Anal. Chem. 1994,66, 1416-1423
Determination of Acidic and Basic/Neutral Pesticides in Water with a New Microliter Flow Rate LWMS Particle Beam Interface Achlile Cappiello,' Giorgio Famlglini, and Fabrirlo Bruner Istituto di Scienze Chimiche, Universith di Urbino, Piazza Rinascimento 6, 6 1029 Urbino, Italy A new method for the determination of 32 baseheutral and 13 acidic pesticides in water, which has been proven to be particularly amenable to high-performanceliquid chromatography, is discussed. A liquid/solid extraction procedure for the isolation of pesticides from aqueous samples is employed. The LC/MS analysis is carried out with a LC-18 reversedphase packed capillary column coupled with a Hewlett-Packard 5989A quadrupole mass spectrometer using a new microliter flow rate particle beam interface. Mobile-phase flow rates, ranging between 1 and 5 ML/min, pass into the electron impact ion source of the mass spectrometer. The aerosol generated by the reduced solvent imput allows improved signal response for high water content mobile phases with better chromatographic performance during gradient analyses. Reliable and reproducible results for positive identificationand quantitation of target analytes are presented. The very low contamination of the instrument satisfies the continuous use demands of routine analysis.
The use of high-performance liquid chromatography (HPLC) methods for the analysis of pesticides in various matrices has become necessary since the introduction of a large series of highly effective and easily degradable polar compounds. However, the lack of specificity of the usual HPLC detectors, and the large number of compounds to be identified and quantitatively analyzed, push toward the use of LC/MS techniques. These techniques can provide high confidence analyte identification in complex environmental mixtures. GC/MS-based analytical methods for nonvolatile organic compounds are less attractive because of their complexity and quantitative uncertainty, as well as the additionai time and cost when derivatization processes are involved. In the last few years an increasing number of publications have contributed to the development of very effective LC/ MS techniques for the analysis of various classes of organic compounds. The main advantage over consolidated GC/MS alternatives is the possibility of injecting the extract directly into the system without further analyte derivatization, allowing for consistent time saving in routine analyses. Although LC/MS coupling techniques have reached a high level of development, each one uses a radically different approach to introduce the chromatographic effluent into the ion source and to promote sample ionization. The enormous difference in chemical properties and molecular weight among molecules suitable for HPLC requires different approaches to transfer the sample from the HPLC environment into the 1410
Analytical Chemistry. Vol. 66, No. 9, May 1, 1994
mass spectrometer. Furthermore, HPLC samples are either thermally unstable or nonvolatile and for these characteristics soft ionization techniques are usually employed. The thermospray interface,' introduced in the mid-l980s, represented a significant breakthrough and gave decisive impetus for a worldwide diffusion of LC/MS instrumentation. Reversed-phase conventional columns, employing high water content mobile phases and volatile buffers, were easily coupled by use of the new interface. Bellar and Budde2 reported an interesting evaluation of the thermospray performance in the analysis of numerous organic compounds in environmental samples. Although having undoubted advantages over previous attempts, thermospray started to show some limitations, these included necessity of different temperature settings for different experimental conditions, ion abundance unstability, and lack of structurally significant fragmentation. Electrospray, which has captured considerable attention for managing delicate high molecular weight compound^,^*^ has been employed successfully for the determination of environmental polar pollutant^.^ The absolute absence of fragment ions requires the use of collision-induced decomposition (CID) in order to collect useful structural information for undoubted identification or confirmation. Recently, highflow ion spray, which also employs CID for structural confirmation, has been used for the determination of some pesticides.6 The particle beam interface, developed by Browner and c o - w o r k e r ~is, becoming ~~~ a viable alternative for the analysis of small molecules with chemical properties which are not suitable for GC/MS. Thousands of biologically and environmentally important compounds can be included in this category. Particle beam is fully compatible with a preexisting electron impact (EI)/CI ion source and may give, for target compounds, the advantages of both worlds: HPLC compatibility with the variety of structurally significant fragment ions characteristic of conventional electron impact ionization mass spectrometry. Since the early reports about a monodisperse aerosol-based interface (MAGIC), a big effort has been devoted to the (1) Vestal, M. L.; Fergusson, G. J. Anal. Chem. 1985, 57, 2373-2378. (2) Bellar, T. A.; Budde, W. L. Anal. Chem. 1988, 60, 2076-2083. (3) Whitehouse, C. M.; Dreyes, R. N.; Yamashita, M.; Fenn, J. B. Anal. Chem. 1985, 57, 6 7 5 4 7 9 . (4) Fenn, J . B.; Mann, M.; Mong, C. K.; Wong, S . F.; Whitehouse, C. M. Mass Specrrom. R ~ G1990, . 9, 37-70. (5) Lin. H . ; Voyksner, R. D. Anal. Chem. 1993, 65, 4 5 1 4 5 6 . (6) Hopfgartner, G.; Wachs, T.; Bean, K.; Henion, J. Anal. Chem. 1993, 65,
439446. (7) Willoughby, R. C.; Browner, R. F. A n d . Chem. 1984, 56, 2626-2631. (8) Winkler, P. C.; Perkins. D. D.: William, W. K.; Browner, R. F. Anal. Chem. 1988, 60. 489,
0003-2700/94/0366-14 18$04.50/0
0 1994 American Chemical Society
development of a more reliable interface. Most of the criticism was addressed to its lower level of sensitivity compared with other LC/MS interfaces, lower response with high water content mobile phases, and unreliable quantitative results. Bellar et al.9J0 have investigated the transport mechanism involved in the particle beam interface and pointed out a relationship between enhanced ion abundance and the use of a given carrier. Calibration curves for quantitative analyses have benefited from a complete understanding of these processes, thus clearing up any perplexity over quantitation results. Many research groups have successfullyemployed particle beam interfaces for the analysis of some pesticides and other compounds of environmental interest."-l7 The methods presented can be considered a valuable improvement in the analysis of nonvolatile pollutants. Recently, our research group presented a radical modification of the nebulization process of a particle beam interface which allows the introduction of a much lower mobile-phase flow rate, on the order of I pL/min,18 into the mass spectrometer ion source. The new coupling device, which is fully compatible with a commercially available desolvation chamber and momentum separator of a Hewlett-Packard 59980B particle beam unit, shows several advantages compared to the conventional device. Benefits include drastic reduction in solvent consumption with negligible contamination by solvent vapors of the pumping system, the ion source, and the analyzer. These characteristics widen the choice of HPLC buffers or mobile phases to those considered potentially harmful to the instrument. Better signal response for high water content mobile phases with improved sensitivity and chromatographic performance during gradient analysis were also noticed. The purpose of this work was to evaluate the possibility of the new device to contribute positively to the analysis of a large number of basic/neutral and acidic pesticides which are particularly amenable with HPLC. The method employs a reversed-phase packed capillary c o l ~ m n ' ~for - ~chromato~ graphic separations and SPE cartridges for the extraction p r ~ c e d u r e . ~The ~ - ~detection ~ limits were generally in the range of 0.1-30 ppb as required for environmental analysis. This method provides sufficient qualitative information for (9) Bellar, T. A.; Behymer, T. D.; Budde, W . L. J. Am. Soc. Mass Specrrom. 1990. 1. 92-98. -. . -,-, -- - -
(10) Ho,J. S.;Behymer, T. D.; Budde, W. L.; Bcllar, T. A. J . Am. Soc. Mass Specrrom. 1992, 3, 662671. (11) Bchymer, T. D.; Bellar, T. A.; Budde, W. L. Anal. Chem. 1990, 62, 1686. (12) Jones,T. L.; Betowski, L. D.;Lwnik, B.; Chiang,T. C.;Teberg, J. E. Enuiron. Sci. Techno/. 1991, 25, 1880-1884. (13) Jones, G. G.; Pauls, R. E.; Willoughby, R. C. Anal. Chem. 1991,63,460463. (14) Incorvia Mattina, M. J. J . Chromatogr. 1991, 542, 385-395. (1 5) Julien-Larose, C.; Voiron, P.; Mas-Chamberlin, C.; Dufour, A. J . Chromnrogr. 1991, 562. 39-45. (16) Kim, I. S.;Sasinos, F. I.; Stephens, R. D.; Wang, J.; Brown, M. A. Anal. Chem. 1991,63, 819-823. (17) Betowski, L. D.; Pace, C. M.; Roby, M. R. J . Am. Soc. MussSpectrom. 1992, 3, 823-830. (18) Cappiello, A.; Bruner, F. Anal. Chem. 1993, 65, 1281-1287. (19) Cappiello, A.; Palma, P.; Mangani, F. Chromnrographia 1991,32,389-391. (20) Crescenthi, G.; Mastrogiacomo, A. R. J . Microcolumn Sep. 1991, 3, 539545. (21) Crescenthi, G.; Bruner, F.; Mangani, F.; Yafeng, G. Anal. Chem. 1988.60, 1659. (22) Palma, P.; Cappiello, A. Ann. Chim. 1992, 82, 371-377. (23) Di Corcia, A.; Marchetti, M.Anal. Chem. 1991, 63, 580-585. (24) Di Corcia, A.; Marchetti, M. J. Chromatogr. 1991, 541, 365-373. ( 2 5 ) Di Corcia, A.; Samperi, R. A n a l . C h e m . 1993, 65, 907-912.
Table 1. Selected Pertlcldes
pesticide
ClaSSU
Baaic/NeutralPesticides (1) oxamyl carbamate (I) (2) methomyl carbamate (I) (3) chloridazon pyridazinone (H) (4) dimethoate phoaphorodithioate (A) (5) aldicarb carbamate (I) (6) metoxuron phenylurea (H) (7) bromacil uracil (H) (8)cyanazine triazine (H) (9) monuron phenylurea (H) (10)metribuzin triazine (H) (11) carbofuran carbamate (I) (12) chlortoluron phenylurea (H) (13) carbaryl carbamate (I) (14) fluometuron phenylurea (H) (15) atrazine triazine (H) (16) isoproturon phenylurea (H) (17) paraoxon organophosphate (I) (18) difenoxuron phenylurea (H) (19) monolinuron phenylurea (H) (20) diuron phenylurea (H) (21) propachlor acetanilide (H) carbamate (I) (22) r" (23) propanil propionalide(H) (24) linuron phenylurea (H) (25) chloroxuron phenylurea (H) (26) chlorbromuron phenylurea (H) (27) chlorpropham carbamate (H) (28) fenitrothion phosphorothioate(I) (29) azinphos-ethyl phosphorothioate(I) (30) parathion-ethyl phosphorothioate(I) (31) coumaphos phosphorothioate(I) (32) phoxim phosphorothioate(I) Acidic Pesticides (1) dicamba methoxybenzoic(H) (2) bentazone thiadiazinone (H) (3) 2,4-D phenoxy acid (H) (4) MCPA phenoxy acid (H) (5) bromoxynil phenol (H) (6) dichlorprop phenoxy acid (H) (7) mecoprop phenoxy acid (H) (8) 2,4,5-T phenoxy acid (H) (9) 2,4-DB phenoxy acid (H) (10) MCPB phenoxy acid (H) (11) 2,4,5-TP phenoxy acid (H) (12) dinoseb phenol (H) (13) dinoterb phenol (H)
CAS RN* 23135-22-0 16752-77-5 1698-60-8 60-51-5 116-06-3 19937-59-8 314-40-9 21725-46-2 150-68-5 21087-64-9 1563-66-2 15545-48-9 63-25-2 2164-17-2 1912-24-9 34123-59-6 311-45-5 14214-32-5 1746-81-2 330-54-1 1918-16-7 122-42-9 709-98-8 330-55-2 1982-47-4 13360-45-7 101-21- 3 122-75-5 2642-71-9 56-38-2 56-72-4 14816-18-3 1918-00-9 25057-89-0 94-75-7 94-74-6 1689-84-5 120-36-5 7085-19-0 93-76-5 94-82-6 94-81-5 93-72-1 88-85-7 1420-07-1
Key: A, acaricide;H, herbicide;I, insecticide. b ChemicalAbatract Service registry number. (I
all compound identification and accurate quantitative results in the range of concentrations considered. Moreover, it does not require costly instrumentation and, although unique, is particularly oriented for routine applications.
EXPER I MENTAL SECTION Particle Beam Interface and Mass Spectrometer. A modified Hewlett-Packard 59980B particle beam interface, coupled with a Hewlett-Packard 5989A quadrupole mass spectrometer, was used in this work. The original supplied nebulizer was replaced with a laboratory-made microneubulizer which has been described in a previous paper.'s This new device does not require modifications in the desolvation chamber, in the momentum separator, or in their original assembly. Nozzle and skimmer holes and their mutual distance were left in their original settings. The final transfer tube of the interface was set in fully retracted position, thus leaving -5 mm of free space prior to the ion source. This gap allows the addition of a third pumping stage for sample AnaiyticalChemlstry, Vol. 66, No. 9,May 1, 1994
1417
a
Abundance
30
20
40
Time (min.)
Flguro 1. UV profile (a) and TIC profile (b) of a chromatographic separation of a standard mixture of 32 basic/neutrai pesticides.
enrichment, operated by the roughing pump of the mass spectrometer. A 50-pm-i.d., 180-pm-0.d. fused-silica capillary tubing was used as the nebulizer tip and to connect the chromatographic column at the opposite end. The mobilephase in-line filter was removed from the liquid path. The nebulizing gas, helium (5.6 purity grade), was obtained from SOL (Milano, Italy). The helium flow rate was -0.2 L/min when maximum signal response was monitored by the ion source with a mobile-phase flow rate of 2 pL/min. This value corresponds to a gas pressure of 30 psi and to a linear velocity at the nebulizer tip of 200 m/s. Because of a redesigned gas delivery tubing inside the nebulizer, a different relation between gas pressure and gas flow was found. The gas temperature was ambient while the desolvation chamber temperature was 40 OC for all the experiments. The pressure was reduced to -0.5 atm in the desolvation chamber, 0.3 Torr in the second stage of the momentum separator, and (5-8) X le5Torr in the manifold of the ion source. The temperature in the ion source was set at 250 OC, while the analyzer was 120 "C.The mass spectrometer tuning and calibration was operated 1410 AnalyticalChemistry, Vol. $6,No. 9, May 1, 1994
automatically using perfluorotributylamine (PFTBA) as the reference compound. The repeller potential was adjusted manually, monitoring fragment ions with m / z close to sample values. Mobile phase flowed into the ion source while the calibration procedure was operating. The mass spectrometer was scanning in the 50-400 amu range with a threshold of 50 counts when operated in TIC mode. The scan speed was 1.2 scan/s, which gave a mean of 10 acquisition samples for each HPLC peak. Peak area values were calculated with automatic integration. The electron energy was set at 70 eV in positive ion mode. Interface PerformanceOptimization. Signal optimization with the new interface was less critical and consistently easier than with the conventional one. A specific combination of the position of the fused-silica capillary inside the coaxial gas tubing and helium pressure offers the highest signal intensity for basic/neutral and acidic pesticides. No further adjustments are usually required as the two species are exchanged. The optimization procedure was carried out in accordance with the flow injection analysis technique (FIA) for prelimi-
-
nary adjustment, followed by HPLC/MS under required chromatographic conditions, with proper buffers and solvents. A unique advantage of the new interface is its excellent performance even with a high concentration of water in the mobile phase. The signal response remains nearly constant over a wide range of mobile phase relative concentrations. Tuning was not affected by the water content in the mobile phase, thus simplifying the overall procedure. All the tuning tests were conducted with a 50% water/acetonitrile solution added to the proper amount of the final buffer. A vernier dial allows shifts of the nebulizer capillary tip, and the most appropriate position was with the tip end aligned with the sharp restriction of the gas tubing or slightly withdrawn. Liquid Chromatography. The packed capillary column used in this work was made in our laboratory from '/Isin.-o.d., 250-pm4.d. poly(ether ether ketone) (PEEK) tubing (Alltech Associates Inc., Deerfield, IL) and packed with C18 reversedphase 5-pm particle size purchased from Phase Sep (Queensferry, UK). The 25-cm-long column had an efficiency of 15 000 theorical plates measured at 1 pL/min flow rate. The liquid chromatography was carried out with a Kontron Instrument 420 dual-pump 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. The splitter allows accurate and stable microliter flow rates and rapid delivery of solvent concentration changes for reliable and reproducible gradients.22 A motor-assisted solvent mixer was placed after the pumps and before the splitter device. For sample injection a zero-volume Valco injector equipped with a 60-nL internal loop was employed (Valco, Houston, TX). Larger loops are not advisable for flow rates lower than 5 pL/min because of their consistent loss of chromatographic efficiency. The 50-pm-i.d., 180-pm-0.d. fused-silica capillary tubing used in the nebulizer was purchased from Polymicro Technologies (Phoenix, AZ). Extraction Procedure. A liquid/solid extraction procedure for the isolation of pesticides from aqueous samples was employed. A cartridge was filled with 250 mg of graphitized carbon black (Carbograph 1) and was capable of sampling up 2 L of water. By taking advantage of the presence of positively charged active centers on the Carbograph surface, a stepwise elution system allowed the complete separation of basic/ neutral pesticides from acidic ones. The extraction cartridge was made using a polypropylene tube, 6.5 X 1.4 cm i.d., packed with 250 mg of Carbograph 1,120-400 mesh (Alltech, Deerfield, IL). Polyethylene frits, 20-pm pore size, were located above and below the sorbent bed. Before the 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 HC1-acidified water (pH 2). The water sample was forced to pass through the trap at a flow rate of 150-160 mL/min using a vacuum apparatus placed below the cartridge. Distilled water (7 mL) was added to the empty trap after all the sample had passed through. Basic/neutral pesticides were eluted by passing 1 mL of methanol, drop by drop, through the trap followed by 6 mL of methylene chloride/methanol (80:20 by volume). Acidic
Table 2. SIM Acquldtkn Paramelere Used for Barlc/Neutral end Acldk Pestkhles
time (min) program 0
1
22
2
40
3
0
1
24
2
-
compound
mlz
dwell(ms)
Basic/Neutral Pesticides oxamyl 72,98 methomyl 88,105 chloridazon 77,105,221 dimethoate 87,125 aldicarb 115,144 metoxuron 72 bromacil 205 cyanazine 198,212 monuron 72,198 metribuzin 144,198 carbofuran 164 chlortoluron 72,132,212 carbaryl 115,144 fluometuron 72,187,232 atrazine 200 isoproturon 72 paraoxon 109 difenoxuron 72,241 monolinuron 61 diuron 72,187,232 propachlor 93,120 propham 93,172 propanil 161 linuron 61,187 chloroxuron 72 chlorbromuron 61 chlorpropham 127 fenitrothion 109 azinphos-ethyl 132 parathion-ethyl 97,109,291 coumaphos 109,226,362 phoxim 77,103 Acidic Pesticides dicamba 173,220 bentazone 119,198 2,4-D 162,164,220 MCPA 107,141,200 bromoxynil 88,277 dichlorprop 162,164,234 mecoprop 107,142,214 2,4,5-T 162,196,254 2,4-DB 162,164,198 MCPB 107,142 2,4,5-TP 162,196,198 dinoseb 163,211 dinoterb 177,225
300 300 300 300 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 120 300 300 300 150 150 150 150 150 150 150 150 150 150 150 500 500
pesticides were collected in a second extraction percolating through the trap, drop by drop, 6 mL of methylene chloride/ methanol (60:40 by volume) alkalinized with 0.016 M KOH. Prior to the concentration step, the extract containing the acidic pesticides was acidified by adding 0.35 mL of 2% (v/v) trifluoracetic acid (TFA) in water. The extracts were then concentrated toobtain a finalvolume of 1OOpL. For a detailed discussion of the extraction procedure see ref 23, Reagents. All solvents were HPLC grade from Farmitalia Carlo Erba (Milano, Italy) and were filtered and degassed before use. Pesticides were purchased from Riedel-De Ha&n (Hannover, Germany). Ammonium acetate and trifluoracetic acid were purchased from Sigma Scientific (St. Louis, MO). Ascorbic acid was supplied by Carlo Erba. Reagent water was obtained from a Milli-Q water purification system (Millipore Corp., Bedford, MA). RESULTS AND DISCUSSION For this study we have selected 32 basic/neutral and 13 acidic pesticides according to the criteria of poor volatility Analytical Chemistry, Vol. 66,No. 9, May I , 1994
1419
a
Abundance 4OooO
-
30000
-
9
IO'
b
61 7
2ooM)-
10000 -
Flgurr 2.
uv
and thermal stability and wide use in agriculture (Table 1). Most of them are not suitable for GC/MS methods or result in distorted GC peak shapes, poor sensitivity,or multiple peaks or show other evidence of partial thermal decomposition. Conventional HPLC analysis and detection techniques are feasible for detecting a few analytes in well-known or noncomplex samples, e.g., production control, but fail in complex environmental mixtures. A mixture of water and acetonitrile was chosen as mobile phase for the separation of both pesticide species. Although methanol gives higher signal response when used at high concentration with the particle beam interface, acetonitrile offers considerably less viscosity and allows more favorable column pressure. Moreover, as already discussed in a previous paper,'* the performance of the new interface is only slightly affected by variations in the organic solvent/water mobilephase composition, thus giving homogeneous responses when gradient analyses are performed. As demonstrated by Bellar and co-~orkers,~JO the addition of certain substances to the mobile phase can enhance both the chromatographic perfor1420 Analyticel Chemlstty, Vol. 66, No. 9, May 1, 1994
pestlckles.
mance and overall particle beam carrier process, with evident advantages in sensitivity and response linearity. A 0.1 M solution of ammonium acetate in water was used for the separation of base/neutral pesticides while 0.05% TFA in water and 0.025% TFA in acetonitrile were used for the acidic ones. TFA is also utilized to lower the pH, as required for acidic pesticide separation using C18 reversed-phase chromatograph. A 25-cm-long, 250-pm4.d. packed capillary column was employed for this work. The column is laboratory made using PEEK tubing. This polymer is rigid but not fragile, has great resistance to chemical and physical agents, and comes in a standard l/la-in.-o.d. size. These features can extend the column lifetime and facilitate its connections and the overall use of HPLC capillary columns. The packing method is also rapid, effective, and allows the preparation of very efficient columns. The use of capillary HPLC hardware does not require particular skills or specifically trained personnel. Simple liquid chromatography expertise is often enough to successfully employ microliter liquid flow rates. HPLC gradients were optimized with a conventional UV detector
a
6000.05400.0
--
4800.0 -4200.0 --
3600.03000.0--
2400.0
--
1800.0 -1200.0-r
600.01
/
0 . o w
;
0
5
I
10
15
25
20
compoundi
30
b
"9 Flgure 3. Linear regression of concentration calibration data for chlortokuon. Meanstandard deviationf8.6 % (averageof five injections for each concentration). peak
20
1.6E41.4E4 --
/
1.2E4 -1.OE4--
6000.0--
0.0
0
1a
/
8000.0_-
5
10
15
20
25 30
35
t 1
2
L 3
4
5
6
7
compounds
Figure 5. Instrument detection limits of basic/neutrai (a) and acidic (b) pesticides. The limits are estimated by SIM detection with a signal: noise ratio of 5:l. 40
45
50
55
60
ng
Flgure 4. Linear regression of concentration calibration data for bentazone. Mean standard deviationf9.6% (average of five injections for each Concentration).
fitted with a down-scaled microflow cell. Figure la,b shows the chromatographic profile of a standard mixture of 32 basic/ neutral pesticides obtained at 230 nm with the UV detector (a) and with the PB-MS in the TIC mode (b). The amount injected is 20 ng for each compound in a 60-nL loop volume, with a mobile-phase gradient from 20% to 80% acetonitrile over 45 min. Because of solvent variation delay, 5 min of initial isocratic elution should be also considered in the solvent program. It can be clearly noticed that no appreciable differences in the peak shape or width can be discerned when the two profiles are compared. No band broadening takes place for extracolumn dead volumes. A 50-cm-long, 50-pmi.d. fused-silica tubing connects the capillary column end and acts like a nebulizer tip on the other side. The delay between the injection of the compound and its appearance as signal peak in the mass spectrometer is 2 min at 2 pL/min mobilephase flow rate, without considering its chromatographic retention. All HPLC connections are standard ' / l a in. with PEEK nuts and ferrules. Methomil, propachlor, and propham gave insufficient response with TIC detection. Totally overlapped peaks, like those obtained from aldicarb and metoxuron or carbaryl and fluometuron, are detected separately by selecting only specific ions. Selected ions for SIM
-
°
detection are shown in Table 2. Three different ion programs are used for basic/neutral detection while two programs are used for acidic analysis. High dwell time is used according to the number of ions and chromatographic peak sampling. Figure 2a,b shows the chromatographic separation of a standard mixture of 13 acidic pesticides with UV detector at 225 nm (a) and with the particle beam inteface in the SIM mode (b). The amount injected is 45 ng for each compound in a 60-nL loop volume, with a mobile-phase gradient from 0% to 40% acetonitrile in 5 min and then to 80% in 25 min. TFA buffer was used in the mobile phase as described above. The evident peak tailing shown by most acidic pesticides during mass spectrometric analysis is due mainly to extra chromatographic processes and, in particular, to the thermal decomposition phenomena activated by the electron impact source environment. An extented discussion by Betowsky et al. is given in ref 17. A calibration curve and an evaluation of result reproducibility using the new interface were performed. A welldectected compound for each pesticide species was chosen for the test. A 60-nL loop of a solution containing a given amount of pesticide was injected five times for each concentration. Each sample was introduced without the column directly into the mass spectrometer via the particle beam interface with a mobile phase composed of equal concentrations of water and acetonitrile. Specific buffers were used for basiclneutral and acidic pesticides. The linear regression of concentration calibration data is reported in Figure 3 for chlortoluron and in Figure 4 for bentazone. The mean standard deviations, AnalytcalChemlstry, Vol. 66, No. 9, May 1, 1994
1421
'**
,.
,
CL
30000
1
on
I
II
I
I I
2,,,]o i 0
-0 1"
30
20
40
TIme(min)
10
20
30
T,,"~")
Figure 6. Baslc/neutrai pesticides extracted from 1 L of river water. Ion chromatogram of chlortoluron (1.3 pg/L). Asterisks Indicate the correspondent mlr value for each Ion chromatogram.
Figure 7. Acidic pesticides extracted from 1 L of river water. Ion chromatogram of bromoxynii (4.4 pg/L). Asterisks indlcate the correspondent m/r value for each Ion chromatogram.
calculated using the average of the peak area values for each concentration experiment, are f8.6% and f9.6%, respectively. The mass spectrometer was operating in SIM a t m / z 212 for chlortoluron and m / z 198 for bentazone. The mobile-phase flow rate was kept at 2 pL/min for all experiments. Excellent linearity can be found in all calibration plots due to the beneficial buffer effect on the interface carrier process. In fact, we have noticed that at very high concentrations of organic solvent in the mobile phase, used for basic/neutral pesticide separation (>80%), the lowest concentrations of chlortoluron were poorly detected. Ammonium acetate, which is water soluble, is decreased in concentration in the mobile phase when the aceonitrile component is increased. When over 80% acetonitrile is present in the mobile phase, ammonium acetate concentration is insufficient to positively influence carrier mechanisms. The calibration curve is thus affected, and quantitation results are unsatisfactory. Thegradient employed in this work for basic/neutral separation does not surpass 80% acetonitrile until after 45 min of linear slope, plus 5 min of isocratic delay, when the last compound is eluted in 43 min. This ensures reliable quantitation results for all analytes considered. Trifluoroacetic acid shows characteristics similar to ammonium acetate in its influence on carrier processes. Acidic pesticides are eluted before 25 min when the acetonitrile concentration is well below 8076,and it should be kept in mind that TFA is also dissolved in the organic solvent and that its
concentration in the mobile phase is only slightly affected by solvent composition. Detection limits for the analytes considered in this work were calculated by injecting diluted concentrations of the standard mixture in operative chromatographic conditions. The mass spectrometer was operating in SIM mode according to the ion program shown in Table 2. The lowest amount was determined for a signal-to-noise ratio of 5 1 . Figure 5a,b shows the estimated instrument detection limits for basic/neutral (a) and acidic pesticides (b). Assuming a 100% extraction recovery for all pesticides from a 2-L water sample, with a concentration factor of 2000 and an injection volume of 60 nL, the instrument detection limits correspond to a method detection limit of 0.2-30 ppb. This range satisfies the detection requirements for environmental pollutants in water and wastewater. Actual recoveries for most pesticides are reported in ref 23. A sample was collected from a river near Urbino, and the pesticide contents were determined as shown in in Figures 6 and 7. The river flows through a hilly rural area, with scattered grain, hay, and sunflower cultures. The sample was collected after an early spring thunderstorm. The spectra reported in the figures were obtained from standard solutions and were injected separately. The peak abundances in the ion chromatograms match the relative abundances of the same ions in the mass spectra.
1422 Analytical Chemistty, Vol. 06, No. 9, May I , 1994
The data obtained in this work may contribute to a promising future for liquid chromatography/mass spectrometry-based methods in environmental applications. The particle beam interface with reduced flow rates offers several advantages in terms of sensitivity and performance with complex HPLC gradients for improved separations. Furthermore, the availability of precise retention times and highquality library-searchable mass spectra with linear calibration plots allows unequivocal identification and quantitation of target and unexpected compounds. The new interface can
better withstand stringent instrumentation requirements when routine analyses are employed.
ACKNOWLEDGMENT The authors are particularly indebted to Pierangela Palma and Arnaldo Berloni for having supplied HPLC capillary columns. Received for review August 3, 1993. Accepted February 15, 1994.' 0
Abstract published in Aduonce ACS Abstracts, March 15, 1994.
Ana&ticalChemistry, Vol. 66, No. 9, May 1, 1994
1423
