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Anal. Chem. 1988, 60,220-226
Postcolumn Photolysis of Pesticides for Fluorometric Determination by High-Performance Liquid Chromatography Carl J. Miles
Department of Agricultural Biochemistry, University of Hawaii a t Manoa, Honolulu, Hawaii 96822
H. Anson Moye* Pesticide Research Laboratory, Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Florida 3261 1
A hlgh-performance llquld chromatography postcolumn reactlon detector that employs UV photolysls with an optional reactlon by using o-phthalaldehyde-2-mercaptoethanol (OPA-MERC) followed by fluorescence detectlon was found to be useful for several classes of pesHdcke. I n the presence of the OPA4ERC reagent, most carbamates, carbamoyl oxImes, carbamothlolc acids, and substituted ureas gave a sensltlve response whlle the response of dlthlocarbamates, phenylamkks, and phenylcarbamates varled. The response of most of the pestkkh tested was signtflcantly affected by the solvent used. Method detection llmits for akllcarb suHoxlde, aldic#b, propoxur, thiram, and neburon In groundwater were 2.5, 2.3, 3.3, 3.8, and 2.0 pg/L, respectlvely. I n the absence of OPA-MERC reagent, several of the substituted aromatlc compounds also gave strong fluorescence after photdyds. This detector Is applicable to a broad range of nltrogenous pestlckles.
Soon after high-performance liquid chromatography (HPLC) was shown to extend the range of pesticides that could be analyzed by chromatographic methods, the search began for detectors that were sensitive and selective. Fluorometric and electrochemical are two of the more sensitive and selective HPLC detectors, but the number of fluorescent and electroactive pesticides is small. Postcolumn reaction detectors allow the chromatographer to modify the analyte's chemical structure in-line, allowing the development of unique analytical methods. Moye and co-workers (1) were the first to report a postcolumn reaction detector that employed alkaline hydrolysis of N-methylcarbamate and carbamoyl oxime pesticides to methylamine followed by condensation with o-phthdaldehyde (OPA) and 8-mercaptoethanol (MERC) to produce a highly fluorescent isoindole. This technique forms the basis of EPA Method 531 (2). This approach was subsequently modified to provide for the analysis of the secondary amine, glyphosate (N-(phosphonomethyl)glycine),by replacing the hydrolytic solution with an oxidative solution of calcium hypochlorite, which produces a primary amine that can be labeled with the OPA-MERC reagent ( 3 , 4 ) . Several investigators have shown that introduction of a UV lamp in-line allows phototransformation of many compounds prior to electrochemical and fluorescent detection and these studies have been reviewed (5). Luchtefeld (6) demonstrated that phenylurea herbicides could be resolved by HPLC and sensitively detected by postcolumn photolysis and labeling with OPA-MERC to form fluorescent products. We have examined several classes of pesticides for fluorescence after UV photolysis and for fluorescence after UV photolysis followed by reaction with OPA-MERC. Water,
and 1:l mixtures of methanol/water and acetonitrile/water, typical reversed-phase mobile phases, were tested as solvents. Chromatographic separations and detection limits for pesticides representing several classes were determined. EXPERIMENTAL SECTION Apparatus. The instrumental system consisted of a PerkinElmer Series 4 solvent delivery system, a Perkin-Elmer ISS-100 autosampler, a UV photolytic reactor, a Milton Roy minipump, a Kratos FS 970 fluorometer, and a Perkin-Elmer LCI 100 integrator (see Figure 1). Reaction of the OPA-MERC reagent with analyk was achieved in a 7.9 m X 0.25 mm i.d. X 1.5 mm 0.d. coiled Teflon tube. During the study, an identical piece of Teflon tubing was woven as described by Engelhardt and Neue (7) and substituted for the coiled tube. A Perkin-Elmer 3 x 3 (0.46 X 3.3 cm; 3 pm) C18 column (room temperature) and a water/acetonitrile gradient were used for all separations (0.4-mL injections); the solvent program was as follows: 1.0 mL/min, linear gradient of 95:5 water/acetonitrile to 5050 water/acetonitrile in 10 min; hold at 50:50 for 5 min; step gradient to 955 water/acetonitrile and equilibrate for 8 min. The photolytic reactor was constructed from a BHK, Inc., Model 80-1178-01jacketed UV lamp (0.9 X 18 cm) inserted in the center of the cylindrical 5.6 m X 0.8 mm i.d. X 1.5 mm 0.d. Teflon coil woven as described by Engelhardt and Neue (7). The UV lamp/Teflon tube assembly was placed in the center of an enclosed section of electrical PVC pipe (6-mm wall thickness, 10 cm i.d.). An exhaust fan (approximately 50 cfm (cubic feet per minute); 1/7 hp (0.105 kW), 3000 rpm motor by Dayton) was connected to the pipe by flexible tubing (5-cm i.d.) inserted through the pipe cap (6-mm wall thickness) for cooling and removal of ozone when an unjacketed UV lamp was used. Holes drilled in the bottom of the PVC pipe allowed cool air to enter the photoreactor. Aluminum foil lined the inside of the PVC pipe to increase photolysis efficiency. A lamp power supply (Model 90-0001-01, BHK, Inc.; 2 W) completed the photoreactor. Molar extinction coefficients at 254 nm were determined from absorbance measurements on a Beckman DU-8 spectrophotometer. Fluorescence spectra were recorded on a Perkin-ElmerMPF 44B spectrophotofluorometer. The OPA-MERC reagent (see below) was metered into the HPLC column effluent with a mixing tee (Rainin No. 200-22) at 0.5 mL/min. Fluorescencewas measured at 418 nm after excitation at 235 nm with a deuterium source. Relative fluorescence was measured without an HPLC column in place using three solvent systems (1.0 mL/min): (1) water, (2) 1:l water/methanol, and (3) 1:l water/acetonitrile. Chemicals. Linuron, monuron, methomyl, and nabam were obtained from Du Pont (Wilmington, DE) while the remainder of the standards came from the U.S. Environmental Protection Agency, Pesticides and Industrial Chemicals Repository (Research Triangle Park, NC). The purities ranged from 95% to 100% with the exceptions of amobam (41%),bunema (40.2%), and metham (33.9%). Benzimidazol-2-onewas synthesized by the method described by Crank and Mursyidi (8). Approximately 1mg/mL solutions of each pesticide were prepared in acetonitrile or water and diluted such that a 1-pL injection provided from 0.1 to 10 nmol. Injections of 1 pL were made to minimize the effects of the pesticide solvent on the photolysis/fluorescence response.
0003-2700/88/0380-0220$01.50/00 1988 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 60, NO. 3, FEBRUARY 1, 1988
221
PERKIN E L M E R
SERIES 4
COLUMN
FLUOROMETER
O.8mm I D x 5 . 6 m KNITTED TEFLON TUBING (2.81~11) SURROUNDING A BHK INC. 0.9 x l 8 c m UV L A M P
Flgure 1. Configuration of HPLC postcolumn UV photolysislOPA-MERC Instrumental system. Optional Items are enclosed with dashed lines.
(Caution: All pesticides, reagents, and solvents are potentially harmful and should be handled with care!) The OPA-MERC reagent was prepared by dissolving 19.1 g of sodium borate (0.05 M) in slightly less than 1L of deionized water followed by addition of 44 mg of o-phthalaldehyde (Aldrich) dissolved in 5 mL of methanol. The reagent was completed by adding 1.0 mL of 2-mercaptoethanol (Kodak) and adjusting the final volume to 1.0 L with water. Photolysis Product Identification. Ammonia and primary amines were determined by cation exchange HPLC with postcolumn fluorogenic labeling with OPA-MERC. A mobile phase of 0.02 M CaClz (pH 7) was delivered at 0.5 mL/min (Altex Model llOA pump) through a Perkin-Elmer0.26 X 25 cm (10 wm) silica A column (room temperature). OPA-MERC reagent was metered in postcolumn at 0.5 mL/min (Milton Roy minipump) and fluorescence was measured by a Gilson Spectra/Glo fluorometer (OPA filters). Photodegraded pesticide solutions were obtained by irradiating 200 wg of each pesticide in acetonitrile in the photoreactor at a flow rate of 0.5 mL/min. Controls were prepared identically with the UV lamp off. Photodegraded and control pesticide mixtures were acylated with trifluoroacetic anhydride (PCR/SCM Specialty Chem; 1 h at 100 "C) and analyzed by gas chromatography/mass spectrometry (0.25 mm X 60 m DB-5; J & W Scientific) on a Finnigan 4500 system. Detection Limit Measurements. Method detection limits (MDL) were determined as recommended by the USEPA (9) and are given by the equation MDL = t(n-l,l-o-0.99)S where t is the Student's t value appropriate for a 99% confidence level and a standard deviation estimate with n - 1 degrees of freedom, and s is the standard deviation for n replications (n = 8). The MDL is defined as the minimum concentration of a substance that can be measured and reported with 99% confidence that the analyte concentration is greater than zero and is determined from analysis of a sample in a given matrix containing the analyte. This procedure was performed on eight separate aliquots of a Floridan groundwater fortified with the test pesticides. Fortified groundwater samples were analyzed without any sample pretreatment. Limits of detection (LOD) of standard solutions were also determined by dilution until the signal-to-noise ratio was 3. The LOD is the lowest concentration or amount of a substance that can be determined to be statistically different (about 99% confidence level) from an analytical blank.
RESULTS AND DISCUSSION Fluorescence a f t e r Photolysis.
Approximately 100
compounds representing several classes of pesticides were examined for fluorescence ( X E ~235 nm; X b > 418 nm) after UV irradiation. Only those compounds that gave a fluorescence response greater than 1% of an equimolar amount of quinine sulfate are listed in Table I. These results show that solvent significantly affects the fluorescence and that a 1:l mixture of water and acetonitrile yields the greatest fluorescence for most of the pesticides for the three solvents tested. Other investigators also have observed that solvent affects the fluorescent response of phototransformation products (10, 11). Most of the compounds in Table I gave a response only when irradiated, indicating that the fluorescence resulted from phototransformation products. Also, all of the compounds in Table I had aromatic character, suggesting that the fluorescence resulted from transformation of this moiety. Fluorescence spectra of photodegraded phenylurea herbicide solutions showed that the excitation and emission maxima were similar for each herbicide (average XEx 340; X b 420) and were similar to the maxima for OPA-MERC-methylamine (A, 345; XEm 455). This suggested that fluorescence was due to a single photodegradation product, possibly an isoindole-type structure similar to the OPA condensation product. Benzimidazol-2-one is such a structure and could be formed by internal cyclization of phenylurea herbicides during photolysis. Synthesis of benzimidazol-2-one and comparison of its fluorescent spectrum (AEx 296; hEm 305) to those observed for ~ the photodegraded phenylureas (average XE= 340; X E 420) showed that it was not a major phototransformation product. Aniline and substituted anilines had fluorescent spectra that were similar to spectra of the photodegradation products of the phenylureas, suggesting that similar structures may be responsible for the fluorescent response observed. Analysis of photodegraded phenylurea herbicide solutions by HPLC with UV detection (ODs with 1:l acetonitrile/O.l M KHzPO,; 200 nm) showed the presence of several peaks, indicating that a mixture of photodegradation products is formed. Also, most of the photodegradation products eluted early in the chromatogram, indicating that they were polar materials. When a fluorometer (OPA filters) was coupled to this HPLC, there was usually more than one peak that showed fluorescence (see Figure 2). These results suggested that within the phenylurea herbicides tested, the fluorescence
222
ANALYTICAL CHEMISTRY, VOL. 60, NO. 3,FEBRUARY 1, 1988
Table I. Relative Fluorescence of Selected Pesticides in Three Solvents
B
relative fluorescence" 1:l 1:l water/ water/ water methanol acetonitrile common name U V o n UV off UVon UVoff U V o n
propoxur carbaryl aminocarb phenmedipham desmedipham propham barban chlorpropham asulam pirimicarb karbutilate benomyl thiophanate 3,4-dichloroaniline bentazon monalide propanil fluoridamid mefluidide propachlor pyracarbolid amdro paraquat diquat difenzoquat isoproturon fluometuron monuron diuron neburon chlorbromuron linuron siduron thidiazuron tolylfluanid antu
-b
0.03 0.01
-
-
-
0.01 0.01
0.19 0.01 -
0.05
0.01 0.10 0.03 0.01
0.01 0.14 0.15 0.02
0.01 0.01 0.01 0.01
0.03 0.04 0.03 0.04 0.02 0.73 0.05 0.23 0.02 0.04
0.01 0.58
0.03 0.08
0.02
-
-
0.04
0.20
0.01
0.01
-
0.04 -
0.01 0.14
0.01 -
0.01
0.01
0.02 0.05 0.01 0.22
0.01 0.17 0.02 0.01 0.01 0.04 0.01 0.03 0.03
0.03 0.05
0.03 0.09
0.07 0.13
0.10 0.16
0.19 0.30 0.12 0.03 0.40
0.15 0.04
0.29
D
0.05 0.04
0.05
0.54 0.03
C
0.46 0.02 0.32
0.05 0.03 0.02 0.05 0.27 0.34 0.03 0.10
0.05 0.10
J
Relative fluorescence is the ratio of the fluorescence response of pesticide to an equilmolar amount of quinine sulfate. Pesticides with a relative fluorescence of less than 0.01 were omitted from this table. bDash is less than 0.01 (1%)relative fluorescence. response produced after photolysis does not result from a single common fluorophore. Connection of an electrochemical detector (+0.9 V) to the same HPLC system showed the presence of several electroactive peaks in the photodegraded phenylurea solutions. This information and the observation that all of the photodegraded phenylurea solutions have a brown color suggested that one or more of these photodegradation products are amino- and/or hydroxy-substituted aromatic compounds. Mass spectra were consistent with this identification. Fluorescence after Photolysis and Labeling with OPA-MERC. Examination of the fluorescence of selected pesticides after photolysis and labeling with OPA-MERC showed that most N-methylcarbamates, carbamoyl oximes, and substituted ureas studied yielded a sensitive response compared to an equimolar amount of methylamine (see Table 11). Aldicarb sulfone was an exception, probably because its molar extinction coefficient at 254 nm is 47 while those of aldicarb and aldicarb sulfoxide were 1205 and 2204, respectively. Addition of acetone, a photosensitizer, to the solvent significantly increased the response of aldicarb sulfone. The relative fluorescence values in Table I1 were not corrected for
ny
I
I
I
I
I
I
0
10 MINUTES
20
0
10 MINUTES
20
Flgure 2. Reversed-phase HPLC chromatograms of fluometuron before and after photolysis on an Alltech C,, column (0.46 X 25 cm) with 1:l 0.1 M KH,PO,/CH,CN at 0.5 mL/min with UV detection (200 nm) [(A)control; (B) UV photodegraded] and fluorescence detection (OPA filters)[(C)control; (D) UV photodegraded].
the responses reported in Table I, since the effect of OPAMERC on the photoinduced or native fluorescence of these compounds is unknown. Relative fluorescence was very dependent upon the solvent used, suggesting that the solute environment was important in the photolysis mechanism. It has been reported that the presence and/or absence of oxygen affects the rate and products formed from some pesticides (12-15). The solvents used here were purged with helium to avoid bubble formation during chromatography, but because of the oxygen permeability of Teflon (16),deoxygenation was not achieved during the entire photolysis reaction. Also, UV irradiation of Teflon produces protons and fluoride ions which may affect phototransformation products. It should be noted that when large percentages (80%-100%) of acetonitrile were used as solvent, the Teflon tubing developed several leaks and appeared to "sweat". Such solvents were avoided because they required that the photolysis coil be replaced. Aldicarb and methomyl have been shown to produce significant amounts of methylamine after photolysis in acetonitrile solutions (12,13). Also, monuron and fenuron produced dimethylamine after photolysis in deoxygenated methanol (14), and Luchtefeld (6) proposed that several phenylurea herbicides produce methylamine upon photolysis. Photodegraded solutions of several of the test pesticides contained methylamine and other primary amines (see Table 111).
(In,
ANALYTICAL CHEMISTRY, VOL. 60, NO. 3, FEBRUARY 1, 1988
Table 11. Relative Fluorescence of Selected Pesticides in Three Solvents (OPA-MERC)
common name aldicarb aldicarb sulfoxide aldicarb sulfone thiofanox methomyl oxamyl propoxur carbaryl aminocarb methiocarb CPMC carbofuran promecarb mexacarbate isoprocarb formetanate HCl glyphosate phenmedipham desmedipham propham barban chlorpropham ferbam thiram CDEC bunema metham amobam nabam thiophanate butylate diallate ethiolate pebulate isoproturon fluometuron monuron diuron neburon chlorbromuron linuron siduron thidiazuron tolylfluanid tebuthiuron antu
class carbamoyl oxime
relative fluorescence' 1:l 1:l water/ water/ acetowater methanol nitrile 0.68 0.55
0.47 0.50
0.33 0.27
0.02
0.03
0.01
0.94 0.59 0.52 N-methylcarbamate 0.74 0.06 0.76 0.47 0.07 0.26 0.31 0.71 0.29 -b
0.77 0.92 0.87 0.42 0.07 0.75 0.32 0.06 0.14 0.20 0.36 0.27 0.01
0.48 0.58 0.62 0.36 0.03 0.39 0.33 0.04 0.13 0.23 0.47 0.29 0.01
0.11 0.01
0.10 0.01
0.17 0.01
0.02
0.10
0.05
0.01 0.01
-
0.01 0.01 0.01
1.52 0.75 0.20 0.03 0.03
1.21 0.67 0.18 0.02 0.02
organophosphate phenylcarbamate
0.01 -
dithiocarbamate
1.75 0.79 0.15 0.01 0.05 0.01
-
-
0.03 0.20 0.03 0.09 0.06 0.23 0.44 0.40 0.12 0.06 0.03
0.01 0.03 0.11 0.01 0.05 0.05 0.25 0.39 0.14 0.13 0.10 0.08
0.01 0.02 0.32 0.08 0.13 0.11 0.38 0.35 0.43 0.38 0.44 0.05
0.03 0.03 0.07 0.36 0.02 0.45
0.09 0.09 0.11 0.39 0.02 0.32
0.05 0.05 0.04 0.51 0.02 0.16
carbamothioic acid
phenylurea
sulfonylurea thiadiazolurea thiourea
223
Table 111. Production of Primary Amines by Photolysis of Selected Pesticides in Acetonitrile molar ratio of amine to pesticiden
~
'Relative fluorescence is the ratio of the fluorescence response of pesticide to an equilmolar amount of methylamine. The fluorescence response of the pesticide is the difference between the average of duplicate measurements with the UV lamp on and a single measurement with the UV lamp off. *Dash is less than 0.01 relative fluorescence. Ammonia, methylamine, ethylamine, propylamine, butylamine, isopropylamine, and isobutylamine could be detected at low nanomole levels with the instrument used. Antu was the only compound tested that produced ammonia upon photolysis. Of the compounds tested, the carbamoyl oximes produced the highest yield of methylamine. Photolysis of compounds with N,N-dialkylamide or N,Ndialkylthiamide structures resulted in the corresponding al-
common name aldicarb aldicarb sulfoxide aldicarb sulfone methomyl oxamyl propoxu r carbaryl methiocarb carbofuran pirimicarb
class carbamoyl oxime
N-methylcarbamate
N,N-dimethylcarbamate carbarnatelurea dithiocarbamate
karbutilate ferbam thiram CDEC dimethyldithiocarbamic acid diethyldithiocarbamic acid butylate carbamothioic acid diallate ethiolate pebulate cycloate vernolate triallate thiobencarb isoproturon fluometuron monuron diuron neburon chlorbromuron linuron siduron monolinuron difenoxuron metoxuron metobromuron chlortoluron diflubenzuron fenuron thidiazuron tolylfluanid tebuthiuron
0.63 0.53 0.29 0.43 0.38 0.53 0.07 0.09 0.14 0.06 0.06 0.04 0.10 0.04 0.21 0.19 (ethylamine) 0.23 (isobutylamine)
0.11 (isopropylamine) 0.18 (ethylamine) 0.12 (ethylamine) and 0.10
(butylamine)
phenylurea
sulfonylurea thiadiazolurea
0.08 (ethylamine) 0.21 (propylamine) 0.05 (isopropylamine) 0.19 (ethylamine) 0.07 0.07 0.14 0.09 0.09 0.10 0.08
