RP-HPLC Method Development and Validation for the Estimation of Dolutegravir In Bulk and Pharmaceutical Dosage Form

Human immunodeficiency virus (HIV) infection is treated with Dolutegravir. Dolutegravir belongs to a group of drugs known as HIV integrase inhibitors. It functions by raising the quantity of immune cells that aid in the body's defence against infections and lowering the level of HIV in blood. ^[1] Dolutegravir is a second-generation HIV Integrase Strand Transfer Inhibitor (INSTI). It stops the HIV virus from multiplying by binding to the active site of the viral enzyme integrase and blocking it, which prevents the virus from inserting its genetic material into the DNA of the host's CD4 immune cells. ^[2] Only a few analytical (limited to 50 mg only) and bioanalytical methods have been developed thus far for the determination of dolutegravir in bulk and pharmaceutical formulation. Dolutegravir's chemical structure ^[3] is shown in Figure 01.

Figure 01: The Chemical structure of Dolutegravir

MATEIALS AND METHODS

Chemicals and Reagents

The working standard drug Dolutegravir (99% purity) was obtained from Hetero Labs Limited, Hyderabad, Telangana. The formulation dosage form having brand name Tivicay PD 5 mg containing 5 mg of Dolutegravir, was purchased from local Pharmacy. HPLC grade Methanol, Water and Acetonitrile & Orthophosphoric acid (OPA) were purchased form Merk chemicals private limited, Mumbai.

Preparation of Mobile Phase

Methanol:0.05% OPA (68:32) at pH 3.0 was prepared. Before being utilised, the mobile phase was filtered through a 0.22 µm nylon filter after being sonicated for 15 minutes to eliminate dissolved gases.  

Preparation of standard drug solution

Dolutegravir, 5 mg was weighed and transfer into a 10mL volumetric flask. It was then dissolved in 5 mL of methanol using a sonicator to make sure it was fully dissolved. The mixture was filtered using a 0.22 µm nylon filter. After that the volume in the flask was adjusted to 10 mL using methanol. This resulted in a stock solution that has 500 µg/mL of Dolutegravir. The stock solution of Dolutegravir was diluted to 50 µg/mL and then to 5µg/mL. The 5µg/mL of Dolutegravir were required for developing and validating a method.

Preparation of formulation solution

After carefully weighing ten Dolutegravir tablets (5 mg according to the label), they were ground into a fine powder. A 10 mL volumetric flask was filled with 5 mg of Dolutegravir tablet powder, the flask was filled with about 5 mL of methanol, and the mixture was sonicated for 15 minutes. After the solution had cooled to room temperature, the volume was adjusted using the same diluent. A sample stock solution of 500 µg/mL was prepared. A 0.22 µm nylon filter was used to filter the sample stock solution, and the first few ml of the filtrate were discarded. A working sample solution of 50 µg/mL was prepared by carefully pipetting 1.0 mL of the filtered sample stock solution into a 10 mL volumetric flask and diluting it to volume with the diluent. To reach the necessary concentration of 5 µg/mL for assay and validation testing, further dilutions were produced from the working sample solution using the same diluent.  

METHOD DEVELOPMENT

Selection of Wavelength

The PDA detector was used to scan reference solutions containing 5μg/mL in order to choose an appropriate wavelength. The maximal wavelength obtained was found to be the optimal wavelength for the detection.

Table 01: Optimized Chromatographic Conditions

Method Validation

In accordance with the ICH standards, the method's specificity, system appropriateness, LOD & LOQ, linearity, accuracy, precision, ruggedness, and robustness were all validated. Duplicate injections of the sample and standard solutions into the column were used for validation. ^[4,5]

RESULTS AND DISCUSSION

Method Development

Figure 02: UV Spectra of Dolutegravir

Figure 03: Optimized Chromatogram of Dolutegravir

Table 02: Results for Optimized Chromatogram

Method Validation

       Specificity

       No inference of diluent & Placebo

Linearity

Table 03: Results for Linearity

Figure 06: Linearity graph for Dolutegravir

LOD & LOQ

Dolutegravir 's LOD was found to be 0.08µg/mL, while its LOQ was determined to be 0.24µg/mL.

Precision

The system and method precision % RSD for dolutegravir were found to be 0.31 and 0.15, respectively. Both the system and method precision %RSDs fell within the permissible range of less than 2. As a result, the developed process was thought to be precisable.

Table 04: Results for Precision

Accuracy

The recovery percentage was found to be between 98.40 and 99.60%. At 50%, 100%, and 150% spiking levels, the percentage RSD was determined to be within the permissible limit for dolutegravir. The results showed that the suggested method was accurate, with an acceptance limit of 98–102% and a percentage RSD of less than two.

Table 05: Results for Accuracy

Ruggedness (Intermediate Precision)

%RSD of less than two is required to characterize ruggedness. In the developed approach, dolutegravir's %RSD was 0.23. The ruggedness of the method is confirmed by results that are within the allowed range.

Table 06: Results for Ruggedness

Robustness

It was confirmed that the Dolutegravir percentage change using the developed approach was within the allowed range of less than 2. Thus, it was demonstrated that when the analytical conditions were significantly altered, the suggested approach was appropriate for the analysis of dolutegravir. This shows that the results are unaffected by even small changes to the analytical conditions.

Table 07: Results for Robustness

Assay

Dolutegravir's test percentage in formulation analysis was 99.30%. As a result, it was demonstrated that the technique was appropriate for routine analysis of Dolutegravir in both bulk and formulation form.

Table 08: Results for Formulation

Figure 07: Chromatogram of Formulation