Anal. Chem. 1997, 69, 2819-2826
Trace Analysis of Sulfonylurea Herbicides in Water: Extraction and Purification by a Carbograph 4 Cartridge, Followed by Liquid Chromatography with UV Detection, and Confirmatory Analysis by an Electrospray/Mass Detector Antonio Di Corcia,* Carlo Crescenzi, Roberto Samperi, and Luisa Scappaticcio
Dipartimento di Chimica, Universita` “La Sapienza” di Roma, Piazza Aldo Moro 5, 00185 Roma, Italy
Seven commonly used sulfonylureas (SUs), i.e., thifensulfuron methyl, metsulfuron methyl, triasulfuron, chlorsulfuron, rimsulfuron, tribenuron methyl, and bensulfuron methyl, were extracted from water by off-line solidphase extraction with a Carbograph 4 cartridge. SUs were then isolated from both humic acids and neutral contaminants by differential elution. Analyte fractionation and quantification were performed by liquid chromatography (LC) with UV detection. Recoveries of SUs extracted from 4 L of drinking water (10 ng/L spike level), 2 L of groundwater (50 ng/L spike level), and 0.2 L of river water sample (250 ng/L spike level) were not lower than 94%. Depending on the particular SU, method detection limits were 0.6-2 ng/L in drinking water, 2-9 ng/L in groundwater, and 13-40 ng/L in river water. A preservation study of SUs stored on the Carbograph 4 cartridge was conducted. Over 2 weeks of cartridge storage, no significant analyte loss was observed when the cartridge was kept frozen as a precaution. Comparing this method with one using a C-18 extraction cartridge, the former appeared to be superior to the latter in terms of sensitivity and, chiefly, of selectivity. This method involves confirmatory analysis by LC-electrospray/mass spectrometry (MS) instrumentation equipped with a single-quadrupole mass filter. MS data acquisition was performed by a timescheduled three-ion selected ion monitoring (SIM) program. The necessary structure-significant fragment ions were obtained by controlled decomposition of SU adduct ions after suitably adjusting the electrical field in the desolvation chamber. Under three-ion SIM condition, limits of detection (S/N ) 3) calculated from the ion current profiles of those fragment or parent ions giving the lowest S/N values ranged between 0.5 (tribenuron methyl) and 3 ng (metsulfuron methyl, thifensulfuron methyl) injected into the LC column. Sulfonylureas (SUs) form a class of herbicides introduced in the 1980s. From a chemical point of view, SUs are labile and weakly acidic compounds. Compared to older herbicides, SUs have much lower use rates and are more rapidly degraded in soil. When present, very low concentrations (low parts-per-trillion region) of these herbicides in environmental waters are then to S0003-2700(96)01164-X CCC: $14.00
© 1997 American Chemical Society
be expected. For this reason and because of the chemical characteristics cited above, monitoring of these herbicides in water is a particularly challenging problem. Although various separation techniques, such as gas chromatography/mass spectrometry (MS) of SU derivatives,1 supercritical fluid chromatography,2 and capillary electrophoresis,3,4 have been proposed to analyze SUs in various matrices, liquid chromatography (LC) is the technique of choice.5-10 LC methods have become even more attractive since the introduction of robust and sensitive devices, such as thermospray (TS) and electrospray (ES), to interface LC to MS. A LC-TS/MS method was developed for analyzing residues of two SUs in soil.11 Volmer et al.12 evaluated the performances of both TS and ES ion sources for determining trace levels of SUs in water. In terms of specificity, they concluded that the latter source, in combination with tandem MS, was superior to the former one. Recently, Henion and co-workers reported that eight SUs in soil could be quantified at 50 ng/kg levels by LC-ES/ MS/MS instrumentation.13 Today, however, LC/MS instrumentation is rarely available in an environmental laboratory. Therefore, sufficiently selective and very sensitive analytical procedures based on inexpensive instrumentation, such as LC with UV detection, are highly desirable for routinely monitoring SUs in water. Eventually, definitive confirmation of tentatively detected SUs could be achieved by sending the extract to a central laboratory equipped with LC/MS instrumentation. Graphitized carbon blacks (GCBs), commercially referred to as Envicarb or Carbograph 1, have been shown to be valuable sorbent materials for solid-phase extraction (SPE) of a variety of (1) Klaffenbach, P.; Holland, P. T. J. Agric. Food Chem. 1993, 41, 388-395. (2) Berger, A. Chromatographia 1995, 41, 133-140. (3) Garcia, F.; Henion, J. J. Chromatogr. 1992, 606, 237-247. (4) Dinelli, G.; Vicari, A.; Bonetti, A. J. Chromatogr. 1995, 700, 195-200. (5) Raiser, R. W.; Barefoot, A. C.; Dietrich, R. F.; Fogiel, A. J.; Johnson, W. R.; Scott, M. T. J. Chromatogr. 1991, 554, 91-101. (6) Howard, A. L.; Taylor, L. T. J. Chromatogr. Sci. 1992, 30, 374-382. (7) Schneider, G. E.; Koeppe, M. K.; Naidu, M. V.; Horne, P.; Brown, A. M.; Mucha, C. F. J. Agric. Food Chem. 1993, 41, 2404-2410. (8) Nilve´, G.; Knutsson, M.; Jo¨nsson, J. A° . J. Chromatogr. 1994, 688, 75-82. (9) Galletti, G. C.; Bonetti, A.; Dinelli, G. J. Chromatogr. 1995, 692, 27-37. (10) Cambon, J. P.; Bastide, J. J. Agric. Food Chem. 1996, 41, 333-337. (11) Shalaby, L. M.; Bramble, F. Q., Jr.; Lee, P. W. J. Agric. Food Chem. 1992, 40, 513-517. (12) Volmer, D.; Wilkes, J. G.; Levsen, K. Rapid Commun. Mass Spectrom. 1995, 9, 767-771. (13) Li, L. Y. T.; Campbell, D. A.; Bennet, P. K.; Henion, J. Anal. Chem. 1996, 68, 3397-3404.
Analytical Chemistry, Vol. 69, No. 14, July 15, 1997 2819
Figure 1. Structures and common names of seven sulfonylureas.
pollutants in water. Carbograph 4 is a new example of GCB which has proved to be remarkably more efficient than Carbograph 1 in extracting polar analytes from large water volumes.14 The uniqueness of the GCB sorbent family is that isolation of acidic compounds from coextracted neutral ones can be easily achieved by differential elution.15-17 When analyzing acidic compounds in complex aqueous environmental matrices, this class fractionation offers the advantage of making the analysis rather selective, as most of the potentially interfering compounds are neutral in nature. This paper describes a method for analyzing traces of seven SUs in natural waters. This method is based on extraction and analyte purification by a single Carbograph 4 SPE cartridge, followed by screening LC/UV analysis. A confirmatory analytical procedure using LC-ES/MS benchtop equipment is also described. EXPERIMENTAL SECTION Reagents and Chemicals. Common names and structures of the seven SUs considered are shown in Figure 1. Triasulfuron was from Alltech (Sedriano, Italy), while the other SUs were kindly supplied by E. I. du Pont de Nemours (Wilmington, DE). Individual standard solutions were prepared by dissolving 20 mg (14) Crescenzi, C.; Di Corcia, A.; Passariello, G. M.; Samperi, R.; Turnes Carou, M. I. J. Chromatogr. 1996, 733, 41-55. (15) Di Corcia, A.; Marchetti, M. Environ. Sci. Technol. 1992, 26, 68-74. (16) Di Corcia, A.; Marchese, S.; Samperi, R. J. Chromatogr. 1993, 642, 163174. (17) Di Corcia, A.; Marcomini, A.; Samperi, R. Environ. Sci. Technol. 1994, 28, 850-858.
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Analytical Chemistry, Vol. 69, No. 14, July 15, 1997
of standard in 20 mL of acetonitrile. Composite working standard solutions were prepared by suitably mixing standard solutions mentioned above. When unused, all standard solutions were stored at 4 °C. Humic acid sodium salts were purchased from Aldrich (Milwaukee, WI). A basified humic acid solution with a dissolved organic carbon (DOC) concentration of 250 mg /L was prepared as reported elsewhere.18 A C10-C13 mixture of linear alkylbenzene sulfonates (LAS) was supplied by Chemische Werke Hu¨ls AG (Marl, Germany). A 2 mg/L stock solution was prepared by dissolving LAS in methanol. For LC, distilled water was further purified by passing it through the Milli-Q Plus apparatus (Millipore, Bedford, MA). Acetonitrile “Plus” of gradient grade was obtained from Carlo Erba (Milano, Italy). When using the LC-ES/MS instrumentation, undesired inorganic ion impurities were removed from acetonitrile by in-glass distillation. Other solvents were of analytical grade (Carlo Erba) and were used as supplied. Trifluoroacetic acid (TFA) was from Aldrich. Apparatus. Extraction cartridges filled with 0.5 g of Carbograph 4 (surface area, 210 m2/g; 120-400 mesh size, Carbochimica Romana, Rome, Italy) were prepared as previously reported.19 The Carbograph 4 cartridge was fitted into a sidearm filtration flask, and liquids were forced to pass through the cartridge by vacuum (water pump). Before water samples were processed, the cartridge was washed with 8 mL of the eluent phase for the analytes (see below), followed by 2 mL of methanol and 20 mL of acidified water (pH 2). Aqueous Samples. Grab samples of river waters and groundwaters were collected in brown bottles and kept at 4 °C in the dark until analysis. Environmental samples were analyzed after not more than a few days of storage, after spiking them with the analytes. Unless they contained suspended materials able to plug the SPE cartridge, such as algae and debris, river water samples were extracted unfiltered (although with restricted flow rates). Drinking water samples were collected from the tap in the laboratory. To eliminate sediments and gas pockets in the pipes, this water was collected after flushing for about 10 min. Before spiking with the analytes, hypochlorite in drinking water samples was eliminated by addition of Na2S2O3‚5H2O, 0.5 g/L. Groundwater samples were obtained from wells located near Rome. Representative samples were obtained after purging 3-10 well volumes to eliminate stagnant water. For recovery studies, a simulated surface water sample was also used. This sample was prepared by adding to distilled water humic acids from the standard solution to create a DOC concentration of 20 mg/L and 0.3 mg/L of the C10-C13 LAS mixture, and then decreasing the pH of this solution to about 8 with diluted HCl. Besides humic acids, LAS surfactants were added to this solution as they are ubiquitous compounds invariably present in real surface waters. Procedure. For recovery studies, aqueous samples were fortified with known amounts of the composite standard solution. Water samples were then agitated firmly for about 1 min and, after 3-4 min, poured in a glass reservoir connected to the sorbent cartridge. Water was forced to pass through the cartridge at flow rates of 70-100 mL/min by reducing the pressure in the vacuum apparatus to the minimum. After the sample was passed through (18) Senseman, S. A.; Lavy, T. L.; Mattice, J. D.; Gbur, E. E. Environ. Sci. Technol. 1995, 29, 2467-2653. (19) Di Corcia, A.; Bellioni, A.; Madbouly, M. D.; Marchese, S. J. Chromatogr. 1996, 733, 383-393.
the trap, the pump was disconnected and the cartridge filled with 20 mL of distilled water, which was allowed to pass through the cartridge at flow rates no higher than 10 mL/min by restoring vacuum. Any void space created by some shrinking of the sorbent bed occurring during the passage of the sample was eliminated by pushing the upper frit against the sorbent bed. Most water was removed from the cartridge by forcing room air through it for 1 min. After the pump was disconnected, neutral compounds coextracted with SUs were washed away by passing through the cartridge 2 mL of methanol, followed by 12 mL of a methylene chloride/methanol solution (80:20 v/v), at a flow rate of ∼8 mL/ min obtained by suitably regulating the vacuum. Thereafter, a suitably drilled cylindrical Teflon piston with one conically indented base and a Luer tip was forced into the cartridge until it reached the upper frit.19 The trap was turned upside-down, a 1.4 cm i.d. glass vial with a conical bottom was placed below it, and analytes were back-eluted by passing through the trap 8 mL of a methylene chloride/methanol (80:20 v/v) solution acidified with acetic acid, 10 mmol/L. The flow rate at which the eluent phase was percolated through the cartridge was ∼6 mL/min, obtained by suitably regulating the vacuum in the apparatus. The last drops of this solvent mixture were collected by a further decrease in the pressure inside the flask. The extract was dried in a water bath at 40 °C under a nitrogen stream. Precaution was taken to not allow the residue-containing vial to stay in the water bath for more than a few minutes after solvents appeared to be completely removed. The residue was reconstituted with 200 µL of a water/ acetonitrile solution (80:20 v/v) acidified with acetic acid, 2 mmol/ L. Then, 50 µL of the final extract was injected into the LC column. LC/UV Analysis. Liquid chromatography was carried out with a Varian (Walnut Creek, CA) Model 5000 instrument equipped with a Rheodyne Model 7125 injector having a 50 µL loop. The analytes were chromatographed on an Alltima 25 cm × 4.6 mm i.d. column filled with 5 µm C-18 reversed-phase packing (Alltech). For fractionating the analytes, phase A was acetonitrile and phase B was water. Both solvents contained TFA, 3 mmol/ L. The initial composition of the mobile phase was 32% A, increased linearly to 62% in 40 min. The flow rate of the mobile phase was 1 mL/min. The UV detector was set at 230 nm wavelength. For recovery studies, the concentrations of the analytes were calculated by measuring peak areas and comparing them with those obtained from standard solutions. These were prepared by dissolving known and appropriate volumes of the working standard solution in the eluent phase used for eluting analytes from the Carbograph 4 cartridge and then following the rest of the procedure reported above. Data were acquired by using Chrom Card software from Fisons Instruments/VG BioTech (Milano, Italy). LC-ES/MS Analysis. Liquid chromatography was carried out with a Varian Model 9010 instrument equipped with a Rheodyne Model 7125 injector having a 50 µL loop. The same type of column as reported above was used. For fractionating the analytes, phase A was again acetonitrile and phase B was water. Both solvents contained TFA, 40 µmol/L. The initial composition of the mobile phase was 30% A, increased linearly to 44% in 15 min and then to 70% in 7 min. Gases in both solvents were removed by sparging with helium. The flow rate of the mobile phase was 1 mL/min, and 40 µL/min of the column
Table 1. Time-Scheduled SIM Conditions for Monitoring Seven Sulfonylureas in Water
compound thifensulfuron methyl metsulfuron methyl chlorsulfuron triasulfuron rimsulfuron bensulfuron methyl tribenuron methyl a
channel mass, abundance)
m/za (relative
retention window, min
cone voltage, V
167 (100), 205 (10), 388 (30)
13-14.2
30
167 (100), 264 (10), 382 (30)
14.2-15.3
30
141 (100), 167 (50), 358 (30) 167 (100), 219 (20), 403 (50) 182 (100), 326 (40), 432 (50) 149 (80), 182 (60), 411 (100)
15.3-16.8 16.8-19 16.8-19 22-25
30 35 35 30
155 (100), 364 (15), 396 (20)
22-25
30
Intact protonated adduct ions are in italics.
effluent was diverted to the ES source. A Fisons VG Platform benchtop mass spectrometer consisting of a pneumatically assisted ES interface and a single quadrupole was used for detecting and quantifying target compounds in the LC column effluent. This was introduced into the ES interface through a 40 cm length of 75 µm diameter PEEK capillary tube. The MS was operated in the positive ion mode by applying to the capillary a voltage of 4 kV. The source temperature was maintained at 70 °C. Under this condition, no thermal degradation of the targeted SUs was observed. Ions were generated using highly pure nitrogen as drying and nebulizing gases at flow rates of 230 L/h and 13 mL/ min, respectively. LC/MS chromatograms were obtained by operating in the time-scheduled three-ion selected ion monitoring (SIM) acquisition mode, under the conditions reported in Table 1. Fragment ions were obtained by controlling the potential difference between sample and skimmer cones. This provides structurally significant fragment ions in the intermediate ion transport region by collision-induced dissociation (CID) of protonated adduct ions with background gas. On a weekly basis, the counter electrode and the sample cone were cleaned as previously reported.20 The mass spectrometry data handling system used was Mass Lynx software from Fisons. RESULTS AND DISCUSSION Matrix Effect. The presence of some active centers bearing a positive charge enables GCBs to behave as both nonspecific and anion exchange sorbents. It follows that anionic organic compounds are specifically adsorbed on the GCB surface via electrostatic forces, and they can be desorbed only by adding a displacing agent to an organic solution. The exchange capacity of Carbograph 4 is far lower than that of conventional exchangers.14 When extracting acidic analytes from relatively large volumes of surface waters rich in humic acids and other acidic species, such as ubiquitous LAS surfactants, it may occur that the above active centers are saturated. In this situation, and depending on their acidity, acidic analytes are to a greater or lesser extent adsorbed also on the nonspecific sorbent sites. This fraction is then washed away, together with nonacidic species, when the neutral eluent phase designed to purify the extract is passed through the GCB cartridge. (20) Crescenzi, C.; Di Corcia, A.; Marchese, S.; Samperi, R. Anal. Chem. 1995, 67, 1968-1975.
Analytical Chemistry, Vol. 69, No. 14, July 15, 1997
2821
Table 2. Recovery (%)a of Seven Sulfonylureas on Extracting Them from Increasing Volumes of a Simulated Surface Water (Spike Level, 4 µg/L) volume, L 0.2 compound thifensulfuron methyl metsulfuron methyl triasulfuron chlorsulfuron rimsulfuron bensulfuron methyl tribenuron methyl a
0.4
0.8
washing eluent washing eluent washing eluent phase phase phase phase phase phase ndb
99
nd
97
10
87
nd
99
nd
98
7
90
nd nd nd nd
96 98 96 97
nd nd nd 4
96 97 95 93
54 7 10 39
42 91 86 57
nd
96
53
43
90
7
Mean values from triplicate measurements. b nd, not detected.
The maximum volume of surface water which could be passed through the cartridge without loss of SUs in the neutral organic effluent was assessed. This investigation was conducted by using a simulated surface water sample (see Experimental Section) instead of a real one. This choice was based on the obvious consideration that no surface water taken at random can be regarded as a model matrix. Surface water samples usually contain concentrations of humic acids significantly lower than 20 mg/L (measured as DOC), and a LAS content of 0.3 mg/L is rarely found in surface waters, provided sampling is accomplished in sites sufficiently distant from raw domestic sewage discharges. Therefore, the simulated river water used in this experiment represents an anomalous aqueous sample which may eventually be encountered while monitoring SUs in surface waters. After spiking with the analytes at the level of 2 µg/L, varying volumes of the simulated surface waters were analyzed in triplicate. Results are reported in Table 2. As expected, the least acidic SUs, i.e., tribenuron methyl, bensulfuron methyl, and triasulfuron, were the most prone to displacement from positively charged sorbent sites by both humic acids and LAS. Anyway, any partial loss of the three above analytes occurring during the purification step should be minimized by submitting no more than 0.2 L of a real surface water sample to the extraction procedure. By taking this precaution, no significant loss of the seven SUs was observed when extracting them from several river water and lake water samples taken between Rome and Florence. Initially, according to a previous work,15 SUs were eluted from the Carbograph 4 cartridge by acidifying the eluent phase with a strong acid, such as TFA. When the simulated surface water samples spiked with the analytes were analyzed, a large fraction of humic acids were coeluted with SUs and produced a huge and tailed chromatographic peak which made correct analyte quantification difficult (Figure 2 A). GCBs behave like conventional exchangers in that stepwise elution of acidic compounds can be obtained on the basis of their acidic strengths by properly selecting displacing agents.16,17 Bearing in mind SUs are weakly acidic compounds, the goal of avoiding coelution of humic acids from the extraction cartridge was reached by replacing TFA with acetic acid (Figure 2B). Evidently, the complex structure of humic acids contains highly acidic functional groups that are able to interact so strongly with positively charged sites on the GCB surface that acetic acid is unable to displace them. 2822 Analytical Chemistry, Vol. 69, No. 14, July 15, 1997
Figure 2. LC/UV chromatograms obtained by extracting seven sulfonylureas added at the level of 2 µg/L to 0.2 L of a simulated surface water sample and reextracting with a CH2Cl2/CH3OH mixture (80:20 v/v) acidified with (A) trifluoroacetic acid and (B) with acetic acid. Peak numbers: 1, thifensulfuron methyl; 2, metsulfuron methyl; 3, triasulfuron methyl; 4, chlorsulfuron; 5, rimsulfuron; 6, bensulfuron methyl; and 7, tribenuron methyl.
Recovery, Precision, and Method Detection Limits (MDLs). Analytical recoveries, repeatability, and detection limits of the method were assessed by spiking 4 L of drinking water, 2 L of groundwater, and 0.2 L of river (Tiber) water respectively with 10, 50, and 250 ng/L each of the seven SUs and then analyzing six times each type of aqueous matrix. Results are reported in Table 3, while a typical LC/UV chromatogram obtained by analyzing SU-fortified drinking water is shown in Figure 3. Analyte recoveries were invariably larger than 90% and were unaffected by the nature of the aqueous matrix in which the analytes were dissolved. At the spike levels considered, within-day precision was between 2.2 and 6.8% for drinking water, 1.0 and 5.6% for groundwater, and 1.6 and 6.9% for river water. MDLs calculated for drinking water and groundwater were between 0.6 and 2 and between 2 and 9 ng/L, respectively. It follows that this method could largely satisfy the stringent requirements imposed by an European Community Directive, setting 100 ng/L as the maximum admissible concentration of an individual pesticide in drinking water. MDLs for surface water ranged between 18 and 51 ng/L. Thus, this method could be effectively used for studying the fate and the impact of SUs in the aquatic ecosystem. Method Comparison. Octadecyl-bonded silica (C-18) has been frequently proposed as a sorbent material to extract SUs from aqueous samples.9,12,21 In terms of selectivity and sensitivity,
Table 3. Percent Recovery, Precision, and Method Detection Limits (MDL, ng/L) on Extracting Seven Sulfonylureas from Three Different Aqueous Matrices drinking water, 4 L (n ) 6), 10 ng/L spike
groundwater, 2 L (n ) 6), 50 ng/L spike
river water, 0.2 L (n ) 6), 250 ng/L spike
compound
recovery
RSDa
MDLb
recovery
RSD
MDL
recovery
RSD
MDL
thifensulfuron methyl metsulfuron methyl triasulfuron chlorsulfuron rimsulfuron bensulfuron methyl tribenuron methyl
95 97 99 99 97 99 95
3.9 2.2 4.8 4.6 6.8 3.8 4.3
1 0.6 2 2 2 1 1
97 97 104 97 103 98 101
1.4 1.8 1.8 1.0 4.2 1.6 5.6
2 3 3 2 6 3 9
98 94 102 95 100 97 96
2.9 1.6 2.2 3.5 6.9 5.5 1.6
22 18 17 26 51 40 13
a
RSD, relative standard deviation. b MDL defined as 3 times the standard deviation calculated at the spike level considered.
Figure 3. LC/UV chromatogram obtained by analyzing 4 L of drinking water spiked with seven sulfonylureas at the individual level of 10 ng/L. Peak numbering is the same as in Figure 2.
this method was compared with one involving the use of a C-18 SPE cartridge. Aliquots of 0.2 L of a river water (Tiber) sample were spiked with 2 µg/L each of the seven SUs and analyzed by extraction with Carbograph 4 and with a disposable 0.5 g C-18 cartridge (Supelco Inc., Bellefonte, PA). Before extraction, the C-18 cartridge was conditioned with 5 mL of methanol followed by 5 mL of water.9 Moreover, water was acidified to pH 3 to suppress analyte dissociation.12 After the passage of the sample, the C-18 cartridge was washed with 10 mL of water. Interstitial water was, in part, eliminated by forcing room air through the cartridge for 5 min. SUs were then eluted with 5 mL of methanol.9 Solvent removal and residue reconstitution were carried out according to our procedure. Experiments were made in duplicate. Resulting LC/UV chromatograms are shown in Figure 4. The sample preparation procedure using the C-18 cartridge, which does not involve any cleanup step, did not avoid the presence in the final extract of two unknown nonacidic compounds which produced peaks that interfered with the analysis of thifensulfuron methyl and triasulfuron (Figure 4A). That the two unknown interfering compounds were nonacidic (or not sufficiently acidic) in nature was indicated by the fact that they were found in the neutral effluent (Figure 4B) designed to remove nonacidic species from the Carbograph 4 cartridge. Comparing chromatograms A and C in Figure 4, it appears that another drawback presented by (21) Zahnow, E. W. J. Agric. Food Chem. 1982, 30, 854-859.
Figure 4. LC/UV chromatograms obtained by extracting seven sulfonylureas added to the level of 2 µg/L in 0.2 L of a river (Tiber) water sample with (A) a 0.5 g C-18 cartridge and with (C) a 0.5 g Carbograph 4 cartridge; (B) chromatogram obtained by injecting into the LC column the washing phase from the Carbograph 4 cartridge. Peak numbering is the same as in Figure 2. Peaks A and B are for two unknown compounds present in the aqueous sample analyzed.
the C-18 cartridge is that SUs are coeluted with trapped fulvic acids. The resulting extremely broad chromatographic peak produced by the latter species makes methods involving a C-18 extraction cartridge not sufficiently sensitive and accurate to detect SUs in surface waters at levels lower than 1 µg/L. Analytical Chemistry, Vol. 69, No. 14, July 15, 1997
2823
Figure 5. S/N of rimsulfuron (60 ng injected into the LC column) vs TFA concentration in the LC mobile phase. Table 4. Effects of Two Storage Treatments on Recovery (%)a of Seven Sulfonylureas Adsorbed on the GCB Surface storage treatment
compound thifensulfuron methyl metsulfuron methyl triasulfuron chlorsulfuron rimsulfuron bensulfuron methyl tribenuron methyl a
7 days 14 days at 18 °C at 18 °C
1 day at 4 °C, 1 day at 4 °C, 6 days 13 days at -18 °C at -18 °C
98
95
99
97
99 101 99 85 97 97
97 98 95 71 89 90
98 102 98 97 99 99
99 101 97 95 98 96
Mean values from triplicate measurements.
Sample and Final Extract Storages. One of the claimed advantages of SPE over solvent extraction is that sampling and extraction of the aqueous sample can be done simultaneously by passing the water sample through a sorbent trap as it is pumped at the sampling site. This procedure could avoid sample contamination and alteration correlated with the traditional sample collection. On the other hand, the original composition of extracts of complex aqueous matrices might be altered after prolonged contact with the sorbent surface owing to some reactions catalyzed by the sorbent itself.22 In addition, SUs are prone to hydrolytic attack4 and could be degraded by residual water remaining in the cartridge even after prolonged air drawing. For these reasons, a stability study of SUs stored on the Carbograph 4 surface was conducted. After a river water sample was spiked with the (22) Crescenzi, C.; Di Corcia, A.; Madbouly, M. D.; Samperi, R. Environ. Sci. Technol. 1995, 29, 2185-2190.
2824 Analytical Chemistry, Vol. 69, No. 14, July 15, 1997
Figure 6. Time-scheduled, three-ion SIM LC/MS chromatograms obtained by analyzing (A) 4 L of a drinking water sample spiked with seven sulfonylureas at the individual level of 3 ng/L and (B) 0.2 L of a river (Tiber) water sample spiked with seven sulfonylureas at the individual level of 60 ng/L. Peak numbering is the same as in Figure 2.
analytes at the individual level of 2 µg/L, the sample was divided in 12 0.2 L aliquots. Analytes were extracted by this method, and water was partially removed from cartridges by forcing room air through them for 1 min. Thereafter, two different cartridge storage treatments were designed. Six cartridges were stored without any precaution at room temperature (16-20 °C), and analytes were reextracted after 7 or 14 days of storage. The second six cartridges were stored 1 day at 4 °C, followed by 6 or 13 days of storage at -18 °C. The rationale behind this combined cartridge storage treatment was that of depicting a practical procedure of field sampling where precautions are in part taken in situ and in part taken upon arrival of samples in the analytical laboratory. Under each storage condition, measurements were made in triplicate, and results are presented in Table 4. As can be seen, a significant loss of rimsulfuron occurred after 2 weeks of storage at room temperature, while it appeared to be stable when the cartridges were kept frozen. This method involves confirmatory analysis by LC-ES/MS instrumentation. If this instrumentation is not available in the same laboratory where screening analysis is performed or the final extract cannot be immediately reanalyzed, the problem of preserving the extract then arises. It has to be considered that the final extract is composed of an acidic watery solution and that some SUs are readily decomposed even when dissolved in moderately acidified water.4 A preservation study of the analytes in the final extract was performed by adding known volumes of the working
Table 5. Effects of Three Storage Treatments on Recovery (%)a of Seven Sulfonylureas Dissolved in the Final Extract storage treatment +18 °C (unneutralized extract)
-18 °C (unneutralized extract)
+18 °C (neutralized extract)
compound
0.12 days
0.25 days
1 days
1 days
7 days
14 days
1 days
2 days
4 days
thifensulfuron methyl metsulfuron methyl triasulfuron chlorsulfuron rimsulfuron bensulfuron methyl tribenuron methyl
100 101 100 99 80 101 40
99 100 103 99 57 103 13
100 99 107 99 13 102 ndb
101 100 99 97 97 98 96
100 99 101 98 99 100 95
98 100 99 100 98 97 94
99 100 99 98 99 97 97
98 100 98 99 97 95 94
100 99 98 100 93 92 90
a
Mean values from duplicate measurements. b nd, not detected.
Table 6. Effect of Increasing the Skimmer Cone Voltage in the Desolvation Chamber on Production of Fragment Ions for Seven Sulfonylureasa skimmer cone voltage, V compound
25
30
35
thifensulfuron methyl
chlorsulfuron triasulfuron
141 (40), 167 (30), 358 (100) 141 (10), 167 (10), 403 (100)
rimsulfuron
182 (10), 326 (10), 432 (100)
141 (50), 167 (100), 205 (10), 270 (10), 388 (30) 141 (40), 167 (100), 199 (10), 264 (10), 382 (30) 141 (100), 167 (50) 358 (40) 141 (70), 167 (80), 219 (10), 284 (10), 403 (100) 182 (80), 251 (10), 326 (50), 432 (100)
141 (20), 167 (100), 205 (15), 388 (10)
metsulfuron methyl
141 (30), 167 (90), 205 (10), 270 (10), 388 (100) 141 (30), 167 (90), 382 (100)
bensulfuron methyl tribenuron methyl
149 (10), 411 (100) 155 (100), 364 (10), 396 (40)
149 (80), 182 (60), 411 (100) 155 (100), 181 (10), 364 (15), 396 (20)
a
141 (25), 167 (100), 199 (10), 382 (10) 141 (100), 167 (70), 358 (20) 141 (90), 167 (100), 219 (20), 284 (10), 403 (50) 178 (15), 182 (100), 299 (10), 326 (40), 432 (50) 149 (100), 178 (15), 182 (50), 411 (60) 155 (100), 181 (20), 396 (10)
Data are reported as m/z (relative abundance). Ions having abundances lower than 10% were not considered.
standard solution to vials each containing 8 mL of the eluent phase designed to elute SUs from the GCB trap and then following the rest of the procedure (see Experimental Section). After residue reconstitution, three extract storage treatments were designed. Two of them involved storage at room temperature and -18 °C, respectively. After 60 µL of the final extract was injected into the LC apparatus, a third storage treatment involved immediate neutralization of the final extract with 2 µL of ammonia, 0.4 mol/ L, 5 days of storage at room temperature, followed by storage at -18 °C. After this storage treatment and before analysis, extracts were reacidified with 2 µL of TFA, 0.4 mol/L. This third set of experiments was designed to simulate a situation in which a final extract is shipped to another laboratory and, upon arrival, is immediately frozen until reanalysis by LC-ES/MS instrumentation. Measurements were made in triplicate, and results are shown in Table 5. Even after only a few hours, extensive degradation of rimsulfuron and tribenuron methyl occurred when the extract was kept at room temperature. The other two storage procedures were both effective in preserving analytes over 2 weeks of storage. Confirmatory Analysis by LC-ES/MS. The electrospray process produced abundant amounts of Na+ and K+ adducts of SUs, in addition to protonated ones. This effect has been already observed in a previous study12 and could be traced to the acidic nature of SU.23 When the MS detector is operated in the SIM mode, this condition is undesired, since it results in signal weakening for the selected [M + H]+ ions. An almost complete (23) Banks, J. F.; Shen, S.; Whitehouse, C. M.; Fenn, J. B. Anal. Chem. 1994, 66, 406-414.
suppression of [M + Na]+ and [M + K]+ ions was obtained by simply distilling acetonitrile in a glass apparatus. Weak ion signals for the analytes considered were obtained when the LC-ES/MS instrumentation was operated with a mobile phase having the same composition as that used for LC/UV analysis. The relatively large concentration of TFA in the electrosprayed solution was responsible for this negative effect. On the other hand, well-retained and sharp peaks, especially for the most acidic SUs, can be obtained only by dissolving a certain amount of an ion suppression agent in the LC mobile phase. With the view of optimizing the chromatographic performance of the analytical column and obtaining a still efficient response of the MS detector for the analytes, the best TFA concentration in the mobile phase was assessed. This set of experiments was performed in the full-scan mode by setting the cone voltage at 25 V and scanning the quadrupole mass filter from 130 to 460 m/z with a 3 s scan. Sixty nanograms of each SU was injected from the composite standard solution into the LC column. At any TFA concentration, total ion current peaks for the analytes were monitored for signal-to-noise ratios (S/N) by measuring peak heights against the average background noise. Measurements were made in duplicate. Since all the analytes behaved analogously, for simplicity, only results for rimsulfuron are shown in Figure 5. According to previous studies,24,25 S/N steadily decreased as the TFA concentration was increased. From a (24) Ikonomou, M. G.; Blades, A. T.; Kebarle, A. T. Anal. Chem. 1991, 63, 19891998. (25) Di Corcia, A.; Crescenzi, C.; Guerriero, E.; Samperi, R. To be published in Environ. Sci. Technol.
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chromatographic point of view, a TFA concentration of 40 µmol/L sufficed to give sufficiently sharp and retained peaks for SUs. With respect to LC/UV chromatographic conditions, the drastic decrease of TFA concentration in the mobile phase resulted in reversing the elution order of the chlorsulfuron-triasulfuron pair. A full-scan mass spectrum affords the highest specificity. However, as can be also deduced from Figure 5, this option could not be exploited to identify a few nanograms of any SU injected into the column. Thus, to meet the criteria of unambiguous and highly sensitive detection of SUs, the MS detector was operated in the three-ion SIM acquisition mode.13 The electrospray process is able to generate only quasi-molecular ions. Anyway, fragment ions can be readily obtained with our instrumentation by suitably controlling the potential difference between the sample and the first skimmer cones. For the seven targeted SUs, the best skimmer cone voltage able to provide a still intense molecular ion plus two fragment ions was evaluated by operating in the fullscan mode (see above) and increasing the cone voltage from 25 to 35 V. This experiment was conducted by injecting 60 ng of each SU into the LC column from a standard solution. At any cone voltage selected, background-subtracted spectra were obtained from the average of the chromatographic peaks. Results are reported in Table 6. Ions having abundances lower than 10% were not considered. When operating at cone voltages lower than 25 V, most of the spectra from SUs displayed only intact protonated adduct ions. At cone voltages higher than 35 V, the situation was reversed. Structures of fragment ions have been already postulated in previous studies by an ES source and tandem MS/MS3,12 or fast atom bombardment.5 As can be seen from Table 6, four of the seven targeted SUs were decomposed by the CID process with production of an ion at m/z 141 from the N-heterocyclic moiety. Unfortunately, one of the most abundant
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background ions had the same m/z value. Therefore, for thifensulfuron methyl, metsulfuron methyl, and triasulfuron, we selected fragment ions (see Table 1) which, though less abundant than that at m/z 141, produced ion current profiles with better S/N values. This was not possible with chlorsulfuron, as only two appreciable fragment ions at m/z 141 and 167 were generated from decomposition of its protonated adduct ion. It has to be pointed out that, as reported elsewhere,25 the repeatability of the CID process in the ion transportation region was no larger than 10% even for the least intense ions. As examples, Figure 6 shows three typical ion SIM LC-ES/ MS chromatograms, obtained by analyzing 4 L of drinking water and 0.2 L of river water fortified respectively with 3 and 60 ng/L each of the seven SUs. Under multiple-ion SIM condition, the limit of detection (LOD) for an analyte is determined from the parent or fragment ion giving the worst S/N value. With this criterion and setting the S/N threshold at 3, LODs for SU ranged between 0.5 (tribenuron methyl, m/z 364) and 3 ng (metsulfuron methyl, m/z 264; thifensulfuron methyl, m/z 205) injected into the LC column from the final extracts of the aqueous matrices considered. From these data and considering the sample preparation procedure involved in this method, it can be deduced that this method can provide definitive and unambiguous confirmation for the presence of the seven targeted SUs at 0.5-3 ng/L levels in drinking water, 1-6 ng/L in groundwater, and 10-60 ng/L in river water. Received for review November 15, 1996. Accepted April 17, 1997.X AC961164U X
Abstract published in Advance ACS Abstracts, June 1, 1997.
