The investigated sample Rucaparib [Maxheal Pharmaceutical Pvt. Ltd.] was procured from local market, Hyderabad. The standard Rucaparib drug was procured from Sanzyme Ltd. Acetonitrile [Merck], Methanol [Merck], Triethylamine [Merck], Water [Merck], Potassium dihydrogen phosphate were of HPLC grade.

Waters HPLC system equipped with 996 photo diode array detector was used for quantitation of Rucaparib. The processing with complete data obtained from Empower 2 software. The samples solubility in mobile phase was enhanced using Sonicator (SE60US model) Labman company. All the samples prepared were filtered using Vacuum filtration kit made by Labman.

The reverse-phase chromatography was performed on Waters HPLC using 996 photo diode array detection. The separation was done by using symmetry C18 ODS (25 cm × 0.46 cm internal diameter) 5 µm analytical column containing Phosphate buffer (0.02 M) and Methanol [65:35% v/v] mobile phase, adjusted the pH to 4.8 by dilute ortho phosphoric acid solution. The method was run at 1 ml∙min⁻¹ at 286 nm UV detection. The elution time for Rucaparib drug was at 5.484 min.

Accurately weighed 10 mg of Rucaparib standard drug was taken into 10 ml volumetric flask, diluted to volume and mixed thoroughly by ultrasonication in order to enhance solubility and to degas the solutions. From the stock solution 0.1 ml was pipetted into 10 ml volumetric flask, diluted to volume mixed thoroughly, treated with 0.45 µl filter paper and finally sonicated for 15 min. The prepared solution was introduced into the flow stream of mobile phase and recorded chromatograms. After so many experimental trials, the successful separation was achieved. Noted the optimized conditions and proceeded for validation as per ICH guidelines.

Twenty tablets of Rubraca (300 mg) were made into fine powder using mortar and pestle. From this, an amount equivalent to 10 mg was taken into clean dry volumetric flask [10 ml], diluted to volume, treated with 0.45 µl filter paper and sonicated for 15 min. From this, further pipetted out 0.1 ml solution, diluted to volume and mixed thoroughly. Injected the standard and sample solutions in triplicates and the % assay was calculated.

For evaluating the suitability of HPLC system and procedure, the Rucaparib standard solution of 10 µg ml⁻¹ concentration of about 20 µl was introduced into the HPLC system and recorded the chromatogram.

In order to determine the method’s accuracy, the drug was spiked at 50%, 100% and 150% levels. The chromatograms were recorded and noted the peak areas. From this, the average recovery of analyte was calculated.

Intra-day assay and intermediate precision were evaluated to determine method’s precision.

The linearity was evaluated by injecting over the range of 6 - 14 µg/ml concentration solutions of Rucaparib. A calibration curve was plotted between analyte concentration and analyte response. Performed regression analysis using least square method and calculated the correlation coefficient (r).

Robustness was evaluated by slightly changing the chromatographic conditions which includes percent organic solvent and flow rate.

The formula used to determine the lowest amount of analyte was,

LOD = 3.3 × standard deviation of response/slope of the calibration curve

The formula used to calculate the quantitation limit was,

LOQ = 10 × standard deviation of response/slope of the calibration curve.

Taken 1ml of Rucaparib stock solution and 1 ml of 2 N HCl, refluxed for 30 min at 60˚C. Later it was neutralized with 1 ml 2 N NaOH and made up to final volume to obtain 10 µg∙ml⁻¹ solution. Cooled to the room temperature and treated with 0.45 µl membrane filter. 20 µl sample was introduced into the HPLC system and recorded the chromatogram.

Taken 1 ml of Rucaparib stock solution and 1 ml of 2 N Sodium hydroxide, refluxed for 30 min at 60˚C. Later it was neutralized with 1 ml 2 N HCl and made up to the final volume to obtain 10 µg∙ml⁻¹ solution. Cooled to the room temperature and treated with 0.45 µm membrane filter. 20 µl sample was introduced into system, and recorded the chromatogram.

Taken 1 ml of Rucaparib stock solution and 1 ml of 20% hydrogen peroxide (H₂O₂) and left for 30 min at 60˚C. Later the solution was made up to the final volume to get 10 µg∙ml⁻¹ solution. Cooled to room temperature, treated with 0.45 µm membrane filter. Finally, 20 µl sample was introduced into the HPLC system, and recorded the chromatogram.

Kept 1 ml of Rucaparib stock solution in an oven at 60˚C for 6 hrs and made up to final volume to get 10 µg∙ml⁻¹concentration. Cooled to room temperature and treated with 0.45 µm member filter. Later, 20 µl sample solution was introduced into the HPLC system and recorded the chromatogram.

1 ml of stock solution of Rucaparib was exposed to Ultra-violet light for 1 day and made up to final volume to get 10 µg∙ml⁻¹ concentration and treated with 0.45 µm membrane filter. Later 20 µl sample solution was introduced into the HPLC system and recorded the chromatogram.

For this method development, various ratios and combination of mobile phases, different stationary phases and flow rates were tried to elute the drug with good peak parameters and to provide good performance in assay. Finally, the best separation was achieved on symmetry C18 ODS column (250 mm × 4.6 mm id, 5 µm) comprising mobile phase of phosphate buffer (0.02M): Methanol [65:35% v/v], adjusted the pH to 4.8 by dilute ortho phosphoric acid. The method was run at a flow rate of 1.0 ml∙min⁻¹ and the eluent was detected at 5.484 min by UV detector at 286 nm. The chromatogram showed the peak with good shape, more theoretical plates and the tailing factor was also found to be within the limits. All the method development trials with various mobile phase compositions, columns and flow rates results were shown in Table 1. The standard chromatogram of rucaparib drug was presented in Figure 1.