Docstoc
EXCLUSIVE OFFER FOR DOCSTOC USERS
Try the all-new QuickBooks Online for FREE.  No credit card required.

Endosulfan absent in worker blood reports

Document Sample
Endosulfan absent in worker blood reports Powered By Docstoc
					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
                                                                         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
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
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