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

Emtricitabine (commonly called FTC, systematic name 2 & 3’-dideoxy-5-ﬂuoro-3-thiacytidine) is a nucleoside reverse-transcriptase inhibitor (NRTI) for the prevention and treatment of HIV infection in adults and children. The UV analysis of the drug was carried out on the 240nm wavelength. A simple, sensitive and accurate RP-HPLC method has been developed& validated for the determination of Emtricitabine in bulk formulation. Present method shows high sensitivity with linearity 10 to 50 (cid:22) g/ml (r2 = 0.9991). Various parameters according to ICH guidelines are followed for validating and testing of this method. Detection limit and quantitation limit were found to be 0.1534 (cid:22) g ml (cid:0) 1 and 0.4649 (cid:22) gml (cid:0) 1 respectively. The results demonstrated that the procedure is accurate, speciﬁc and reproducible and also being simple, cheap and less time consuming and appropriate for the determination of Emtricitabine in bulk formulation. This is an Open Access (OA) journal, and articles are distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike


Introduction
Emtricitabine is an analogue of cytidine. The drug works by inhibiting reverse transcriptase, the enzyme that copies HIV RNA into new viral DNA. By interfering with this process, which is central to the replication of HIV, emtricitabine can help to lower the amount of HIV, or "viral load", in a patient's body and can indirectly increase the number of immune system cells (namely T cells/CD4+ T-cells), [1][2][3] Both of these changes are associated with healthier immune systems and decreased likelihood of serious illness. Literature review reveals that various analytical methods like UVSpectrophotometry, 3,4 HPLC [5][6][7] have been developed. However, no stability indicating UV methods are reported but one stability indicating HPLC methodwas reported for the estimation of emtricitabine. Hence, an attempt was made to develop simple, accurate and precise stability indicating UV and RP-HPLC methods for the estimation of emtricitabine in pure and their marketed formulations. [4][5][6][7]

Material and Method
All reagents used were of analytical reagent (AR) grade. HPLC grade methanol and water was used throughout analysis. Emtricitabine sample was kindly provided by Hetero drugs ltd.

UV-visible spectrophotometric method 2.1.1. 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. [8][9][10][11][12][13][14][15] 2.2.1. 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 = 3.0σ S LOQ = 10 σ S Where, σ = the standard deviation of the y-intercept S = slope of calibration curve of analyte

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.
1. Effect of variation in Flow rate: A study was conducted to determine the effect of change in flow rate. Test solution prepared as per the test method and injected 3 concentrations of test solution into HPLC system with flow rate 0.7 ml/min. and 0.9 ml/min. The system suitability parameters were evaluated as per the test method.

Effect of variation in Wavelength:
A study was conducted to determine the effect of change in wavelength. Test solution prepared as per the test method and injected 3 concentrations of test solution into HPLC system with wavelength of 238 -242 nm.
The system suitability parameters were evaluated as per the test method.

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.

System suitability study
System suitability is an integral part of many analytical procedures. The tests are based on the concept that the equipment's, electronics, analytical operations and sample to be analysed constitute an integral system that can be evaluated as such. System Suitability test parameters to be established for particular procedure depends on the type of procedure being validated. Tests are carried out on freshly prepared solution of Emtricitabine with six replicates. 16

Thermal degradation
Thermal degradation was performed at 60 0 C 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.

First order derivative Spectroscopy
The wavelength maxima was found to be 228 nm and are shown in Figure 2

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.

Development of HPLC method for Emtricitabine
High performance liquid chromatographic method was developed and validated for determination of Emtricitabine in bulk form. Mobile phase consists of Methanol: Water

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 3

.2.5. 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 3

Specificity
Excipients and impurities were not interacting with the standard drug, hence method is specific. Results of specificity are shown in Table 9 3.2.9. 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 4

. Conclusion
In the present research work, a successful attempt was made for determination of Emtricitabine in Bulk form by UV-Visible Spectrophotometric, HPLC degradation study. The method was developed by experimentation, based on literature survey. The simplicity, rapidity, reproducibility and economy of the proposed method completely fulfil the objective of this research work. The HPLC method was developed and validated for estimation of Emtricitabine. The mobile phase was consisting of Methanol: water (60:40) at pH3. Detection was done at 240nm. The method was found to be simple, linear, rapid, accurate, precise, reproducible and robust. The % RSD was found within limit. The result showed that proposed method was suitable for the accurate, precise and rapid determination of Emtricitabine in its bulk form. Forced degradation studies were performed on Acid, Alkali, Peroxide, Thermal and Photolytic conditions. Degradation studies were performed on solutions containing 50 µg/ml of Emtricitabine. The result showed that proposed method was suitable for the accurate and rapid determination of Emtricitabine in its bulk form.

Acknowledgment
I am thankful to the principal and management of Sandip Institute of Pharmaceutical Sciences, Nashik for their help, without which the work would not be completed.

Source of Funding
None.

Conflict of Interest
None.