Method stability indicating method development and validation for Emitricitabine by UV spectroscopic and RP-HPLC methods

Zero order spectrophotometric method

Standard stock solution of Emtricitabine was prepared by accurately weighing 100 mg of Emtricitabine to 100 ml volumetric flask with 100 ml of water. The drug was sonicated and volume was made up to mark with water to get the concentration of 1000 μg/ml.

First order derivative

Standard stock solution of Emtricitabine was prepared by accurately weighing 100 mg of Emtricitabine to 100 ml volumetric flask with specific volume of water. The drug was sonicated and volume was made up to mark with water to get the concentration of 1000 μg/ml.

Linearity

The chromatographic conditions were set as per the optimized parameters and mobile phase was allowed to equilibrate with stationary phase as was indicated by the steady baseline. Test solutions of different concentration were injected separately and the chromatograms were recorded. A series of test preparations of Emtricitabine (10-50µg/ml) were prepared by taking 0.1, 0.2, 0.3, 0.4, 0.5ml from the stock solution in five 10 ml volumetric flask and final volume make up to the mark with mobile phase. A 20 µl volume of each concentration was injected into HPLC, three times under the optimized chromatographic conditions.

Accuracy

Samples are prepared normally covering 50 % to 150 % of the nominal sample preparation concentration. These samples are analyzed and the recoveries of each are calculated. For this study, three preparation of each 50 %, 100 % and 150 % level are prepared and injected in to the chromatography, form the data obtained individual recovery, mean recovery and %RSD is calculated.

Precision study procedure: Intraday and interday precision

Intraday precision study was carried out by preparing test solution of same concentration and analyzing it at two different times in a day. The same procedure was followed for two different days to determine interday precision. The result was reported as %RSD. The precision result showed a good reproducibility with percent relative standard deviation less than 2.Three concentration selected 10,30,50 ug/ml.

LOD and LOQ

LOD and LOQ determined by the following formula by taking the standard deviation of y-intercept and slope from the linearity curves.

LOD=3.0σS

LOQ=10 σS

Robustness

The effect of small deliberate change in optimized method was studied by robustness evaluation. To evaluate robustness of the developed method, parameter was deliberately varied. This parameter included variation of flow rate and variation wavelength. Factor selected was changed at two levels, for change in the flow rate in ml/min and change in wavelength in nm. In significant differences in peak areas and less variability in retention time were observed.

Ruggedness

It is the degree of reproducibility of the test results under variety of conditions like different analyst, different instrument.

Specificity

The specificity of method was determined by checking the interference of excipients with analyte. Study was done by spiking known amount of excipients stock solution to the standard solution and area was measured at 235-245 nm.

Acid induced-degradation

10.0 mg Emtricitabine was transferred to 100.0 mL volumetric flask, added 50.0 mL mobile phase, sonicated for 20.0 min., volume was made up to the mark with mobile phase. From this stock solution take 1 ml of solution into 20 ml volumetric flask then add 10 ml of mobile phase and then add 1 ml of 1N HCl and stressed the samples at condition mentioned in water bath, cooled at room temperature and neutralize of acid with base of same concentration and volume. Make up the volume with mobile phase and mixed. Filter the solution through 0.45 µ membrane filter and filtrate use as such. Then these filtrates were chromatographed under optimized chromatographic system.

Base induced-degradation

10.0 mg Emtricitabine was transferred to 100.0 mL volumetric flask, added 50.0 mL mobile phase, sonicated for 20.0 min., volume was made up to the mark with mobile phase. From this stock solution take 1 ml of solution into 20 ml volumetric flask then add 10 ml of mobile phase and then add 1 ml of 1N NaOH and stressed the samples at condition mentioned in water bath, cooled at room temperature and neutralize of base with acid of same concentration and volume. Make up the volume with mobile phase and mixed. Filter the solution through 0.45 µ membrane filter and filtrate use as such. Then these filtrates were chromatographed under optimized chromatographic system.

Peroxide degradation

10.0 mg Emtricitabine was transferred to 100.0 mL volumetric flask, added 50.0 mL mobile phase, sonicated for 20.0 min., volume was made up to the mark with mobile phase. From this stock solution take 1 ml of solution into 20 ml volumetric flask then add 10 ml of mobile phase and then add 1 ml of 3% w/v H₂O₂ and stressed the samples for 4 hours at 60^oC in water bath cooled at room temperature Then this sample was chromatographed under optimized chromatographic system.

Thermal degradation

Thermal degradation was performed at 60⁰C for 24 hours. The mobile phase used was Methanol: Water (60:40) at pH 3. The concentration used was 50ppm at wavelength of 240nm.

Photolytic degradation

Photolytic degradation was performed at room temperature for 24 hours. The concentration used was 50ppm at 240nm. The mobile phase used was Methanol: Water (60:40) at pH 3.

Zero order spectrophotometric method

UV Spectrophotometric method was developed for estimation of Emtricitabine in bulk form. The wavelength maxima were found to be 240 & 280 nm and are shown in Figure 1

Figure 1

Zero order spectra of Emtricitabine

First order derivative Spectroscopy

The wavelength maxima was found to be 228 nm and are shown in Figure 2

Figure 2

First order derivative spectra of Emtricitabine

Area under curve method

The absorption spectra show the absorbance maxima 240 at nm and area under curve in absorption spectra was measured between 235-245 nm.

Figure 3

Area under curve spectra of emtricitabine

Linearity

Drug was found to be linear in the concentration range of 10-50 μg/ml. Results obtained are shown inTable 1 and calibration plot obtained was shown in Figure 5

Figure 5

Calibration curve for Emtricitabine

Optical characteristics

Optical characteristics and statistical data of linearity for Emtricitabine by HPLC method are summarized in Table 2

Accuracy

Accuracy was studied by standard addition method and % recovery found was within acceptable limit. Results of recovery study are shown inTable 3 and statistical validation is shown inTable 4

Precision

Intraday and interday precision assures the repeatability of test results. The % RSD found to be less than 2. Result of intraday and interday precision was shown in Table 5

Limit of detection and limit of quantitation

The LOD and LOQ are found to be 0.1534µg/ml and 0.4649µg/ml respectively

Robustness

Robustness was studied by different deliberate variations in the chromatographic conditions. Results are shown in Table 7

Ruggedness

Ruggedness was studied by differentanalyst. Resultsobtainedareshownin Table 8

Specificity

Excipients and impurities were not interacting with the standard drug, hence method is specific. Results of specificity are shown in Table 9

System suitability

System suitability parameters were measured to verify the system, method and column performance. Standard solution of Emtricitabine was injected in to the system for five times and system suitability parameters were checked.

Degradation studies

Stress testing of the drug substance can help to identify the likely degradation products, the stability and specificity of the analytical procedure. Degradation studies were performed on solutions containing 50µg/ml of Emtricitabine. Results of the forced degradation studies are summarized in Table 11