL/min), generated by a conventional HPLC binary system, into microliter-per-minute flow rates. A two-step splitting of the main stream of solvents generates a 2-µL/min mobile phase flow rate with a splitting ratio of 100:1. The splitting device accurately reproduces at lower scale any solvent concentration gradient generated at higher flow rate in the pumping system. For large-volume sample injection, a six port valve Valco injector was employed (Valco, Houston, TX) connected to laboratory-made 10 µL external loop. A fused silica tubing was used for the loop preparation and was purchased from Polymicro Technologies. Final loop volume was calculated from the length and the internal diameter values supplied by the manufacturer and for a 10-µL volume it measured 124 × 0.32 mm. A 60-nL internal loop injector was used for FIA experiments. A Spectra 100 UV/vis detector (Spectra-Physics, San Jose´, CA) was placed online between the chromatographic column and the LC/MS interface for additional peak detection. A laboratory-made packed capillary column was used for the chromatographic separations. These columns are routinely made in our laboratory from 1/16 in. o.d., 250µm i.d. PEEK tubing (Alltech Associates Inc., Deerfield, IL) and are packed with C18 reversed-phase 5-µm particle size purchased from Phase Sep (Queensferry, UK). A 25-cm long column has a mean of 20.000 theoretical plates at 1-µL/min flow rate, and no appreciable loss of efficiency is observed for flow rates up to 5-µL. Acetonitrile was used as organic solvent in the mobile phase. Acetonitrile was preferred over methanol because of its lower viscosity, a parameter that is crucial in micro-HPLC. To achieve a correct solute focusing at the head of the column in large volume injection conditions every sample solution was prepared in water. IIR-HPLC usually makes use of conventional C18 stationary phase dynamically modified in isocratic conditions by the ion-interaction reagent present in the eluent. Concentration gradient elution of the organic modifier are generally not recommended since it leads to an unstable baseline and to an unsatisfactory reproducibility of the results. In this work a successful attempt was done to perform the IIR-PLC on a capillary column (25-cm × 0.25-mm i.d.), employing a solvent concentration gradient. Two eluents were used: a 5-mM hexylamine aqueous solution (A) and a 5-mM hexylamine solution in H2O-ACN (90:10, v/v) (B) both adjusted at pH 6.5 with o-phosphoric acid. The elution was carried out starting at 100% of eluent A and ending at 20% of eluent A in 20 min. The flow rate was kept constant at 2-µL/min. When using the large volume injection (10-µL), to avoid any modification of the ion-interaction double electrical layer established on the stationary phase, hexylammonium phosphate was added to sample at the same concentration found in the mobile phase. 3906

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TABLE 1: Selected Pesticides and Their Instrument Detection Limits (IDL)

compd

CAS-RN

2,4-D bromoxynil dichlorprop 2,4,5-T 2,4-DB bromacil cyanazine Monuron isoproturon diuron

94-75-7 1689-84-5 120-36-5 93-76-5 94-82-6 314-40-9 21725-46-2 150-68-5 34123-59-6 330-54-1

m/z

time scheduled (min)

162, 164, 220 start-21.8 88, 277 162, 234 162, 196, 254 162, 164 205, 207 21.8-27.5 172, 240, 225 198 206, 146 27.5-end 187, 232

IDL (ng) 0.10 0.40 0.05 0.25 0.07 0.07 0.50 0.10 0.10 0.70

Particle Beam and Mass Spectrometry. All the experiments were carried out with a Hewlett-Packard 59980B particle beam unit coupled with a Hewlett-Packard 5989A mass spectrometer. The original nebulizer was replaced by a laboratory-made microflow nebulizer (1). This device generates a mobile phase aerosol using flow rates as low as 1 µL/min. A 50-µm i.d., 180 µm o.d. fused silica capillary tubing (Polymicro Technologies, Phoenix, AZ) was used as the nebulizer tip and to connect the chromatographic column. The nebulizing gas was helium 5.6 purity grade (>99.9996%) and was purchased from SOL (Milano, Italy). The helium pressure needed was 70-90 psi to supply 0.1 L/min. The desolvation chamber temperature was kept at 40 °C. The operating pressures were 0.5 Torr in the desolvation chamber, 0.3 Torr in the second stage of the momentum separator, and 8 to 10 × 10-5 Torr in the manifold of the ion source. Mass spectrometer tuning and calibration were performed automatically using perfluorotributylamine (PFTBA) as a reference compound and monitoring m/z 69, 219, 502. The repeller potential was adjusted manually. A mobile phase composed of buffer A:buffer B (50:50) was allowed into the ion source during calibration. The dwell times during selected ion monitoring (SIM) analyses were adjusted in order to obtain 0.5 cycles/s and a mean of 10 acquisition samples for each HPLC peak. A two- or three-ion detection was used in SIM mode. The ions were selected on the basis of the intensity in the mass spectrum (Table 1), discarding the lowest m/z values. The final transfer tube, prior to the ion source, was shifted to a fully retracted position after the tuning procedure allowing a third pumping stage for a further sample enrichment. A Teflon insert was placed on the vaporization surface of the ion source to minimize adsorption and thermal decomposition of phenoxy acid herbicides. No signs of thermal decomposition were observed in these conditions. Assuming an average mobile phase concentration of buffer A:buffer B (50:50) and simulating different operating conditions of analysis and preanalyzis, the absolute hexylammonium phosphate introduction is approximately 1 µg/min. It has to be considered that during every preanalyzis elution the isolation valve between the interface and the mass spectrometer remains closed, while the rest of the interface operates normally. This means that only during the analysis the reagent is admitted into the ion source, thus reducing substantially its impact in such a critical zone. Extraction Procedure. A liquid-solid extraction procedure for the isolation of the pesticides from aqueous samples was employed. This procedure has been slightly modified with respect to that previously described (9) and adapted to the large volume injection in ion-interaction conditions. All the labware, except the LSE cartridge, was made of Teflon including the water samples container and the connection tubings and relative hardware. Although it has a higher cost, a Teflon surface is less prone to adsorbing analyte molecules

FIGURE 1. Chemical structures of the selected pesticides. at very low concentration and avoids the trapping 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. Polyethylene frits, 20 µm pore size, were located above and below the sorbent bed. Before a water sample was extracted, 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 ascorbic acid in HCl-acidified water (pH 2). Water samples were forced through the trap at a flow rate of approximately 150 mL/min by using a vacuum apparatus placed below the cartridge. Distilled water (7 mL) was passed through the trap after the entire sample was passed through. Forcing a stream of nitrogen through the cartridge for 30 min dried out the residues of water. The extraction solution was prepared with methylene chloride:methanol (60:40 by volume) basified with KOH 0.016 M. Six milliliters of this solution was passed through the cartridge and collected in a Teflon vial. The eluate was brought to neutrality by adding 70 µL of a 1.92-M solution of H3PO4 in water. Two hundred thirty microliters of water was added to the sample in order to bring the total water content to 300 µL. The sample was heated at 40 °C, and a gentle stream of nitrogen rapidly evaporated the organic solvents over the liquid surface. At the end of the evaporation process a 300-µL aqueous solution of the extracted pesticides was found in the bottom of the vial as required by large volume injection conditions. Because of the ion-interaction mechanism, there was no need for lowering the final extract pH. Reagents and Chemicals. All solvents were HPLC grade from Farmitalia Carlo Erba (Milano, Italy) and were filtered and degassed before use. Also o-phosphoric acid was a Carlo Erba chemical. Hexylamine was purchased from Fluka Chemie AG (Buchs, Switzerland).

Ten pesticides (Figure 1) were selected for this work on the basis of their presence in the environment and their particular amenability by HPLC. As reported in Table 1, they own to various chemical classes such as phenols and phenoxy acids, phenylureas triazines, etc., are easily degradable by sunlight or heat and often show hard-to-solve drawbacks during the analysis. They were purchased from Riedel-De Haen (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).

Results and Discussion Figure 1 reports the structural formula of the selected pesticides. 2,4-D, dichlorprop, 2,4,5-T, and 2,4-DB are phenoxy acid herbicides. Bromoxynil is a phenol, bromacil is a uracil, and cyanazine is a triazine, while Monuron, isoproturon, and diuron belong to the class of phenylureas. All but phenoxy acids are neutral or basic pesticides. They represent a wide range of chemical and functional classes and a good test stand for the validation of this method. The capability of the capillary-scale particle beam interface of withstanding a continuous intake of nonvolatile salts has been investigated (18). In that case, the interface and mass spectrometer were exposed to a mobile phase enriched with a 10-mM phosphate buffer for a total of 35 days under heavy duty conditions, and none of the crucial parameters involved in the mass spectrometry operation was affected. Only a modest deposition of salts was found, as soon as the work was concluded, in the nozzle/skimmer area and inside the ion source and was easily removed during a routine cleaning procedure. As discussed in the paper by Gennaro et al. (1), the retention of the analytes are influenced by several parameters such as pH, organic modifier concentration, and ioninteraction reagent concentration. It was demonstrated that each factor may have an influence on the effect of the others, and only a balanced coordination may lead to a fruitful VOL. 33, NO. 21, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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separation. In this work, a method that was originally developed for a conventional 4.6-mm i.d. analytical column under isocratic conditions was transposed to a micro-HPLC column in gradient conditions. The increasing concentration of the organic modifier further complicates an already complex picture, and, as consequence of that, its use in IIRHPLC remains controversial. It can be pointed out, that most common drawbacks related to the use of gradients in liquid chromatography such as the baseline drift are usually not observed in LC/MS. The use of an acetonitrile gradient was imposed by the necessity of modulating the extent of the reversed-phase retention for a larger number of species and, more important, for creating the conditions of a solute focusing under large volume injection conditions. In fact, the volume normally injected into a 0.25-mm i.d. packed capillary column is only 60 nL, while a larger volume lead to band broadening and poorer resolution. In real-world applications, a 60-nL sample impairs the ability of the system to detect very dilute analytes compromising the overall method sensitivity even in the presence of low IDLs. Under IIR conditions, the capacity factor logarithm ln k′ linearly increase as the acetonitrile concentration decreases and, for a 100% water mobile phase, k′ ≈ ∞. If a mobile phase composed of 100% water is used at the start of the chromatographic run and to initially condition the column, up to 10-µL of sample can be injected without alteration of the original chromatographic profile. At 2-µL/min flow rate a 10-µL sample is displaced into the column in 5 min. As soon as the sample reaches the column, the solvent in which the analytes are dissolved temporarily replaces the original mobile phase that precedes and follows the sample plug. Since the ion-interaction reagent is in dynamic equilibrium with the reversed stationary phase, the passage of the sample plug through the column would represent an alteration of the IIR conditions into a moving zone, thus disturbing the pesticide separation. To keep the electrical double layer constantly in place and to promote solute focusing, the sample was prepared in a 5-mM hexylammonium phosphate water solution as found in eluent A. The 60-nL internal-loop valve was replaced by a conventional six-port injection valve equipped with a 10-µL external loop. Any connection between the splitter and the column was carefully inspected for dead volumes, particularly troublesome at this flow rate level. The 10-µL external loop was shaped in a long fused silica capillary tubing to avoid liquid turbulence and promoting a prompt loop emptying. pH of the mobile phase affects acid-base equilibrium of the pesticides and influences retention through a double mechanism: electrical ion interaction and reversed-phase hydrophobic affinity. In addition, the extent of such an influence depends on the chemical structure and the acidic or basic behavior of the analytes. For example, 2,4-D, 2,4DB, and 2,4,5-T are relatively strong acids and are heavily influenced by the H3O+ concentration; bromacil and bromoxynil are almost neutral species and are sensitive only to the acetonitrile concentration; retention of basic pesticides, such as Monuron and diuron, depends on pH but in opposition respect to the acidic ones. The best compromise for our mixture was found at a pH of 6.5. Sample pH different from 6.5 worsens solute focusing in large volume conditions, and it has to be adjusted prior to injection. In these conditions the acidic pesticides were eluted before the basic ones as shown in the reconstructed ion chromatogram obtained from a 10-µL injection of a 10-ng standard solution and reported in Figure 2. Mobile phase flow rate was kept at 2-µL/min constantly. All pesticides are fully separated except bromoxynil and dichlorprop, which are overlapped. Completely different ion profiles allow mass spectrometric separation of the two analytes. 3908

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FIGURE 2. Reconstructed ion chromatogram realtive to a SIM acquisition of 10-ng standard solution of the selected pesticides. Injection volume 10 µL, flow rate 2 µL/min: (1) 2,4-D, (2) bromoxynil, (3) dichlorprop, (4) 2,4,5-T, (5) 2,4-DB, (6) bromacil, (7) cyanazine, (8) Monuron, (9) isoproturon, and (10) diuron.

FIGURE 3. Ion profiles relative to the on-column detection of 100 pg of 2,4-D. Injection volume 10 µL, flow rate 2 µL/min. Mass spectrometric detection was performed in SIM using one, two, or three characteristic ions chosen on the base of the highest abundance and mass-to-charge ratio. The chromatogram was subdivided in three parts, and for each one was assigned only those m/z values relative to the group of pesticides eluted during that time window. In this way, dwell times increased by a factor of 3 and sensitivity could be maximized as well. Switching times are found at 21.8 and 27.5 min. SIM conditions are reported in Table 1. Due to its poor fragmentation, only Monuron is identified by a single ion signal at m/z 198. When possible, a three-ion detection has been allowed. A corrected analyte identification is verified by matching retention times, fragment ions, and relative abundance among sample and standard peaks. Instrument detection limits were obtained in actual chromatographic and mass spectrometric conditions and injecting diluted solutions of the standard mixture. Each IDL was assigned when the least intense ion signal, relative to a specific pesticide, reached a signal-to-noise ratio of 5:1. In the authors’ opinion, this procedure reduces the chance of presenting too optimistic detection data that are often far from the effective ability of detecting analytes in real-world applications. Figure 3 reports the ion profiles relative to the detection limit of 2,4-D obtained injecting 100 pg of the compound. This plot demonstrates an excellent overall LC/MS response even at very low concentrations, with a symmetrical and narrow chromatographic peak well perceivable from the

TABLE 2: Retention Time and Peak Area Reproducibility peak no.

inj 1

inj 2

inj 3

inj 4

inj 5

inj 6

xj

RSD (%)

1 ret time (min) 1 area 2 ret time (min) 2 area 3 ret time (min) 3 area 4 ret time (min) 4 area 5 ret time (min) 5 area 6 ret time (min) 6 area 7 ret time (min) 7 area 8 ret time (min) 8 area 9 ret time (min) 9 area 10 ret time (min) 10 area

19.78 61603 20.05 127692 20.48 62210 21.33 69035 23.18 67812 24.13 42445 25.13 175772 26.03 26126 29.33 62960 29.91 32439

19.49 58742 19.7 114036 20.12 57995 20.87 56116 22.74 66529 23.55 41049 24.55 172876 25.42 25528 28.72 61245 29.3 31416

19.87 62409 20.1 122197 20.55 62726 21.34 60746 23.35 67261 24.2 42659 25.2 178578 26.1 29051 29.37 63211 29.95 31283

20.57 64456 20.08 129775 21.23 66823 22.02 69388 24.07 70582 25.07 45253 26.03 183800 26.93 34347 30.23 64579 30.82 31314

21.11 74801 21.5 128902 21.77 72745 22.52 71612 24.32 70261 24.64 49869 25.59 193.868 26.47 36472 29.82 69281 30.37 33212

21.1 68374 21.47 130145 21.84 64348 22.59 65193 24.44 66653 24.72 51348 25.67 193221 26.57 37069 29.89 65601 30.45 32223

20.32 65064 20.48 125457 21.0 64474 21.78 65348 23.68 68183 24.38 45437 25.36 183019 26.25 31432 29.56 64479 30.13 31981

3.44 8.83 3.81 5.02 3.42 7.72 3.21 9.07 2.91 2.63 2.21 9.36 2.01 4.87 1.98 16.48 1.79 4.31 1.75 2.44

TABLE 4: Recoveries of the Selected Pesticidesa

TABLE 3: Concentration Calibration Statistic Data compd bromacil 2,4-D

range (ng) 0.1-2.0 0.1-2.0

y ) ax + b

RSD (yEr, ()

r

y ) 125.3x - 5.8 y ) 148.7x - 15.4

11.5 6.3

0.994 0.974

baseline. The baseline itself is the product of a limited electric noise. Instrument detection limits relative to the rest of the analytes are reported in Table 1. To evaluate the reproducibility of this method, the LC/ MS analysis of the standard solution was repeated six times acquiring retention times and peak areas for each pesticide. Data relative to the peak sequence are reported in Table 2. Average values (xh ) and relative standard deviation (RSD) are also reported. The results demonstrate a substantial reproducibility in terms of chromatographic retention and solute transmission. The slightly worse RSD values for the peak no. 1 are probably due to a poorer focusing of the less retained, more hydrophilic compounds. Concentration calibration experiments were performed for 2,4-D and bromacil in a range of concentrations (0.1-2.0 ng injected) for both compounds. The experiment was carried out in FIA using eluents A and B in equal proportion. The flow rate was set at 2 µL/min, and the mass spectral acquisition was obtained in SIM (Table 1). Linear regression equations and mean standard deviation data were calculated on the basis of five replicates for each concentration. The results are reported in Table 3. Because of its higher polarity, 2,4-D shows a slightly limited range of linearity with respect to that of bromacil. Probably, its higher affinity to water delayed, at lower concentrations, the desolvation of the droplets and affected, to a certain extent, the transport efficiency of the interface. For the validation of the method, 1 L of tap water was spiked with 50 µL of 2.2-ng/µL methanolic solution of the pesticides in order to obtain a final concentration in water of 0.11 µg/L (ppb w/v). This level is very close to the limit issued by the European Community for the presence of each pesticide in a drinking water. The sample was extracted as specified in the Experimental Section and collected in a final 300-µL aqueous solution. As previously discussed, the sample was adapted, prior to injection, to the needs of the large volume injection and the ion-interaction reagent. The recovery efficiency of this method was carefully evaluated for both acidic and base-neutral pesticides using six replicates at a very low concentration (ppt) in order to match the lowest

recovery (%)

a

compd

n)6

RSD (%)

2,4-D bromoxynil 2,4,5-T dichlorprop bromacil Monuron cyanazine 2,4-DB isoproturon diuron

86.7 104.3 98.2 120.0 101.7 106.7 112.8 128.9 94.1 104.5

16.5 13.8 11.8 12.2 12.3 11.5 8.4 9.8 8.6 8.8

Sample volume: 1 L. Analyte concentration: 110 ppt (w/v).

allowed concentration of these pollutants within the European Community (0.1 ppb w/v). The recovery data were collected comparing each peak area of the extracts with the correspondent peak in the standard ion chromatogram. For this purpose, 10 µL of a 0.36-ng/µL standard solution of the pesticides was injected after and prior to the extraction sample. The recovery data are reported in Table 4. Recoveries varied from 86.7% to 128.9% with a RSD of approximately 10%. In our opinion, the extremely low concentration range considered in this work can safely justify the observed fluctuation in the recovery values. Considering this, the values for recoveries and standard deviations are excellent. Figure 4 shows the reconstructed ion chromatogram relative to the LC/MS analysis of the spiked water. The acquisition was carried out in the same conditions reported for the analysis of the standard solution. A blank injection, relative to a sample of nonspiked tap water, was also performed and gave no signal for any of the analytes considered. A direct comparison of the peak area may give sufficient facts for a preliminary evaluation of the method efficiency. For all the pesticides considered, this method would ensure undoubted detection and quantitation even at a level below 0.1 ppb in water. The overall recovery, although not yet supported by enough data, is satisfactory with values ranging between 72% to over 100%. The peak relative to Monuron, which would show a recovery much higher than 100%, has been clearly affected by a sort of contamination. In conclusion, the ability of a capillary-scale interface to deal with nonvolatile or exotic chromatographic eluents, VOL. 33, NO. 21, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 4. Reconstructed ion chromatogram relative to 0.1 ppb spiked tap water sample. Injection volume 10-µL, flow rate 2 µL/ min: (1) 2,4-D, (2) bromoxynil, (3) dichlorprop, (4) 2,4,5-T, (5) 2,4-DB, (6) bromacil, (7) cyanazine, (8) Monuron, (9) isoproturon, and (10) diuron. without interfering with electron ionization, opens the door to novel applications. It has been demonstrated the possibility to analyze simultaneously functionally different pesticides at a scaled-down flow rate and at ultratrace concentrations. Particle beam interfacing can take full advantage of the separation possibility offered by the IIR-HPLC, which is normally forbidden to mass spectrometric detection.

Literature Cited (1) Gennaro, M. C.; Giacosa, D.; Abrigo, C.; Simonetti, S. J. Chromatogr. Sci. 1996, 34, 334.

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Received for review March 25, 1999. Revised manuscript received July 14, 1999. Accepted August 16, 1999. ES990340O