A rapid and sensitive analytical method for the quantificationof residues of endosulfan in bloodAtmakuru Ramesh* and Peruma...
Fig. 3 Total ion chromatogram of endosulfandiol (5.71), alpha-                                                            ...
Table 1 Recovery of total endosulfan (alpha-endosulfan 1 beta-                Table 3 Residues of total endosulfan in huma...
17 M. F. Zaranyika and P. M. Mugari, J. Environ. Sci. Health, Part        34 R. P. Singh, Pestic. Res. J., 1997, 9, 54.   ...
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Endosulfan absent in worker blood reports

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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.

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Endosulfan absent in worker blood reports

  1. 1. A rapid and sensitive analytical method for the quantificationof residues of endosulfan in bloodAtmakuru Ramesh* and Perumal Elumalai RaviDepartment of Pesticide Chemistry, Fredrick Institute of Plant Protection and Toxicology(FIPPAT), Padappai, Chennai 601 301, Tamil Nadu, India. E-mail: raamesh_a@hotmail.comReceived 21st November 2001, Accepted 14th December 2001First published as an Advance Article on the web 11th February 2002A new sensitive analytical procedure has been developed for the determination of residues of endosulfan inhuman blood samples. The method involves the extraction of residues of endosulfan from blood samples by theaddition 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 ofalpha-endosulfan, beta-endosulfan, endosulfan sulfate and endosulfandiol. The influence of temperature duringthe 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 toquantify the residue of total endosulfan up to the 1.0 ng ml21 level. The recovery was 92% with a calculatedrelative 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 itsmetabolites in blood samples collected from the exposed population. The data obtained has been confirmed byGC-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 theform of residues in the environment, food and agriculturalsubstrates has become an important issue in analytical science. ExperimentalFurther, there is growing concern regarding the potential Apparatustoxicity and/or ecotoxicity of the transformation productsassociated with these residues, which is demanding the A Shimadzu gas chromatograph supplied by Shimadzudevelopment of appropriate analytical techniques for their Corporation, Tokyo, Japan, model GC-14B with ECDmonitoring. To a large extent this is the consequence of interfaced to a computer for data acquisition throughincreased consumer concern about food quality, and has led to Communication Bus Module 101 supported by Class GC-10the establishment of numerous and lower maximum residue software was used. A DB-5 megabore column of length 15 m 6limits (MRLs). Thus a greater demand has been placed on the 0.53 mm id with film thickness 1.5 mm was used forcurrent 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 5.0hexachloro-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 withresidues,1 environmental fate and behavior,2–7 metabolites in film thickness 0.25 mm was used for quantification. Classfruits 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 Eventhough endosulfan is a well established pesticide, a literaturesurvey clearly shows the scarcity of information regarding Operating conditions. Column: initial 180 uC; hold forhuman 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, floware 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 endosulfancomplexity of the substrate. Thus the present investigations are sulfate 11.9 min. The specific fragment ions monitored foraimed at two objectives: (i) to develop a suitable analytical confirmation purposes in SIM mode (GC-MS-EI) includemethod for the determination of residues of endosulfan and its endosulfandiol at m/z 241, 271, and 307, alpha-endosulfan atmetabolites, endosulfan sulfate and endosulfandiol, in human m/z 160, 195, and 245, beta-endosulfan at m/z 159, 195, and 235blood; and (ii) to establish the impact of long term spray and endosulfan sulfate at m/z 229, 272, and 387 (Fig. 1). Aexposure to endosulfan in terms of monitoring the residues of signal-to-noise ratio of 1 : 3 is maintained throughout theendosulfan 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. 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 acidMeyzieu, France, with automatic temperature recorder and 60% (10 uC) solution in the order 1.5 ml 1 1.5 ml 1 2.0 ml withdisplay facility was used for storing the samples at an interval of 10–15 s between each addition and this was245 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 aboutReagents 10 min at 3000 rpm. The solvent layer was collected and the process repeated thrice using 10 ml of 9 : 1 hexane–acetoneAll the chemicals and reagents used in the studies were orga- mixture. The hexane–acetone layer was collected each time andnic trace analysis grade unless stated otherwise. They were combined and then evaporated to 3.0 ml under a stream ofpurchased from E. Merck, Darmstadt, Germany. Reference nitrogen at 45 uC. Utmost care is needed to ensure that duringanalytical standards of alpha-endosulfan, beta-endosulfan, the extraction the temperature of the sample should not riseand endosulfan sulfate were supplied by Dr. Ehrenstorfer- beyond 10 uC.Schafers, Augsburg, Germany. Stock standard solutions ofeach containing 10 mg ml21 were prepared in acetone and Collection of blood samplesstored at 245 uC. Known volumes of these solutions weremixed 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 practicedRecovery and fortification for several years. All the samples were coded and received in dry ice pack with the details of the donors. Donors consistsFor experimental purposes, heparinized blood samples were of both females and males of various age groups from 18 tocollected from the donors and stored in the deep freezer at 70 years. Informed consent was obtained from the donors or215 uC. 20 ml of reference analytical working standard from the head of the family from whom blood was collected forsolutions of endosulfandiol, alpha-endosulfan, beta-endosulfan, the study and the same documented in archives. About 5 mlendosulfan sulfate were spiked into 2 ml of blood sample and of blood was collected from each donor for experimentalvigorously shaken for homogeneity. Various known concen- purposes. All the samples were processed and analyzed astrations 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. 3. Table 1 Recovery of total endosulfan (alpha-endosulfan 1 beta- Table 3 Residues of total endosulfan in human blood samplesendosulfan 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 ml21ng 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) —aAverage 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 ofAll the blood samples collected from the exposed population various analytical parameters in determining false positive orwere analyzed for residues of endosulfan. The results showed low recoveries of endosulfan. The analysis of blood samplesthat 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 incomplex substrates is always an indication of the appropriatetechnology and expertise utilized in plant protection and has Acknowledgementgreater importance at national and international level. Anynon-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 studyhas been confirmed by analyzing all the blood samples usingGC-MS in a selective ion monitoring mode. The results showed Referencesthat there is no presence of accumulation of concentrations of 1 H. Goebel, S. Gorbarch, W. Knauf, R. H. Rimpau andendosulfan 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. 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