Evaluation of Residues of Chlorpyrifos and its metabolite in green apples

 

Tentu Nageswara Rao¹*, Botsa Parvatamma², Karri Apparao¹, Jakka Mastan¹

¹Department of Chemistry, Krishna University, Machilipatnam, Andhra Pradesh, India.

²Department of Organic Chemistry, Gayathri PG College, Vizianagaram, Andhra Pradesh, India.

*Corresponding Author E-mail:

A simple and inexpensive method was developed using solid-phase extraction, together with gas chromatographic method and high performance liquid chromatographic method for determination of chlorpyrifos and its metabolite O,O-diethyl triphosphate residues in green apples.  The evaluated parameters include the extracts by silica gel packed column using methanol and water solvent mixture (90:10) and ethyl acetate solvents. The method was validated using green apple fruit samples spiked with chlorpyrifos and its metabolite (O,O-diethyl triphosphate) at different fortification levels (0.01 and 0.1 mg/kg). Average recoveries (using each concentration six replicates) of range 86-95%. The calibration solutions concentration in the range 0.01-5.0 mg/L and limit of detection (LOD) and limit of quantification (LOQ) were 0.003mg/kg and 0.01mg/kg respectively. Finally the green apple fruit residue samples were analyzed by GC and HPLC.

 

 

 

INTRODUCTION:

Chlorpyrifos is a broad spectrum insecticide, a chemical used to kill a wide variety of insects. It was introduced in 1965 (1). Even as initially used frequently to kill mosquitoes in the immature, larval stage of development, chlorpyrifos is no longer registered for this use¹. Chlorpyrifos is powerful in controlling a diffusion of insects, consisting of cutworms, corn rootworms, cockroaches, grubs, flea beetles, flies, termites, fire ants, and lice. it's far used as an insecticide on grain, cotton, discipline, fruit, nut and vegetable plants, and well as on lawns and ornamental plants.

 

Chlorpyrifos act upon contact, ingestion, and inhalation.^2,3 The US Environmental Protection Agency (EPA) has assessed the risks of chlorpyrifos and reached an Interim Reregistration Eligibility Decision (IRED) for this organophosphate (OP) pesticide.

 

Numerous opportunities for public comment were offered as this decision was being developed. In addition, the chlorpyrifos IRED has been issued with a public comment period. The European legislation has included chlorpyrifos in Annex I till 2016, with a pre–harvest interval (PHI) of 60, 30, 30, and 21 days and maximum residue level (MRL) of 0.2, 0.3, 0.5, and 0.5 mg kg−1 for peaches, oranges, grapes, and tomatoes, respectively.

 

This study has been undertaken to develop an improved method for analysis of chlorpyrifos and its metabolite O,O-diethyl triphosphate  to determine the residues in green apples.

 

EXPERIMENTAL:

Standards, Reagents and samples:

The analytical standards of chlorpyrifos (99.0%) and O,O-diethyl triphosphate (99.9%) were obtained from Sigma Aldrich. Acetonitrile and methanol were purchased from rankem, New Delhi, analytical grade solvents i.e., ethyl acetate, acetone and silica gel sorbet were supplied from Merck Limited and green apples were purchased from local market.

Standard stock solutions:

The chlorpyrifos and O,O-diethyl triphosphate  standard stock solutions were individually prepared in acetone and acetonitrile at a concentration level 1000 µg/mL and stored in a freezer at -18°C. The stock standard solutions were used for up to 3 months. Suitable concentrations of working standards were prepared from the stock solutions by dilution using acetone and acetonitrile, immediately prior to sample preparation.

 

Sample preparation:

Representative 50.0 g portions of green apple fortified with 0.1 mL of working standard solutions. The sample was allowed to stand at room temperature for one hour, before it was kept at refrigerator condition, until analysis.

 

Extraction procedure:

Weighed 50g of green apple fruit sample in to a 250 mL separatory funnel and 50 mL of  deionized water and shake vigorously. The mixture was vertex-mixed and centrifuged at 3000 rpm for 5 minutes. The supernatant was decanted and the above step was repeated twice and pooled sample was concentrated to 1 mL at 45°C under a steam of nitrogen.

 

Clean-up procedure:

Prepared a pre-washed silica gel chromatographic column by placing a glass wool in the bottom of the column, added 50 ml methanol. Rinsed the sides of the column with solvent methanol and placed a 15g layer of Sodium sulphate over the silica gel. Again rinsed the sides of the column with methanol and allowed the solvent to drain to the top of the column.

 

The sample in a small volume of methanol is adsorbed on the top of the column.  Elute the column with 50 ml of ethyl acetate. The eluate was evaporated to dryness under a gentle stream of nitrogen at water bath temperature 45°C. Final samples were prepared in suitable solvent for chromatographic analysis.

 

Chromatographic separation parameters for chlorpyrifos:

 

Chromatographic separation parameters for O,O-diethyl triphosphate 

 

Method validation:

Method validation ensures analysis credibility. In this study, the parameters accuracy, precision, linearity and limits of detection (LOD) and quantification (LOQ) were considered. The accuracy of the method was determined by recovery tests, using samples spiked at concentration levels of 0.01 and 0.1 mg/kg. Linearity was determined by different known concentrations (0.01, 0.1, 0.5, 1.0, 2.0 and 5.0 mg/L) were prepared by diluting the stock solution. The limit of detection (LOD, mg/kg) was determined as the lowest concentration giving a response of 3 times the baseline noise defined from the analysis of control (untreated) sample^4,5,6. The limit of quantification (LOQ, mg/kg) was determined as the lowest concentration of a given fungicide giving a response of 10 times the baseline noise.

 

RESULTS AND DISCUSSION:

Specificity:

Aliquots of chlorpyrifos and its metabolite (O,O-diethyl thiophosphate) standard solutions, spiking sample solution, fruit control, extracted solvents and mobile phase solvents were assayed to check the specificity. There were no matrix peaks in the chromatograms to interfere with the analysis of residues shown in (Figure 1 and 2). Furthermore, the retention times of chlorpyrifos and O,O-diethyl thiophosphate were constant at 5.2 ± 0.2 and 4.6 ± 0.2, minutes.

 

Fig.1. Representative Chromatogram of chlorpyrifos in green apple at fortification level of 0.1 mg/kg  

 

Fig.2. Representative chromatogram of O, O-diethyl thiophosphate in green apple at   fortification level of 0.1 mg/kg

 

Linearity:

Preparation of Chlorpyrifos standard stock solution:

Accurately weighed 10.10 mg of reference analytical standard of chlorpyrifos (Purity 99.0 %) in 10ml volumetric flask separately and the volume was made upto the mark using acetone, sonicated and made upto the mark with the same solvent. The concentration of the stock solution was 1000 mg/LL.

 

Preparation of O, O-diethyl thiophosphate metabolite stock solution:

Accurately weighed 10.01 mg of reference standard of O, O-diethyl thiophosphate (Purity 99.9 %) in 10 mL volumetric flask and dissolved in acetonitrile, sonicated and made upto the mark with the same solvent. The concentration of the stock solution was 1000 µg/mL.

 

Preparation of Calibration solutions:

Different known concentrations of standard solutions (0.01, 0.1, 0.5, 1.0, 2.0 and 5.0 mg/L) were prepared in acetone and acetonitrile by diluting the above stock solutions. The serial dilution details were presented in Table 1. Injected the standard solutions and measured the peak areas. The calibration curve has been plotted of concentration of the standards injected versus area observed and the linearity of method was evaluated by analyzing six solutions^7,8,9. The peak areas obtained from different concentrations of standards were used to calculate linear regression equations. These were Y=32428X + 68.35 and Y=22560 + 38.45 with correlation coefficients of 0.9999 and 0.9998 for chlorpyrifos and O, O-diethyl thiophosphate respectively. The calibration curves were showed in Figure 3 and Figure 4. 

 

 

 

Table 1. Serial dilutions of linearity standard solutions for chlorpyrifos and O, O-diethyl thiophosphate      

Stock solution concentration (µg/mL)	Volume taken from stock solution (mL)	Final make up volume (mL)	Obtained concentration (µg/mL)
1000	1.000	10	100
100	0.500	10	5
100	0.200	10	2
100	0.100	10	1
10	0.5	10	0.5
10	0.1	10	0.1
1	0.1	10	0.01

 

 

Fig.3. Representative Calibration curve of chlorpyrifos Fig.4. Representative Calibration curve of O, O-diethyl thiophosphate

 

Table 2. Mean recoveries of the chlorpyrifos and O, O-diethyl thiophosphate from fortified green apple sample (n=6)

Compound Name	Analytical method	LOQ level in  Mean Recovery (%)	LOQ level x 10 times in  Mean Recovery (%)
Chlorpyrifos	GC-ECD	86	91
O,O-diethyl thiophosphate	HPLC-UV	89	94

 

 

Accuracy and Precision:

Recovery studies in gren apples were conducted by fortifying different concentrations of pesticide standards in the range chlorpyrifos (0.01 and 0.1 mg/L), its metabolite O,O-diethyl thiophosphate (0.01 and 0.1 mg/L). The samples were homogenized, extracted and analysed, as described in the extraction procedure.  

 

Recovery studies were carried out at 0.01 and 0.1 mg/kg fortification levels for chlorpyrifos and O,O-diethyl thiophosphate in green apple. The mean recovery % values obtained by this method are summarized in Table 2.

 

Detection and Quantification Limits:

The limit of quantification was determined to be 0.01 mg/kg. The quantitation limit was defined as the lowest fortification level evaluated at which acceptable average recoveries (86-94%, RSD<2%) were achieved. This quantitation limit also reflects the fortification level at which an analyte peak is consistently generated at approximately 10 times the baseline noise in the chromatogram^10,11,12. The limit of detection was determined to be 0.01 mg/kg at a level of approximately three times the back ground of control injection around the retention time of the peak of interest.

 

Calculations:

Residue (chlorpyrifos its metabolite O,O-diethyl thiophosphate     content (mg/kg)

 

                    A x B x C

           = ------------------x F

                      D x E

 

Where,                  

A   -        Peak area of  chlorpyrifos/ its metabolite in sample (µV-sec)      

B    -        Volume of the sample (mL)

C    -        Concentration of the standard (mg/L)             

D    -        Peak area of chlorpyrifos/ its metabolite in standard (µV-sec)

E     -       Weight of the sample (g)

F     -       Dilution Factor

P     -       Purity of Test item

 

CONCLUSIONS:

This paper describes a fast, simple sensitive analytical method based on SPE-GC and SPE-HPLC methods were determination of chlorpyrifos and O,O-diethyl thiophosphate residues in green apple. The SPE extraction procedure is very simple and inexpensive method for simultaneous extraction of chlorpyrifos and O,O-diethyl thiophosphate residues in green apple. The
mobile phase Acetonitrile and HPLC grade water showed good separation and resolution and the analysis time required for the chromatographic determination of the O,O-diethyl thiophosphate was very short (around 15 min for a chromatographic run). Satisfactory validation parameters such as linearity, recovery, precision and very low limits were obtained and according to the SANCO guidelines. Therefore, the proposed analytical procedure could satisfactorily be useful for regular monitoring of chlorpyrifos and O,O-diethyl thiophosphate residues on a large number of leaf, seed, oil, fruit, water and soil samples.

ACKNOWLEDGEMENT:

The authors are thankful to the Dr. Gowtham Prasad, S.V.V University, Hyderabad for his keen interest and help.

 

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7.        T Parvathamma, A Ramesh, TNageswara Rao. Determination of paclobutrazol residue in mango fruit using a matrix solid-phase dispersion method coupled to high performance liquid chromatography with ultraviolet detection. International Journal of ChemTech Research. 4; 2012: 1473-1477.

8.        Tentu Nageswara Rao, T Benarji Patrudub, MV Rao, Karri Apparao. Determination of Ethoxysulfuron Residues in Sugarcane Juice followed by HPLC-PDA Detection and Confirmation of Residues by LC-MS/MS. Eurasian Journal of Analytical Chemistry. 10; 2015:187-194.

9.        Botsa. Parvatamma, Tentu. Nageswara Rao. A New Validated HPLC method for determination of Cyantraniliprole and its metabolite residues in tomato fruit. Asian Journal of Research in Chemistry. 8 (6) : 2015: 383-388.

10.     Tentu. Nageswara Rao, D. Sreenivasulu, T.B. Patrudu and E.G. Sreenivasula Reddy. Simultaneous Extraction and detection of six fungicide residues in mango fruit Followe by new Validated HPLC-UV method. Scholars Academic Journal of Bioscience 1(3); 2013: 80- 84.

11.     Tentu Nageswara Rao, et al. Persistence study of pyraclostrobin and epoxiconazole fungicide formulation in groundnut plant followed by HPLC-UV method. International  journal of current microbiology and applied sciences. 2(9); 2013: 5-13.

12.     SANCO Guidelines. Method validation and quality control procedures for pesticide residues analysis in food and feed. Document NO. SANCO/10684/2009.

 

 

 

 

 

Received on 14.05.2017         Modified on 06.06.2017

Accepted on 20.06.2017         © AJRC All right reserved