Analytical method development

Selection of wavelength

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

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 optimisation. The procedure was continued until obtaining a sharp peak. The sharp peak was obtained at 50:50(v/v) of ACN and Millipore water.

Preparation of standard stock solution

The standard stock of Dichlorvos was prepared by dissolving 10mg of the standard drug in 10ml of HPLC grade acetonitrile to obtain 1mg/ml concentration. From the stock solution, the standard stock solutions of 10, 20, 30, 40, and 50µg/ml were prepared. All dilutions were made up by using HPLC grade acetonitrile.

Sample preparation

0.131ml of marketed formulation (Action-3) containing 76% of Dichlorvos was diluted to 100ml by using HPLC grade acetonitrile to form 1mg/ml solution. From the above sample solution, pipette out 0.2ml and make-up to 10ml by using HPLC grade acetonitrile to get 20µg/ml solution. The above resolution was passed through 0.20µm syringe filter and injected to RP-HPLC.

Optimization of the method for Dichlorvos

Study of the effect of various parameters in developing method was carried out. Initially, the solubility of Dichlorvos in multiple solvents was tested. Then a suitable column for separation is selected for the proposed method. To achieve a proper separation of eluted compounds in HPLC, the chromatographic conditions were optimised. Initially, different diluent was tested to elute the drug.

Flow rate and mobile phase choice are determined based on peak parameters like tailing factor or asymmetry, run time, resolution. Acetonitrile and Millipore water in ratio 50:50 (v/v) was used as mobile phase at a flow rate of 1.5ml/min. The blank chromatogram was shown in Figure 3.

The standard and sample chromatogram of Dichlorvos at 2.9 min were shown in Figure 4 and Figure 5 respectively. Chromatographic conditions used for the method is shown in Table 1.

Bio-analytical method development

Preparation of standard stock

Accurately weigh 10mg of pure Dichlorvos into 10ml volumetric flask dissolve and make-up 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 a 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 precipitating protein agent. To the Eppendorf tube add 100µl of human serum and 100µl of a 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. The blank chromatogram was shown in Figure 6. The standard and sample chromatogram of Dichlorvos at 2.9 min were shown in Figure 7 and Figure 8 respectively. Chromatographic conditions used for the method is shown in Table 1.

Analytical method validation

System suitability

The standard stock of Dichlorvos was injected six times for testing system suitability parameters, The results were shown in Table 2.

Linearity

It is the ability to obtain experimental results equal to the analyte content in the specimen. The calibration curve was attained by using five different concentrations in triplicate 10, 20, 30, 40, and 50µg/ml and the linearity was established by applying linearity expression y=mx+c, and the slope was calculated. The calibration curve for Dichlorvos is shown in Figure 9. The concentration and peak area was established in Table 3.

Precision

The repeatability of the method was validated by using different concentration of the drug 10, 30 and 50 µg/ml. The above solutions were prepared from the stock solution and used to inject in interday and intraday for the evaluation of precision. The concentrations were prepared at three different times in a day for intraday studies. The results for accuracy was shown in Table 4 and Table 5.

Accuracy

It is the closeness of the obtained value to the true value of the sample, to check the accuracy of the method, the formulation was spiked with 50%, 100% and 150% of Dichlorvos standard drug. The results were analysed to find the % recovery of the Dichlorvos. The result for accuracy was given in Table 6.

Limit of detection and limit of quantification

The LOD and LOQ for the HPLC method were determined by using a calibration standard. LOD can be calculated as per the ICH guidelines by using the formula LO D=3.3×N÷S, N is the standard deviation and S is the slope. LOQ can be calculated by the formula LOQ=10×N÷S where N is the standard deviation and S is the slope. The results for LOD and LOQ was shown in Table 7.

Robustness

A method can stay unchanged when small differences in parameters are applied. The robustness of the suggested technique was verified by increasing and decreasing the wavelength, flow rate, column temperature and 30μg / mL concentration were injected. The result of the robustness was shown in Table 8.

Ruggedness

An experimental procedure's ruggedness is its ability to remain unaffected by minor or intentional changes in system parameters. The ruggedness of the proposed procedure is validated by changing analyst and instrument. The result was shown in Table 9.

Bioanalytical method validation

Calibration curve

It consisted of a 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 a coefficient of correlation 0.9934. The calibration curve for Dichlorvos is shown in Figure 10. The concentration and peak area was shown in Table 10.

Specificity/selectivity

An analytical technique can differentiate and quantify the analyte in the presence of other components in the sample. For selectivity blank plasma of two different lots were taken and analysed. Selectivity was assessed by comparing the 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 to %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 the acceptance limit. The results for precision is shown in Table 11 and Table 12.

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%. Results for recovery is shown in Table 13.

Stock solution stability

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

Bench top stability

Low and high QC was prepared and kept at the benchtop 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 an extended period in freeze state and extracted then analysed. The response is compared with a fresh calibration standard response. Results for stability is shown in Table 14.