Introduction

According to the ICH recommendations, stress testing is designed to discover potential degradation products, which assists in estimating the inherent stability of the molecule and developing degradation routes, as well as confirming the stability indicating methodologies utilised. However, in the case of forced degradation, these guidelines are very general. The chemical stability of the molecule influences drug product safety and efficacy. The information on molecule stability is used to select the best formulation, package, storage conditions, and shelf life (Alsante, Hatajik, Lohr, Santafianos, & Sharp, 2003; Harmon & Boccardi, 2005). Many pharmaceutical drug substances and products are photosensitive and undergo photodegradation upon exposure to outdoor or indoor photon sources (Baertschi, Thatcher, Piechocki, & Thoma, 2006; Brummer, 2011), they have been estimated the drug molecule intrinsic stability. Forced degradation studies can be used to estimate the intrinsic stability of a drug molecule. Forced degradation studies, which create degradation products that may be analysed to identify the molecule's stability, can be used to examine potentially polymorphic or enantiomeric compounds, as well as differences in drug degradation and excipient interferences (Dorman, Lorenz, Occolowitz, Spangle, & Collins, 1997; Rockville, 2004) showed the ICH guidelines and degradation routes for stress testing to potential degradation products, which aids in determining the molecule's intrinsic stability. Photostability studies were carried out to generate primary degradants of drug substances through UV or fluorescent exposure. Some recommended photostability testing conditions were described in the ICH guidelines. Drug substance and solid/liquid drug product samples should be exposed to a minimum of 1.2 million lx h and 200 W h/ m2 light, with the most commonly accepted wavelength of light causing photolytic degradation being 300-800 nm (European Medicines Agency, 2003; ICH Q3C (R3) Impurities, 2002). Degradation of the drug product and new drug substance by forced degradation under conditions is more severe and accelerated. Stress testing, stress studies, stress decomposition studies, forced decomposition studies etc., are known to be the introduction of forced degradation studies. It is necessary to follow ICH recommendations while conducting forced degradation tests under proper circumstances such as light, oxidation, dry heat, acidic, basic, hydrolysis, and so on. Moreover, from the degradation products, the drug gets separated. The time and different environmental factors varies the quality of a drug substance and drug product obtained from forced degradation studies and it should be mandated by FDA and ICH guidance (Kovařı́ková, Klimeš, Dohnal, & Tisovská, 2004; Maheswaran, 2012). The HPLC method stability indicating nature has been assessed by detecting the solution state and the solid state samples were degraded and then the photo diode-array detector was analyzed (Oyler et al., 2012; Prekodravac, Damm, & Kappe, 2011) have been expressed that the forced degradation is promoted during phase I of a clinical trial to allow for the identification of degradation products, structural elucidation, and optimization of stress settings. The determination of an assay by HPLC method of Tenofovir disoproxil orotate has been developed and conducted the analytical method as per the guidelines of ICH and performance of the method characteristics met the requirements for the analytical application (Ranjit & Rehman, 2012; Singh & Bakshi, 2000). The identification of each contaminant is essential in terms of both chemistry and safety prospects. Chemical possibilities include impurity categorization and identification, report creation, an impurity catalogue in the specification, and a brief description of analytical methodologies. The safety prospects contain explicit instructions for qualifying those contaminants that were not present at much lower levels in a batch of a new therapeutic ingredient (Trabelsi, Hassen, Bouabdallah, Bouzouita, & Safta, 2005).

Materials and Methods

Reagents and chemicals

Triethylamine HPLC grade, acetonitrile and Orthophosphoric acid (AR grade), Sodium hydroxide, hydrochloric acid, Hydrogen peroxide (LR grade) were obtained from Qualigen (Mumbai, India). The HDPE bottle and the polypropylene cap meet the general Pharmaceutical European requirements for plastic primary packaging material. Tenofovir disoproxil orotate standard and Tenofovir disoproxil orotate sample in house material (Baertschi & Alsante, 2005).

Instrumentation and Chromatographic Conditions

Waters Alliance HPLC system module 2695 with 2998 PDA detectors with wavelength range 190-700 and an automatic sample injector were used In this study. The data processing and management for chromatographic peak integration was obtained by using the Empower 2 software. YMC Make Column YMC Pack ODS-AQ (250mm x4.6mm x 5μm particle size) was used in this study as a stationary phase. Analytical Balance Mettler Toledo, photostability chamber newtronics, and PH meter Metrohm.

Sample standard solution

About 50 mg of Tenofovir disoproxil orotate is accurately weighed and transferred to a 50 ml volumetric flask for dilution with the diluents. Again 10.0ml of this solution is further diluted to 50 ml with diluent.

Studies on forced degradation

Studies on forced degradation are undertaken to degrade the sample consciously; these studies are helpful to evaluate analytical methods ability to appraise the functioning ingredient and its products of degradation without interference. In order to determine the HPLC stability indicating nature, the Tenofovir disoproxil orotate sample will be stressed by acid, base, hydrogen peroxide, heat, light (Fluorescent overall illumination with UV) to produce about 10% - 30% degradation of active pharmaceutical ingredient. The breakdown samples will be analyzed for determining the peak purity by using a photodiode array detector. The degradation study will be designed in the following parts.

Solution state degradation

Acid Hydrolysis

10 ml of this solution is required to transfer into a 50 ml volumetric flask, add 15 ml of diluent and 2.5 ml of 1.0 N Hydrochloric acid solution (final strength of an acid is equivalent to about 0.1N HCl). Keep the solution at room temperature for 24 hours and then neutralize with 2.5 ml of 1.0 N Sodium hydroxide solution and dilute to volume with diluents and then results are listed in Table 2 and Figure 11; Figure 10; Figure 9.

Base Hydrolysis

10 ml of this solution is required to transfer into 50 ml volumetric flask, add 15.0 ml of diluent, add 2.5 ml of 1.0 N Sodium hydroxide solution (final strength of Base is equivalent to about 0.1N NaOH). Keep the solution at room temperature for 24 hours and then neutralize with 2.5 ml of 1.0 N Hydrochloric acid solution and dilute to volume with diluents and results are then listed in Table 3 and Figure 14; Figure 13; Figure 12.

Oxidation

10 ml of this solution is required to transfer into a 50 ml volumetric flask, add 15 ml of diluent, add 2.5 ml of 20% Hydrogen peroxide (final strength of Hydrogen peroxide is equivalent to about 6%). Keep the solution at room temperature for 24 hours and dilute to volume with diluents and results are then listed in Table 4 and Figure 8; Figure 7.

Heat Degradation

10 ml of this solution is required to transfer into a 50 ml volumetric flask, add 15.0 ml of diluents. Keep the solution at 60°C temperature for 24 hours and dilute to volume with diluents and then the results are listed in Table 5 and Figure 8; Figure 7.

Blank Preparation

For the above degradations studies, prepared a blank solution without addition of sample, keep under the same condition and analyze as per the method.

Solid state degradation

Perform the solid state degradation study by exposing the Tenofovir disoproxil orotate sample to light (an overall illumination of not less than 1.2 million lux hours and an integrated near ultraviolet energy, not less than 200-watt hours/square meter fluorescent light and UV light), heat (at 105°C for 24 hours) and humidity (90% + 5), Analyze the thermal and photo exposed samples along with control sample for determining the peak purity by using a photodiode array detector.

Note: if the sample is prone to degrade in any stress condition, the degradation will be carried out between 10% - 30% degradation in that condition (European Medicines Agency, 2003).

Method development

System Suitability

System suitability is an integral part of the method of analysis. A standard solution was prepared and analysed as per the proposed method. Inject blank followed by standard solution six times into the chromatograph and record the chromatograms. The system is suitable, if and only if the number of theoretical plates for Tenofovir disoproxil peak should be not less than 5000. The tailing factor should be not more than 2.0. The %RSD for the Tenofovir disoproxil peak area should be not more than 0.85. System suitability results are listed in Table 1.

Photostability degradation

Transfer about 200 mg of the sample in a Petri dish, spread it uniformly and completely wrap with aluminium foil and display to the illumination of light at least 1.2 million lux hours an integrated near UV energy of at least 200-watt hours/ square meter. After exposure, about 50 mg of Tenofovir disoproxil orotate sample is accurately weighed and transferred into 50 ml volumetric flask and dilute to volume with diluent. 10 ml of this solution is required to transfer into a 50 ml volumetric flask and make up to the mark for dilution.

Heat at 105°c by exposure

Transfer the sample around 200 mg into a Petri dish, spread it uniformly, expose it to 105°c heat for one day; after this, transfer accurately weighed around 50 mg sample of Tenofovir disoproxil orotate into a 50 ml volumetric flask to dissolve and dilute with diluent to the volume. Again 10.0 ml of this solution is diluted to 50 ml with diluent.

Humidity stress study

Transfer the sample around 200 mg into a Petri dish, spread it uniformly and expose it In a desiccator at 25°C With 90% RH+ 5% RH for 24 hrs. After exposure, transfer accurately weighed around 50 mg sample of Tenofovir disoproxil orotate into a 50 ml volumetric flask to dissolve dilute with diluent to the volume. Again 10.0 ml of this solution is diluted to 50 ml with diluent.

Solid State stability

Significant degradation was observed when Tenofovir disoproxil orotate sample solutions were exposed to light (1.2 million Lux hours and 200 Wh/sq.mt exposure to white fluorescent light and UV light) and heat at 105^oC and Humidity at 90% RH for the typically degraded sample, the API peak purity test. The Tenofovir disoproxil peak passed the peak purity testing, leading that the peak is spectrally homogeneous. In other words, none of the degradants formed during the stress study co-elute with the Tenofovir disoproxil peak. The results are listed in Table 6 and Figure 6; Figure 5; Figure 4; Figure 3; Figure 2; Figure 1.

Results and Discussion

Method development

System Suitability

System suitability is an integral part of the method of analysis. A standard solution was prepared and analysed as per the proposed method. Inject blank followed by standard solution six times into the chromatograph and record the chromatograms.

The system is suitable, if and only if the number of theoretical plates for Tenofovir disoproxil peak should be not less than 5000. The tailing factor should be not more than 2.0. The %RSD for the Tenofovir disoproxil peak area should be not more than 0.85. System suitability results are listed in Table 1.

Forced Degradation Studies

The HPLC method stability indicating nature can be able to assess by taking the Tenofovir disoproxil orotate sample and stressed by acid, base, oxidation, heat, light, humidity, the degraded samples were analyzed using a photodiode array detector. Passed the testing of peak purity for the tenofovir disoproxil orotate peak; this understands that the peak is spectrally homogeneous. In other words, during the stress study, no measurable degradants was formed, co-elute with the Tenofovir disoproxil orotate peak.

Conclusions

Significant degradation has been observed. The peak purity of Tenofovir disoproxil orotate is passing purity angle is less than purity threshold. There is no interference of degradant peaks with the Tenofovir disoproxil peak. By compiling the results of degradation studies obtained, the prominent way to develop degradation pathways and to identify the degradation products of the active ingredients are forced degradation studies; further, it facilitates the elucidation of the structure of degradants. The chemical and physical stability analysis of drug substances and drug products can be facilitated by forced degradation studies. The forced degradation studies help during the development of formulation, manufacturing conditions, storage conditions and also determines the date of expiry of a drug formulation.

Funding Support

The authors declare that they have no funding support for this study.

Conflict of Interest

The authors declare that they have no conflict of interest for this study.