This analysis deals with finding the residues of Endosulfan in blood. Researchers, Atmakuru Ramesh and Perumal Elumalai Ravi tested the blood samples of workers and people exposed to Endosulfan for a long time. The study concludes the absence of endosulfan residues in the blood reports.

1. 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.
Introduction samples collected from a directly exposed population. Details
are presented in this paper.
In recent years the consequence of widespread and indis-
criminate 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. Experimental
Further, there is growing concern regarding the potential
Apparatus
toxicity and/or ecotoxicity of the transformation products
associated with these residues, which is demanding the A Shimadzu gas chromatograph supplied by Shimadzu
development of appropriate analytical techniques for their Corporation, Tokyo, Japan, model GC-14B with ECD
monitoring. To a large extent this is the consequence of interfaced to a computer for data acquisition through
increased consumer concern about food quality, and has led to Communication Bus Module 101 supported by Class GC-10
the establishment of numerous and lower maximum residue software was used. A DB-5 megabore column of length 15 m 6
limits (MRLs). Thus a greater demand has been placed on the 0.53 mm id with film thickness 1.5 mm was used for
current regulatory and environmental monitoring programs quantification. The operating conditions are as follows: oven,
resulting in government and industry laboratories searching for 180 uC; injector, 220 uC; detector, 230 uC; gas flow rate,
fast, sensitive and reliable analytical methods to determine the nitrogen, 10 ml min21; split ratio, 1 : 5; retention time/min,
residues of pesticides at trace levels. Endosulfan (1,4,5,6,7,7- endosulfandiol 1.5, alpha-endosulfan 3.3, beta-endosulfan 5.0
hexachloro-8,9,10-trinorborn-5-en-2,3-ylenebismethylene) sul- and endosulfan sulfate 6.8.
fite, a cyclodiene insecticide is composed of a mixture of two For confirmation a Shimadzu Quadrupole GC-MS 5050 QP,
stereoisomers alpha-endosulfan (64–67%) and beta-endosulfan was used. GC-MS was operated in EI mode.
(29–32%). The compound has been extensively studied for its A DB-5 capillary column of length 30 m 6 0.32 mm id with
residues,1 environmental fate and behavior,2–7 metabolites in film thickness 0.25 mm was used for quantification. Class
fruits and vegetables,8–27 meat,28 dairy and milk products,29–32 GC-MS 5000 software system was used for data acquisition.
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 Operating conditions. Column: initial 180 uC; hold for
human exposure due to application of endosulfan. In addition 3.0 min; increase at 10 uC min21 to 230 uC; hold for 5 min.
to this, various extraction techniques published in the literature Injector: 260 uC. Interface: 280 uC. Carrier gas: helium, flow
are found to be difficult to apply to the determination of 1.2 ml min21. Retention times: endosulfandiol 5.7 min, alpha-
residues of endosulfan in human blood samples due to the endosulfan 8.7 min, beta-endosulfan 10.4 min and endosulfan
complexity of the substrate. Thus the present investigations are sulfate 11.9 min. The specific fragment ions monitored for
aimed at two objectives: (i) to develop a suitable analytical confirmation purposes in SIM mode (GC-MS-EI) include
method for the determination of residues of endosulfan and its endosulfandiol at m/z 241, 271, and 307, alpha-endosulfan at
metabolites, endosulfan sulfate and endosulfandiol, in human m/z 160, 195, and 245, beta-endosulfan at m/z 159, 195, and 235
blood; and (ii) to establish the impact of long term spray and endosulfan sulfate at m/z 229, 272, and 387 (Fig. 1). A
exposure to endosulfan in terms of monitoring the residues of signal-to-noise ratio of 1 : 3 is maintained throughout the
endosulfan and its metabolites, if present, in human blood experiment. An Artic 380 deep freezer supplied by Froilabo,
190 J. Environ. Monit., 2002, 4, 190–193 DOI: 10.1039/b110687m
This journal is # The Royal Society of Chemistry 2002

2. Fig. 3 Total ion chromatogram of endosulfandiol (5.71), alpha-
endosulfan (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. Extraction of endosulfan residues from blood samples
To a blood sample were added the following: cold sulfuric acid
Meyzieu, France, with automatic temperature recorder and
60% (10 uC) solution in the order 1.5 ml 1 1.5 ml 1 2.0 ml with
display facility was used for storing the samples at
an interval of 10–15 s between each addition and this was
245 uC. Representative chromatograms are presented in
mixed well in a separatory funnel; 10 ml of a 9 : 1 hexane–
Fig. 2 and 3.
acetone mixture was then quickly added. After vigorous
shaking for 2 min the sample was centrifuged for about
Reagents 10 min at 3000 rpm. The solvent layer was collected and the
process repeated thrice using 10 ml of 9 : 1 hexane–acetone
All the chemicals and reagents used in the studies were orga-
mixture. The hexane–acetone layer was collected each time and
nic trace analysis grade unless stated otherwise. They were
combined and then evaporated to 3.0 ml under a stream of
purchased from E. Merck, Darmstadt, Germany. Reference
nitrogen at 45 uC. Utmost care is needed to ensure that during
analytical standards of alpha-endosulfan, beta-endosulfan,
the extraction the temperature of the sample should not rise
and endosulfan sulfate were supplied by Dr. Ehrenstorfer-
beyond 10 uC.
Schafers, Augsburg, Germany. Stock standard solutions of
each containing 10 mg ml21 were prepared in acetone and
Collection of blood samples
stored at 245 uC. Known volumes of these solutions were
mixed and diluted to obtain the working standard solutions. Blood samples were collected from a population where intense
use of endosulfan for agricultural purposes had been practiced
Recovery and fortification for several years. All the samples were coded and received in
dry ice pack with the details of the donors. Donors consists
For experimental purposes, heparinized blood samples were of both females and males of various age groups from 18 to
collected from the donors and stored in the deep freezer at 70 years. Informed consent was obtained from the donors or
215 uC. 20 ml of reference analytical working standard from the head of the family from whom blood was collected for
solutions of endosulfandiol, alpha-endosulfan, beta-endosulfan, the study and the same documented in archives. About 5 ml
endosulfan sulfate were spiked into 2 ml of blood sample and of blood was collected from each donor for experimental
vigorously shaken for homogeneity. Various known concen- purposes. All the samples were processed and analyzed as
trations were fortified and stored in the deep freezer before described earlier.
analysis.
Results and discussion
The presence of pesticide residues in food and environmental
substrates may have both legally and commercially important
implications. Therefore, reproducibility, reliability, and integ-
rity 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
Fig. 2 GC-ECD chromatogram of 10 ng ml21 of endosulfan. purpose resulted, surprisingly, in very high recoveries of
J. Environ. Monit., 2002, 4, 190–193 191

3. Table 1 Recovery of total endosulfan (alpha-endosulfan 1 beta- Table 3 Residues of total endosulfan in human blood samples
endosulfan 1 endosulfan sulfate) in human blood samples
Sample Age (sex) Residuea/ Sample Age (sex) Residuea/
Spiked concentrationa/ Recovery Relative standard code of donor ng ml21 code of donor ng ml21
ng ml21 (%) deviation
E1 35 (F) — E28 45 (F) —
1.00 92 1.94 E2 32 (F) — E29 56 (F) —
5.00 92 1.99 E3 36 (F) — E30 46 (F) —
10.00 94 1.73 E4 31 (M) — E31 46 (F) —
20.00 96 1.53 E5 38 (M) — E32 55 (F) —
30.00 95 1.88 E6 45 (M) — E33 45 (F) —
40.00 94 1.56 E7 45 (F) — E34 56 (F) —
50.00 94 1.50 E8 55 (F) — E35 50 (F) —
a
Average of six replicates. Correlation coefficient: 0.9999. E9 56 (F) — E36 62 (M) —
E10 46 (F) — E37 55 (F) —
E11 51 (F) — E38 52 (M) —
endosulfan. Anticipating false positive results due to inter- E12 56 (F) — E39 52 (F) —
E13 55 (F) — E40 44 (F) —
ference associated with the purity of solvents, trace organic E14 57 (F) — E41 50 (F) —
analysis grade or residue solvents were used to minimize these E15 56 (F) — E42 53 (M) —
interferences and to obtain good recoveries. Under the E16 56 (M) — E43 48 (F) —
established conditions recovery studies showed that the E17 49 (M) — E44 38 (F) —
method is found suitable to quantify residues of alpha- E18 53 (M) — E45 48 (M) —
endosulfan, beta-endosulfan and endosulfan sulfate up to E19 48 (M) — E46 40 (F) —
E20 50 (F) — E47 37 (F) —
1.0 ng ml21 and endosulfandiol up to 0.02 ng ml21 in human E21 53 (M) — E48 18 (F) —
blood samples. The recoveries are more than 92% (Table 1). E22 45 (M) — E49 70 (F) —
The relative standard deviations (RSDs) and correlation E23 53 (M) — E50 41 (M) —
coefficients were calculated. Further the method was also E24 50 (M) — E51 36 (M) —
found suitable for the determination of residues of endosulfan E25 52 (M) — E52 56 (F) —
and it metabolites in blood samples collected from animals. E26 54 (F) — E53 55 (M) —
E27 48 (M) — E54 35 (F) —
No major deviations were observed in the recovery (Table 2). a
Results below detection limit.
Application to real samples
origin. Present investigations clearly show the influence of
All the blood samples collected from the exposed population
various analytical parameters in determining false positive or
were analyzed for residues of endosulfan. The results showed
low recoveries of endosulfan. The analysis of blood samples
that none of the blood samples contains residues of endosulfan
collected from an exposed populations clearly indicated the
(alpha-endosulfan 1 beta-endosulfan 1 endosulfan sulfate) or
absence of accumulation of residues of endosulfan.
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
Acknowledgement
greater importance at national and international level. Any
non-scientific way of conducting the studies and projecting the The authors thank the management of FIPPAT, the Director,
results will always give adverse effects on society and on the and friends for their immense support in conducting this work.
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 References
that there is no presence of accumulation of concentrations of
1 H. Goebel, S. Gorbarch, W. Knauf, R. H. Rimpau and
endosulfan or its metabolites in blood samples collected from H. Huttenbach, Residue Reviews, Springer-Verlag, New York,
the village population due to endosulfan exposure. 1982, vol. 83.
2 N. Olea, F. Olea-Serrano, P. Lardelli-Claret, A. Rivas and
A. Barba-Navarro, Toxicol. Ind. Health, 1999, 15, 151.
Conclusions 3 A. C. Araujo, D. L. Telles, R. Gorni and L. L. Lima, Bull Environ.
Contam. Toxicol., 1999, 62, 671.
From the above studies it can be concluded that the present 4 J. Ceron and C. Gutierrez-Panizo, J. Environ. Sci. Health, Part B,
method fills the gap with respect to the need for an analytical 1995, B30, 221.
method for the determination of residues of endosulfan in 5 E. Papadopoulou-Mourkidou and A. Milothridou, Bull. Environ.
blood samples. Further, the method is simple and suitable for Contam. Toxicol., 1990, 44, 394.
6 National Research Council of Canada, NRCC Associate Commit-
the analysis of residues of endosulfan from human blood tee on Scientific Criteria for Environmental Quality, Report No. 11,
samples and also is applicable to blood samples of animal NRCC, Ottawa, ON, 1975, pp. 1–100.
7 N. Chopra and A. M. Mahfouz, J. Agric. Food Chem., 1970, 25,
Table 2 Effect of temperature on the recoveries of total endosulfan 32.
8 L. Rosenblum, T. Hieber and J. Morgan, J. AOAC Int., 2001, 84,
Spiked Recovery of Recovery of 891.
Temperature/ concentrationa/ total endosulfan endosulfandiol 9 R. Gaidano and R. Fabbrini, Ital. J. Food Sci., 2000, 12, 291.
uC mg ml21 (%) (%) 10 M. Volante, M. Pontello, L. Valoti, M. Cattaneo, M. Bianchi and
L. Colzani, Pestic. Manage. Sci., 2000, 56, 618.
0 0.2 98 0.2 11 N. Ahmad, G. Buguenu, L. Guo and R. Marolt, J. Environ. Sci.
10 0.2 98 0.4 Health, Part B, 1999, 34, 829.
15 0.2 98 1.2 12 J. Cook and M. Engel, J. AOAC Int., 1999, 82, 313.
20 0.2 72 26 13 D. Tsipi, M. Triantafyllou and A. Hiskia, Analyst, 1999, 124, 473.
30 0.2 34 69 14 R. R. Roy, P. Wilson, R. R. Laski, J. I. Roberts, J. A. Weishaar,
40 0.2 13 88 R. L. Bong and N. J. Yess, J. AOAC Int., 1997, 80, 883.
50 0.2 — 96 15 W. Dejonckheere, W. Steurbaut, S. Drieghe, R. Verstraeten and
a
Average of six replicates. H. Braekman, J. AOAC Int., 1996, 79, 520.
16 E. Neidert and P. W. Saschenbrecker, J. AOAC Int., 1996, 79, 549.
192 J. Environ. Monit., 2002, 4, 190–193

4. 17 M. F. Zaranyika and P. M. Mugari, J. Environ. Sci. Health, Part 34 R. P. Singh, Pestic. Res. J., 1997, 9, 54.
B, 1996, B31, 485. 35 S. Navarro, A. Barba, J. C. Segura and J. Oliva, Pestic. Manage.
18 R. A. Lovell, D. G. Mcchensey and W. D. Price, J. AOAC Int., Sci., 2000, 56, 849.
1996, 79, 544. 36 A. Boyd-Boland, S. Magdic and J. B. Pawliszyn, Analyst, 1996,
19 R. Garcia Repetto, I. Garrido and M. Repetto, J. AOAC Int., 121, 929.
1996, 79, 1423. 37 AOAC Official Methods of Analysis, AOAC, Gaithersburg, MD,
20 S. J. Lehotay, N. Aharonson, E. Pfeil and M. A. Ibrahim, J. AOAC 1995, pp. 13–16.
Int., 1995, 78, 831. 38 G. H. Tan, Analyst, 1992, 117, 1129.
21 M. Gopal and I. Mukherjee, Pestic. Sci., 1993, 37, 67. 39 W. E. Cotham and T. F. Bidleman, J. Agric. Food. Chem., 1989,
22 H. M. Pylypiw, J. AOAC Int., 1993, 76, 1369. 37, 824.
23 H. Sekita, K. Sasaki, Y. Kawamura, M. Takeda and 40 C. M. Lino, C. B. Azzolini, D. S. Nunes, J. M. Silva and
M. Uchiyama, Eiscei Shikenjo Hokoku, 1985, 103, 129. M. I. D. Silveira, J. Chromatogr., B, 1998, 716, 147.
24 D. S. Pokharkar and M. D. Dethe, J. Environ. Sci. Health, Part B, 41 D. S. Rupa, P. P. Reddy and O. S. Reddi, Mutat. Res., 1989, 222,
1981, 16, 439. 37.
25 P. S. Wilker, J. Assoc. Off. Anal. Chem., 1981, 64, 1203. 42 C. S. Daniel, S. Agarwal and S. S. Agarwal, Toxicol. Lett., 1986,
26 E. Cwiertniewska and K. Potrzebnicka, Rocz Panstw Zakl Hig, 32, 113.
1979, 30, 261. 43 F. D. Griffith Jr. and R. V. Blanke, J. Assoc. Off. Anal. Chem.,
27 L. R. Mitchell, J. Assoc. Off. Anal. Chem., 1976, 59, 209. 1974, 57, 595.
28 B. Novak and N. Ahmad, J. Environ. Sci. Health, Part B, 1989, 44 D. M. Holstege, D. L. Scharberg, E. R. Tor, L. C. Hart and
B24, 97. F. D. Galey, J. AOAC Int., 1994, 77, 1263.
29 D. Bennett, A. C. Chung and S. M. Lee, J. AOAC Int., 1997, 80, 45 D. P. Goodspeed and L. I. Chestnut, J. Assoc. Off. Anal. Chem.,
1065. 1991, 74, 388.
30 M. Saleh, A. Kamel, A. Ragab, G. El-Baroty and A. K. El-Sebae, 46 P. K. Gupta, Toxicology, 1978, 9, 371.
J. Environ. Sci. Health, Part B, 1996, 31, 241. 47 J. Demeter, A. Heyndrickx, J. Timperman, M. Lefevere and
31 I. Cok, A. Bilgili, M. Ozdemir, H. Ozebek, N. Bilgili and S. Burgaz, J. D. Beer, Bull. Environ. Contam. Toxicol., 1977, 18, 110.
Bull. Environ. Contam. Toxicol., 1987, 59, 577. 48 D. Roberts, Bull. Environ. Contam. Toxicol., 1975, 13, 170.
32 I. Graca, A. M. Silva Fernandes and H. C. Mourao, Pestic. Monit. 49 T. S. Kathpal and R. S. Dewan, J. Assoc. Off. Anal. Chem., 1975,
J., 1974, 8, 148. 58, 1076.
33 T. S. Kathpal, A. Singh, S. Dhankhar and G. Singh, Pestic. Sci.,
1997, 50, 21.
J. Environ. Monit., 2002, 4, 190–193 193