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Anal. Chem. 1984, 56, 2465-2468
Determination of N-Methylcarbamate Pesticides in Well Water by Liquid Chromatography with Postcolumn Fluorescence Derivatization Kenneth M. Hill,* Richard H. Hollowell, and Leo A. Dal Cortivo Division of Medical-Legal Investigation and Forensic Sciences, Suffolk County Department of Health Services, Veterans Memorial Highway, Hauppauge, New York 11788
Nine N-methylcarbarnate and U-(methylcarbamoyl)oxime pesticides and transformation products in groundwaters are assayed employlng a sensitlve, selective method whlch encompasses separatlon by reversed-phase liquid chromatography, postcolumn hydrolysis to yield methylamlne and formatlon of an intense fluorophore with o-phthalaldehyde and mercaptoethanol. Increased sensltlvlty Is achieved by excltatlon of the product at 230 nm using a deuterium source. Fluorescence intensity is measured wlth a 418-nm filter. The process Is automated and requlres vlrtually no sample preparatlon. Mean recoveries for ail nlne substances at the approximate concentrations of 8 and 40 ppb are In excess of 95%.
Discovery of the 0-(methylcarbamoy1)oximepesticide, aldicarb, and its two oxidation products, aldicarb sulfoxide and aldicarb sulfone, in the groundwater aquifers of agricultural areas has created a demand for a sensitive and selective method for their analysis which is at the same time rapid and economical. Gas chromatography (GC) employing flame photometric detection (1) suffers the serious disadvantage of poor selectivity because such methods require the conversion of parent aldicarb and its sulfoxide to the sulfone, thus providing no information regarding concentrations of the three compounds. Residue characterization of the parent pesticide and its two metabolites is critical to the proper evaluation of the degree of contamination of potable waters since the sulfone reportedly is 25-30 times less toxic than aldicarb and its sulfoxide (2). I n addition, GC methods require preconcentration by timeconsuming liquid-liquid extraction processes to achieve adequate sensitivity. Mede (3) determined aldicarb and its two oxidation products by reverse-phase liquid chromatography (LC) using an ultraviolet detector at 200 nm. However, necessary sensitivity was achieved only after extensive specimen preparation. Moye et al. (4)reported a LC procedure in which several N-methylcarbamate pesticides were chromatographically separated followed by postcolumn alkaline hydrolysis of each eluted species to yield methylamine and production of an intense fluorophore by reaction of the amine with o-phthalaldehyde and 2-mercaptoethanol. The fluorescent material was identified as (1-hydroxyethylthio)-2-~ethylisoindole by Simons and Johnson (5). In his elegant studies, Krause (6, 7)evaluated this procedure and modified postcolumn conditions to achieve greater sensitivity. He employed a noncommercially available postcolumn system with reagent solution reservoirs of approximately 300 mL. At the recommended flow rate of 0.5 mL/min, the reagents would be exhausted in about 10 h, thus prohibiting multiple analyses to proceed unattended overnight. Also, the hydrolysis step is carried out at 100 "C, necessitating the application of a back pressure to prevent boiling of the alkaline hydrolysis solution. 0003-2700/84/0356-2465$01.50/0
Table I. Chromatographic and Postcolumn Conditions
methanol/water; flow rate, 1.5 mL/min; T1, 25-70%, 10 min, linear; T2, 70-25%, 5 min, linear; Tequar25%, 7 min analytical column temp 31 O C sample size, 500 pL; injection cycle, 24 min auto sampler hydrolysis Step: flow rate, 0.5 mL/min; postcolumn reaction coil, 1.0 mL; temp, 85 "C fluorophore step: flow rate, 0.3 mL/min; reaction coil, 0.5 mL excitation wavelength, 230 nm; emission detector filter, 418 nm cutoff; time constant, 3; energy source, deuterium lamp Hewlett-Packard 3354 laboratory automation data collection system mobile phase
The objective of Krause's work was to carefully assess chromatographic and postcolumn experimental parameters and, accordingly, the method was not applied to actual well water samples. We describe a fluorescence procedure for aldicarb, its oxidation products, and six other compounds of interest using a commercially available postcolumn reaction system which provides sensitivity sufficient for potable water assays and which is amenable to unattended, overnight analyses of multiple specimens. In addition, our procedure allows on-column injection of large volumes of filtered but otherwise unprocessed well water thus providing increaged sensitivity while significantly decreasing overall analysis time.
EXPERIMENTAL SECTION Apparatus. The chromatographicsystem employed is shown in Figure 1. A Perkin-Elmer Series 3B high-pressure liquid
chromatograph was interfaced with a Waters Associates 710B WISP autosampler. The standard 0.25-mLsyringe on the sampler was replaced with one of 1.0 mL volume (Waters,no. 76457). The sample loop volume was 0.7 mL. A Du Pont Zorbax C8 (25.0 cm X 4.6 mm X 6 pm) reversed-phase analytical column was used in conjunction with a Brownlee RP-8 guard column (3.0 cm X 4.6 mm X 10 wm). Column temperature was maintained at 31 OC employing a Beckman Instruments oven (part no. 235781). Derivatization was accomplished with a Kratos URS 051 postcolumn reaction system (PCRS). The detector was a Kratos FS 970 L.C. fluorometer equipped with a 5-pL flow cell and an automatic overload reset (FSA 986). Detector signal monitoring and data reduction were performed with a Hewlett-Packard 3354 laboratory automation system. Chromatographic conditions appear in Table I. Reagents. Methanol was HPLC grade from Burdick and Jackson Laboratories (Muskegon, MI). HPLC grade water was used throughout and was obtained by processing all glass distilled water in a Barnstead Nanopure system (Model D2782). All pesticide and transformation product standards were supplied by the United States Environmental Protection Agency. The hydrolysis reagent was prepared by dissolving 3.6 g of potassium hydroxide (Fisher Scientific) in 1 L of water, filtering through a 0.45 pm membrane filter (Gelman 66068), and degassing with helium at a flow rate of 1500 mL/min for 15 minutes. 0 1064 American Chemical Soclety
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 P
Water HPLC Pumps
Methanol
Autosampler
Guard Column
R P - CS Analytical Column
Hydrolysis coil (S5’C)
KOH
f:FSD
10%
OPA Reagent
I
Derlvotlzation coil
J
20
I
15
10
5
TIME (min.)
1 Waste Figure 1.
Schematic diagram of the automated HPLC/PCRS.
o-Phthalaldehyde reagent (OPA) was prepared by dissolving 70 mg of OPA (Dionex) in 10 mL of methanol. This solution was added to 1 L of borate buffer of pH 9.1. After this mixture was degassed with helium, at a flow rate of 1500 mL/min for 15 min, 0.5 mL of 2-mercaptoethanol (American Scientific Products, 1208-100) was added and mixed with a 30-5 purge of helium at a flow rate of 600 mL/min. The borate buffer was prepared by dissolving 8.0 g of KzB407.4Hz0(Fisher Scientific) in 1L of water and filtering through a 0.45 pm membrane. Preparation of Standards. Approximately 10 mg, accurately weighed, of aldicarb, aldicarb sulfoxide, aldicarb sulfone, oxamyl, methomyl, carbofuran, 3-hydroxycarbofuran, carbaryl, and 1naphthol, was dissolved in 10 mL of methanol to provide stock standards. These solutions are stable for at least several months when stored in a -10 O C freezer. A mixed concentrated standard of the nine compounds was prepared by addition of appropriate amounts of each stock solution to methanol yielding a final concentration of about 8000 ppb for each. This concentrated standard was prepared monthly. Fresh working references were prepared by dilution of the stock solution with water to furnish standards of approximately 80 ppb, 40 ppb, 8 ppb, and 0.8 ppb for each substance. Sample Preparation and Analysis. Unless examined immediately, samples were stored in a -10 O C freezer. Fresh or thawed specimenswere filtered by using a 5O00-pL macropipettor (Supelco,no. 5-8471) with a disposable tip (Supelco, no. 5-8461) fitted with a 2-pm porosity filter (Supelco, no. 5-8498). The samples were then transferred to disposable autosampler vials (Wheaton, no. 224802) for loading in the autosampler. Samples of 500 p L volume were injected sequentially every 24 min. Prior to each automated run, the 40 ppb mixed reference solution was injected and the detector sensitivity adjusted so that total area counts for the methomyl peak were in the range 90 i 10 K. The areas observed for each peak in this run were then used by the computer to generate the day’s response factors. The automated run was then initiated, the f i s t specimen being a 40 ppb standard mixture. Thereafter, every sixth specimen vial consisted of a mixed standard. The sequence of standard runs was 8 ppb, 0.8 ppb, and 40 ppb, thus providing continuous quality control. The last reference solution to be run consisted of an 80 ppb mixture to assure linearity.
Chromatogram of a mixed standard. Peaks are (1) aldlcarb sulfoxide (41 ppb), (2)aldlcarb sulfone (40 ppb), (3) oxamyl (41 ppb), (4) methomyl(40 ppb), (5) 3-hydroxycarbofuran (43 ppb), (6) aldicarb (40 ppb), (7) carbofuran (41 ppb), (8) carbaryl (42 ppb), and (9) 1naphthol (48 ppb). Figure 2.
Table 11. Retention Times for Carbamate Pesticides compound
retention time” (std dev), min
aldicarb sulfoxide aldicarb sulfone oxamyl methomyl 3-hydroxycarbofuran aldicarb carbofuran carbaryl 1-naphthol
7.58 (0.0083) 7.99 (0.0096) 9.05 (0.0074) 9.94 (0.0094) 12.42 (0.0067) 14.63 (0.0067) 15.89 (0.0079) 16.37 (0.0063) 16.90 (0.0092)
‘Mean of nine (9) determinations for each compound.
RESULTS AND DISCUSSION Chromatographic separation of the nine compounds in the mixture appears in Figure 2. Each compound was present at an approximate concentration of 40 ppb. The sample size analyzed was 500 pL. Despite this large volume, it is evident that peak shape is not detrimentally affected and resolution is satisfactory. In nine repetitive injections of the standard mixture the maximum deviation from the mean retention time (Rt) for any compound was
