INTRODUCTION:

Organophosperous compounds are synthesized in the 1800s, later they are used as insecticides in the late 1930’s and early 1940’s¹. The German scientist Gerhard Schrader is known for the creation of the basic chemical structure of anticholinesterase organophosphate compounds and development of the first commercialized Organophosperous insecticide¹. Such chemicals are anticholinesterase insecticide commonly used in agriculture and horticulture². To a lesser extent they are used for domestic use. Due to the absence of biopersistance in organophosphates most of the western countries opted to substitute organochlorines with organophosphates³. Organophosphate pesticides are commonly used around the world, and contamination by these compounds are a serious public health concern in developing countries⁴. Toxicokinetics and toxicodynamics of OP poisoning not only differ with path or level of exposure. But also the agent’s chemical composition².

The toxicity mechanism of organophosphates is by suppression of acetylcholinesterase, which results in building up of acetylcholine neurotransmitter and the continues activation of acetylcholine receptors^3.2,5,6. The recommended treatment comprises of reactivating blocked acetylcholinesterase with an oxime antidote and suppressing acetylcholine’s action on receptor with atropine^3,1. Patient who received appropriate diagnosis usually recover from acute toxicity. Dichlorvos is the active component of many insecticidal formulations^6,5. The toxicity of Dichlorvos was reported by FAO/WHO at joint meeting on pesticide residues (1965, 1967, 1968 and 1970) and a permissible daily intake of 0.004 mg /kg was suggested⁶. high concentration exposure over the short-term also up to 50 days of exposure on monkeys and rat to 0.1-0.5mg. The only effect seen were depression of cholinesterase. Dichlorvos is a cholinesterase inhibitor exhibiting stomach, contact and systemic mode of action⁷. Dichlorvos is broad spectrum organophosphate compound having insecticidal activity. In the premature days, most organophosperous insecticides were also dangerous to mammals, including humans, i.e. they were not selectively toxic to insects. Chromatographic techniques are commonly used for the chemical isolation, detection and quantification of as many pesticides. The HPLC, GC or TLC methods were used to determine Dichlorvos, which are having some advantages and disadvantages^8,9. In order to increase food production, pesticide applications have become totally vital. India is the world’s fifth pesticide consumer. The proposed RP-UFLC method for detection of Dichlorvos is having short retention time compared to other HPLC methods. Dichlorvos chemical structure is shown in figure1.

Figure 1. Chemical structure of Dichlorvos

MATERIALS AND METHODS:

Instrumentation and Chromatographic conditions

For the current study, HPLC LC-20AD with PDA detector is used. The separation was achieved by using Phenomenex luna C18 column (250 mm X 4.60 mm 5µ)¹⁰. Run time was set to 10min. Acetonitrile (ACN) and Millipore water (50:50 v / v) at a flow rate of 1.5 ml / min are used as mobile phase⁸. Temperature of the column was set at 40°C. The wavelength of detection was set at 200 nm. Phenex PTFE 0.02μm syringe sensor is used for filtration purposes.

Chemicals and reagents:

Dichlorvos standard was procured from Sigma Aldrich, Bengaluru. Action-3 which is a Dichlorvos marketed formulation manufactured by Jayakrishna pesticides private limited. was procured from local market. All chemicals used were analytical grade purchased from Merck pharmaceuticals. HPLC grade ACN and Millipore water is used as mobile phase. HPLC grade ACN was used as the diluent for preparation of the solutions.

Method Development:

Selection of wavelength:

The λ max of Dimethoate was determined by using UV-visible spectrophotometer 1800. UV spectrum for Dichlorvos is shown in figure 2.

Figure 2: UV spectrum

Mobile phase selection and preparation:

Dichlorvos being less polar, different mobile phase combinations of various ratios were tried for the selection of mobile phase. The standard Dichlorvos drug was injected with various combination of mobile phase at different ratios and flow rate for the peak optimization. The procedure was continued till obtaining a sharp peak. The sharp peak was obtained at 50:50(v/v) of ACN and Millipore water.

Preparation of standard stock:

Accurately weigh 10mg of pure Dichlorvos in to 10ml volumetric flask dissolve and makeup the volume by using HPLC grade acetonitrile to get 1mg/ml concentration.

From the above solution prepare 75, 150, 225, 300 and 375µg/ml so that after diluting it with serum and ACN final concentration will be 10, 20, 30, 40 and 50µg/ml.

Optimized extraction procedure:

After trying with different combination of serum and drug volume for protein precipitation method by using acetonitrile. The following method is finalized.

In this procedure acetonitrile act as a protein precipitating agent. To the Eppendorf tube add 100µl of human serum and 100µl of drug, Vortex the above mixture in a vortex meter for 20 seconds. To the mixture add acetonitrile and make-up the volume to 1.5ml then centrifuge it at 9500 RPM for 10 minutes at 4˚C. the supernatant is filtered through 0.20µm syringe filter and injected to RP-UFLC.

Optimized chromatographic conditions.

Table 1: Chromatographic conditions

Instrument used	Shimadzu LC-20AD equipped with an auto sampler and PDA detector.
Column	Phenomenex luna C₁₈ column (250 mm X 4.60 mm 5µ)
Wavelength	200nm
Flow rate	1.5ml/min
Detector	PDA
Injection volume	10µl
Column oven temperature	40°c
Mobile phase	ACN and Millipore water 50:50 (v/v)
Retention time	2.9 min

Figure 3: standard chromatogram of Dichlorvos.

Figure 4: Sample chromatogram of Dichlorvos.

Figure 5: chromatogram of blank serum.

BIO-ANALYTICAL METHOD VALIDATION:

Calibration curve:

It consisted of matrix sample processed without analyte and matrix sample with calibration standards. It is showing good linearity over the range of 10 to 50µg/ml with coefficient of correlation 0.9934

Table 2: Calibration data of Dichlorvos

Sl. No	Conc. (µg/ml)	Peak area
1	0	0
2	10	312932
3	20	615335
4	30	907831
5	40	1095066
6	50	1513468

Figure 6: Linearity graph of Dichlorvos

Specificity/selectivity:

It is the capability of an analytical technique to differentiate and quantify the analyte in presence of other components in the sample. For selectivity blank plasma of two different lots were taken and analysed. Selectivity was assessed by comparing extracted blank plasma response with extracted LLOQ. At the RT of the drug No significant interference from the blank plasma was observed.

Sensitivity:

This parameter was evaluated by injecting six different aliquots of extracted LLOQ concentration. Percentage deviation from the nominal concentration and percentage CV were calculated. The developed method was found to be sensitive with % CV.

Accuracy and precision:

Within run and between run accuracy were performed by five replicates of LLOQ, LQC, MQC and HQC. Between run accuracy was assessed by analysing sample on different days. The accuracy and precision for all batches at LLOQ, LQC, MQC and HQC levels were calculated. Mean percentage nominal concentration and CV for all the batches were found to be within acceptance limit.

Table 3: Results showing precision for Dichlorvos (within run)

concentration	Mean peak area	Mean concentration	SD	%CV
10 (LLOQ)	305015.4	9.9026	0.8183	8.2643
20 (LQC)	585185.0	19.962	0.8725	4.3708
30 (MQC)	861875.4	29.897	1.0632	3.5563
40(HQC)	1136209.2	39.748	1.9101	4.8055

Table 4: Results showing precision for Dichlorvos (between run)

concentration	Mean peak area	Mean concentration	SD	%CV
10 (LLOQ)	307135.8	9.97877	0.7763	7.7795
20 (LQC)	581728.6	19.8809	0.8446	4.2483
30 (MQC)	862136.0	29.9069	1.0649	3.5609
40(HQC)	1134797.2	39.6973	1.8912	4.7640

Recovery:

After spiking the extracted QC samples were analysed and percentage recovery at each level was calculated by comparing the peak area of low, medium and high QC levels. Mean recovery across all the QC levels is found to be 66.0%

Stock solution stability:

Both main stock and spiking stock of Dichlorvos were found to be stable at 2-10°c for 20 days (long term) and for 8 hours at room temperature. (Table 5)

Bench top stability:

low and high QC were prepared and kept at bench top at room temperature for a minimum of 4 hrs (stability samples). Then analysed the response is compared with the freshly prepared calibration standard responses. Mean percentage change was calculated

Freeze and thaw stability:

The samples were exposed to three freeze thaw cycles. The peak area response is then compared with standard calibration responses. Mean percentage change was calculated and verified against acceptance criteria.

Long term stability:

The stability of the sample is evaluated by keeping it for long period of time in freeze state and extracted then analysed. The response is compared with fresh calibration standard response.

Table 5: Results showing recovery for Dichlorvos.

Standards	Concentration	Analytical Peak Area	Bioanalytical Peak area	% Recovery
LLOQ	10	549627	312932	56.93533978
LQC	20	937318	615335	65.64847789
MQC	30	1271496	907831	71.39865167
HQC	40	1670206	1095066	65.56472675
UQC	50	2147712	1513468	70.46885243

CONCLUSION:

The proposed RP-UFLC method for Dichlorvos was validated and it offers good accuracy, sensitivity and selectivity. It can be used for the detection of Dichlorvos in biological fluids. The proposed method is showing acceptable values of recovery. The developed chromatogram has a well resolved peak without any interference. From the result we conclude that the developed method can be applied in Toxicokinetics and bioequivalence Toxicokinetics studies with desired precision and accuracy.

CONFLICT OF INTEREST:

The authors have no conflict of interest.

ACKNOWLEDGEMENT:

We would like to thank the Principal, JSS College of Pharmacy, Mysuru and JSS Academy of Higher Education and Research, Mysuru for providing the facilities in successfully completion of the research work.

REFERENCE:

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Received on 18.11.2019 Modified on 15.01.2020

Research J. Pharm. and Tech. 2020; 13(8):3725-3728.

DOI: 10.5958/0974-360X.2020.00659.9

Stability	Standards	Concentration µg/mL	Mean recovered concentration µg/mL)	SD	%CV
Bench-top	LQC	20	19.9169	0.5020	2.5205
Bench-top	HQC	40	39.2690	0.7317	1.8633
Freeze and thaw	LQC	20	19.9370	0.2522	1.2650
Freeze and thaw	HQC	40	39.7585	0.7294	1.8347
Long term stability	LQC	20	19.9120	0.2621	1.3166
Long term stability	HQC	40	38.3938	0.0700	0.1827