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INTRODUCTION The toxicological impact of insecticidal

compounds is increasing day by day as aconsequence of their rapid and widespreadapplication. It is used as ectoparasiticide inanimal, production of agricultural crops andalso public health programme. The consequenceof indiscriminate use include apart from healthhazards to non-target organisms, pollution ofecosystem and accidental poisoning. In fieldcondition the pesticides poisoning is commondue to accidental, malicious poisoning andimproper use of pesticides by farmer, but thedetection is very rare or undetected due to lackof analytical laboratory for identification and

quantification of residue of pesticides and othertoxin. Present study was undertaken to ascertainwhether the cause of death of large number ofgoats was due to pesticide poisoning and if so,the identification and quantification of thepesticides by HPLC method.

MATERIALS AND METHODSThe viscera of six (6) animals (random

sampling) like stomach contents and liver, lungsalso blood smear were received from projectco-coordinator, Animal Disease ResearchInstitute, Cuttack, Orissa for diagnosis of causesof death of goats in the farm. It was suspectedthat, surrounding the farm field the insecticide

Research Article

HPLC WITH SOLID PHASE EXTRACTION FORIDENTIFICATION AND DIAGNOSIS OF

ORGANOPHOSPHOROUS POISONING IN GOATS

S. Manna* B.B. Roy, A.G. Bandopadhyay1

Institute of Animal Health & Veterinary Biologicals, 37 Belgachia Road, Kolkata -700037, West Bengal,India.1Additional Director (Health) ARD, Govt. of West Bengal, India.*Corresponding author. e-mail: drmannasukumar@gmail.com.

ABSTRACT: High performance liquid chromatographic determination of organophosphorouscompound has been done by reverse phase chromatography in goats. The goats were dying showingthe symptoms of organophosphorous poisoning. The viscera and stomach contents sample were receivedfrom Project Co-Ordinator, Animal Disease Research Institute, Phulnakhara, Cuttack, Orissa. Theanalysis of samples by HPLC with UV detector after cleaning up in Solid Phase Extraction (SPErevealed presence of malathion that was later quantified.

Key words: HPLC, SPE, Malathion, Goats.

Explor Anim Med Res,Vol.4, Issue - 2, 2014, p. 235-239

ISSN 2277- 470X (Print), ISSN 2319-247X (Online)Website: www.animalmedicalresearch.org

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Exploratory Animal and Medical Research, Vol.4, Issue 2, December, 2014

Fig. 1: LC Calibration report of Malathion.

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Fig. 2: Chromatogram of unknown sample.

may be sprayed and after the return from grazingthe animals showed frothy salivation, withnervous symptoms, colic, constriction of pupil,tetany etc. and out of 150 goats, there was deathof 90 goats. The blood smears were examined.The liver and lungs were taken for

histopathological examination.The liquid chromatography consisted of

Millipore Waters (Milford, USA) model 1525pumps, 2487UV detector. The column was aWaters reversed phase C18 (Nova pack)stainless steel column (150mm × 3.9mm I.D,

HPLC with solid phase extraction for identification and diagnosis....

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Exploratory Animal and Medical Research, Vol.4, Issue 2, December, 2014

and also this method (HPLC) is now widelyused according to Watanabe et al. (2014).Tissues (lungs and liver) were extracted for 4min with acetonitrile and anhydrous sodiumsulfate (0.4%) using a homogenizer. The extractwas filtered through anhydrous sodium sulfate(0.5 g) and the tissues were re-extracted twicewith acetonitrile . The extract was clarified bycentrifugation and filtered through anhydroussodium sulfate. The combined acetonitrileextracts were concentrated to 20 ml andpartitioned with hexane. The hexane phaseswere discarded and the acetonitrile phase wasevaporated to dryness using a rotary vacuumevaporator. The volumes were finally adjustedto 5 ml with acetonitrile for HPLC estimation

5µm) equipped with a waters guard packcolumn module. Solid Phase Extraction (SPE,Waters) was used for purification and clean upof the sample. The analytical method wasmodified and developed according to methodof Abu-quare et al. (2001).

Reagents: HPLC grade acetonitrile, waterand all other reagents / chemicals were procuredfrom E. Merck (India). The standard compoundof pesticides like malathion, etc were obtainedfrom (Supelco-Sigma-Accustandard) andresidue of organophosphorus compound insample was compared to external standard.

Residue level determination: The tissueresidue level of the unknown compound wasestimated by the method of Manna et al. (2006),

Fig. 4. Photomicrograph of liver of goat showingmild congestion of the blood vessels (H&E, 100 X).

Fig. 3. Photomicrograph of lungs of goat show-ing congestion (H&E, 100 X).

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after purified and clean up by solid phaseextraction (SPE). A stock solution of 1 mg perlitre of malathion (analytical grade >99%) wasprepared as an external standard. Acetonitrileand water were used as mobile phase in gradientmanner and the retention time (RT) ofmalathion was 7.46 min. The data wererecorded and calibration curve of malathion hasbeen done (Fig. 1). The method has beenrepeated 4 times for validation of the assay With10 ml Cap. Hamilton Syringe, 2 ml of standardand samples were injected into HPLC.

Histopathological examination: Smallpieces of lungs, liver, were fixed in 10% neutralbuffered formalin. Sections of 3-5m thicknesseswere cut and stained with haematoxylin andeosin (H&E) for observation under lightmicroscope.

RESULTS AND DISCUSSION The antimortem symptoms were frothy

salivation, nasal discharge, nervous symptoms,colic, constriction of pupil etc. The postmortemsymptoms were bloat and inflammation instomach and intestine, haemorrhages (Fig. 3,4) in lungs and intestine. The histopathologicalchanges in liver and lungs showed congestion,hemorrhages. The examination of blood smearshowed no pathogenic organisms. The analysisconfirmed the presence of malathion and themethod of extraction –purification gave verygood results without fortification of the sample(Fig.2) The detected residue level (averagewithin six samples) of malathion was 5277ppb,

and the death of the animals were due toorganophosphorous compound poisoning(malathion The oral LD50 for malathion in rathas been reported to 5400mg/kg in male and5700mg/kg in female according to Zeid et al.(1993).

The detection of pesticides and itsmetabolites in animal tissue more accuratelyand specifically using Mass Spectrometry (MS)may be taken into consideration in future.

REFERENCES

Abu-Quare AW, Abu-Donia MB (2001)Simultaneous determination of malathion,permethrin, DEET (N-N-diethyl-m-toluamide) theirmetabolites in rat plasma and urine using highperformanace liquid chromatography. J PharmBiomed Anal 26(2): 291-299.

Watanabe E, Kobara Y, Baba K, Eun H (2014)Aqueous acetonitrile extraction for pesticide residueanalysis in agricultural products with HPLC-DAD.Food Chem 154: 7–12.

Manna S, Bhattacharyya D, Mandal TK, Dey S(2006) Neuropharmacological effects ofdeltamethrin in rats. J Vet Sci 7(2): 133-136.

Zeid MMA, El-Barouty G, Adbdel-Reheim E,Blancato, J, Dary C, El-Sebae A H, Saleh M (1993)Malathion’s disposition in dermally and orally treatedrats and its impact on the blood serum acetylcholineesterase and protein profile. J Environ Sci HealthPart B 28 (4): 413-430.

* Cite this article as: Manna S, Roy BB, Bandopadhyay AG (2014) HPLC with solid phaseextraction for identification and diagnosis of organophosphorous poisoning in goats.Explor Anim Med Res 4(2): 235-239.

HPLC with solid phase extraction for identification and diagnosis....