ANALYTICAL SCIENCES DECEMBER 2003, VOL. 19 1605 2003 © The Japan Society for Analytical Chemistry

Development of a Simultaneous Analysis Method for Carbofuran and Its Three Derivative Pesticides in Water by GC/MS with Temperature Programmable Inlet On-column Injection

Tatsuhiko KAWAMOTO† and Nobuko MAKIHATA

Water Environment Division, Hyogo Prefectural Institute of Public Health and Environmental Sciences, 2-1-29, Arata, Hyogo, Kobe 652Ð0032, Japan

A simultaneous analytical method was examined for carbofuran and its derivative pesticides in water. Since carbofuran derivatives are hydrolyzed to carbofuran in water, the liquid-liquid extraction method was used to obtain an accurate concentration value. Moreover, since these compounds are easily decomposed at the GC/MS injection port, temperature programmable inlet on-column injection was used. By combining the two methods, a sensitive analytical method was established without hydrolysis and thermal decomposition. As a result of recovery experiments using distilled water, river water and tap water, acceptable recovery rates and favorable reproducibility were obtained. This method was used in a field investigation to determine carbofuran and its derivative pesticides in river water taken from three points of the Y river over a period of one year. Carbofuran, benfuracarb, and carbosulfan were detected and corresponded to the period when these pesticides were used in the area. Although benfuracarb and carbosulfan using traditional methods are believed to easily hydrolyze and thermally decompose during the analytical process, by using our method they can be detected.

(Received May 16, 2003; Accepted September 29, 2003)

simultaneous analytical method is required. To date, no Introduction indications of such a method have been reported. Since carbamate pesticides can thermally decompose easily at the GC For the safety of drinking water, raw and tap water are injection port, it is supposed that HPLC-fluorescence detection thoroughly inspected for pesticide residues at a number of is a more suitable method for their analysis than GC.13Ð15 The research organizations, including Hyogo Prefecture.1Ð6 In order author of this paper has, however, previously succeeded in to investigate pesticide contamination in water at an early stage, analyzing thermally unstable trichlorfon and methyl dymron a sensitive and accurate analytical method is required. using GC/MS by temperature programmable inlet (TPI) on- Carbofuran, a carbamate pesticide that is included in four column injection.16 Using this method, it is possible to obtain a pesticides (carbofuran, bentazone, 2,4-D, triclopyr), have mass spectrum with a specificity higher than the fluorescence recently been added to the list of surveillance items under the spectrum obtained by the fluorescence detection method. In Water Supply Law set by the Ministry of Health and Welfare on addition, although the LC/MS analysis of carbamate pesticides June 29, 1999.7 Carbofuran has the strongest acute toxicity in showed high sensitivity, as previously reported, variations were the four pesticides8,9 and it is also suspected of being an observed in the ion signal intensities due to various adduct ions endocrine disrupter.10 In Japan, although carbofuran has neither generated under the influence of a matrix.12 Therefore, we have been produced nor used, its derivatives, namely carbosulfan, looked to establish an analytical method with higher furathiocarb and benfuracarb, are produced and used as reproducibility by using the GC/MS method, which would not pesticides. Since these pesticides degrade in the environment to generate adduct ions during the process. carbofuran, they pose a potential contamination risk to tap Based on these experiments, a simultaneous analytical method water. The Ministry has, therefore, set a guideline value for has been developed for the four carbamate pesticides using carbofuran.7 Solid-phase extraction HPLC-fluorescence liquid-liquid extraction to avoid hydrolysis during concentration detection, or HPLC-postcolumn reaction fluorescence detection, of the pesticides from water, and a TPI on-column GC/MS has been used as a standard method for the analysis of method to avoid thermal decomposition. Using this analysis carbofuran in drinking water.11 However, when solid-phase method, we investigated the concentration of carbofuran and extraction is used the carbofuran derivatives are hydrolyzed in a three derivative pesticides in river water over a 13-month solid-phase extraction process and become carbofuran.12 As a period. result, the concentration value for carbofuran may indicate a larger value than its actual value. Therefore, for an accurate concentration measurement of carbofuran in water, including Experimental carbofuran derivatives, the establishment of an accurate Reagents and chemicals † To whom correspondence should be addressed. Carbofuran (purity > 99.9%) and furathiocarb (purity > 99%) 1606 ANALYTICAL SCIENCES DECEMBER 2003, VOL. 19

Table 1 GC/MS operating conditions [GC] Injection method (1): Conventional splitless injection, Injection volume: 1 µl Injection temp.: 40, 80, 120, 160, 200, 240, 280, 300ûC Injection method (2): TPI on-column injection, Injection volume: 1 µl Injection temp.: 40ûC(1 min) → 80ûC/min → 240ûC(42 min) → 80ûC/min → 300ûC(2.5 min) Coolant: liquid CO2 Oven temp: 40ûC (4 min) → 20ûC/min → 180ûC → 2ûC/min → 200ûC (5 min) → 2ûC/min → 240ûC → 20ûC/min → 300ûC(4 min) Carrier gas: Helium, 40 cm/s (EPC) Column: DB-5MS (film thickness: 0.1 µm, L: 30 m, i.d.: 0.25 mm) [MS] Ion source temp.: 200ûC (EI), Transferline temp: 275ûC Emission current: 285 µA, Ionization volt: 70 eV Mass range: 50 Ð 450 m/z (a) Mode: Full scan Monitor ion: Carbofuran 221, 164, Carbofuranphenol 164, 121, Carbosulfan 323, 160, Furathiocarb 382, 325, 194, Benfuracarb 190, 164, 144

Fig. 2 Sampling points in Hyogo Prefecture. S, K and M, river water; S′, intake point for drinking water; , golf links.

were obtained from Riedel-de Haën (Seelze, Germany). Carbosulfan (purity > 98%) and benfuracarb (purity > 98%) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Carbofuranphenol (10 mg/l in acetonitrile; purity > 99.9%) was obtained from Dr. Ehrenstorfer GmbH (Augsburg, Germany). The chemical structure and GC/MS spectra of carbofuran and its pesticide derivatives tested in this study are shown in Figs. 1-a and 1-b, respectively.

Fluoranthene-d10 (purity > 98%) was obtained from Cambridge Isotope Laboratories (Andover, USA). Pesticide-grade dichloromethane, acetone, hexane and methanol were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Pesticide-grade sodium chloride and sodium sulfate were (b) obtained from Wako Pure Chemical Industries, Ltd. and were heated to 500ûC for 5 h. Water was distilled using an Autostill WG220 (Yamato Scientific Co., Ltd., Tokyo, Japan), and was further purified by passing it through a Milli-Q SP.TOC (Nihon Millipore Ltd., Yonezawa, Japan).

Fig. 1 (a) Chemical structures of pesticides tested in this study. Standard solutions (b) GC/MS spectra of pesticides tested in this study. Individual standard solutions were prepared by dissolving 10 ANALYTICAL SCIENCES DECEMBER 2003, VOL. 19 1607

Fig. 3 Variation of thermal decomposition products for pesticides with an increasing GC/MS injection temperature. This injection method was conventional splitless, but other GC/MS operating conditions were the same as in Table 1. The concentration of each pesticide was 1 mg/l. CF, Carbofuran; CF-PH, carbofuranphenol; CS, carbosulfan; FC, furathiocarb; BC, benfuracarb.

mg of each pesticide in 10 ml of acetone. Carbofuranphenol total of 53 times). Point S is near to the source of the tap water; was diluted with acetone to 5 mg/l. A working standard point M is at a branch in which the river upstream is dotted with solution for each pesticide was prepared by diluting the standard paddy fields and other fields; and point K is at a junction at the solutions with n-hexane. For recovery experiments, the branch which included point M in its main stream and has a golf standard solutions were diluted with methanol. course upstream.

Apparatus GC/MS was carried out using the ion-trap type with a TPI Results and Discussion system GCQ (Thermo Quest, Manchester, UK). Generation of decomposition products in the GC injection port Analytical procedure Figure 3 shows the variation in the peak area of the Sodium chloride (100 g) and dichloromethane (25 ml) were compounds observed with increasing injection temperature from added to a 500 ml water sample in a separatory funnel. After 40ûC to 300ûC. When the injection temperature reached 120ûC, shaking using a mechanical shaker for 5 min, the sample was the generation of carbofuranphenol from carbofuran was left to calm for further 5 min. The extraction procedure was observed, and the peak area value increased with rising repeated for dichloromethane (25 ml). After the second temperature. With regard to carbosulfan and furathiocarb, the extraction, each extract was combined, and anhydrous sodium generation of carbofuran and carbofuranphenol were observed sulfate (ca. 10 g) was added to dehydrate the extract and at 160ûC and 200ûC, respectively. For benfuracarb, the evaporated almost to dryness under a nitrogen stream. generation of carbofuran and carbofuranphenol were observed

Fluoranthene-d10 (0.5 µg) was added to the residue and the at 300ûC and 280ûC, respectively. Since it was observed that volume was adjusted to 0.5 ml with hexane prior to analysis. the four pesticides decomposed with increasing GC injection The GC/MS conditions for both TPI on-column and the temperature, the application of a TPI on-column GC/MS conventional splitless method are given in Table 1. A subject method that could control the injection temperatures was was quantified by referring to the mass pattern of the peak considered. (within ±10 s of the retention time of the standard at a signal to noise ratio of three or more) obtained from the total ion Application of the TPI on-column GC/MS method chromatogram with a full scan. By using selected ion Figure 4 illustrates the total ion chromatogram of carbofuran monitoring to each pesticide, quantitative analysis was and its derivatives by the TPI on-column method. Using the performed by adjusting the sensitivity of Fluoranthene-d10 to an conditions shown in Table 1, each compound clearly separated. internal standard. Table 2 shows comparison results for the generation rate of the thermal decomposition products for each pesticide (1 ng) using Sampling of river water either the TPI on-column or the conventional splitless method at The Y river in the northern part of Hyogo prefecture was 200ûC and 300ûC, respectively. For all of the pesticides selected for monitoring the concentration of the four pesticides examined using the TPI on-column method, the decomposition in river water (Fig. 2). A particular feature in this area is the was less than 1%, exhibiting a favorable result. The standard expanse of paddy fields spread throughout the valley. From solutions for each compound with a concentration of 1, 5, 12.5, three designated points (S, M and K), river water was sampled 25, 50, 125, 250 and 500 µg/l were then analyzed. Table 3 weekly from 11th May, 1997, through to 10th May, 1998, (a shows the regression equation, correlation coefficient and limits 1608 ANALYTICAL SCIENCES DECEMBER 2003, VOL. 19

Table 2 Generation rate (%) of thermal-decomposition products using different GC/MS injection temperatures

Pesticide Injection temp. Carbofuranphenol Carbofuran Carbofuran 40 Ð 300ûCa <1 — 200ûCb 3 — 300ûCb 22 — Carbosulfan 40 Ð 300ûC<1<1 200ûCb 5 1 300ûCb 37 21 Furathiocarb 40 Ð 300ûCa <1 <1 200ûCb 6 <1 300ûCb 22 2 Benfuracarb 40 Ð 300ûCa <1 <1 200ûCb <1 <1 300ûCb 6 7

Fig. 4 Total ion chromatogram of carbofuran and its derivatives by a. TPI on-column injection method was used. the TPI GC/MS method. The concentration of each pesticide was 1 b. Conventional splitless injection method was used. mg/l. The left panel shows the chromatogram from Rt. 8.63 to 39.51 The concentration of each pesticide was 1 mg/l. min, and the right panel shows the chromatogram from Rt. 8.43 to 14.23 min. benfuracarb were 100%, 52%, 92% and 71%, respectively.12 Although carbofuran and furathiocarb generally showed good of detection (LOD) and quantitation (LOQ) for the compounds. recovery rates, and hydrolysis was not observed under these A correlation coefficient linearity of 0.993, or higher, was experiment conditions, 18% of carbosulfan and 6% of determined in the concentration range for each compound. The benfuracarb hydrolyzed into carbofuran. The solid-phase LODs (S/N = 3) and the LOQs (S/N = 10) of compounds extraction process consisted of concentration by adsorption of without a concentration procedure were 0.9 Ð 4.2 µg/l and 2.9 Ð the pesticides to a solid phase and elution from a solid phase. 13.9 µg/l, respectively. According to the standard method, the We have already reported in detail the hydrolysis reaction of LOQs of carbofuran, without a concentration procedure, HPLC- carbosulfan and benfuracarb in water with time.17 Based on fluorescence detection method was 250 µg/l11 and the LOQs of these studies, it is thought that these pesticides would hydrolyze carbofuran using a TPI on-column method was 5.7 µg/l without to carbofuran during solid-phase extraction. However, in a a concentration procedure. These contrasting results showed liquid-liquid extraction process the extraction time is extremely that the TPI on-column method had a capability for analyzing short compared with the solid-phase extraction time. Therefore, low concentration levels (as low as 44 times). The LOD and it was thought that the pesticides were stable and did not LOQ by the previous LC/MS were 0.03 Ð 0.9 µg/l and 0.4 Ð 5.4 hydrolyze in the extraction solvent, and thus a good recovery µg/l, respectively.12 However, the high sensitivity was rate was obtained. With regard to the reproducibility, as considered to be attributable to the injection volume for LC/MS, reported, in the LC/MS analysis the variations in the ion which was ten-times that of GC/MS. In a future study, a large intensity due to adduct ions generated during the process were volume injection system needs to be introduced for GC/MS to observed, and the CV for the four pesticides were 6.2 Ð 12.0%.12 exhibit higher sensitivity. On the other hand, in the liquid-liquid extraction-TPI on- These experimental results revealed that the TPI on-column column GC/MS method, the CV were 1 Ð 4%, which were method was effective for analyzing the four pesticides at low significantly favorable compared with the LC/MS method. concentration levels. In addition to the favorable results Giving these thoughts, by combining the liquid-liquid detection obtained using the TPI on-column method to analyze thermally method that avoids hydrolysis and the TPI on-column GC/MS unstable pesticides, such as trichlorfon and methyl dymron,16 method that avoids thermal decomposition, an accurate these present results revealed that this method was also effective analytical method for four pesticides was developed. for the simultaneous analysis of carbofuran and its derivatives. Survey of four pesticides in river water Recoveries using the liquid-liquid extraction method The concentrations of the four pesticides in river water were Using three different water samples, namely distilled water, determined using the liquid-liquid extraction TPI on-column river water and tap water, to which ascorbic acid sodium salt GC/MS method, as shown in Fig. 5. The water samples were was added to remove residual chlorine, an experiment was collected from points S, K, and M of the Y river (Fig. 2). conducted to quantify the four pesticides. Then, 0.5 µg of each Sampling was performed weekly from May, 1997, to May, pesticide was added to 500 ml of each water sample and liquid- 1998. At point S, carbofuran (maximum 0.84 µg/l, frequency liquid extraction was performed, with the experimental 19/53) was detected from May to July, 1997, and during May, procedure repeated five times. During the extraction procedure, 1998. Benfuracarb (maximum 0.05 µg/l, frequency 5/53) was no hydrolyte was observed. The recovery rate of the four detected in September and October, 1997. At point K, pesticides showed 96 Ð 104% of high value from the three types carbofuran (maximum 0.18 µg/l, frequency 19/53) was detected of water samples. In addition, significant reproducibility was from May to August, 1997, and carbosulfan (maximum 0.03 demonstrated for all water samples with coefficient of variations µg/l, frequency 1/53) was detected during May, 1997. At point (CVs) of 1 Ð 4%, as shown in Table 4. Our past experimental M, carbofuran (maximum 0.20 µg/l, frequency 13/53) was results, obtained using solid-phase extraction, showed that the detected during May to August, 1997, and in May, 1998. recovery rates of carbofuran, carbosulfan, furathiocarb and Furathiocarb was never detected at any of the sampling points. ANALYTICAL SCIENCES DECEMBER 2003, VOL. 19 1609

Table 3 Regression equation, correlation coefficient (r), limit of detection (LOD) and limit of quantitation (LOQ) of carbofuran and its derivatives without a concentration procedure

Compound Rt. Regression equation r LOD/µg LÐ1 LOQ/µg LÐ1 Carbofuran 12.58 y = 40228x + 86416 0.998 1.7 5.7 Carbofuranphenol 9.43 y = 31726x 0.995 0.9 2.9 Carbosulfan 29.01 y = 45227x Ð 159290 0.993 4.2 13.9 Furathiocarb 31.59 y = 73109x + 192228 0.994 3.8 12.5 Benfuracarb 35.45 y = 43915x Ð 511074 0.996 2.0 6.6

Table 4 Recovery of carbofuran and its derivatives Dist. water Tap water River water Pesticide R, %CV, % R, %CV, % R, % CV, %

Carbofuran 98 1 96 2 103 1 Carbosulfan 98 4 104 4 103 1 Furathiocarb 99 2 100 4 100 2 Benfuracarb 102 3 104 3 100 4

0.5 µg of each pesticide was added to 500 ml of distilled water, tap water, and river water. R: recovery, CV: coefficient of variation. Dist. water: pH 7.0, SS; <1.0 mg/l, n = 5. Tap water: pH 6.9, SS; <1.0 mg/l, n = 5. River water: pH 7.1, SS; 2.0 mg/l, n = 5.

Although the guideline value for the surveillance item under the Water Supply Law was only set for carbofuran, and not for the three pesticides,7 the concentration levels of the four pesticides were all below the regulated value for carbofuran (5 µg/l). The current pesticide use by farmers in the surrounding vicinity of the Y river was investigated by both a survey of the pesticide shipments to Hyogo Prefecture17 and interviewing the farmers. The result showed that benfuracarb was used mainly to prevent damage from insect pests in the rice cultivations. Fig. 5 Concentration of four pesticides in river water at sampling points S, K, and M from 11th May, 1997 to 10th May, 1998. Carbosulfan was used as an insecticide in the fields, but was used less frequently than benfuracarb, and furathiocarb was never used. Around the golf course area, carbofuran derivative pesticides were not sprayed. Given this background, most of the investigations carried out by Kondo et al. and Yoshizawa et the benfuracarb and carbosulfan applied from spring through al. could be: (1) carbosulfan had already hydrolyzed in the river autumn in the farm land around the sampling point was believed water or (2) carbosulfan had hydrolyzed during the solid phase to have degraded to carbofuran in the environment. Moreover, extraction process or (3) due to heat decomposition at the GC it became clear that non-decomposed benfuracarb and injector. carbosulfan also existed in the water. It is known that As a result, it is considered that benfuracarb and carbosulfan benfuracarb easily photodegrades,18 but no reports on its are pesticides that are hard to detect due to their degradation in concentration in water have been publicized. Kondo et al.19 environmental water and hydrolysis during the analytical investigated a multicomponent pesticide that was sprayed on process. We applied our analysis method, established in this farmland in the Ishikari River basin once a week, or more, over research, to a field investigation, and could successfully detect a two-year period. As a result, carbofuran was detected, but not benfuracarb, carbosulfan and carbofuran in river water. carbosulfan. In this investigation, a solid-phase (disk-type) extraction was used to extract pesticides from the water, and GC/MS was used as the analysis apparatus with a GC injection Conclusions temperature of 240ûC. Furthermore, Yoshizawa et al.20 reported a pesticide investigation conducted once a month over a one- We have newly established a highly sensitive simultaneous year period in a water source region used for public use in analytical method for carbofuran and its three derivative Chiba Prefecture. In this experiment, solid-phase extraction and pesticides. The method was developed by combining two GC/MS were used as the analysis apparatus, and the recovery techniques: TPI on-column GC/MS to avoid thermal rates of carbofuran were extremely low (52%). However, the decomposition and a liquid-liquid extraction method to avoid authors of this paper have already confirmed that both hydrolysis of the subject during the extraction process. In order benfuracarb and carbosulfan hydrolyze with time to carbofuran to evaluate the method, we conducted recovery experiments in water, as determined by HPLC17 and LC/MS.12 Given these using distilled water, tap water, and river water. The overall results, the reasons why carbosulfan was not detected in experimental results indicated acceptable recovery rates and 1610 ANALYTICAL SCIENCES DECEMBER 2003, VOL. 19 reproducibility, even at low concentrations. Moreover, we 10. N. C. Rawlings, S. J. Cook, and D. Waldbillig, J. Toxicol. conducted water-quality testing using this method on river water Environ. Health, Part A, 1998, 54, 21. collected from three points near to the source of the drinking 11. Water Supply and Environmental Sanitation Department, water. Carbofuran, benfuracarb and carbosulfan were detected Environmental Health Bureau, Ministry of Health and corresponding to the period when benfuracarb or carbosulfan Welfare, “Standard Method for the Examination of Water were used in the area. Benfuracarb and carbosulfan, which are (with Commentary)”, 2001, Japan Water Works considered to easily hydrolyze and thermally decompose during Association, Tokyo, 598. the analytical process, were also detected using our method. 12. N. Makihata, T. Kawamoto, and K. Teranishi, Anal. Sci., 2003, 19, 543. 13. R. J. Argauer, K. I. Eller, M. A. Ibrahim, and R. T. Brown, References J. Agri. Food. Chem., 1995, 43, 2774. 14. D. Chaput, J. Assoc. Off. Anal. Chem., 1998, 71, 542. 1. Y. Takahashi, S. Onodera, M. Morita, and Y. Terao, J. 15. M. J. Page and M. French, J. Assoc. Off. Anal. 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