Development of A Novel Method for the Detection of 54 Pesticides in Gcms-Ms-Tqd

A. Harinathareddy¹, N. B. L Prasad², K. Lakshmi Devi³*^, D. Ravindranath⁴, B. Ramesh⁵

¹Department of Bio technology, JNTUA, Anantapuramu.

²OTRI, JNTUA, Anantapuramu.

³Dept of Biochemistry, SK. University, Anantapuramu.

⁴Department of Bio technology, JNTUH, Hyderabad.

⁵Department of Bio chemistry, SV University, Tirupathi.

Corresponding Author Email: klakshmidevi2000@yahoo.co.in

DOI : http://dx.doi.org/10.13005/ojc/310345

ABSTRACT:

Study the residues of 54 pesticides in vegetables recovered from the developed method been validated for the extraction of pesticides of various chemical classes from vegetables such as tomato. Mixtures of 54 pesticides amenable to gas chromatography were quantitatively recovered from spiked tomato determined by using gas chromatography– Mass spectroscopy. The sample preparation approach is known as QuEChERS, which stands for “quick, easy, cheap, effective, rugged and safe”. As expected, the results are excellent and showed overall average of 98% recoveries with 10% RSD. The method involved extraction with acetonitrile, liquid–liquid partition with addition of NaCl followed by MgSO₄ and Primary secondary amine (PSA) and the analyses were carried out with GC–MS equipment. It was a rapid, simple and cost effective procedure. The spiking levels for the recovery experiments were 0.05, 0.25, 0.5 mg kg−1. Adequate pesticide quantification and identity confirmation were done. The recovery ratios, the very good accuracies and precisions, as well as the good matches between the observed ion ratios. the percent recovery of OCs in the range of 83.65%–102.92% in 0.05 mg/kg, in the range of 78.78–99.66% in 0.25 mg/kg.and in the range of 81.18–108.50% in 0.5 mg/kg respectively The percent recovery of OPs in the range of 81.45–100.60% in 0.05 mg/kg, in the range of 81.27–103.72% in 0.25 mg/kg.and in the range of 79.07–102.77% in 0.5 mg/kg respectively. The percent recovery of SPs in the range of 91.00–98.69% in 0.05 mg/kg, in the range of 76.45–110.29% in 0.25 mg/kg.and in the range of 90.56–107.40% in 0.5 mg/kg respectively. the percent recovery of Herbicides and Fungicides in the range of 93.57–98.72% in 0.05 mg/kg, in the range of 87.34–97.53% in 0.25 mg/kg.and in the range of 79.10–93.07% in 0.5 mg/kg respectively.

KEYWORDS:

Method Development; GC-MS/MS-TQD; Multi residue Method; Pesticides; Organo chlorines; Organophosphates; Synthetic Pyrethroids; Herbicide and Fungicide mixtures

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Copy the following to cite this article: Harinathareddy A, Prasad N. B. L, Devi K. L, Ravindranath D, Ramesh B. Development of A Novel Method for the Detection of 54 Pesticides in Gcms-Ms-Tqd. Orient J Chem 2015;31(3).

Copy the following to cite this URL: Harinathareddy A, Prasad N. B. L, Devi K. L, Ravindranath D, Ramesh B. Development of A Novel Method for the Detection of 54 Pesticides in Gcms-Ms-Tqd. Orient J Chem 2015;31(3). Available from: http://www.orientjchem.org/?p=10874

Six different concentrations of each category (OCs, OPs, SPs, Herbicide and Fungicide mixture) of pesticides were prepared separately for building a calibration curve. Each concentration level was injected six times, and the calculated mean value was used as the calibration point. LOD, LOQ and % RSD values were also calculated for each pesticide.

Figure 3: Linearity curve for Organo phosphates  Click here to View figure

 

Six calibration standard solutions (0.05, 0.1, 0.15 , 0.20 ,0.25 ,0.30 ppm) of OCs, Alpha HCH ,Aldrin, Dieldrin, Beta HCH, Gamma HCH, Delta HCH, Heptachlor, Dicofol, 2,4 DDE, Alpha endosulfan 4,4 DDE ,2,4 DDD, Beta endosulfan,4,4 DDD, 2,4 DDT, 4,4 DDT, Endosulfan sulphate. OPs Dichlorvos, Monocrotophos, Diazinon, Phorate, Methamidophos, Dimethoate, Methyl parathion, Chloropyrifos methyl, Fenitrothion, Malathion, Azinphosethyl, Triazophos, Chloropyrifos, Quinalphos,Profenophos, Phosphomidon, Chlorfenvinphos, Parathion, Fenamiphos, Ethion, ,Phosalone, Synthetic Pyrethroids(SPs) Bifenthrin, Fluvalinate, Fenvalarate, Fenpropathrin, Deltamethrin, Lamdacyhalothrin, Cypermethrin, Alpha cypermethrin,Permethrin, Cyfluthrin, Herbicides Atrazine, Alachlor, Butachlor Fungicides Hexaconazole Trifloxystrobin, Fipronil of other insecticides were prepared by adding different volumes of the composite standard solution and injected on GC-MS/MS-TQD. Tomatoes were collected from the field of Student farm, College of Agriculture, Professior Jayasankhar Telangana state Agricultural university, Rajendranagar, Hyderabad, Telangana, India.

Figure 4: Linearity curve for Synthetic Pyrethroids  Click here to View figure

 

Extraction and Cleanup

The collected fresh vegetable sample (100 g) was chopped, and grind in warring blander. 15 g sample of each vegetable in triplicate was taken for multi-pesticide residue analysis by QuEChERS method. Fifteen grams of macerated sample was mixed with 30 ml acetonitrile and 3gms of Nacl was added and centrifuged at 2500rpm then 9 gms of Sodium sulphate was added to removes water content, and vortexed for 10 min at 50 rpm on rotospin test tube mixture. The extract was centrifuged for 10 min at 10,000 rpm. One milliliter aliquot of supernatant extract was cleaned with the mixture of 0.4 gmsPSA,1.2gms anhy. MgSO4 and 10 mg activated charcoal. The extract was again shaken for 10 min at 50 rpm on rotospin and centrifuged for 10 min at 10,000 rpm. The 2ml of supernatant was collected and evaporated with Turbo vap and finally made up to 1ml with Hexane. One microliter of clean extract was used for the multi pesticide (OCs, SPs, OPs and Herbicides and Fungicides) residues analysis.

The final sample were analyzed on (Brukers 436 GCMS) equipped with fused silica capillary column Factor Four (30 mt×0.25 mm id) coated with 1% phenyl-methyl polysiloxane (0.25 µm film thickness) using Brukers 5ms column. General operating

conditions were as follows: Column temperature program: initially 90⁰C hold for 3min; increase to 150⁰C @20⁰C hold for 6 min; increase to 220⁰C @ 20 C hold for 5min; increase to 280⁰ c @ 50c/min hold for 5min. Total 63 min. injection volume: 1 µl nitrogen flow rate 1 ml/min and makeup 25 ml/min with split ratio 1:10; using carrier gas Helium 99.9%; Injector port temperature 260^◦C, Detector parameters are Source- Triple Quadrupole, Mass Range – 50-400, Transfer line temp -250⁰C, Source temp -220⁰C, Manifold temp- 40⁰C.

Figure 5: Linearity curve for Herbicides, Fungicides and other pesticides  Click here to View figure

 

Results and Discussion

Eight point linearity curve was drawn by injecting OC mix, OP mix, SP mix, Herbicide and Fungicide mix. Regression values were also calculated from Linearity for each pesticide. All the concentration mentioned above were injected six times in order to calculate % RSD values given in Table (2).

Table 2: Pesticide Correlation coefficient and Relative standard deviation values    Click here to View table

 

It is seen that in Table (2) 54 GCMS six concentrations injected six times and calculated the Regression value and % RSD value. In OCs, Alpha HCH , Aldrin, Dieldrin, Beta HCH, Gamma HCH, Delta HCH, Heptachlor, Dicofol, 2,4 DDE, Alpha endosulfan 4,4 DDE ,2,4 DDD, Beta endosulfan, 4,4 DDD, 2,4 DDT, 4,4 DDT, Endosulfan sulphate shows the Regression value ranged from 0.991-0.999 and percentage of Relative standard deviation is in between 0.29-6.14( Table 2) . LOD of OC pesticides varied in the range of 0.001–0.005 mg kg−1.Similarly, the percent recovery of OCs in the range of 83.65%–102.92% in 0.05 mg/kg, in the range of 78.78–99.66% in 0.25 mg/kg.and in the range of 81.18–108.50% in 0.5 mg/kg respectively (Table 3).

Table 3: Fortification and recovery in Tomato in the level of 0.05 mg kg-1, 0.25 mg/kg-1, 0.5 mg/kg-1  Click here to View table

 

In OPs, Dichlorvos, Monocrotophos, Diazinon, Phorate, Methamidophos, Dimethoate, Methyl parathion,Chloropyrifos Methyl,Fenitrothion, Malathion, Azinphos ethyl, Triazophos,Chloropyrifos, Quinalphos, Profenophos, Phosphomidon, Chlorfenvinphos, Parathion,Fenamiphos, Ethion, Phosalone shows the Regression value ranged from 0.991-0.998 and percentage of Relative standard deviation is in between 0.31-6.02 (Table 2). LOD of OP pesticides varied in the range of 0.001–0.005 mg kg−1. Similarly, the percent recovery of OPs in the range of 81.45–100.60% in 0.05 mg/kg, in the range of 81.27–103.72% in 0.25 mg/kg.and in the range of 79.07–102.77% in 0.5 mg/kg respectively (Table 3) .

In SPs, Bifenthrin, Fluvalinate,Fenvalarate,Fenpropathrin,Deltamethrin,, Lamda cyhalothrin, Cypermethrin, Alpha cypermethrin,Permethrin, Cyfluthrin shows the Regression value ranged from 0.991-0.999 and percentage of Relative standard deviation is in between 0.58-8.24 ( Table 2) Similarly, the percent recovery of SPs in the range of 91.00–98.69% in 0.05 mg/kg, in the range of 76.45–110.29% in 0.25 mg/kg.and in the range of 90.56–107.40% in 0.5 mg/kg respectively (Table 3) .

In Herbicides and fungicides Alachlor, Butachlor, Atrazine, Hexaconazole Fungicides Trifloxystrobin, Fipronil of other insecticide shows the Regression value ranged from 0.994-0.999 and percentage of Relative standard deviation is in between 0.77-3.79 (Table 2) . Similarly, the percent recovery of Herbicides and Fungicides in the range of 93.57–98.72% in 0.05 mg/kg, in the range of 87.34–97.53% in 0.25 mg/kg.and in the range of 79.10–93.07% in 0.5 mg/kg respectively (Table 3) .

The use of acetone in place of acetonitrile in QuEChERS method has many advantages, but it has low recovery compared to acetonitrile and also it is difficult to analyse in LC. The use of acetonitrile in QuEChERS method has good recovery including its ability to separate from water upon the addition of salt without the addiction nonpolar solvent and amenability with GC and LC applications.

Mixtures of 17 OCs at 6 different levels six times were injected in to GC-MS for drawing the Linearity curve, Regression value ranged from 0.991-0.999 and percentage of Relative standard deviation is in between 0.29 to 6.14 for the all Organo chlorines.

Mixture of 21 OPs at 6 different levels were injected 6 times in to GC-MS for drawing linearity curve. Regression value ranged from 0.991-0.999 and percentage of Relative standard deviation is in between 0.31-6.02 for all the organo phosphates.

Mixture of 10 SPs at 6 different levels was injected 6 times GC-MS for drawing Linearity curve. Regression value ranged from 0.991-0.999 and percentage of Relative standard deviation is in between 0.58-8.24 for all the Synthetic pesticides.

Mixture of Herbicides and Fungicides at 6 different levels were injected 6 times in to GC-MS for drawing linearity curve. Regression value ranged from 0.994-0.999 and percentage of Relative standard deviation is in between 0.77-3.79 for all the Herbicides and Fungicides and Other Pesticides.

Conclusion

Multi residue Method for determination of 54 pesticides of Different categories Viz, OCs, OPs, SPs, Herbicide and Fungicide mix has been developed. All the pesticides were separated and could be analyzed on single method. Linearity curve with all the Regression values ≥ 0.940 and % RSD values in permissible range from 0.29 to 8.24 were calculated.

Acknowledgments

The authors express their gratitude to the Head if the Department, AINP on pesticide residues providing the research facilities and support.

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