HPLC Waters 2695 separation mode with T-60 UV-Visible spectrophotometer and a Rheodyne injector valve fitted with 10µL sample loop was used. Chromatographic separation was performed on Agilent C18 column (4.6 x150mm, 5µm). The column effluent was monitored with PDA detector set at 260nm and column temperature at 30^oC

All the chemicals and reagents in this experiment were of analytical grade. Water was double distilled and filtered with a membrane filter. Acetonitrile and potassium dihydrogen orthophosphate (Rankem) were used to prepare mobile phase. Pharmaceutical grade standard drugs

The accurately weighed 1.42gm of potassium dihydrogen orthophosphate was transferred to a 1000ml volumetric flask and 900ml of mille-Q water was added and sonicated. Finally, volume was made up with water and pH was adjusted to 4.8 with a dil. orthophosphoric acid solution.

Accurately measured 700ml (70%) of buffer solution, 300ml (30%) of HPLC grade acetonitrile were mixed and degassed in an ultrasonic water bath for 10 minutes and then filtered through 0.45µm filter under vacuum filtration.

Based on the solubility of the drugs acetonitrile and water in the ratio of 50:50 v/v was used as a diluent.

Accurately weighed 50mg of cabotegravir and 75mg of rilpivirine were transferred to 50 ml clean dry volumetric flask separately and 3/4th of diluents was added to the flask and sonicated for 10min. Volume was made up to the mark with diluent and labelled as standard stock solution (1000µg/mL of cabotegravir and 1500µg/mL of rilpivirine)

1mL from each stock solution was pipetted out and taken into a 10mL volumetric flask and volume was made up to the mark with diluent (100µg/mL of cabotegravir and 150 µg/mL of rilpivirine).

The sample solutions were prepared from injections vials of the drugs Cabotegravir (CAB) 600mg in 3ml inj vial & Rilpivirine (RILP) 900mg in 3mL vial. From injection vials 1ml of Rilpivirine

and Cabotegravir samples were transferred to a 100 volumetric flask, and 50ml of diluent was added to the flask and sonicated for 25 min, further the volume was made up with diluent and filtered using HPLC filter of 0.45µm pore size.

0.5 mL from the above stock solution was pipetted out and was transferred into a 10 mL volumetric flask and made up to the mark using diluent to give 100µg/mL of cabotegravir and 150µg/mL of rilpivirine respectively.

1 ml of 20% hydrogen peroxide (H₂O₂) was added separately to 1 ml of rilpivirine and cabotegravir stock solution. The solutions were kept at 600 degrees Celsius for 30 minutes. The resultant solution was diluted to obtain 150 µg/mL and 100 µg/mL solutions for HPLC analysis, and 10 µl were injected into the system and chromatograms were recorded. The percentage degradation was then calculated to determine the sample's stability.

Acid- and base-induced degradation was attempted by separately adding 1 ml of 2N Hydrochloric acid to the 1 ml of stock solutions of rilpivirine and cabotegravir, and refluxed for 30mins at 60^oC. The resultant solution was diluted to obtain 150µg/mL & 100µg/mL solution and 10 µL solutions were injected into the system and the chromatograms were recorded to assess the stability of sample.

To 1 ml of stock solution rilpivirine and cabotegravir, 1 ml of 2N sodium hydroxide was added and refluxed for 30mins at 60^oC. The resultant solution was diluted to obtain 150µg/mL & 100µg/mL solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

The standard drug solution was placed in oven at 105°C for 1 h to study dry heat degradation. For HPLC study, the resultant solution was diluted to 150µg/ml & 100µg/ml solution and 10µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

The photochemical stability of the drug was also studied by exposing the 1500µg/ml & 1000µg/ml solution to UV Light by keeping the beaker in UV Chamber for 1days or 200 Watt hours/m² in photo stability chamber. For HPLC study, the resultant solution was diluted to obtain 150µg/ml & 100µg/ml solutions and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of sample.

Stress testing under neutral conditions was studied by refluxing the drug in water for 1h at a temperature of 60^oC. For HPLC study, the resultant solution was diluted to 150µg/ml & 100µg/ml solution and 10 µl were injected into the system and the chromatograms were recorded to assess the stability of the sample.

The system suitability parameters were determined by preparing standard solutions of cabotegravir (100µg/mL) and Rilpivirine (150µg/mL) and the solutions were injected six times and the parameters like peak tailing, resolution and plate count were determined. The results were depicted in Table 2.

Specificity of the method was determined by injecting blank to check whether peaks in the blank are eluting with drugs peaks and the chromatogram was shown in Figure 4.

Figure 4

The linearity of cabotegravir and rilpivirine was in the concentration range of 25-150 µg/mL and 37.5-225µg/mL respectively with correlation coefficient (r²) of 0.9999 for both the drugs. The results are given in Table 3 and calibration curves are shown Figure 5, Figure 6.

Figure 5

Figure 6

The precision of an analytical procedure is usually expressed as relative standard deviation (coefficient of variation) of a series of measurements

It was performed by injecting a standard solution of cabotegravir and rilpivirine at working concentration of 100% six times that is 100µg/mL of cabotegravir and 150 µg/mL of rilpivirine). The % RSD was calculated for peak area and R_t and results are given in Table 4.

Six working sample solutions with the same concentrations were produced after multiple sampling from a sample stock solution. Each injection was given from a different working sample solution, and obtained areas were mentioned in the above table. Average area, standard deviation and % RSD were calculated for two drugs and obtained as 0.5% and 0.6% respectively for rilpivirine and cabotegravir. Data of method precision was given in Table 4.

The degree to which test findings acquired using an analytical method that are near to the actual value defines the method's accuracy. It is done by measuring the amount of pure drug recovered at three different concentrations (50%, 100% and 150%) in triplicate. The results are given in Table 5.

Robustness of the proposed method has been evaluated by small deliberate changes in the system parameters such as flow rate, mobile phase composition, and temperature It was discovered that none of the aforementioned parameters changed the peak area, retention duration, or USP tailing even when the flow rate was changed by 0.1mL, the mobile phase was changed by 5%, or the temperature was changed by 5°C. The % RSD was found to be within the limits and the method was found to be robust. The robustness results were shown in Table 6.

Table 6

S. No.	Parameteres	Cabotegravir			Rilpivirine
		RT	Plate coun t	TF	RT	Plate coun t	TF
	Flow Rate 0.9	3.16	6156	1.2	2.28	4294	1.2
1		5		6	3		6
	Flow Rate 1	3.18	6134	1.3	2.3	4348	1.2
		7					8
	Temperature	3.17	6037	1.2	2.27	4540	1.2
2	27°C			5	8		6
	Temperature	3.18	6134	1.3	2.3	4348	1.2
	30°C	7					8
3	Mobile Phase (- 5%)	3.16 8	6181	1.2 7	2.28 6	4458	1.2 8
	Mobile Phase (+5%)	3.17 8	6111	1.2 4	2.28 8	4502	1.3

RT - Retension rime, TF - Tailing factor

The validated RP-HPLC method was successfully applied for the assay of cabotegravir and rilpivirine in marketed formulations. The chromatogram for sample is shown in Figure 8. Assay results are represented in Table 7.

Figure 7

According to ICH guidelines, the drug product underwent forced degradation in order to determine the method's ability to indicate stability, learn more about the degradation products, and demonstrate how the quality of a drug substance and drug product changes over time under the influence of various stressful conditions. According to ICH guidelines, the percentage of rilpivirine and cabotegravir that degraded was between 5% and 20%. The results of stress studies were shown in Table 8. The chromatograms of Forced Degradation studies were shown in Figure 8.

Figure 8

The simultaneous estimation of cabotegravir and rilpivirine in pharmaceutical dosage form was achieved using a simple, accurate, and precise method. Cabotegravir's retention time was determined to be 2.30 minutes, while retention time of Rilpivirine's was found to be 3.187 minutes, with %RSD of 0.3 and 0.4 respectively. For cabotegravir and rilpivirine recovery rates was 100.25% and 99.79% respectively. The technique created was straightforward and affordable, and it could be used for standard quality control tests in industries

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