Method development and validation for Cabotegravir and Rilpivirine by using HPLC and its degradants are characterized by LCMS and FTIR

Using a Symmetry C18 (4.6 × 150 mm, 3.5) column, a high-performance liquid chromatographic method for quantification of Rilpivirine and Cabotegravir in active pharmaceutical ingredients was developed and validated. The mobile phase is made up of buffer, acetonitrile, and 0.1 percent formic acid in a 20:80v/v ratio. The flow rate was kept constant at 1.0 ml/min, and detection was accomplished through absorption at 231 nm with a photodiode array detector. The calibration curve was linear, with a regression coefficient (R2) value of 0.999 and concentrations ranging from 30 to 450 g/ml of Rilpivirine and 20–300 g/ml of Cabotegravir. The method's LOD and LOQ were 0.375 g/ml, 1.238 g/ml, and 0.25 g/ml, 0.825 g/ml for Rilpivirine and Cabotegravir, respectively. In the forced degradation studies, the degradants were characterized by using LCMS and FTIR. The current application was found to be simple, economical, and suitable, and validated according to ICH guidelines.


Background
Cabotegravir is a medicine utilized for the treatment of acquired immunodeficiency syndrome [1,2]. It is available in tablet form and as an intramuscular injection [3,4], as well as an injectable blend with Rilpivirine under the brand name Cabenuva. The injection forms are used once every month or every two months. Cabotegravir in combination with Rilpivirine has been shown to treat human immunodeficiency virus type 1 in adults (Human immune virus-1). And, if the virus has not developed resistance to the inhibitors, the combination injection is planned for the treatment of adults who do not have detectable human immune virus levels in their blood despite their current antiretroviral treatment [5,6] and integrase strand transfer inhibitors [7]. Before beginning injection therapy, the tablets are used to see if a person responds to the medication. The two drugs are the first antiretroviral drugs to be available in an injectable formulation with a long half-life. This implies that rather than a day by day pills, individuals get intramuscular injections month to month.
Rilpivirine, also known as Edurant and Rekambys, is a Tibotec prescription medication which is used to treat human immune virus/acquired immune deficiency syndrome [8]. It is a second-generation non-nucleoside reverse transcriptase inhibitor with a lower side-effect profile, higher strength, and a longer half-life than older non-nucleoside reverse transcriptase inhibitors like efavirenz [9]. The well-known side effects of the injectable formulation include reactions at the injection site (in

LC-MS/MS conditions
In the stress degradation study, HPLC was linked to a mass spectrophotometer with the splitter placed before the ESI source, allowing only 35% of eluent to enter. The following were the standard operating source conditions for Cabotegravir and Rilpivirine MS scans on positive ESI mode: the fragmented voltage was set at 80 V, the capillary at 3000 V, the skimmer at 60 V, nitrogen was used as a drying and nebulizing gas (45 psi), and highly filtered nitrogen gas was used as collision gas.

FTIR conditions
FTIR was utilized to get insights concerning the presence of different functional groups like keto, Aldehydes, cyano alcohol, and amides present in the degradation samples. Perkin Elmer range 100 models were utilized with KBr as a dispersion medium to make the specimen pellets. Approximately1.5-3.5 mg of specimens were mixed with 4.5 mg of KBr for a complete examination.

Buffer preparation
1 mL formic acid is dissolved in 1lt high-performance liquid chromatography-grade water and filtered through 0.45 filter paper.

Mobile phase preparation
After thoroughly mixing buffer and acetonitrile in 20:80 ratios, the mixture was sonicated for 5 min and filtered through a 0.22 m membrane filter. The HPLC analysis was carried out on a reversed phase-HPLC system with isocratic elution mode using a mobile phase of 0.1 percent formic acid and acetonitrile (80:20) on a Symmetry C18 column (150 × 4.6 mm, 3.5) with a flow rate of 1 mL/min and a photodiode array detector at 231 nm. As a diluent, the mobile phase was used.

Standard solution preparation (Rilpivirine 300 g/ml and Cabotegravir 200 g/ml)
Weigh out 300 mg of Rilpivirine and 200 mg of Cabotegravir working standards into a 100 ml volumetric flask, add 70 ml of diluent, sonicated for 30 min to dissolve it, and makeup to the mark with diluent. Transfer 5 ml of this stock solution to a 50 ml volumetric flask and dilute to the desired concentration with diluent.

Chromatographic condition optimization
Different mobile phase combinations were tested to determine theoretical plate count, resolution, tailing, and other system suitability parameters. Finally, the separation was accomplished using a freshly prepared mobile phase composed of buffer: acetonitrile in a 20:80 ratio with a flow rate of 1.0 ml/min. The injection volume of 10 l and the ambient temperature were kept constant throughout the process to obtain the symmetric peak of Rilpivirine and Cabotegravir at 231 nm.

Results
To achieve the best chromatographic conditions, various columns such as C18, C8, and CN-propyl, as well as mobile phases, were tested. The best chromatographic separation occurred on a Symmetry C18 column with acetonitrile and 0.1 percent formic acid in (80:20) mobile phase at a flow rate of 1 ml/min and PDA detection at 231 nm (Fig. 2). Table 1 depicts the optimized chromatographic conditions.

System suitability
Six replicates of a standard solution containing 300 g/ml of Rilpivirine and 200 g/mL of Cabotegravir were used to assess system suitability. The results show that the system's suitability is within the acceptable range. Table 2 and Fig. 3 show the results.

Linearity
The current application's linearity was determined by plotting a graph between concentration and corresponding peak area for Rilpivirine and Cabotegravir over concentration ranges of 30-450 g/ml and 20-300 g/mL, respectively ( Fig. 4) For both drugs, the correlation coefficient was found to be 0.999. Table 3 contains a summary of the linearity results.

Limit of Detection (LOD) and Limit of Quantification (LOQ)
The LOD and LOQ for Rilpivirine and Cabotegravir were 0.375 g/mL, 1.238 g/mL, and 0.25 g/mL, 0.825 g/mL, respectively. Table 4 summarizes the findings. Precision

Repeatability (or) method precision
The percentage of RSD value for six replicate injections of known concentrations of Rilpivirine and Cabotegravir performed on the same day was discovered to be 2%, indicating that the method precision is within the limit. Table 5 summarizes the findings.

Accuracy
Rilpivirine and Cabotegravir were prepared in three concentration levels: 50%, 100%, and 150%. The recovery percentage was found to be within the acceptable range of 98-102 percent. Based on these findings, it was determined that the developed method is precise and accurate. Table 6 summarizes the findings.

Robustness
The current method's robustness was tested by varying the mobile phase composition and flow rate. The percentage of RSD was discovered to be within an acceptable range. Table 7 contains a summary of the robustness results.

Degradation effects and its characterization
Rilpivirine and Cabotegravir samples were subjected to a variety of stress degradation conditions to observe the drugs partial degradation. The stress studies revealed the conditions under which the drug becomes unstable; these measures can be implemented during formulation to avoid potential instabilities. LC-MS and FTIR are used to characterize these degradation products.

Acid degradation
Initially, no degradant peaks were formed when Rilpivirine and Cabotegravir were studied in 0.1 N HCl. When the acid concentration was increased to 1 N HCl and heated at 60 °C for 30 min, 16.5 percent of Rilpivirine and 15.3 percent of Cabotegravir were degraded, and two degradations were observed. (DP1and DP2) products were formed on acid hydrolysis.

Alkali degradation
Rilpivirine and Cabotegravir were stressed under in 0.1 N NaOH, and no degradant products are formed. When the strength of alkali was increase to 1 N NaOH and heated at 60 °C for 30 min, 12.5% of Rilpivirine and 12.1% of Cabotegravir degradation were observed and one degradation (DP3) product was formed on alkali hydrolysis.

Oxidative degradation
Rilpivirine and Cabotegravir were studied in 10% peroxide condition, and no degradant peaks are formed. Then, the strength of peroxide was increased to 30% and refluxed for 3 h, one degradant (DP4) was formed.

Reductive degradation
The first trial of Rilpivirine and Cabotegravir were studied in 10% sodium bisulfate solution, for reduction degradation process, and no degradant was formed. After that, the above solution refluxed for 3 h, and no degradants were formed.

Thermal degradation
For thermal degradation, the sample was exposed at 105 °C for 3 h, and no degradant products are formed. After that, the above solution was refluxed for 3 h, and no degradation peaks were formed.

Photolytic degradation
For the first trial of photolytic degradation, the sample was exposed to UV light for 6Hrs and the exposed sample was analyzed, and no degradants peaks are formed. After that, the exposed sample was refluxed for 3 h, and no degradants peaks were formed. The details of degradation products and chromatograms are represented in Table 8 and Fig. 5. FTIR spectra show the frequencies (Fig. 6) of all different functional groups like acid, amide, cyano, and keto. The details of FTIR spectra for all degradation products are provided in Table 9.     and DP4 are displayed in Fig. 7, and fragmentation pathway mentioned in Fig. 8

Acid degradation product (DP1) characterization
The

Alkali degradation product (DP3) characterization
The

Collision-induced dissociation and FTIR details of Cabotegravir and Rilpivirine:
The degradation products of DP1, DP2, DP3, and DP4, which were observed under acid, alkali, and peroxide conditions. The ESI spectrum showed most intense

Discussion
We developed a high-performance liquid chromatography method for indicating stability, and forced degradation products are characterized using LC-MS and FTIR under ICH guidelines [19]. Very few articles were reported in the last few decades for determining the Cabotegravir and Rilpivirine by using HPLC [20][21][22]. In the present study, we intended to explore a specific, sensitive, and new HPLC method towards the analysis of Cabotegravir, Rilpivirine, and characterization of its degradation products by LC-MS and FTIR.

Conclusion
Till today there is no HPLC method to estimate the combination of Rilpivirine and Cabotegravir. A single HPLC method was validated and developed under ICH