K. Karunambigai et al. / JPBMS, 2012, 22 (20)

1 Journal of Pharmaceutical and Biomedical Sciences © (JPBMS), Vol. 22, Issue 22

Available online at www.jpbms.info JPBMS

JOURNAL OF PHARMACEUTICAL AND BIOMEDICAL SCIENCES

Reverse phase High Performance Liquid Chromatographic determination of residual

Carbendazim (fungicide) in raw and cooked Indian cereals and pulses

K. Karunambigai*, C. Saravanan, C. A. Sureshkumar, K. Kaveri, M. Thamizhmozhi

Aadhibhagawan College of Pharmacy, Rantham, Cheyyar-604407, Thiruvannamalai, Tamil Nadu, India. Abstract: The present work describe a reverse phase HPLC method for line quantification of carbandazim in raw and cooked Indian Cereals as pulses the retention time of carbandazim is 2.85minutes in RP HPLC using 10%V/V phosphate butter in acetonitrile water in ratio 17.37:46 as mobile phase and c18 (5u 250X4.6mm)by hypersil column as stationary phase . The effluent was monitored by UV detection at 230nm the method was validated for accuracy, precision, specificity in this study 15 samples of cereals by pulses of different varieties were analyzed for residues of carbandazim in raw as well as cooked forms. The maximum residual limit for cerbandazim residues in cereals and pulses is 0.5 mg/kg. The analyzed amount of carbandazim residues in raw sample of cereals and pulses was 0.0320-18.8171 mg/kg by collected samples 0.003-0.6776 mg/kg. The carbandazin plots were within range of 0.1 to 100 mg /ml (R.0.9995), the observed percentage recovery of carbandazim was found to be 90.09-102-82% which indicated good accuracy and reproducibility of the method. Key words: RP-HPLC, Carbendazim, Cereals, Pulses, Determination

Introduction: The most obvious and rational concern has been for the presence of pesticides in human food and feed. In order to limit the contamination of food with chemical residues, it has been customary to fix administrative action levels to gauge whether chemicals have been used in accordance with registered directions and good agricultural practices. Maximum Residue Limits (MRL) shows the maximum residue that could result when the chemical is used according to approved directions, when the crop is harvested, on the stored grain or when the cereal product is processed[1,2]. Residues greater than the MRL shows that the chemical has been misused or good agricultural practice has not been followed. Carbendazim or MBC [methyl-2-benzimidazole carbamate] is a widely used systemic fungicide that controls a wide range of pathogens on a broad range of field crops like paddy, cereals, fruits, vegetables[3-14], etc. In India, its consumption is 600-700 tonnes/year and it is marketed under more than fifteen brand names. It is also a most common degradation product of benomyl [methyl-1-butylcarbomoyl-2-benzimidazole carbamate]. Several analytical techniques like spectroscopy and gas liquid chromatographic methods were developed for determination of carbendazim in various substrates. HPLC methods were also reported for analysis of carbendazim[15-

20] in various substrates like cow milk, urine, faeces, etc. Carbendazim usage specifies rates from 0.2 to 2 kg ai/ha and application is once in year by spraying with intervals of 7 to 14 days. It is formulated as an aqueous dispersion or suspension, wettable powder or dispersible powder. The maximum residue limits for carbendazim are given by

Food & Agricultural organization and World Health Organization. Materials and Methods: Collection of Cereals and Pulses: About 15 varieties of cereals and pulses were collected from different parts of Tamilnadu. The collected samples were stored in well closed container without any addition of preservatives. The sample collection was done during June–September (2002). The varieties of cereals and pulses analysed for carbendazim are presented in table 1. Reagents and Chemicals: All the chemicals used were of analytical grade and procured from E. Merck, India. The chemicals used for the study were Ethyl acetate, Chlorofrom, Sulphuric acid, Sodium hydroxide, Dichloromethane, Anhydrous sodium sulphate, Acetonitrile HPLC grade and Triethyl amine. Triple glass distilled water was used. Carbendazim (reference standard) was generously gifted by international Institute of Biotechnology and Toxicology, Padappai 601301 India. Instrumentation: The instruments employed for residual analysis of carbendazim were High performance liquid chromatography Shimadzu, LC6A, SPD-10ATVP with UV detector, Injection: Rheodyne needle with hamiltone loonadzu schweinge syringewith 20ul loop, Column: C18 hypersil, 250x 4.6mm, 5u particle size, Electronic single pan balance - (shimadzu Libror AEG, 220), Digital PH meter – Model IIIE, Rotary vacuum evaporator.

ISSN NO- 2230 – 7885

CODEN JPBSCT NLM Title: J Pharm Biomed Sci.

Research article

K. Karunambigai et al. / JPBMS, 2012, 22 (20)

2 Journal of Pharmaceutical and Biomedical Sciences © (JPBMS), Vol. 22, Issue 22

Instrumentation and operational conditions for RP- HPLC Mode of operation : Isocratic Temperature : Ambient Flow rate : 1ml/min Load : 20ul UV detection : 282 nm Injection interval : 10 min

Mobile phase: The mobile phase used was 10% w/v PH 7 Phosphate buffer in acetonitrile water in the ratio of 17:37:46. Preparation of buffer: The phosphate buffer was prepared by mixing solutions of 0.067 M disodium hydrogen phosphate (Na2 HPO4) and 0.67M potassium dihydrogen orthophosphate (KH2 PO4) AT the ratio of 3: (v/v), so that the ph of the prepared buffer was 6.98 = 0.05. Preparation of standard solution: An accurately weighed quantity of 100 mg of carbendazim was dissolved in minimum quantity of o.1 N hydrochloric acid and made up 100 ml with acetonitrile to obtain 1000 g/ ml of carbendazim solution. The stock solutions were further diluted to get different concentration and a calibration curve was plotted. Quantitati Ve Analysis of Carbendazim: Linearity and calibration: Calibration of instruments is essential in obtaining accurate analysis. The linearity of an analytical procedure is its ability to obtain test results within a given rage which are directly proportional to the concentration of analyte in the sample. A minimum of 5 concentrations are required for linearity. It is evaluated by linear regression analysis of the plot of signals as a function of analyte concentration. The correlation coefficient and slope of regression line should be satisfactory. Varying quantities of stock solution of carbendazim was diluted with acetonitrile to give different concentration and the injections were made at an interval of 10 minutes. The peak areas were measured and tabulated. The detector response (UV AT 282 nm) of the developed RP-HPLC protocol by standard solutions of carbandazim (0.1to 100 g/ml in mobile phase) were measured. Quantitative Analysis of Carbendazim in Raw Cereals and Pulses: About 20 g of sample was weighed followed by extraction and clean up process. Extraction: About 20 g of the sample was weighed and homogenized in a heavy duty waring blender and then extracted with 100 ml of ethylactate and filtered. The solid residue was re-extracted with 50 ml of ethylacetate and again filtered. The extracts were combined and evaporated to dryness on a rotary vaccum evaporator. The residues were dissolved in 3x 20 ml of 0.5 N sulphuric acid and passed through a funnel with a glass wool plug into a separating tunnel for further clean up.

Clean up: The acid extract was washed with 3x40 ml of chloroform and the chloroform layers were discarded after phase separation. The PH of the acidic aqueous layer was adjusted between 8.5 to 9 with 6% V/V aqueous sodium hydroxide. Then the solution was extracted with 2x25 ml of dichloromethane. The extracts were combined and dried by passing it through anhydrous sodium sulphate and evaporated to dryness on a rotary vacuum evaporator. The residues were immediately dissolved in 5 ml of water: acetonitrile (55:45) solution filtered through 0.2m membrane filter and analysed by RP-HPLC. Quantitative Analysis of Carbendazim in Cooked Cereals and Pulses: About 20 g of sample was weighed and cooked under steam. The extraction and clean up procedure was done as per that of the raw samples. the sample solutions were then filtered through 0.2 m membrane filter and analysed by RP-HPLC. The sample were fortified with 1 g of carbendazim and injected. Each solution was run thrice at an interval of 10 min to ensure elution of earlier injection. The amount of carbendazim present in both raw and cooked cereals was calculated by comparing the sample peak area with the standard peak area. Amount of Carbendazimin in each sample per kg= Sample peak area × Concentration of standard× Dilutiion factor × 1000

Standard peak area × weight taken Results and Discussion: The UV spectrum of carbendazim is presented in Fig.1. The chromatogram of carbendazim standard, in raw cereals and pulses and in cooked cereals and pulses (RP-HPLC) under the experimental condition were presented in Fig .2, 3 & 4 respectively.

Figure 1. UV Spectrum of Carbendazim

Figure 2. Chromatogram of Carbendazim standard

K. Karunambigai et al. / JPBMS, 2012, 22 (20)

3 Journal of Pharmaceutical and Biomedical Sciences © (JPBMS), Vol. 22, Issue 22

Figure 3. Chromatogram of Carbendazim in raw cereals and pulses

Figure 4. Chromatogram of Carbendazim in Cooked cereals and pulses

The linearity data of carbendazim is presented in Table 2. The system sutability data is presented in table 3. The LOD and LOQ of carbendazim are presented in Table 4. Table 5 represents quantitative data and statistical analysis for raw cereals and pulses after extraction and cleanup for determination of carbendazim. Table 6 represents the quantitative data and statistical analysis for cooked cereals and pulses samples after extraction and cleanup for the determination of carbendazim after fortification with 1 ug of carbendazim. Table 7 presents the recovery data of carbendazim in the developed protocol. The comparison of

carbendazim content is raw and cooked cereals and pulsesis presented in Table 8. Table 1. Varieties of Cereals and Pulses samples analysed for Carbendazim

Sample no Common name

I Horse gram

II Millet

III Green gram

IV Red gram

V Groundnut

VI Bengal gram

VII Barley

VIII Oats

IX Maize

X Black gram

XI Ragi

XII Sorghum

XIII Chickpea (Black)

XIV Wheat

XV Chickpea (White )

Table 2. Linearity data of Carbendazim

S.no Concentration ( g/ml) Peak area

1 0.1 133796

2 0.2 266180

3 0.3 452383

4 0.4 593819

5 0.5 762128

6 1 1272409

7 2 2625585

8 3 4357796

9 4 5230370

10 5 7004253

11 10 9390596

12 20 21527424

13 30 32554304

14 40 44767305

15 50 56555117

16 100 114672844

Table 3. System suitability data of the developed method

Parameter Carbendazim Nuber of Theoritical Plates 1357

Tailing factor 1.12 Table 4. Limit of detection and Limit of quantification data of Carbendazim in present study

Table 5. Quantitative analysis of Carbendazim in raw cereals and pulses

Sample Peak Areas

Amount Mg/kg

Standard Deviation

X10-3

Relative Standard Deviation

Standard Error of

Mean x 10-3

I 40343224 18.8171 0.252 0.134 0.145

II 9995829 2.0276 0.289 0.014 0.231

III 2072117 0.9521 0.4 0.042 0.176

IV 6363779 2.9612 0.306 0.013 0.176

Parameter Carbendazim ug/ml Limit of Detection 0.0825

Limit of Quantification 0.2751

K. Karunambigai et al. / JPBMS, 2012, 22 (20)

4 Journal of Pharmaceutical and Biomedical Sciences © (JPBMS), Vol. 22, Issue 22

V 588189 0.2736 0.252 0.0092 0.145

VI 669525 0.3101 0.252 0.081 0.145

VII 69257 0.0320 0.153 0.477 0.882 VIII 2072810 0.9732 0.351 0.036 0.203

IX 232377 1.0956 0.212 0.019 0.122

X 318709 0.1488 0.321 0.202 0.173

XI 2642259 1.2277 0.208 0.017 0.120

XII 773906 0.3635 0.3 0.083 0.173

XIII 253633 0.1171 0.2 0.171 0.115

XIV 289658 0.1350 0.154 .0113 0.088

XV 259208 0.1219 0.361 0.296 0.208

Table 6. Quantitative analysis of Carbendazim in cooked cereals and pulses

Sample Peak Areas

Amount Mg/kg

Standard Deviation

X10-3

Relative Standard Deviation

Standard Error of

Mean x 10-3

I 176416 0.0484 0.257 0.535 0.148 II 547796 0.2579 0.222 0.086 0.128

III 731513 0.3621 0.252 0.069 0.145 IV 121482 0.0174 0.265 1.521 0.153 V 317552 0.1289 .0252 0.195 0.145 VI 308340 0.1211 0.268 0.219 0.154 VII 100329 0.0054 0.264 0.049 0.153

VIII 664291 0.3034 0.400 .0132 0.231 IX 757172 0.3759 0.202 0.053 0.115 X 126578 0.0201 0.158 0.787 0.091 XI 251739 0.6776 0.252 0.037 0.176 XII 251736 0.0896 0.306 0.341 0.176

XIII 93613 0.0016 0.233 14.446 0.134 XIV 154470 0.0358 0.252 0.707 0.145 XV 91465 0.0003 0.153 41.659 0.0882

*Each value is mean of three readings

Table 7.Recovery studies of Carbendazim Sample Area of plain

Cooked samples Area of spiked

Samples Area of plain And standard %Recovery

I 513426 1058610 1057823 100.07

II 2764829 3284630 3309226 99.26 III 3882468 4386588 4426865 90.09

IV 174832 728382 719229 101.27

V 1434978 1907790 1979375 96.38 VI 1267853 1848196 1812250 101.98

VII 55982 599436 600379 99.84

VIII 3452620 3925698 3997017 98.21 IX 3894321 4542678 4438718 102.34

X 7025261 7749564 7569658 95.18 XI 7025261 7749564 7569658 95.18

XII 979423 1507320 1523820 98.92

XIII 17632 559044 562029 99.46 XIV 354382 546978 547339 99.93

Area of standard Carbendazim (1g/ml) = 544397. Table 8. Comparison of Carbendazim content in raw and cooked cereals and pulses

Sample Amount in raw Samples (mg/kg )

Amount in cooked Samples (mg/ kg )

I 18.8171 0.484* II 2.0276 0.2579* III 0.9521 0.3621*

IV 2.9612 0.0174* V 0.2736 0.1289* VI 0.3101 0.1211* VII 0.0320 0.0054* VIII 0.9732 0.3034* IX 1.0956 0.3759*

X 0.1488 0.201* XI 1.2277 0.6776* XII 0.3635 0.0896*

XIII 0.1171 0.0016* XIV 0.1350 0.0358*

XV 0.1219 0.003* The wavelength for quantification of carbendazim was selected as 282 nm. The linearity lies in the range of 0.1-100 ug/ ml (r=0.9995). The retention time of carbendazim is 2.85 minutes in RP-HPLC using 10%v/v phosphate butter in acetonitrile – water in the ratio of 17:37:46 as

K. Karunambigai et al. / JPBMS, 2012, 22 (20)

5 Journal of Pharmaceutical and Biomedical Sciences © (JPBMS), Vol. 22, Issue 22

mobile phase and C18 (5u , 250x 4.6mm) hypersil column as stationary phase. In the present study, 15 samples of cereals and pulses of different varieties were analysed for residues of carbendazim in raw as well as cooked forms. The maximum residual limit for carbendzim residues in cereals and pulses was 0.5 mg / kg. The amount of analysed carbendazim residues in raw samples of cereals and pulses was 0.0320-18.8171mg/kg, and in cooked samples of cereals and pulses is 0.003-0.6776 mg/kg, Out of the 15 raw cereals and pulses samples taken for study only 8 samples are found to have their residues within the maximum residual limit. The remaining 7 samples have their residues beyond the maximum residual limit specified by Food & Agricultural Organisation and World Health Organization. It was found that cooking reduces the residues of carbendazim significantly (p<0.001). From the 15 samples analysed, only one lies outside the limits. Though in raw samples 7 samples exceeded their limits, cooking reduced the amount and only on exceeded the limit. The accuracy, precision, specificity, suitability and validity of the developed RP=HPLC method was further verified by recovery studies. The observed percentage recovery of carbendazim was found to be 90.09-102.82 % which indicated good accuracy and reproductibility of the method. Summary and Conclusion: In the present study, RP-HPLC method was developed for the analysis of residual carbendazim in raw and cooked samples of cereals and pulses. The developed RP-HPLC method utilises 10 % v/v phosphate buffer in acetonitrile and water in the ratio of 17:37:46 as mobile phase and hypersil C 18 (5u, 250 x 4.6mm) colum as a stationary phase. The eluted carbendazim was detected at 282 nm. It was observed that in raw samples, 7 samples exceeded their maximum residual limits specified by Food &Agricultural Organisation and World Health Organization. The process of cooking reduced the carbendazim content significantly (p<0.001) and only one sample exceeded the maximum uses of the fungicide carbendazim. The recovery studies showed that the percentage recovery was found to be 90.09 -102.82 %. From the above analytical data and recovery studies it was found that the developed RP-HPLC method for the quantification of residual carbendazim was accurate and precise. Acknowledgements: The authors are thankful to our chairman and directors, Aadhibhagawan College of pharmacy, Rantham, Cheyyar, Thiruvannamalai district, Tamilnadu for providing necessary facilities to carry out this research work. Reference: 1. Sharma, J. Pesticides. Anal. Chem. 1993; 63: 40R-50R. 2. Clarke, S. Isolation and identification of drugs. 2nd edn.

The Pharmaceutical Press, London. 1986; 70-86. 3. Banarjee, K. Sawant, S.D; Sawant, I.S. Persistence of

benomyl in grapes. Pesticide Res J, 2001; 13(1): 106-108.

4. Ott, D.E; Gunther, F.A. Colorimetric method for field screening above-tolerance parathion residues on and in citrus fruits. J.Assoc. Off. Anal. Chem., 1983; 66(1): 108-10.

5. Appaiah, K.M; Kapur, O; Nagaraja, K.V. Colorimetric determination of propoxur and its residues in vegetables. J.Assoc. Off. Anal. Chem., 1983; 66(1): 105-7.

6. Startin, J. R.; Sykes, M.D; Taylor, J.C; Hird, S.J; Jackson, K; Fussell, R.J; Hill, A.R.C. Determination of residues of pirimicarb and its desmethyl-formamido metabolites in fruits and vegetables by liquid chromatography- electrospray/ mass spectrometry. Journal of AOAC International, 2001; 83(3): 735-41.

7. Valenzuela, A.I; Pico, Y; Font, G. Determination of five pesticide residues in oranges by matrix solid-phase dispersion and liquid chromatography to estimate daily intake of consumers. Journal of AOAC International, 2001; 84(3): 901-9.

8. Corcia, A.D; Crescenzi, C; Lagana, A. Evaluation of a method based on liquid chromatography / electrospray / mass spectrometry for janalyzing carbamate insecticides in fruits and vegetables. J.Agric. Food. Chem., 1996; 44(7): 1930-8.

9. Fernandez-Alba, A.R; Tejedor, A; Aguera, A. Determination of imidacloprid and benzimidazole residues in fruits and vegetables by liquid chromatography-mass spectrometry after ethyl acetate multi residue extraction. Journal of AOAC International, 2000; 83(3): 748-55.

10. Torres, G.M; Pico, Y; Marin R; Manes J. Evaluation of organophosphorous pesticide residues in citrus fruits from the valencian community (Spain). Journal of Chromatography, 1997; 80(5): 1122-8.

11. Bushway R.J. High performance liquid chromatographic determination of carbaryl in fruit juices. Journal of Chromatography, 1988; 457: 437-441.

12. Ishii Y; Kobori I; Araki Y; Kurogochi S; Iwaya K; Kagabu S. HPLC determination of new insecticide imidacloprid and behaviour in rice and cucumber. J.Agric.Food.Chem, 1994; 42(12): 2917-21.

13. Maeda M; Tsuji A. Determination of benomyl and 2-(4-thiazolyl) benzimidazole in plant tissues by High Performance Liquid Chromatography using fluorimetric detection. Journal of Chromatography, 1976; 120: 449-55.

14. Chiba M; Veres D.F. High Performance Liquid Chromatographic method for simultaneous determination of residual benomyl and methyl -2- benzimidazole carbamate on apple foliage without cleanup. J.Assoc. Off. Anal. Chem, 1980; 63: 1291-95.

15. Sharma D, Aswathi M.D. High Performance Liquid Chromatogralphyic method for analysis of Carbendazim residues in fruits and vegetables. Pesticide Res. J, 1991; 11: 123-6.

16. Aharonson N; Kafkafi U. Adsorption of benzimidazole fungicides on montmorillonite and kaolinate clay surfaces. J.Agri.Food.Chem, 1975; 23: 434-7.

17. Austin D.J; Briggs G.G; Lord K.A. Problems in the assay of residues of Carbendazim and its precursors. Proc. 8th Brit. Insec. Fung. Conf, Brighton, 1975.

K. Karunambigai et al. / JPBMS, 2012, 22 (20)

6 Journal of Pharmaceutical and Biomedical Sciences © (JPBMS), Vol. 22, Issue 22

18. Lerouse P; Gredt M. Adsorption of methyl benzimidaziloe -2- yl carbomate by corn roots. Pestic. Biochem. Physiol, 1975; 5: 507-14.

19. Rouchaud J.P; Decallonne J.R; Meyer J.A. Metabolic fate of methyl -2- benzimadazole carbamate in melon plants. Phytopathology, 1974; 64: 1513-7.

20. Baker P.B; Hoodless R.A. Analytical methods for the detection and determination of residues of systemic fungicides. Pestic. Sci, 1974; 5: 465-72.

Corresponding Author:- Mr.C.Saravanan, Asst. Professor, Aadhi Bhagawan College of Pharmacy, Rantham, Cheyyar-604407, Thiruvannamalai District, Tamil Nadu

Quick Response code (QR-Code) for mobile user to access JPBMS website electronically. Website link:- www.jpbms.info