Anal. Chem. 1994,66, 4444-4449
Determination of Organophosphorus Pesticides in Water by Solid-Phase Extraction Followed by Liquid ChromatographyMigh-Flow Pneumatically Assisted Electrospray Mass Spectrometry C. Molina, M. Honing, and D. BarcelV Department of Environmental Chemistty, ClD-CSlC, c/ Jordi Girona 18-26, 08034 Barcelona, Spain
Priority organophosphoruspesticides in water samples were subjected to analysis by solid-phase extraction followed by liquid chromatography/high-flow pneumatically assisted electrospray mass spectrometry (LC/EsPMS). The operational parameters of ESP optimized by use of an eluent flow rate of 0.3 d m i n and at a source temperature of 150 "C were as follows: dryins gas flow rate, 250-300 Vh; ESP nebulizinggas flow, 5-20 Vh; ESP voltage, 2-3.5 kV; HV lens voltage, 0.2-1.0 kV; extraction voltage, 20-130 V; focus voltage, and the ion energy. Water samples (500 mL) were preconcentrated by use of either Empore disks of CIS and styrenedivinylbenzene or monofunctional CIS cartridges in an ASPEC XL system. No thermal degradation was observed for the organophosphorus pesticide triclorfon, which usually causes a problem under W/MS using thermospray interface. The method detection limit was 0.01 pg/L for most of the organophosphoruspesticides used employing SIM of the [M + Nal+ ion. The analysis of organophosphoruspesticides in water samples is performed with either liquid-liquid extraction or solid-phase extraction (SPE) followed by gas chromatography (GC) with nitrogen-phosphorus or mass spectrometric During the last few years, liquid chromatographic (LC) techniques have grown in this application field due to the possibility of determining thermally labile and polar compounds that are not GC amenable. Since several organophosphorus pesticides, e.g., dichlorvos and trichlorfon, do not exhibit a good chromophore under conventional LC/UV detection? the use of liquid chromatography /mass spectrometry (LC/MS) with a thermospray (EP) interface has been However, still difficulties arise for the determination of organophosphorus pesticides, as pointed in several LC/MS publications; e.g., this is the case for the organophosphorus pesticide (1) Lacorte, S.;Molina, C.; Barcel6, D. Anal. Chim. Acfa 1993,281,71-84, (2) Barcelo , D. j. Chromafogr. 1993,643,117-143. (3) De la Colina, C.; Peiia Heras, A; Dios Cancela, G.; Sanchez-Rasero, F. j. Chromatogr. A 1993,665,127-132. (4) Barcelo, D.Biomed. Environ. Mass Specfrom. 1988,17,363-369. 1430-1434. (5) Betowski, L.D.;Jones, T. L. Enuiron. Sci. Technol. 1988,22, (6) Volmer, D.;F'reiss, A; Levsen, K; Wiinsch, G. j. Chromafogr. 1993,647, 235-259. (7) Volmer, D.;Levsen, IC; Wiinsch, G. j. Chromatogr. A , 1994,660, 231248. (8)Barcelo, D.;Albaiges, J. j. Chromafogr. 1989,474,163-178. (9) Vreeken, R J.; Van Dongen, W. D.; Ghijsen, R T.; Brink", U. A Th. Int. J. Enuiron. Anal. Chem. 1994,54, 119-145.
4444 Analytical Chemistry, Vol. 66,No. 24, December 15, 1994
TSP
(PI) DICH LOR VOS
TRICHLORFON
F:
CH,O-P-CH-CCI, I
1
CH30 OH
HCI
0
CH,O-P-CCCI, CH,O
OH
-
0
CH,O-P-0-CKCCI, CH,O
Flgure I. Degradation pathway of trichlorfon to dichlorvos under LCTTSP-MS.
which is a compound that has been shown to suffer thermal degradation under a TSP process caused by the probe tip and gas-phase temperatures of >200 "C. Trichlorfon is converted to dichlorvos (-16% with a rearrangement and HC1 elimination which was confirmed by isotopic abundance ( F i r e 1).
Another difficulty encountered with many organophosphorus pesticides, e.g. trichlorfon, is determination at the 0.1 pg/L level in water. This has been pointed out in a recent report of the Commission of the European Communities affecting the Drinking Water Directive (CEC-DWD).'O Analytical methods for the determination of few priority organophosphoruspesticides within the CEC-DWD, e.g., trichlorfon, demeton-s-methyl, oxydemeton methyl, and dimethoate, used in amounts higher that 50 tons in the different European Community countries and that are potential leachers to groundwater need to be developed (see Table 1). Recently3recoveries below 5%have been achieved for dimethoate in water samples using SPE followed by GC. Many of the pesticides that have analytical difficulties in water exhibit a high water solubility, e.g., > 10 g/L and as a consequence are not easily trapped in Clstype materials. At present there is no good analytical method that facilitates the extraction and analysis of certain priority organophosphoruspesticides from water samples at the 0.1 pg/L level. The use of LC with electrospray ionization (ESPI)12-14 may facilitate its determination since the method does not provide (10)Fielding, M.; Barcel6, D.; Helweg, A; Galassi, S.; Torstensson, L.; van Zoonen, P.; Wolter, R; Angeletti, G. Pesticiides in Ground and Drinking Water, Water Pollution Research Report 27: Commission of the European Communities: Brussels, Belgium, 1992; p 1-136. (11) Wauchope, R D.;Buttler, T. M.; Homsby, A G.; Augustijn-Beckers, P. W. M.; Burt, J. P. Rev. Enuiron. Contam. Tmicol. 1992,123,1-156. (12) Voyksner, R D.Enuiron. Sci. Technol. 1994,28,118A-127A (13) Voykner, R D.;Pack, T. Rapid Commun. Mass Specfrom. 1991,5, 263268. (14) Ikonomou, M. G.;Blades, A T.; Kebarle, P. Anal. Chem. 1990,62, 957967. 0003-2700/94/0366-4444$04.50/0 Q 1994 American Chemical Society
Table 1. Priority Organophosphorus Pesticideswithin CEC-DWD in Europe in Amounts over 50-500 Tons per Annum Classified as Probable or Transient Leachers'
chlorpyrifos diazinon dimethoate
demeton-s-methylb
fenamiphos
ethoprophos
oxydemeton-methyl trichlo$on
a Priority organophosphorus pesticides for which no satisfactory analytical method appears to exist at 0.1 pg/L are in italics. Organophosphorus pesticides in amounts over 50 tons per annum for which there was insufficient data to evaluate probability of leaching and for which no suitable analytical method at 0.1 pg/L appears to exist.
I thermal degradation of the compounds and it has been reported to be more sensitive than TSP by factor of -10O.l2 LC/ESP-MS has been scarcely used in environmental analysis, as pointed out recently by Budde in the ASMS conference in Chicago.15 From the 54 presentations of LC/MS in environmental analysis, only 5 use ESP, with atmospheric pressure chemical ionization (APCI) being the most popular LC/MS technique. Recently, high-flow ESP has been developed and is commercially available. It has an advantage over conventional ESP in that it does not require working at low flow rates (10 pL/min) that need either split-when using conventional LC pumps-or syringe LC pumps, at the cost of time- and extra money. In addition, the ideal situation of an environmental laboratory would be to perform the same analysis under conventional LC/W or LC diode array conditions using LC/MS. In the present paper we have used-high flow LC/ESP-MS for the determination of thermally labile and polar organophosphorus pesticides in water samples at the 0.1 pg/L level. The aim of the present paper will be achieved by (i) the optimization of the different ESP parameters (extraction voltage, ESP voltage, etc.) in high-flow LC/ESP-MS and (ii) the combination of various SPE approaches (Empore disks and automated ASPEC XL with cartridges) followed by LC/ESP-MS.
Analyser f
$.
Turbo
Aecula:
PUmpS
I +
Rotary
P?llllpS
-4
I
Exhaust (Black)
4
Drying Gas (White)
II
3.
Nebuliser Gas (Red)
Figure 2. Coaxial high-flow pneumatically assisted ESP source for VG Platform.
Chemicals. HPLC-grade water and methanol were obtained from J. T. Baker (Deventer, The Netherlands) and were passed through a 0.45pm filter before use. The organophosphorus pesticides were purchased from Promochem (Wesel, Germany). Liquid Chromatography/Mass Spectrometry. The eluent was delivered by a gradient system from Waters 616 pumps controlled by Waters 600s Controller from Waters-Millipore (Milford, MA). The LC eluent conditions varied from 28:72 (15 min isocratic conditions) to 60:40 methanol-water in 32 min at 0.3 mL/min. The column was 150 mm x 2.1 mm id., packed with 5pm particles from Zorbax, Rockland Technologies Inc. (Nuenen, The Netherlands) coated with a cyanopropyl stationary phase. This gradient LC system was connected to a VG Platform ESP from Fisons Instruments (Manchester, U.K.) equipped with a Megaflow ESP probe. The design of this ESP consists of a coaxial flow probe (see Figure 2). After the LC separation,the sample is introduced into the ESP source together with a nebulizing gas, which flows directly through the probe tip, maximizing the
efficiency of the nebulization. A drying gas is added to flush out any solvent that may have entered the gas line by capillary action. There also exists the possibility of a triaxial flow probe that incorporates a sheath tube that allows additional solvent to be transported to the probe tip and mixed co-axially with the sample flow immediately before spraying. This is particularly beneficial when one attempts to spray with mobile phases approaching 100% water, which is often required in gradient elution LC/MS. In this case, the organic solvent can be added triaxially just before entering the ESP source, which will be beneficial for the enhancement of response since ESP ionization is more sensitive when more modifier is used.16 This will also be shown in Figure 4 of this paper. The instrument control and data processing utilities included the use of the MassLynx application software installed in a Digital DEC PC 466. The high-flow pneumatically assisted ESP using a VG Platform instrument has been used at a flow rate of 0.3 mL/min and at a source temperature of 150 "C. This parameter was not changed, although we have performed a few experiments (not reported here) that showed a substantial decrease in sensitivity when the flow rate increased to 0.5-0.6 mL/min. We found 0.3 mL/min was an acceptable flow rate for our experiments since it permits use of 2.1-mm-i.d. LC columns and conventional LC pumps without any specific restriction. Sample Preparation. The method involving the Empore disks [CISor styrene-divinylbenzene (SDB) ] has been described previous1y.l A standard Millipore 47-mm filtration apparatus was used. The membrane extraction disks were manufactured by 3M (St. Paul, MN) under the trademark Empore and are distributed by J. T. Baker (Deventer, The Netherlands). The disks used in these experiments were 47 mm in diameter and 0.5 mm thick. Each disk contains about -500 mg of CISbonded silica or SDB. The extraction procedure used is as follows: After spiking 500 mL of water with different pesticides, resulting in an analyte
(15) Budde, W. L. Proceedings, 42nd ASMS Conference on Mass Spectrometry and Allied Topics; Chicago, IL, May 29-June 3,1994; p 872.
(16) Ikonomou, M. G.; Blades, A. T.; Kebarle, P. Anal. Chem. 1991,63,19891998.
EXPERIMENTAL SECTION
Analytical Chemistry, Vol. 66, No. 24, December 15, 7994
4445
analyte
calibration equation
oxydimeton-methyl trichlorfon dimethoate dichlorvos demeton-s-methyl fenamiphos sulfoxide fenitrooxon fenamiphos sulfone fenamiphos
Y = 6544X + 531755 Y = 1087X + 80914 Y = 2076X Y = 2098X Y= 5 8 8 s Y = 2386X Y = 1606X Y = 2127X Y = 4839X
+ 305343 + 54824 + 859013 + 77674 + 3423 + 100906 + 1130145
+ 7
Table 2. Calibration Data for Organophosphorus Pesticldes from 0.1 to 60 ng (0.01 2-6 &I.).
R2
LOD @g)
0.998 0.998 0.999 0.999 0.997 0.999 0.999 0.998 0.998
100 30 60 20 100 200 100 10
10
E
*
3
‘f 3t-
4
g 2
I---
-
a Exceptions: oxydemeton methyl, demeton-s-methyl,dichlorvos, and fenamiphos were linear from 0.02 to 60 ng. Calibration was performed by plotting peak intensity area (Y)versus amount injected ar).
concentration of 0.1 pg/L, the solution was prefiltered using 0.45 pm PTFE fiberglass lilters (Millipore Corp. Bedford, MA) to eliminate particulate matter. The disk, placed in the conventional Millipore apparatus, was washed with 2 x 10 mL of methanol with the vacuum on. The disk was not allowed to become dry, and immediately 500 mL of water was extracted with the vacuum adjusted to yield a 20-min extraction time. After this operation, the pesticides trapped on the disk were collected with the 2 x 10 mL of methanol. After careful evaporation of part of the solvent, methanol was added up to a volume of 500 and 10 p L was injected onto the LC/ESP-MS, using SIM conditions. An automatic sample preparation with extraction column (ASPEC) XL, fitted with an extemal 306 LC pump for the dispensing of samples through the SPE cartridges and with a 817 switching valve for the selection of samples, was a gift from Gilson (Viers-leBel, France). Disposable 6mL cartridge columns from ISOLUTE International Sorbent Technology (Hengoed, Mid Glamorgan, U.K.) packed with 1 g of monofunctional CU bonded silica were used. An optimum flow rate of 20 mL/min was used (at 40 mL/min we have noticed losses in certain compounds). Quantitation. External calibration was used with the quantitation of the extract after SPE with an standard. The system was linear in most of the cases using seven to nine points from 0.1 to 60 ng (0.012, 0.025, 0.060, 0.12, 0.61, 1.23, and 6 pg/L). The calibration equations for the different pesticides analyzed are shown in Table 2. An example of the linearity of the system for trichlorfon is shown in Figure 3. The quantitation of the water extracts was achieved by using timescheduled selected ion monitoring using [M Na]+ ion for each organophosphorus pesticide (see Table 3). The LODs were calculated by using a signal-to-noiseratio of 3-6 (the ratio between the peak intensity with SIM conditions and intensity of the noise was used). For the studied organophosphorus pesticides, LODs varied from 10 to 100 pg, depending on the compound, and are shown in Table
Table 4. Optimization of the Different Operating Parameters under High-Flow Pneumatically Assisted ESP
flow rate (for CH30H-HzO (5050) (mL/min) drying gas flow rate (L/h) ESP nebulizing gas flow (l/h) ESP voltage (kV) HV lens voltage (kV) extraction voltage (V) focus voltage (V) source temp (“C)
ion energy (V)
0.3 250-300 10 (5-20) 3.1 (2-3.5) 0.3 (0.2-1.0) 20,40 (20- 130) 27,47 150 0.9 (-5, +5)
Numbers in parentheses in column 2 are the optimized range.
+
2. RESULTS AND DISCUSSION
Optimization of ESP parameters. The different parameters optimized for the characterization of organophosphoruspesticides are shown in Table 4. The eluent clusters were also investigated under ESP conditions using an eluent of methanol-water (50 50). Major cluster ions were found at m / z 33, 55, 65, 87,97, and 119 corresponding to [CHsOH HI+, [CHsOH + Nal+, [2CHr OH + HI+, t2CH3OH + Nal+, [3CH30H + HI+, and [~CHBOH
+
+
4446 Analpica1 Chemistry, Vol. 66, No. 24, December 15, 7994
Nal+. These clusters can reach fi = 6 (m/z215) and are generally used for the ESP calibration of the instrument reaching a m / z value of 215. This is a better calibrant than a poly(ethy1ene glycol) (PEG) 300600:1000:1500mixture since when compounds like organophosphorus pesticides are analyzed, it permits one to achieve a more accurate m / z characterization of the different fragment ions at low m / z values. Trichlorfon showed no degradation under ESP conditions. In Table 5, the mass spectral values of trichlorfon and other organophosphorus pesticides using extraction voltages of 20 and 40 V are reported. Generally an increased fragmentation is observed when working at 40 V, with the exceptions of fenamiphos and its metabolites. [M Na]+ ion is always obtained as the base peak at 20 V. The Na+ ions are due to the sodium ions present as an impurity in the methanol solution, and it means that more
+
Table 5. Important Mass Spectral Fragments, Relative Intensities of OrganophosphorusPesticides Using an LC Eluent of Methanol-Water (50:50),and Extractlon Voltages of 20 and 40 V under ESP-MS
Mw
m/z
246
164 169 191 269
256
229
220 230
109 133 279 125 230 252 109 243 89 109 169 253
319
320 342 358
261
109 262 284
335
358
303
304 326
compounds and ions tentative identifn oxydemeton-methyl [(CH30)zPOS + Nal+ [ (CH30)zPOSCzH41+ [CzHsSOCzH&l+ [M + Na]+ trichlorfon [ (CH30)zPOl+ [(CH30)zPOH + Na]+ [M + Na]+ dimethoate [(CH30)zPSI+ [M [M
+ HI+ + Na]+
dichlorvos [(CH30)zPOl+ [M + Na]+ demeton-s-methyl [CzHsSCzH41+ [(CH30)zPOl+ (CH30)zPOSCzH41+ [M + Na]+ fenamiphos sulfoxide [M + HI+ [M Na]' [M Kl+
+ +
fenitrooxoi [(CH30)zPOl+ [M + HI+ [M + Nal+
fenamiphos sulfone [M + Na]+ fenamiphos [M [M
+ HI+ + Na]+
re1 intens 2OV
40V
100
30 19 34 100
22 100
50 89 100
3
6
I
i2
15
RETENTION TIME (MIN.)
28 7 100
100
100
55 100
24 20 100
100 20 10 50
11 100 41
10 100 20
38
100
100 21 21
100
100
11 100
9 100
than 90% of the observed ions are due to ions present in solution.14 At the highest extraction voltage, a slight loss in sensitivity was observed, so further experiments regarding sensitivity and optimization of the SPE of organophosphorus pesticides from water samples were performed at 20 V. The fragments ions shown in Table 5 correspond to typical ions of organophosphorus pesticides together with the molecular weight information, generally with [M Nal+ ion as the base peak. The fragmentation found for most of the organophosphorus pesticides investigated agrees with previous papers.1~4-~ The first thiig to notice is that no dichlorvos is formed from trichlorfon under ESP conditions, which is already an important point of the analysis. This can be seen by the fact that no fragment ions corresponding to HCl losses are observed in the trichlorfon spectrum. That means that no degradation of trichlorfon occurs under the ESP conditions reported in this paper and represents an improvement as regards to previous work using TSP interface.4-6 A remarkable fact from the data observed in Table 5 is that typical diagnostic ions of organophosphorus pesticides under electron impact ionization are observed in the data pre~ented.',~ This corresponds to diagnostic ions of m / z 109,110,125,and 141 or to adducts of these diagnostic ions with Na+, such as m / z 133 and 164. This a very important feature of ESP since it is a technique that can be used for identification purposes of unknown pesticides, contrary to most of the TSP that usually gives very
+
0
RETENTION TIME
(MIN.)
Figure 4. Effect of the water percentage on the response of oxydemeton-methylin LC/ESP-MS. Water-methanol, (A) 90:10, (B) 5050,and (C) 10:90.Successive injections of the samples using a mobile phase flow rate of 0.3 mumin and a LC column of cyanopropyl (see Experimental Section). Retention time of the oxydemeton-methyl in (A-C) was 2.5, 1.2, and 1 min, respectively.
poor structural information and consequently it is used for confirmation purposes of target analytes. Another point to consider from the data of Table 5 is that at 40 V more structural information is obtained as compared to 20 V of extraction voltage, and consequently, this higher voltage will be recommended when identification of unknown pesticides in environmental samples is required. The repeatability and the long-term reproducibility (n = 9) for the organophosphoruspesticides varied from 12-17% to 22-30%, respectively, when 1 ng of each compound was injected. Such values can be considered as good values since usually repeatability in LC/TSP-MSis performed by injecting higher amounts, e.g., 0.2 pg. Recently it has been reported that for TSP the reproducibility often exceeds 2O%,' which is a value that is in the range of the values obtained by us using ESP. The effect of the water percentage on the ESP response was also studied. It has been observed that using 50%and 10%water in the LC eluent the response was enhanced by a factor of 2 and 3.5, respectively as compared to 90%water. Figure 4 shows the differences in sensitivity by this effect when oxydemeton-methyl was analyzed. The use of low water percentages under ESP conditions favors the response in contrast to TSP.8These values are of importance for knowledge of the mechanism of LC/ESPMS. It has been reportedI6that the percentage of methanol has Analytical Chemistry, Vol. 66, No. 24, December 15, 1994
4447
01 0
I
,
5
10
r 20
15
RETENTION TIME
25
30
35
(MIN.)
Figure 5. LC/ESP-MS SIM chromatogram obtained after preconcentration of 500 mL of water spiked at 0.1 pg/L. For compound numbers, see Table 6.
Table 0. R-overies (%) with Empore Disks C18 and SOB and 1-g (6 mL) Cartrldges (ASPEC XL) MF CIS Using SO0 mL of Water Spiked at 0.1 p a a
no.
Empore disks ion compounds (d~) Cis SDB oxy dimeton-methyl 269 70 60 trichlorfon 279 30 53 252 42 87 dimethoate 243 12 14 dichlorvos demeton-s-methyl 253 93 85 fenamiphos sulfoxide 342 96 95 fenitrooxon 284 93 98 358 96 93 fenamiphos sulfone 326 78 94 fenamiphos
AspEc xL MFCis 95 23 58 28 78 106 88 96 70
a Coefficient of variation (n = 6) varied between 10%and 15%except for trichlorfon and dichlorvos (20%and 27%,respectively). The ions monitored under LC/ESP-MS usingSIM and 20 Vas extraction voltage are indicated and correspond to [M + Na]+.
a high innuence on the signal of conventionalESP. The sensitivity and signal stability decreased under ESP by a factor of 10 when the methanol percentage was decreased from 75%to lo%,which is a greater difference that we have obsemed in the high-flow ESP. This would be also the case for high-flow ESP used here and leads to the conclusions that the system used in this work has similarities in performance to ion spray, also called pneumatically assisted ESP but using a lower flow rate of -50 pL/min. This can probably be attributed to a relatively minor change in droplet size for pneumatically assisted ESP techniques such as ion spray and high-flow ESP. Off-LineSPE Followed by LC/ESP-MSfor tfie Determination of Organophosphorus Pesticides in Water. A typical chromatogram of a water extract after preconcentration on CIS Empore disks is shown in Figure 5. Table 6 shows the recovery values obtained for a variety of organophosphorus pesticides spiked at the 0.1 pg/L level using various sorbent materials: Empore disks of CIS and styrenedivinylbenzene and cartridges containing 1g of monofunctional cl8.
The determination at the 0.1pg/L level in water of oxydemeton-methyl, demeton-s-methyl , dimethoate, and fenamiphos was solved with recovery values higher than 70%. This is an improve4448 Analyfical Chemistry, Vol. 66, No. 24, December 15, 1994
ment in regard to previous data33 because these priority pesticides posed a problem in water since no satisfactory analytical method appeared to exist at this LOD. For trichlorfon, a recovery up to 53%is noted, which can help its detection in water samples. Trichlorfon degrades under GC and LC/TSP-MS conditions to dichlorvos, as mentioned, and also has a poor chromophore, in a way similar to dichlorvos-with a maximum of
