A rapid and sensitive analytical method for the quantification of residues of endosulfan in blood Atmakuru Ramesh* and Perumal Elumalai Ravi Department of Pesticide Chemistry, Fredrick Institute of Plant Protection and Toxicology (FIPPAT), Padappai, Chennai 601 301, Tamil Nadu, India. E-mail: raamesh_a@hotmail.com Received 21st November 2001, Accepted 14th December 2001 First published as an Advance Article on the web 11th February 2002 A new sensitive analytical procedure has been developed for the determination of residues of endosulfan in human blood samples. The method involves the extraction of residues of endosulfan from blood samples by the addition of 60% sulfuric acid at 10 uC, liquid/liquid partitioning by using hexane and acetone mixture (9 : 1) and quantification by using GC-ECD. Residues of endosulfan in blood samples were quantified as the sum of alpha-endosulfan, beta-endosulfan, endosulfan sulfate and endosulfandiol. The influence of temperature during the extraction has been studied. Recovery experiments were conducted over the concentration range 1.0– 50 ng ml21 and the relative standard deviation calculated. The method was found to be sufficiently sensitive to quantify the residue of total endosulfan up to the 1.0 ng ml21 level. The recovery was 92% with a calculated relative standard deviation of 1.96%. Conversion of endosulfan to endosulfandiol is found to be less than 0.5% under the defined conditions. The method was applied to the analysis of residue contents of endosulfan and its metabolites in blood samples collected from the exposed population. The data obtained has been confirmed by GC-MS-EI in selective ion monitoring (SIM) mode.
samples collected from a directly exposed population. Details are presented in this paper.
Introduction In recent years the consequence of widespread and indiscriminate use of pesticides, i.e., their subsequent presence in the form of residues in the environment, food and agricultural substrates has become an important issue in analytical science. Further, there is growing concern regarding the potential toxicity and/or ecotoxicity of the transformation products associated with these residues, which is demanding the development of appropriate analytical techniques for their monitoring. To a large extent this is the consequence of increased consumer concern about food quality, and has led to the establishment of numerous and lower maximum residue limits (MRLs). Thus a greater demand has been placed on the current regulatory and environmental monitoring programs resulting in government and industry laboratories searching for fast, sensitive and reliable analytical methods to determine the residues of pesticides at trace levels. Endosulfan (1,4,5,6,7,7hexachloro-8,9,10-trinorborn-5-en-2,3-ylenebismethylene) sulfite, a cyclodiene insecticide is composed of a mixture of two stereoisomers alpha-endosulfan (64–67%) and beta-endosulfan (29–32%). The compound has been extensively studied for its residues,1 environmental fate and behavior,2–7 metabolites in fruits and vegetables,8–27 meat,28 dairy and milk products,29–32 soil,33,34 water,35–39 and plant and animal tissues.40–49 Even though endosulfan is a well established pesticide, a literature survey clearly shows the scarcity of information regarding human exposure due to application of endosulfan. In addition to this, various extraction techniques published in the literature are found to be difficult to apply to the determination of residues of endosulfan in human blood samples due to the complexity of the substrate. Thus the present investigations are aimed at two objectives: (i) to develop a suitable analytical method for the determination of residues of endosulfan and its metabolites, endosulfan sulfate and endosulfandiol, in human blood; and (ii) to establish the impact of long term spray exposure to endosulfan in terms of monitoring the residues of endosulfan and its metabolites, if present, in human blood 190
Experimental Apparatus A Shimadzu gas chromatograph supplied by Shimadzu Corporation, Tokyo, Japan, model GC-14B with ECD interfaced to a computer for data acquisition through Communication Bus Module 101 supported by Class GC-10 software was used. A DB-5 megabore column of length 15 m 6 0.53 mm id with film thickness 1.5 mm was used for quantification. The operating conditions are as follows: oven, 180 uC; injector, 220 uC; detector, 230 uC; gas flow rate, nitrogen, 10 ml min21; split ratio, 1 : 5; retention time/min, endosulfandiol 1.5, alpha-endosulfan 3.3, beta-endosulfan 5.0 and endosulfan sulfate 6.8. For confirmation a Shimadzu Quadrupole GC-MS 5050 QP, was used. GC-MS was operated in EI mode. A DB-5 capillary column of length 30 m 6 0.32 mm id with film thickness 0.25 mm was used for quantification. Class GC-MS 5000 software system was used for data acquisition. Operating conditions. Column: initial 180 uC; hold for 3.0 min; increase at 10 uC min21 to 230 uC; hold for 5 min. Injector: 260 uC. Interface: 280 uC. Carrier gas: helium, flow 1.2 ml min21. Retention times: endosulfandiol 5.7 min, alphaendosulfan 8.7 min, beta-endosulfan 10.4 min and endosulfan sulfate 11.9 min. The specific fragment ions monitored for confirmation purposes in SIM mode (GC-MS-EI) include endosulfandiol at m/z 241, 271, and 307, alpha-endosulfan at m/z 160, 195, and 245, beta-endosulfan at m/z 159, 195, and 235 and endosulfan sulfate at m/z 229, 272, and 387 (Fig. 1). A signal-to-noise ratio of 1 : 3 is maintained throughout the experiment. An Artic 380 deep freezer supplied by Froilabo,
J. Environ. Monit., 2002, 4, 190–193 This journal is # The Royal Society of Chemistry 2002
DOI: 10.1039/b110687m
Fig. 3 Total ion chromatogram of endosulfandiol (5.71), alphaendosulfan (8.78), beta-endosulfan (10.39) and endosulfan sulfate (11.92) in spiked blood at 5.0 ng ml21. Fig. 1 Structural representation of alpha-endosulfan, beta-endosulfan, endosulfan sulfate and endosulfandiol.
Meyzieu, France, with automatic temperature recorder and display facility was used for storing the samples at 245 uC. Representative chromatograms are presented in Fig. 2 and 3. Reagents All the chemicals and reagents used in the studies were organic trace analysis grade unless stated otherwise. They were purchased from E. Merck, Darmstadt, Germany. Reference analytical standards of alpha-endosulfan, beta-endosulfan, and endosulfan sulfate were supplied by Dr. EhrenstorferSchafers, Augsburg, Germany. Stock standard solutions of each containing 10 mg ml21 were prepared in acetone and stored at 245 uC. Known volumes of these solutions were mixed and diluted to obtain the working standard solutions. Recovery and fortification For experimental purposes, heparinized blood samples were collected from the donors and stored in the deep freezer at 215 uC. 20 ml of reference analytical working standard solutions of endosulfandiol, alpha-endosulfan, beta-endosulfan, endosulfan sulfate were spiked into 2 ml of blood sample and vigorously shaken for homogeneity. Various known concentrations were fortified and stored in the deep freezer before analysis.
Extraction of endosulfan residues from blood samples To a blood sample were added the following: cold sulfuric acid 60% (10 uC) solution in the order 1.5 ml 1 1.5 ml 1 2.0 ml with an interval of 10–15 s between each addition and this was mixed well in a separatory funnel; 10 ml of a 9 : 1 hexane– acetone mixture was then quickly added. After vigorous shaking for 2 min the sample was centrifuged for about 10 min at 3000 rpm. The solvent layer was collected and the process repeated thrice using 10 ml of 9 : 1 hexane–acetone mixture. The hexane–acetone layer was collected each time and combined and then evaporated to 3.0 ml under a stream of nitrogen at 45 uC. Utmost care is needed to ensure that during the extraction the temperature of the sample should not rise beyond 10 uC. Collection of blood samples Blood samples were collected from a population where intense use of endosulfan for agricultural purposes had been practiced for several years. All the samples were coded and received in dry ice pack with the details of the donors. Donors consists of both females and males of various age groups from 18 to 70 years. Informed consent was obtained from the donors or from the head of the family from whom blood was collected for the study and the same documented in archives. About 5 ml of blood was collected from each donor for experimental purposes. All the samples were processed and analyzed as described earlier.
Results and discussion
Fig. 2 GC-ECD chromatogram of 10 ng ml21 of endosulfan.
The presence of pesticide residues in food and environmental substrates may have both legally and commercially important implications. Therefore, reproducibility, reliability, and integrity of analytical data is of utmost important. The literature1 clearly shows that endosulfan rapidly gets converted to endosulfandiol in the presence of sulfuric acid. Our initial experiments43 showed very low recoveries. When conducting experiments using sulfuric acid solution stored at room temperature (25 uC) emulsion formation was observed. This made the matrix unsuitable to proceed further. Further, the rise in temperature during the extraction process also resulted in the formation of endosulfandiol. Hence subsequent studies were conducted by using cold sulfuric acid and by maintaining the temperature below 10 uC during extraction. Under these defined conditions conversion of endosulfan to endosulfandiol is found to be very low (v0.5%). It was also found that the quality of reagents has a great influence on the recovery of the analytes. Use of analytical reagent grade solvents for extraction purpose resulted, surprisingly, in very high recoveries of J. Environ. Monit., 2002, 4, 190–193
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Table 1 Recovery of total endosulfan (alpha-endosulfan 1 betaendosulfan 1 endosulfan sulfate) in human blood samples Spiked concentrationa/ ng ml21
Recovery (%)
Relative standard deviation
1.00 5.00 10.00 20.00 30.00 40.00 50.00
92 92 94 96 95 94 94
1.94 1.99 1.73 1.53 1.88 1.56 1.50
a
Average of six replicates. Correlation coefficient: 0.9999.
endosulfan. Anticipating false positive results due to interference associated with the purity of solvents, trace organic analysis grade or residue solvents were used to minimize these interferences and to obtain good recoveries. Under the established conditions recovery studies showed that the method is found suitable to quantify residues of alphaendosulfan, beta-endosulfan and endosulfan sulfate up to 1.0 ng ml21 and endosulfandiol up to 0.02 ng ml21 in human blood samples. The recoveries are more than 92% (Table 1). The relative standard deviations (RSDs) and correlation coefficients were calculated. Further the method was also found suitable for the determination of residues of endosulfan and it metabolites in blood samples collected from animals. No major deviations were observed in the recovery (Table 2).
Table 3 Residues of total endosulfan in human blood samples Sample code
Age (sex) of donor
Residuea/ ng ml21
Sample code
Age (sex) of donor
Residuea/ ng ml21
E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 E27
35 32 36 31 38 45 45 55 56 46 51 56 55 57 56 56 49 53 48 50 53 45 53 50 52 54 48
— — — — — — — — — — — — — — — — — — — — — — — — — — —
E28 E29 E30 E31 E32 E33 E34 E35 E36 E37 E38 E39 E40 E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 E54
45 56 46 46 55 45 56 50 62 55 52 52 44 50 53 48 38 48 40 37 18 70 41 36 56 55 35
— — — — — — — — — — — — — — — — — — — — — — — — — — —
(F) (F) (F) (M) (M) (M) (F) (F) (F) (F) (F) (F) (F) (F) (F) (M) (M) (M) (M) (F) (M) (M) (M) (M) (M) (F) (M)
(F) (F) (F) (F) (F) (F) (F) (F) (M) (F) (M) (F) (F) (F) (M) (F) (F) (M) (F) (F) (F) (F) (M) (M) (F) (M) (F)
a
Results below detection limit.
Application to real samples All the blood samples collected from the exposed population were analyzed for residues of endosulfan. The results showed that none of the blood samples contains residues of endosulfan (alpha-endosulfan 1 beta-endosulfan 1 endosulfan sulfate) or endosulfandiol (Table 3). Investigations on pesticide residues in complex substrates is always an indication of the appropriate technology and expertise utilized in plant protection and has greater importance at national and international level. Any non-scientific way of conducting the studies and projecting the results will always give adverse effects on society and on the environment. Hence, the data obtained in the present study has been confirmed by analyzing all the blood samples using GC-MS in a selective ion monitoring mode. The results showed that there is no presence of accumulation of concentrations of endosulfan or its metabolites in blood samples collected from the village population due to endosulfan exposure.
Conclusions From the above studies it can be concluded that the present method fills the gap with respect to the need for an analytical method for the determination of residues of endosulfan in blood samples. Further, the method is simple and suitable for the analysis of residues of endosulfan from human blood samples and also is applicable to blood samples of animal Table 2 Effect of temperature on the recoveries of total endosulfan Temperature/ uC
Spiked concentrationa/ mg ml21
Recovery of total endosulfan (%)
Recovery of endosulfandiol (%)
0 10 15 20 30 40 50
0.2 0.2 0.2 0.2 0.2 0.2 0.2
98 98 98 72 34 13
0.2 0.4 1.2 26 69 88 96
—
a
Average of six replicates.
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origin. Present investigations clearly show the influence of various analytical parameters in determining false positive or low recoveries of endosulfan. The analysis of blood samples collected from an exposed populations clearly indicated the absence of accumulation of residues of endosulfan.
Acknowledgement The authors thank the management of FIPPAT, the Director, and friends for their immense support in conducting this work.
References 1 H. Goebel, S. Gorbarch, W. Knauf, R. H. Rimpau and H. Huttenbach, Residue Reviews, Springer-Verlag, New York, 1982, vol. 83. 2 N. Olea, F. Olea-Serrano, P. Lardelli-Claret, A. Rivas and A. Barba-Navarro, Toxicol. Ind. Health, 1999, 15, 151. 3 A. C. Araujo, D. L. Telles, R. Gorni and L. L. Lima, Bull Environ. Contam. Toxicol., 1999, 62, 671. 4 J. Ceron and C. Gutierrez-Panizo, J. Environ. Sci. Health, Part B, 1995, B30, 221. 5 E. Papadopoulou-Mourkidou and A. Milothridou, Bull. Environ. Contam. Toxicol., 1990, 44, 394. 6 National Research Council of Canada, NRCC Associate Committee on Scientific Criteria for Environmental Quality, Report No. 11, NRCC, Ottawa, ON, 1975, pp. 1–100. 7 N. Chopra and A. M. Mahfouz, J. Agric. Food Chem., 1970, 25, 32. 8 L. Rosenblum, T. Hieber and J. Morgan, J. AOAC Int., 2001, 84, 891. 9 R. Gaidano and R. Fabbrini, Ital. J. Food Sci., 2000, 12, 291. 10 M. Volante, M. Pontello, L. Valoti, M. Cattaneo, M. Bianchi and L. Colzani, Pestic. Manage. Sci., 2000, 56, 618. 11 N. Ahmad, G. Buguenu, L. Guo and R. Marolt, J. Environ. Sci. Health, Part B, 1999, 34, 829. 12 J. Cook and M. Engel, J. AOAC Int., 1999, 82, 313. 13 D. Tsipi, M. Triantafyllou and A. Hiskia, Analyst, 1999, 124, 473. 14 R. R. Roy, P. Wilson, R. R. Laski, J. I. Roberts, J. A. Weishaar, R. L. Bong and N. J. Yess, J. AOAC Int., 1997, 80, 883. 15 W. Dejonckheere, W. Steurbaut, S. Drieghe, R. Verstraeten and H. Braekman, J. AOAC Int., 1996, 79, 520. 16 E. Neidert and P. W. Saschenbrecker, J. AOAC Int., 1996, 79, 549.
17 M. F. Zaranyika and P. M. Mugari, J. Environ. Sci. Health, Part B, 1996, B31, 485. 18 R. A. Lovell, D. G. Mcchensey and W. D. Price, J. AOAC Int., 1996, 79, 544. 19 R. Garcia Repetto, I. Garrido and M. Repetto, J. AOAC Int., 1996, 79, 1423. 20 S. J. Lehotay, N. Aharonson, E. Pfeil and M. A. Ibrahim, J. AOAC Int., 1995, 78, 831. 21 M. Gopal and I. Mukherjee, Pestic. Sci., 1993, 37, 67. 22 H. M. Pylypiw, J. AOAC Int., 1993, 76, 1369. 23 H. Sekita, K. Sasaki, Y. Kawamura, M. Takeda and M. Uchiyama, Eiscei Shikenjo Hokoku, 1985, 103, 129. 24 D. S. Pokharkar and M. D. Dethe, J. Environ. Sci. Health, Part B, 1981, 16, 439. 25 P. S. Wilker, J. Assoc. Off. Anal. Chem., 1981, 64, 1203. 26 E. Cwiertniewska and K. Potrzebnicka, Rocz Panstw Zakl Hig, 1979, 30, 261. 27 L. R. Mitchell, J. Assoc. Off. Anal. Chem., 1976, 59, 209. 28 B. Novak and N. Ahmad, J. Environ. Sci. Health, Part B, 1989, B24, 97. 29 D. Bennett, A. C. Chung and S. M. Lee, J. AOAC Int., 1997, 80, 1065. 30 M. Saleh, A. Kamel, A. Ragab, G. El-Baroty and A. K. El-Sebae, J. Environ. Sci. Health, Part B, 1996, 31, 241. 31 I. Cok, A. Bilgili, M. Ozdemir, H. Ozebek, N. Bilgili and S. Burgaz, Bull. Environ. Contam. Toxicol., 1987, 59, 577. 32 I. Graca, A. M. Silva Fernandes and H. C. Mourao, Pestic. Monit. J., 1974, 8, 148. 33 T. S. Kathpal, A. Singh, S. Dhankhar and G. Singh, Pestic. Sci., 1997, 50, 21.
34 R. P. Singh, Pestic. Res. J., 1997, 9, 54. 35 S. Navarro, A. Barba, J. C. Segura and J. Oliva, Pestic. Manage. Sci., 2000, 56, 849. 36 A. Boyd-Boland, S. Magdic and J. B. Pawliszyn, Analyst, 1996, 121, 929. 37 AOAC Official Methods of Analysis, AOAC, Gaithersburg, MD, 1995, pp. 13–16. 38 G. H. Tan, Analyst, 1992, 117, 1129. 39 W. E. Cotham and T. F. Bidleman, J. Agric. Food. Chem., 1989, 37, 824. 40 C. M. Lino, C. B. Azzolini, D. S. Nunes, J. M. Silva and M. I. D. Silveira, J. Chromatogr., B, 1998, 716, 147. 41 D. S. Rupa, P. P. Reddy and O. S. Reddi, Mutat. Res., 1989, 222, 37. 42 C. S. Daniel, S. Agarwal and S. S. Agarwal, Toxicol. Lett., 1986, 32, 113. 43 F. D. Griffith Jr. and R. V. Blanke, J. Assoc. Off. Anal. Chem., 1974, 57, 595. 44 D. M. Holstege, D. L. Scharberg, E. R. Tor, L. C. Hart and F. D. Galey, J. AOAC Int., 1994, 77, 1263. 45 D. P. Goodspeed and L. I. Chestnut, J. Assoc. Off. Anal. Chem., 1991, 74, 388. 46 P. K. Gupta, Toxicology, 1978, 9, 371. 47 J. Demeter, A. Heyndrickx, J. Timperman, M. Lefevere and J. D. Beer, Bull. Environ. Contam. Toxicol., 1977, 18, 110. 48 D. Roberts, Bull. Environ. Contam. Toxicol., 1975, 13, 170. 49 T. S. Kathpal and R. S. Dewan, J. Assoc. Off. Anal. Chem., 1975, 58, 1076.
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