INTRODUCTION:

Olaparib is used to treat various tumours such as prostate, pancreatic, ovarian and breast cancers¹.Olaparib has the molecular formula C₂₄H₂₃FN₄O₃ (Mol. wt. 434.4628) (Figure 1). FDA had approved Olaparib in December 2014⁶ and Bevacizumab in 2004 for the treatment of cancer.Bevacizumab (Figure 2) is a monoclonal antibody used for the treatment of cancers². Bevacizumab was estimated using HPLC³, size-exclusion chromatography⁴ and Olaparib was estimated by using HPLC^5-8, UPLC⁹, LCMS¹⁰, LC-MS/MS¹¹ in pharmaceutical dosage form as well as biological fluids. A new stability indicating RP-HPLC technique has been developed and validated for the simultaneous assay of Olaparib and Bevacizumab.

Figure 1: Structure of Olaparib

Figure 2: Structure of Bevacizumab

MATERIALS AND METHODS:

Waters Alliance HPLC system equipped with a 2695 pump with Empower 2 software, UV detector, auto-injector, Shimadzu UV-Visible spectrophotometer, Ohus Electronic balance, Eutech pH Meter and Phoenix 4.5 L digital ultrasonic cleaner were used for the present study. Olaparib and Bevacizumab API samples obtained from Pharma Life Research facility in Hyderabad, India and other chemicals were of AR-grade (Rankem Chemicals, India).

Procedure

Accurately weighed quantities of 150 mg Olaparib and 25 mg Bevacizumab working standard into a clean, dry 100 mL volumetric flask. Few mL of diluent was added and sonicated to dissolve the substances completely. Solution was made up to final volume with the same diluent (stock solution). 5mL of stock solution was transferred into a 50 mL volumetric flask and the solution was made up to the final volume to get 150 μg/mL of Olaparib and 25 μg/mL of Bevacizumab.

Sample solution preparation

279 mg of the Olaparib sample and 1 mL of the Bevacizumab sample were accurately measured and transferred into a 100mL clean and dry volumetric flask. A small quantity of diluent was added and sonicated for 30 min. Mixture was centrifuged for 30 min. Final volume was made up to the mark with the diluent and solution was filtered through 0.45 µ filter to get stock solution. 5 mL of the stock solution was diluted to 50 mL to get 15 μg/mL of Olaparib, 25 μg/mL of Bevacizumab working standard solutions.

Method validation¹²

The process of method validation involves conducting a series of tests that are based on the analytical method. These tests are used to determine and document the performance characteristics of the method, as well as to determine if the method is suitable for a specific analytical purpose. The performance characteristics of LC methods include specificity, selectivity, precision, linearity, robustness, recovery, range, limit of detection, limit of quantification, and ruggedness. The focus of validation should be on the precision of the analytical system, rather than just the method itself. This includes having a well-defined method protocol, concentration ranges for the analyte, and a specified type of test material. The method validation protocol will be prepared by carefully considering the analytical system as a whole and the analytical procedure.

Linearity, Precision, Accuracy, Robustness

The specificity of the proposed method was confirmed by the absence of interfering peaks in the blank, placebo, and sample during the retention periods of Bevacizumab and Olaparib. In Figures 5 and 6, the chromatograms of a placebo and blank are displayed. The retention times for Olaparib and Bevacizumab were found to be 2.336 min and 4.873 min respectively. Figure 4 displays the optimized chromatogram.

Assay and Forced degradation studies¹³

The HPLC was used to acquire the chromatogram by injecting 5 µL of the standard solution and sample solution separately. The quantification of the medicines in solution was accomplished by evaluating the peak regions of the obtained chromatograms.

Stress testing was suggested by ICH guidelines as a means to determine the intrinsic stability of drug substances. Acid degradation (2 N HCl, refluxed at 60 °C for 30 minutes), alkali degradation (2 N NaOH, refluxed at 60 °C for 30 minutes), peroxide degradation (20% hydrogen peroxide heated at 60°C for 30 minutes), thermal degradation (samples arranged in a hot air oven at 105 °C for 1 hour), photolytic degradation (sample in an ultraviolet chamber for one day), and hydrolysis were all applied to the solution of the standard in these investigations. The peak areas of the so-stressed samples were determined and compared to the standard's peak areas.

RESULTS AND DISCUSSION:

A new stability indicating RP-HPLC technique has been developed and validated for the simultaneous assay of Olaparib and Bevacizumab using Inertsil ODS column and a mixture of acetonitrile and OPA (30:70, v/v) as mobile phase (Detection wavelength 258 nm) within a run time of 6 mins. The UV spectrum of Olaparib and Bevacizumab was shown in Figure 3. The retention time of Olaparib and Bevacizumab was found to be 2.336 min & 4.873 min (Figure 4) respectively.

Figure 3: UV spectrum of Olaparib and Bevacizumab

Linearity, Precision, Accuracy, Robustness studies

The linearity of the proposed method was observed within the concentration ranges of 6.25-37.50 μg/mL for Bevacizumab and Olaparib, and 37.50-225 μg/mL for Olaparib and the regression equations for Bevacizumab and Olaparib were found to be y=14512.58x+2387.04 and y=16815.19x+22410.75 respectively (Table 1). The calibration curves for Bevacizumab and Olaparib, were shown in Figures 5A and Figure 5B. The LOD values for Bevacizumab and Olaparib were 0.15 μg/mL and 0.9 μg/mL, respectively and the LOQ values for Bevacizumab and Olaparib were 0.5 μg/mL and 3.00 μg/mL, respectively. The % RSD in precision (Table 2), accuracy (Table 3) and robustness (Table 4) studies was found to be less than 2.0 indicating that the proposed method is precise, accurate and robust.

Blank

Placebo

Figure 4: Optimized chromatogram

Table 1: Linearity study

Sample	Olaparib		Bevacizumab
	Conc. (μg/mL)	*Peak area	Conc. (μg/mL)	*Peak area
1	37.50	639653	6.25	93123
2	75.00	1295299	12.50	189437
3	112.50	1963147	18.75	274437
4	150.00	2541563	25.00	363524
5	187.50	3188539	31.25	451032
6	225.00	3770639	37.50	549932
Slope	16815.19		14512.58
Intercept	22410.75		2387.04
R²	0.9997		0.9998

*mean of three replicates

A-Olaparib

B- Bevacizumab

Table 2: Precision study

Peak area	Repeatability (For sample)		Intermediate precision (Method precision)
Peak area	Olaparib	Bevacizumab	Olaparib	Bevacizumab
1	2537472	362893	2537472	363430
2	2545965	364560	2537472	362320
3	2512625	366195	2545965	360461
4	2530410	363591	2512625	362312
5	2505364	365786	2530410	365761
6	2520205	364268	2505364	364521
Mean	2525340	364549	2526367	363756
SD	15397.15	1263.22	16983.21	1483.32
% RSD	0.61	0.347	0.672	0.408

Table 3: Accuracy study

Level	Peak area		% Recovery (%RSD)
Level	Olaparib	Bevacizumab	Olaparib	Bevacizumab
50%	1267441 1263234 1246126	183053 181336 180124	99.7 (0.89)	99.8 (0.81)
100%	2525921 2536023 2517308	366406 368384 362467	100.0 (0.37)	100.5 (0.81)
150%	3772308 3761124 3753564	545237 547364 543759	99.3 (0.25)	99.9 (0.34)

Table 4: Robustness study

Olaparib
Condition	Flow rate (−) 0.90ml/min	Flow rate (+) 1.1ml/min	MP (−) 27:73 (v/v)	MP (+) 33: 67 (v/v)
Peak area	2724125	2239920	2081154	2857986
	2739254	2255843	2062754	2842715
	2727558	2243254	2078485	2835456
Mean	2730312	2246339	2074131	2845386
SD	7931.67	8397.82	9942.74	11499.98
% RSD	0.291	0.374	0.479	0.404
Bevacizumab
Peak area	383866	323829	406612	304442
	383214	322654	405564	303214
	385654	322187	406412	302787
Mean	384245	322890	406196	303481
SD	1263.31	846.06	556.39	859.2
% RSD	0.329	0.262	0.137	0.283

Assay and Forced degradation studies

The HPLC was used to inject 5 µL of standard solution (of pure drugs) and sample solution (extracted from Capsules) separately, in triplicate, to record the chromatograms. The percentage assay of the sample was determined by comparing the areas of standard and sample peaks. The results for the assay of Olaparib and Bevacizumab can be found in Table 5. The results of degradation studies for Olaparib and Bevacizumab obtained were shown in Tables 6 (Figure 6).

Table 5: Assay data for Olaparib and Bevacizumab

Drug	Avg sample area (n=5)	Std. Conc. (µg/ml)	Sample Conc. (µg/ml)	Label amount (mg)	Std purity	*Amount found (µg/ml)	% Assay
Olaparib	2524138	150	150	150	99.8	149.93	99.9
Bevacizumab	361714	25	25	25	99.9	24.85	99.4

*mean of three replicates


Acid Degradation	Alkali Degradation

Hydrolysis Degradation	Peroxide Degradation

Thermal Degradation	Photolytic Degradation
Figure 6: Typical chromatograms obtained during the forced degradation studies

Table 6: Forced degradation studies

Degradation condition	% Drug degradation*		Peak purity of Olaparib		Peak purity of Bevacizumab
Degradation condition	Olaparib	Bevacizumab	Purity angle	Purity threshold	Purity angle	Purity threshold
Acid degradation	12.7	13.2	0.407	4.035	1.53	6.341
Alkali degradation	14	12.6	0.101	4.055	1.442	6.195
Peroxide degradation	16.8	15.6	0.373	4.054	1.365	6.742
Thermal degradation	1.2	0.6	0.122	4.057	1.154	6.323
Photolytic degradation	3.4	2.2	0.154	4.021	1.253	6.264
Hydrolysis degradation	2.5	1.4	0.136	4.079	1.183	6.357

*mean of three replicates

CONCLUSION:

The HPLC technique that was developed is characterized by its simplicity, speed, cost-effectiveness, specificity, and reliability in estimating the quantities of Olaparib and Bevacizumab. The approach was deemed satisfactory and may be effectively utilized in normal laboratory analysis to simultaneously estimate Olaparib and Bevacizumab in both bulk and pharmaceutical dose forms.

REFERENCES:

1. Bochum S, Berger S and Martens UM. Olaparib. Recent Results Cancer Res. 2018; 211: 217-233.

2. Kazazi-Hyseni F, Beijnen JH and Schellens JH. Bevacizumab. Oncologist. 2010; 15(8): 819-825.

3. Sousa F, Gonçalves VMF and Sarmento B. Development and validation of a rapid reversed-phase HPLC method for the quantification of monoclonal antibody Bevacizumab from polyester-based nanoparticles. J Pharm Biomed Anal. 2017; 142: 171-177.

4. Oliva A and labrés M. Validation of a size-exclusion chromatography method for Bevacizumab quantitation in pharmaceutical preparations: application in a biosimilar study. Separations 2019; 6(3): 43.

5. Chaudhary A, Tonk R, Dagur P, Dey S and Ghosh M. Stability indicating assay method for the quantitative determination of Olaparib in bulk and pharmaceutical dosage form. Turk J Pharm Sci. 2022; 19(5): 488-497.

6. Priyanka W, Preeti K, Smita G, Vijaya Kumar M, Raman Mohan S and Bhaskar V. Method development and validation for quantitative estimation of Olaparib in tablet dosage. World Journal of Pharmaceutical Research. 2021; 10(12): 2162-2174.

7. Daumar P, Robin D, Clemence D and Emmanuel M. Development and validation of a high-performance liquid chromatography method for the quantitation of intracellular PARP inhibitor Olaparib in cancer cells. J Pharm Biomed Anal. 2018; 152: 74-80.

8. Antima C, Rajiv D, Pankaj D and Sudhasatya D. Stability indicating assay method for the quantitative determination of Olaparib in bulk and pharmaceutical dosage form. Turk J Pharm Sci. 2022; 19(5): 488-497.

9. Kavitapu D, Arthanareeswari M, Devikala S, Arockia S, Tamil selvin M, Sudershan M and Pradeep Kumar G. New rapid stability indicating RP-UPLC method for the determination of Olaparib, its related substances and degradation products in bulk drug and dosage form. Materials Today: Proceedings. 2019; 2(14): 492-503.

10. Nijenhuis C.M, Lucas L, Rosing H, Schellens J.H, Beijnen J.H. Development and validation of a high-performance liquid chromatography-tandem mass spectrometry assay quantifying Olaparib in human plasma. J Chromatogr B Analyt Technol Biomed Life Sci. 2013; 940: 121-125.

11. Ottria R, Ravelli A, Miceli M, Casati S, Orioli M and Ciuffreda P. Quantitative characterization of Olaparib in nano delivery system and target cell compartments by LC-MS/MS. Molecules. 2019; 24(5): 989.

12. ICH Validation of analytical procedures: Text and methodology, Q2 (R1), International Conference on Harmonization, 2005.

13. ICH Q1A (R2) Stability testing of new drug substances and products (2003).

Received on 24.11.2023 Modified on 28.12.2023

Research J. Pharm. and Tech 2024; 17(2):910-914.

DOI: 10.52711/0974-360X.2024.00141