Drug Elevating Efficiency: Validated RP-HPLC Method for Concurrent Detection of Cabotegravir and Rilpivirine in Medicine

Received: 02-Sep-2024, Manuscript No. JDAR-24-146450 ; Editor assigned: 04-Sep-2024, Pre QC No. JDAR-24-146450 (PQ); Reviewed: 18-Sep-2024, QC No. JDAR-24-146450; Revised: 23-Sep-2024, Manuscript No. JDAR-24-146450 (R); Published: 30-Sep-2024, DOI: 10.4303/JDAR/236406

Introduction

Antiretroviral medication cabotegravir is a structural counterpart of dolutegravir. Inhibiting strand transfer of the viral genome into the host genome and stopping virus replication, cabotegravir interacts to the active site of HIV integrase. Due to the daily oral tablet administration and the monthly intramuscular suspension administration, the medication has a protracted period of action. Rilpivirine belongs to the class of compounds known as diary pyrimidines, which are similar to the pyrimidine nucleotides present in DNA. Rilpivirine is a non-nucleoside reverse transcriptase inhibitor (NNRTI) used to treat HIV-1 infections in patients who have never received treatment [1]. Figures 1 and 2 represent the chemical structures of cabotegravir and rilpivirine respectively. According to literature survey, it was found that RP-HPLC, LC-MS, UPLC methods were the works performed till date for both drugs in combination [1-9]. The study’s major objective was to create an easy, precise RP-HPLC method for the measurement of cabotegravir and rilpivirine in both pure and pharmaceutical dosage forms.

Figure 1: Structure of cabotegravir

Figure 2: Structure of rilpivirine

Materials and Methods

Chemicals and reagents

Spectrum pharma research solutions (Hyderabad) provided a free sample of rilpivirine and cabotegravir. Orthophosphoric acid, potassium dihydrogen ortho phosphate (KH₂PO₄) buffer, acetonitrile and methanol were purchased from Rankem Laboratories Pvt.Ltd. Millipore Milli Q water was prepared in the laboratory.

Instruments and equipment

Waters 2695 HPLC system equipped with quaternary pumps and photodiode array detector was used. The pH of the solutions was calculated by a pH meter (BVK enterprises, India). All analytical measurements were done on analytical balance (Denver).

Method of analysis

Preparation of standard stock solutions: 37.5 mg of rilpivirine and 25 mg of cabotegravir working standards were accurately weighed and placed into a fifty millilitres clean dry volumetric flask. 10 millilitres of diluents were added and sonicated for ten minutes. Diluents were used to make up the final volume. The obtained standard stock solution was 1000 μg/ml.

Preparation of sample working solutions: A volumetric flask of capacity 10 ml was filled with 100 μg/ml of diluent after 1 ml of each stock solution was pipetted out and added.

Preparation of diluents: Diluent was prepared using acetonitrile and water (50:50 v/v).

Preparation of sample stock solutions: 1 ml of the rilpivirine and cabotegravir injection sample was pipetted into a 100 ml volumetric flask along with 50 ml of diluents. The mixture was then subjected to 25 minutes of sonication. Finally, diluent was added to the volume (1000 μg/ml) and filters were used to remove impurities.

Preparation of sample working solutions: After being filtered, 0.5 ml of the sample stock solution was transferred to a 10 ml volumetric flask and diluted with diluents.

Chromatographic conditions: The RP-HPLC method development and validation of rilpivirine and cabotegravir was carried on Kromasil C₁₈ column. Mobile phase consisted of 0.1N KH₂PO₄: Acetonitrile in the ratio of 70:30 v/v with a flow rate of 1 ml/min. The sample injector volume was 10 μl. Temperature of column was ambient. The wavelength of rilpivirine and cabotegravir from the UV spectrum was 257 nm. Analyte eluted was observed at 257 nm. Table 1 listed the chromatographic conditions, and Figure 3 represented the optimised chromatogram.

Table 1: Optimized chromatographic conditions

Figure 3: Optimized chromatogram of cabotegravir and rilpivirine

Method development

Trial 1 was carried out by using water: Methanol in the ratio of 50:50. Trial 2 was conducted out by using OPA: Methanol in the ratio of 50:50. Trial 3 was involved out by using acetonitrile: 0.1N KH₂PO₄ in the ratio of 50:50. Trial 4 was included out by using acetonitrile: KH₂PO₄ in the ratio of 75:25. The method optimization was done in the trial 5 by 0.1N KH₂PO₄: Acetonitrile.

Method validation

System suitability parameters: 6 duplicate injections of the drug standard solution of 10μg/ml were injected into HPLC system and the system suitability parameters were determined. Peak tailing, USP theoretical plates count, and the resolution were noted.

Specificity: The specificity was evaluated by loading samples into the HPLC system. Drug solution was compared to the blank solution. The output chromatograms were examined for the interference between a drug peak response and a blank response.

Linearity: Dissimilar drug standard solutions were made to evaluate the linearity by diluting the drug stock solutions with diluents in different concentrations of rilpivirine and cabotegravir ranging from 18.75 μg/ml to 112.5 μg/ml and 12.5 μg/ml to 75 μg/ml respectively. The linearity plot of the calibration curve was assessed by linear regression analysis.

Sensitivity: The following equations, which were based on the slope of the calibration and the standard deviation of the responses using various concentrations of the standard stock solution, were used to calculate the limit of detection and limit of quantification.

Limit of detection=3.3 × standard deviation of the response/ slope of calibration curve of the analyte.

Limit of quantification=10 × standard deviation of the response/slope of calibration curve of the analyte.

Accuracy: Accuracy was determined at 50%, 100% and 150% by adding an acknowledged amount of sample stock solution of rilpivirine and cabotegravir to the standard stock solution. The percent recoveries were calculated.

Precision: Precision was studied as system precision, intraday and inter day. Intraday precision was determined by injecting 6 different concentrations of standard solutions in the same day. The peak area was measured, and the %RSD was computed. Inter-day precision was measured by injecting 6 different concentrations of standard solutions 3 times a week for 3 days. The peak area was measured, and the %RSD was computed.

Robustness: The determination of the robustness was done by injecting the samples by varying the mobile phase ratio and flow rate.

Forced degradation studies

Oxidation: 0.1 ml of the solution was pipetted out from the standard stock solution of rilpivirine and cabotegravir, 20% of 1 ml H₂O₂ was added to it. The solutions were heated at 60°C for 30 mins in a water bath. The solutions were cooled and brought to room temperature. Diluent was used to make up the volume. Solution of 10 μl was injected into the HPLC system.

Acid degradation: 1 ml of the stock mixture of rilpivirine and cabotegravir was mixed with 1 ml of 2N HCl. Then, it was refluxed for 30 minutes at 600°C. The resultant solution was made up with diluents and 10 μl solution were injected into the system.

Alkali degradation: To 1 ml of stock solution of rilpivirine and cabotegravir, 1 ml of 2N NaOH was added and refluxed for 30 mins at 60°C. To determine the sample’s stability, the resulting solution was diluted with diluents. The system was injected with 10 μl, and the chromatograms were obtained.

Thermal degradation: The standard sample solution was heated at 105°C for 6 hours. Diluent was used to prepare the solution. After the system has cooled, 10 μl of the solution was added.

Photolytic degradation: 1500 μg/ml and 1000 μg/ml solution was exposed to UV light by keeping the beaker in UV Chamber for 1 day or 200-Watt hours/m² in photo stability chamber and 10 μl were injected into the system.

Results

Method optimization of chromatographic conditions

UV spectroscopic analysis of the drug showed maximum absorbance at 257 nm. An appropriate and precise HPLC technique for analysis of cabotegravir and rilpivirine was employed after several trials with different mobile phases were done. The first trail started with water and methanol in the ratio of 50:50. This trail was not selected as only cabotegravir peak was eluted but not rilpivirine. The 2^nd trail was with orthophosphoric acid and methanol in the ratio of 50:50. This trail was not selected as broad peak shape was eluted for cabotegravir. The 3^rd trail was with acetonitrile and 0.1N KH₂PO₄ in the ratio of 50:50. This was not selected as both the peaks were eluted in void volume range. The 4^th trail was with acetonitrile and KH₂PO₄ in the ratio of 75:25. This was not selected as both peaks were eluted with more elution time. The 5^th trail was optimised by 0.1N KH₂PO₄ and acetonitrile in the ratio of 70:30 and the obtained chromatogram was found to be in good shape.

Method validation

System suitability parameters: The parameters for the rilpivirine and cabotegravir revealed that the theoretical plates were >2000 and the tailing factor was <2. Table 2 displayed statistics on system appropriateness.

Table 2: System suitability parameters

Linearity: Six linear concentrations of rilpivirine (18.75 μg/ml-112.5 μg/ml) and cabotegravir (12.5 μg/ml-75 μg/ ml) were injected in a duplicate manner. Linearity equations obtained for cabotegravir was y=7596.9x+1542.1 and of rilpivirine was y=7517.8x+5409.4. Correlation coefficient obtained was 0.999 for the 2 drugs. The linearity data was provided in Table 3. Figures 4 and 5 showed the calibration curves of cabotegravir and rilpivirine respectively.

Table 3: Linearity data of cabotegravir and rilpivirine

Figure 4: Calibration curve of cabotegravir

Figure 5: Calibration curve of rilpivirine

Accuracy: Three injections were given for each degree of accuracy. The mean percent recovery for cabotegravir and rilpivirine, respectively, was found to be 100.06% and 99.46%. Tables 4 and 5 showed the accuracy data of cabotegravir and rilpivirine respectively.

Table 4: Accuracy data of Cabotegravir

Table 5: Accuracy data of Rilpivirine

Repeatability: Average area, SD, and percent RSD were calculated for rilpivirine and cabotegravir. They were found to have respective values of 0.6% and 0.7%. Data on repeatability are displayed in Table 6.

Table 6: Repeatability of Cabotegravir and Rilpivirine

Intermediate precision: Chromatogram values for intermediate precision were found to be 0.4 and 1.2 for rilpivirine and cabotegravir respectively. The outcomes were shown in Table 7.

Table 7: Intermediate precision of Cabotegravir and Rilpivirine

Robustness: The %RSD of flow+, flow-, mobile phase+, mobile phase-, temperature+ and temperature-were found to be 0.4%, 1.1%, 0, 0.9%, 0.4%, 1% respectively for cabotegravir and 0.4, 0.3, 0.5, 0.4, 0.4, 0.4 for rilpivirine respectively. Table 8 provided an illustration of the outcomes.

Table 8: Robustness data of Cabotegravir and Rilpivirine

Specificity: Interference was not detected. The specificity data was shown in Table 9. The chromatogram of blank was shown in Figure 6.

Table 9: Specificity

Figure 6: Chromatogram of blank sample

Sensitivity: Cabotegravir and rilpivirine LOD was found to be 0.25 μg/ml and 1.79 μg/ml respectively and cabotegravir and rilpivirine LOQ was found to be 0.77 μg/ml and 5.44 μg/ml.

Assay: Average %assay for rilpivirine and cabotegravir obtained was 99.93% and 99.88% respectively.

Forced degradation studies: Cabotegravir and rilpivirine were subjected to acid degradation (2.65%, 2.53%), base degradation (2.58%, 1.85%), peroxide degradation (4.42%, 4.77%), thermal degradation (2.63%, 2.67%), UV (1.38%, 1.27%), water (0.69%, 0.78%). Table 10 represented the forced degradation studies data. Figures 7-12 listed the chromatograms for the many types of degradation.

Table 10: Forced degradation studies data of Cabotegravir and Rilpivirine

Figure 7: Chromatogram of acidic degradation of cabotegravir and rilpivirine

Figure 8: Chromatogram of alkali degradation of cabotegravir and rilpivirine

Figure 9: Chromatogram of peroxide degradation cabotegravir and rilpivirine

Figure 10: Chromatogram of thermal degradation cabotegravir and rilpivirine

Figure 11: Chromatogram of UV degradation cabotegravir and rilpivirine

Figure 12: Chromatogram of water degradation cabotegravir and rilpivirine

Discussion

RP-HPLC technique for the quantification of cabotegravir and rilpivirine in the pure and tablet dosage forms was developed and validated as per ICH guidelines. The analyte separation was obtained by Kromasil C₁₈ column. Mobile phase was in the ratio (70:30 v/v) of 0.1N of KH₂PO₄ and acetonitrile Flow rate of 1 ml/min was used. The wavelength of the drug was at 257 nm. The development of the method required a system suitability test that ensured the system was appropriate for the analysis of cabotegravir and rilpivirine. A proper protocol was established to ensure that the HPLC equipment executed techniques that produced findings with an acceptable level of accuracy and precision prior to the analysis of samples from each day. With the proposed HPLC approach, linearity was attained at a concentration range of 18.75 μg/ml-112.5 μg/ml for rilpivirine and 12.5 μg/ml-75 μg/ml for cabotegravir. When the correlation coefficient was found to be within accepted limits, acceptable linearity was indicated. The conventional addition technique was used to obtain accuracy samples at three levels (50%, 100%, and 150%). The method’s high recovery rates demonstrated that the suggested approach can be used for quality control examination. The repeatability of the chromatograms was found to be within the predetermined limit (%RSD not more than 2.0%). As a result, it proved that the method was found to be repeatable. The limit specified (%RSD not more than 2.0%) for chromatogram data with intermediate precision was determined to be met. Thus, it demonstrated that the procedure was determined to be effective. The robustness was evaluated by introducing small, deliberate changes to the chromatographic conditions, which include the ratios of 0.1N KH₂PO₄ and acetonitrile in the mobile phase (65B:35A) and (75B:25A), and the flow rate of the mobile phase (0.9 ml/min and 1.1 ml/min). The %RSD was discovered to be robust.

Specificity was examined by introducing a blank solution into the HPLC apparatus. This showed that there was no interference in the blank sample at the retention time of the standard cabotegravir and rilpivirine sample. At the retention time of the typical cabotegravir and rilpivirine, there was no interference with the blank sample. So, the procedure was specific, as can be seen. LOD was defined as about S/N 3 and LOQ as the minimal verified concentration with (%) RSD and (%) error 20%, all of which were taken into consideration when calculating LOD and LOQ. LOD and LOQ were observed to be sensitive. As per the label claim, the amount of drug content obtained from the sample solutions values was in the permissible 90%-110% range. Cabotegravir and rilpivirine were subjected to acid degradation (2.65%, 2.53%), base degradation (2.58%, 1.85%), peroxide degradation (4.42%, 4.77%), thermal degradation (2.63%, 2.67%), UV (1.38%, 1.27%), water (0.69%, 0.78%). The results were less than 10% indicating that cabotegravir and rilpivirine were more resistant towards all forced degradation conditions applied. The system suitability parameters were also within limits. Table 11 represents the summary of the approach that was created and verified.

Table 11: Summary

Conclusion

The proposed method was authenticated for a various parameter, included accuracy, precision, linearity, specificity, system suitability, and robustness, as specified by ICH requirements. The results attained fit the criteria for approval. The method can therefore be used successfully for the routine analysis of cabotegravir and rilpivirine in bulk and pharmaceutical dose forms as it is easy to use, precise, inexpensive and safe.

Acknowledgement

The management of GITAM (Deemed to be University), Visakhapatnam, Andhra Pradesh, India, is gratefully acknowledged by the authors for providing the facilities needed to conduct the research.

Funding

None.

Conflict Of Interest

Regarding the research and authorship of this publication, the authors state that there are no conflicts of interest.

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