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 2001First 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

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.Further, there is growing concern regarding the potentialtoxicity and/or ecotoxicity of the transformation productsassociated with these residues, which is demanding thedevelopment of appropriate analytical techniques for theirmonitoring. To a large extent this is the consequence ofincreased consumer concern about food quality, and has led tothe establishment of numerous and lower maximum residuelimits (MRLs). Thus a greater demand has been placed on thecurrent regulatory and environmental monitoring programsresulting in government and industry laboratories searching forfast, sensitive and reliable analytical methods to determine theresidues of pesticides at trace levels. Endosulfan (1,4,5,6,7,7-hexachloro-8,9,10-trinorborn-5-en-2,3-ylenebismethylene) sul-fite, a cyclodiene insecticide is composed of a mixture of twostereoisomers alpha-endosulfan (64–67%) and beta-endosulfan(29–32%). The compound has been extensively studied for itsresidues,1 environmental fate and behavior,2–7 metabolites infruits 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 Eventhough endosulfan is a well established pesticide, a literaturesurvey clearly shows the scarcity of information regardinghuman exposure due to application of endosulfan. In additionto this, various extraction techniques published in the literatureare found to be difficult to apply to the determination ofresidues of endosulfan in human blood samples due to thecomplexity of the substrate. Thus the present investigations areaimed at two objectives: (i) to develop a suitable analyticalmethod for the determination of residues of endosulfan and itsmetabolites, endosulfan sulfate and endosulfandiol, in humanblood; and (ii) to establish the impact of long term sprayexposure to endosulfan in terms of monitoring the residues ofendosulfan and its metabolites, if present, in human blood

samples collected from a directly exposed population. Detailsare presented in this paper.

Experimental

Apparatus

A Shimadzu gas chromatograph supplied by ShimadzuCorporation, Tokyo, Japan, model GC-14B with ECDinterfaced to a computer for data acquisition throughCommunication Bus Module 101 supported by Class GC-10software was used. A DB-5 megabore column of length 15 m60.53 mm id with film thickness 1.5 mm was used forquantification. 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 5.0and 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. ClassGC-MS 5000 software system was used for data acquisition.

Operating conditions. Column: initial 180 uC; hold for3.0 min; increase at 10 uC min21 to 230 uC; hold for 5 min.Injector: 260 uC. Interface: 280 uC. Carrier gas: helium, flow1.2 ml min21. Retention times: endosulfandiol 5.7 min, alpha-endosulfan 8.7 min, beta-endosulfan 10.4 min and endosulfansulfate 11.9 min. The specific fragment ions monitored forconfirmation purposes in SIM mode (GC-MS-EI) includeendosulfandiol at m/z 241, 271, and 307, alpha-endosulfan atm/z 160, 195, and 245, beta-endosulfan atm/z 159, 195, and 235and endosulfan sulfate at m/z 229, 272, and 387 (Fig. 1). Asignal-to-noise ratio of 1 : 3 is maintained throughout theexperiment. 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

Meyzieu, France, with automatic temperature recorder anddisplay facility was used for storing the samples at245 uC. Representative chromatograms are presented inFig. 2 and 3.

Reagents

All the chemicals and reagents used in the studies were orga-nic trace analysis grade unless stated otherwise. They werepurchased from E. Merck, Darmstadt, Germany. Referenceanalytical standards of alpha-endosulfan, beta-endosulfan,and endosulfan sulfate were supplied by Dr. Ehrenstorfer-Schafers, Augsburg, Germany. Stock standard solutions ofeach containing 10 mg ml21 were prepared in acetone andstored at 245 uC. Known volumes of these solutions weremixed and diluted to obtain the working standard solutions.

Recovery and fortification

For experimental purposes, heparinized blood samples werecollected from the donors and stored in the deep freezer at215 uC. 20 ml of reference analytical working standardsolutions of endosulfandiol, alpha-endosulfan, beta-endosulfan,endosulfan sulfate were spiked into 2 ml of blood sample andvigorously shaken for homogeneity. Various known concen-trations were fortified and stored in the deep freezer beforeanalysis.

Extraction of endosulfan residues from blood samples

To a blood sample were added the following: cold sulfuric acid60% (10 uC) solution in the order 1.5 ml1 1.5 ml1 2.0 ml withan interval of 10–15 s between each addition and this wasmixed well in a separatory funnel; 10 ml of a 9 : 1 hexane–acetone mixture was then quickly added. After vigorousshaking for 2 min the sample was centrifuged for about10 min at 3000 rpm. The solvent layer was collected and theprocess repeated thrice using 10 ml of 9 : 1 hexane–acetonemixture. The hexane–acetone layer was collected each time andcombined and then evaporated to 3.0 ml under a stream ofnitrogen at 45 uC. Utmost care is needed to ensure that duringthe extraction the temperature of the sample should not risebeyond 10 uC.

Collection of blood samples

Blood samples were collected from a population where intenseuse of endosulfan for agricultural purposes had been practicedfor several years. All the samples were coded and received indry ice pack with the details of the donors. Donors consistsof both females and males of various age groups from 18 to70 years. Informed consent was obtained from the donors orfrom the head of the family from whom blood was collected forthe study and the same documented in archives. About 5 mlof blood was collected from each donor for experimentalpurposes. All the samples were processed and analyzed asdescribed earlier.

Results and discussion

The presence of pesticide residues in food and environmentalsubstrates may have both legally and commercially importantimplications. Therefore, reproducibility, reliability, and integ-rity of analytical data is of utmost important. The literature1

clearly shows that endosulfan rapidly gets converted toendosulfandiol in the presence of sulfuric acid. Our initialexperiments43 showed very low recoveries. When conductingexperiments using sulfuric acid solution stored at roomtemperature (25 uC) emulsion formation was observed. Thismade the matrix unsuitable to proceed further. Further, the risein temperature during the extraction process also resulted in theformation of endosulfandiol. Hence subsequent studies wereconducted by using cold sulfuric acid and by maintainingthe temperature below 10 uC during extraction. Under thesedefined conditions conversion of endosulfan to endosulfandiolis found to be very low (v0.5%). It was also found that thequality of reagents has a great influence on the recovery of theanalytes. Use of analytical reagent grade solvents for extractionpurpose resulted, surprisingly, in very high recoveries ofFig. 2 GC-ECD chromatogram of 10 ng ml21 of endosulfan.

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.

J. Environ. Monit., 2002, 4, 190–193 191

endosulfan. Anticipating false positive results due to inter-ference associated with the purity of solvents, trace organicanalysis grade or residue solvents were used to minimize theseinterferences and to obtain good recoveries. Under theestablished conditions recovery studies showed that themethod is found suitable to quantify residues of alpha-endosulfan, beta-endosulfan and endosulfan sulfate up to1.0 ng ml21 and endosulfandiol up to 0.02 ng ml21 in humanblood samples. The recoveries are more than 92% (Table 1).The relative standard deviations (RSDs) and correlationcoefficients were calculated. Further the method was alsofound suitable for the determination of residues of endosulfanand it metabolites in blood samples collected from animals.No major deviations were observed in the recovery (Table 2).

Application to real samples

All the blood samples collected from the exposed populationwere analyzed for residues of endosulfan. The results showedthat none of the blood samples contains residues of endosulfan(alpha-endosulfan 1 beta-endosulfan 1 endosulfan sulfate) orendosulfandiol (Table 3). Investigations on pesticide residues incomplex substrates is always an indication of the appropriatetechnology and expertise utilized in plant protection and hasgreater importance at national and international level. Anynon-scientific way of conducting the studies and projecting theresults will always give adverse effects on society and on theenvironment. 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 showedthat there is no presence of accumulation of concentrations ofendosulfan or its metabolites in blood samples collected fromthe village population due to endosulfan exposure.

Conclusions

From the above studies it can be concluded that the presentmethod fills the gap with respect to the need for an analyticalmethod for the determination of residues of endosulfan inblood samples. Further, the method is simple and suitable forthe analysis of residues of endosulfan from human bloodsamples and also is applicable to blood samples of animal

origin. Present investigations clearly show the influence ofvarious analytical parameters in determining false positive orlow recoveries of endosulfan. The analysis of blood samplescollected from an exposed populations clearly indicated theabsence 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.

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Table 1 Recovery of total endosulfan (alpha-endosulfan 1 beta-endosulfan 1 endosulfan sulfate) in human blood samples

Spiked concentrationa/ng ml21

Recovery(%)

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192 J. Environ. Monit., 2002, 4, 190–193

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