Method development and validation by UV Visible spectrophotometer

Method development and validation by UV Visible spectrophotometer Olaparib

1. “UV- SPECTROPHOTOMETRIC METHOD DEVELOPMENT AND VALIDATION FOR THE QUANTATIVE ESTIMATION OF OLAPARIB IN API FORM ” Dissertation Submitted to Faculty of Pharmacy PALAMURU UNIVERSITY In partial fulfilment for the award of the degree of BACHELOR OF PHARMACY Submitted by M.PALLAVI (3119881009) B.UDAY KUMAR (3119881023) K.SRAVANI (3119881027) N.AKHILANDESWARI (3119881029) S.SUSHIMITHA (3119881036) S.PRASHANTHI (3119881046) TP.RUBY DEV (3119881052) Under the guidance of A.RAJAMANI, M.Pharm Assistant Professor Department of Pharmaceutical analysis DHANVANTHRI COLLEGE OF PHARMACEUTICAL SCIENCES MAHABUBNAGAR, TELANGANA STATE-509001 June -2023

2. LIST OF CONTENTS INTRODUCTION DRUG PROFILE LITERATURE REVIEW AIM AND OBJECTIVES PLAN OF WORK EXPERIMENTAL DETAILS METHOD DEVELOPMENT METHOD VALIDATION RESULTS AND DISCUSSION CONCLUSION BIBILOGRAPHY

3. INTRODUCTION Spectroscopy: It is the branch of science that deals with the study of interaction of matter with light. [or] It is the branch of science that deals with the study of interaction of electromagnetic radiation with matter. UV-Visible spectroscopy: Ultraviolet (UV) spectroscopy is a physical technique that involves the use of ultraviolet (UV) light. Light in the visible, ultraviolet, and near-infrared wavelengths is used in optical spectroscopy.

4. PRINCIPLE: The basic principle behind the UV spectroscopy is absorption of visible and UV radiation (200– 400 nm) is associated with excitation of electrons, in both atoms and molecules, from lower to higher energy levels. It is important to note that the difference in the energies of the ground state and excited state of the electron is always equal to the amount of ultraviolet radiation or visible radiation absorbed by it . P-electrons or nonbonding electrons in molecules can absorb ultraviolet light to increase the anti-bonding of these electrons from a low-energy state to a high-energy state.

5. Electromagnetic radiation: Electromagnetic radiation consist of discrete packages of energy which are called as photons. A photon consists of an oscillating electric field (E) & an oscillating magnetic field (M) which are perpendicular to each other .The relationship between wavelength and frequency can be written as: C= νλ As photon is subjected to energy ,so E=h ν = hc / λ

6. Frequency: It is defined as the number of times electrical field radiation oscillates in one second. The unit for frequency is Hertz (Hz). 1 Hz= 1 cycle per second Wavelength ( λ ): It is the distance between two nearest parts of the wave in the same phase i.e. distance between two nearest crest or troughs . Interaction of EMR with matter: When the molecules absorb UV-Visible light from EMR ,one of the outermost bond /lone pair electron is promoted to higher energy state such as E1,E2,........En, etc is called as electronic transition and the difference is as: ∆E=h ν =En-E

7. INSTRUMENTATION : LIGHT SOURCE : Light because they span the entire UV spectrum, lights with tungsten filaments, and Hydrogen Deuterium lamps are the most extensively utilized and appropriate lighting sources. Tungsten filament lamps release a lot of red radiation, namely 375 nm, whereas hydrogen-Deuterium lamps have a lower intensity and emit less red radiation . MONOCHROMATOR: It is a type of prism and slits make up this structure. Double beam spectrophotometers make up the majority of the spectrophotometers

8. The slits choose the various wavelengths of the light source that have been separated by the prism. Consequently, as a result of the prism's rotation, a series of wavelengths of steadily increasing wavelengths pass through the prism . SAMPLE AND REFERENCE CELL: Slits Cells as well as the sample reference the sample solution is passed through one of the two separated beams, while the reference solution is passed through the other. The cells hold both the test and the reference solution. Silica or quartz are used to make these cells. DETECTOR: Detectors used in UV/visible spectrophotometers can be called a photometric detectors. When a radiation is passed through a sample cell part of it is being absorbed by the sample solution and the rest is being transmitted. This transmitted radiation falls on the detector and the intensity of absorbed radiation can be determined or displayed. In these detector the light energy is converted to electrical signal which can be read recorded. The most commonly used detectors are:

9. APPLICATIONS : Qualitative analysis Quantitative analysis Detection of impurities Structure elucidation of organic compounds Molecular weight determination Calibration curve method Determination of elements, ions or functional groups Determination of organic substance and pharmaceuticals Barrier Layer cell or Photo Voltaic cell Photo tubes or photo emissive cells Photo Multiplier tubes

10. INTRODUCTION Anti cancer drug : A nticancer or Antineoplastic drugs are used in inhibiting the abnormal cell growth or killing the cancer cells. TYPES OF CANCERS : Ovarian cancer. Lung cancer. Breast cancer. Bronchial Cancer. Colon and Rectal Cancer. Breast Cancer. Pancreatic Cancer. Prostate Cancer. Liver and Intrahepatic Bile Duct Cancer.

11. IUPAC NAME: 4-[[3-[4-( cyclo propane carbonyl) piperazine-1-carbonyl]-4-fluoro phenyl] methyl]-2H-phthalazin-1-one MOLECULAR FORMULA: C 24 H 23 FN 4 O 3 MOLECULAR WEIGHT : 434.4628g/mol SOLUBILITY: Olaparib was found to be Soluble in Methanol (soluble), DMSO(soluble), Acetonitritle (soluble), Ethanol (slightly soluble),Water (poorly soluble). CATEGORY: Anti-Cancer Agent DRUG PROFILE :OLAPARIB Fig No:1 :-Structure of olaparib

12. BIOAVAILABILITY: Low oral bioavailability of this drug requires increase in the dose and frequency causing haematological toxicity in the patients. MECHANISM OF ACTION: Poly (ADP-ribose) polymerases (PARPs) are multifunctional enzymes comprising 17 members. They are involved in essential cellular functions, such as DNA transcription and DNA repair. PARPs recognize and repair cellular DNA damage, such as single-strand breaks (SSBs) and double-strand breaks (DSBs). Different DNA repair pathways exist to repair these DNA damages, including the base excision repair (BER) pathway for SSBs and BRCA-dependent homologous recombination for DSBs. HALF -LIFE : Following a single oral dose in patients with cancer, the mean terminal half-life was 6.10 hours.

13. Volume of distribution: The mean (± standard deviation) apparent volume of distribution of olaparib is 158 ± 136 L following a single 300 mg dose. Clearance : Following a single oral dose in patients with cancer, the mean apparent plasma clearance was 4.55 L/h. Adverse effect /Side effects: May occur that usually do not need medical attention. These side effects may go away during treatment as your body adjusts to the medicine. Diarrheea Joint pain Headache Nausea Painful or difficult in urination, Rapid shallow breathing shivering, Unusual bleeding, Vomiting.

14. S. No. Drug Name Label Claim Brand Name Company 1 Olaparib 50mg Lynparza Capsules Astra Zeneca MARKETED FORMULATION OF OLAPARIB :

15. LITERATURE REVIEW : Yuanyuan Yang et al, (2022): Ovarian cancer (OC) is the fifth most common cancer worldwide and is one of the most fatal gynecological cancers in women . According to GLOBOCAN 2020 data, there are 313,959 new cases of OC and 207,252 fatalities worldwide each year . Most OC cases (90%) are assumed to be caused by epithelial cells, and more than 70% are identified as high-grade OC, which usually manifests at an advanced stage (III and IV) . The primary therapies for OC are usually surgery and platinum-based chemotherapy Qiming Wang et al, (2022): Olaparib, an orally active inhibitor, has undergone comprehensive clinical evaluation as either single or combination therapy in several solid tumors. To explore the efficacy and safety of Olaparib versus placebo in the treatment of platinum sensitive recurrent ovarian cancer, we conducted a systematic review and meta-analysis.

16. 3.Priyanka Waje , et al. (2021): A simple and new isocratic high-performance liquid chromatography (HPLC) method was developed for quantitative determination of Olaparib in its tablet dosage form. The chromatographic separation was achieved on Thermo Scientific C18 column (250mm x 4.6mm i.d.5µ). The mobile phase selected was 0.1% Trifluoroacetic buffer: Acetonitrile in the ratio of 60: 40 v/v at flow rate 1.0ml/min with column temperature maintained at 35°C and 10µl injection volume. The detection was carried out at 276nm. The retention time of Olaparib was found to be at 5.14 min. The developed HPLC method was validated as per ICH (Q2R1) guideline. The HPLC method was linear over range of 25-75 µg/ml with regression coefficient 0.9991.

17. 4. Antima Chaudhary, et al. (2022 ): Olaparib is an orally active poly (ADP-ribose) PARP (polymerases) inhibitor known to destroy cancer cells with BRCA1 or BRCA2 deficiency. An authentic, fast, distinct, and reliable reverse phase-high performance liquid chromatography (RP-HPLC) method was developed and promptly validated in tablet formulations for Olaparib estimation. Materials and The proposed method focuses on the separation of Olaparib in reverse phase mode using a Waters symmetry C18 (150 x 4.6 mm, 5 μm ) analytical column with a flow rate of 1.0 mL/min and the injection volume was kept at 20μL. The optimized mobile phase consists of ammonium acetate buffer (pH adjusted to 3.5 by glacial acetic acid): methanol in the ratio of 50:50v/v.

18. 5.Pierre Daumar , et al. (2018): Olaparib is a potent PARP inhibitor in clinical use for cancer therapy. A bioanalytical assay was developed and validated for quantitation of intracellular level of Olaparib in cells exposed to the drug. The assay involves an optimized and straightforward sample pretreatment with acetonitrile for Olaparib solubilization , cell lysis and protein precipitation, and a high performance liquid chromatography (HPLC) method with ultraviolet detection. Several parameters in both the sample preparation and the detection steps were investigated. Optimal chromatographic conditions were achieved with a 5μL injection on a Nova-Pak® C18 column (150 × 3.9 mm, 4 μm ) using a mobile phase consisting of acetonitrile and ultra-pure water in gradient mode, at a constant 1.2 mL /min flow rate, at 35 °C. Detection was carried out at 254 nm and a diode array detector was used to insure purity of the Olaparib peak. The method was validated according to Food and Drug Administration guidelines. Linearity, accuracy and precisions were satisfactory over the concentration range of 200-2000 ng / mL. Limits of detection and quantification for Olaparib were 50ng/ mL and 200ng/ mL , respectively. Good stability was showed in three relevant analytical conditions.

19. 6.Graham R Williamson et al,(2019): A Bckground The Poly (ADP-ribose) polymerase inhibitor olaparib , acts against cancer cells in people with breast cancer pre-disposition gene mutations ( BRCAm ). Despite US and EU approval as a therapy for ovarian cancer patients with BRCAm , but research into olaparib therapy for breast cancer patients with BRCAm is in its infancy. Objective As no systematic review has yet been undertaken to synthesise clinBical trials looking at olaparib as a therapy for breast cancer patients with BRCAm , this systematic review aims to establish the current effectiveness of olaparib as a treatment for these patients. Methods CINAHL, MEDLINE, Royal College of Nursing, Cochrane Library, Joanna Briggs Institute, Centre for Reviews and Dissemination, Internurse , Embase , Google Scholar and PubMed databases were searched, supplemented by a grey literature search, hand searching and cross-referencing. Authors independently reviewed and graded the studies also using Kmet et al . scoring system. Results One long-term case study and six clinical trials were included. Heterogeneity prevented statistical meta-analysis, meaning only narrative synthesis was possible. The overall clinical benefit of olaparib appears to be greater and longer lived in BRCAm carriers compared to BRCAwt , and also compared to standard chemotherapy treatments.

20. 7. ManiK Ghosh et al,(2022): whenPARP inhibitors hold a lot of therapeutic potential and will likely be employed in many cancer therapies in the future. However, preclinical and clinical studies have revealed that tumor cell sensitivity to PARP inhibitors varies significantly, indicating that treatment efficacy must be enhanced. Because PARP is an intracellular target, a crucial element influencing tumour cell sensitivity and the efficacy of a PARP-targeted treatment is the quantity of PARP inhibitors reaching the intracellular compartment. PARP inhibitors, like any other intracellular target medicine, are affected by processes such as excretion, metabolism, drug absorption, and expression/ upregulation of transmembrane drug efflux transporters. The latter, which is particularly significant for PARP inhibitors, was discovered as a key resistance mechanism during early preclinical trials.Analytical method validation assures that diverse high performance liquid chromatography (HPLC) analytical procedures provide consistent and reproducible results; it is an important stage in the development of novel dosage forms since it provides information on accuracy, linearity, precision, detection, and quantification limits.

21. 8.Pierangela Ciuffreda et al; (2019): Having a good assay method is important to optimize the formulation of Ola-loaded nanoparticles and to study in vitro release profiles during the development of drug delivery systems, and to determine drug distribution in target and non-target organs or cells compartments. Several clinical trials, containing HPLC/MS-MS Ola quantification for pharmacological studies, have been published, but no technical details on analytical method employed were given . In the last decade, a few methods for Ola quantification in human plasma (100 uL ), characterized by a calibration range of 10–5000 ng/mL and high limit of quantification (10 ng/mL) or expensive and time consuming solid phase extraction procedure]were published. Interestingly, Roth and coworkers developed an HPLC/MS-MS method for Ola quantification in human plasma with large applicability to clinical trials due to its high sensitivity (0.5 ng/mL) and a large range of calibration (0.5–50000 ng/mL) Only a very recent article presents an HPLC coupled with Ultraviolet Diode Array Detector (HPLC-UV-DAD) method for Ola quantification inside cancer cells .

22. 9. Eunha Kim et al, (2019):   Modified Olaparib with TCO for target identification of the drug. In addition, they further developed a cleavable enrichment linker containing Tz (for click chemistry), biotin (for pull down assay), and 2-(4′-hydroxy-2′-alkoxy phenylazo )benzoic acid (as a cleavable site). They first confirmed the activity of Olaparib -TCO against recombinant PARP1 proteins and confirmed that Olaparib -TCO still had a nano -molar range of IC 50 . Later, MHH-ES1 Ewing's sarcoma cells and A2780 ovarian cancer cells were treated with Olaparib -TCO. Proteins labeled with TCO drugs were then pulled down by two-step bioorthogonal magnetic separation. To release proteins from magnetic beads, sodium dithionite was used to cleave the linker that allowed the specific release of small-molecule captured proteins while leaving non-specifically bound proteins on the solid support. Interestingly, LC/MS-MS data revealed a list of 24 different proteins including PARP1 proteins. During a follow-up in-depth study, they identified the protein TOP2A as another binding partner of Olaparib with an estimated  K d  of 3.7 nM .

23. 10. Roberta Ottria et al, (2019): The present analytical method was applied to develop a new delivery system for Ola, two different loading method were assessed to introduce the drug in ferritin nano -carriers, as described elsewhere . The first method, that plan the opening of the nano -carrier and its re-assembly at different pH values gave Ola concentrations of 240 ± 40 ng / mL (mean ± sd , 15 loading assay), while the second method, that plan the Ola complexation with copper before loading, gave Ola concentrations of 1117 ± 816 ng / mL (mean ± sd , 10 loading assay). Since the second loading method allows to encapsulate high quantities of the drug in the nano -carrier leading to Ola concentrations higher than the calibration range of the method (25–750 ng / mL ), accuracy and precision were assessed also on LQC2, MQC2, and HQC2, loading samples diluted 50 fold, 100 fold, and 200 fold The method was also successfully used to determine the amount of Ola in both cell culture media and lysates after incubation of free or H-Fn encapsulate Ola  report chromatograms relative to a real sample of cytoplasmic extract and two matrices, blank, and spiked at the LOQ (cell nuclei and cytoplasmic fraction), respectively.

24. 11. Joseph Garcia et al, (2020 ): Cancer cells differ fundamentally from normal cells, as a result of having acquired hallmark capabilities that enable tumor growth and progression. Due to their high metabolic demands, growing solid tumors depend on vascularization for provision of nutrients and oxygen and disposal of metabolic waste products. Vascularization can be promoted by angiogenesis, i.e. the generation of new blood vessels by sprouting from existing blood vessels. In normal physiology, angiogenesis plays a vital role in the generation of new vasculature during embryogenesis, but it is mostly quiescent in the adult body, with transient activation during wound healing and the female reproductive cycle. While angiogenesis is tightly controlled by an intricate interplay of pro- and anti- angiogenic factors, it can be activated by growing solid tumors; this so-called “ angiogenic switch” is recognized as a hallmark of solid tumors . Indeed, it was shown in animal models that blood vessels were essential to support tumor growth beyond the size allowed by oxygen diffusion alone.

25. 12. Christine C. Davis et at,(2019): The recent approval of olaparib , a poly (ADP-ribose) polymerase (PARP) inhibitor, in HER2-negative, metastatic breast cancer provides an additional treatment option for patients with a g BRCA m . Inhibition of PARP results in the trapping of the PARP-DNA complex at replication forks, causing single-strand breaks to become double-strand breaks (DSBs). PARP trapping and the accumulation of DSBs ultimately leads to cell apoptosis. Cells deficient in BRCA1/2 are particularly sensitive to the effects of PARP inhibition, as cells lacking these functional proteins are unable to repair DSBs, resulting in synthetic lethality. The phase III OlympiAD trial showed a progression-free survival benefit but no overall survival benefit, leading to the US Food and Drug Administration approval of olaparib . The purpose of this article is to describe current data regarding the use of olaparib in metastatic breast cancer, its role in the treatment of patients with a g BRCA m , and the clinical implications of its approval for oncology advanced practitioners.  

26. 13 . Robin dufour et al ;( 2018): . A bioanalytical assay was developed and validated for quantitation of intracellular level of olaparib in cells exposed to the drug. The assay involves an optimized and straightforward sample pretreatment with acetonitrile for olaparib solubilization , cell lysis and protein precipitation, and a high performance liquid chromatography (HPLC) method with ultraviolet detection. Several parameters in both the sample preparation and the detection steps were investigated. Optimal chromatographic conditions were achieved with a 5 µL injection on a Nova-Pak® C18 column (150 x 3.9 mm, 4 µm) using a mobile phase consisting of acetonitrile and ultra-pure water in gradient mode, at a constant 1.2 mL /min flow rate, at 35 °C. Detection was carried out at 254 nm and a diode array detector was used to insure purity of the olaparib peak. The method was validated according to Food and Drug Administration guidelines. Linearity, accuracy and precisions were satisfactory over the concentration range of 200 2000 ng / mL. Limits of detection and quantification for olaparib were 50 ng / mL and 200 ng / mL , respectively. Good stability was showed in three relevant analytical conditions

27. AIM AND OBJECTIVES AIM: The aim of investigation was to develop a Simple UV-visible Spectrophotometric method for the determination of Olaparib in its pure form and pharmaceutical formulations, further to validate the developed method. UV spectrophotometry combines the advantages of low cost and simplicity with the possibility of achieving high sensitivity and selectivity with good precision, accuracy and reliability.

28. OBJECTIVES: After observing the literature review we concluded that the few spectroscopic developed methods are available like HPLC and LC-MS. Those methods are used for the determination of Olaparib in API and tablet dosage form. To develop a simple, precise, accurate, rapid & economical analytical methods for quantitative estimation of anticancer drugs (Olaparib) in combined dosage form using UV spectrophotometric method.

29. PLAN OF WORK :

30. EXPERIMENTAL DETAILS LIST OF INSTRUMENTS USED S NO INSTRUMENTS /EQUIPMENTS/APPARATUS 1 UV- Visible spectrophotometer ( ELICO SL-159 ) 2 Electronic Balance ( SHIMADZU ATY224 ) 3 Ultra sonicator ( Wensar wuc-2L )

31. LIST OF CHEMICALS AND REAGENTS : S NO NAME PURITY GRADE MANUFACTURER/ SUPPLIER 1 Methanol 99.9% A.R Loba chem;Mumbai 2 Ethanol 96% L.R Sd fine-chem ltd; Mumbai 3 DMSO 99.9% HPLC Loba chem;Mumbai 4 Acetonitritle 93.9% L.R Sd fine-chem ltd;Mumbai 5 Doubled distilled water _ _ Sd fine-chem ltd;Mumbai SPECIFICATIONS

32. SOLUBILITY STUDY : REAGENTS SOLUBILITY Methanol soluble DMSO soluble ACN soluble Ethanol Slightly soluble water Poorly soluble

33. METHOD DEVELOPMENT PARAMETERS . Instrumentation The Spectroscopic analysis was carried out using Single beam UV-Visible Spectrophotometer ELICO SL-159 with I mm path length matched quartz cells was used for analytical purpose. Preparation of standard stock solution of olaparib . PRIMARY SOLUTION:- 10mg drug ( olaparib ) dissolved in 10 ml of Methonal [V.F] (1000PPM) SECONDARY SOLUTION :- Take 1ml of solution from above primary solution dissolved in 10 ml of Methanal [V.F] (100PPM) TERTIARY SOLUTION :- Take 1ml of solution from above secondary solution dissolved in 10 ml of Methonal [V.F] (10PPM)

34. Take 2ml of solution from above tertiary solution and make up the volume up to 10 ml (20PPM). Take 4ml of solution from above tertiary solution and make up the volume up to 10 ml (40PPM). Take 6ml of solution from above tertiary solution and make up the volume up to 10 ml (60PPM). Take 8ml of solution from above tertiary solution and make up the volume up to 10 ml (80PPM). Take 10ml of solution from above tertiary solution and make up the volume up to 10 ml(100PPM).

35. VALIDATION PARAMETERS: 1.RANGE: Range of an analytical method is the interval between the upper and lower levels (including these levels) that have been demonstrated to be determined with precision, accuracy and linearity using the method as written. It includes working range, linearity range and target range and 100% concentration or test concentration . The range of Olaparib drug is 10-60 μg/ml. UV SPECTRUM OF OLAPARIB AT 278 nm

36. 2.ACCURACY: The accuracy is nothing but the comparison of obtained value with the standard value. After completion of analysis of olaparib containing 3 group 3 replicates with the API form. Method: The accuracy of the developed method can be studied by preparing the solutions of various concentrations i.e. 80%, 100% and 120%. The prepared solutions in triplicates and here determined is percentage recovery of pure drug. Level of Recovery Sample Conc. (µg/ml) Standard Conc. Added (µg/ml) Total Conc. (µg/ml) Amount Recovered (µg/ml) % Recovery Mean %Recovery ±SD   % RSD 80% 8 10 18 18.025 100.138 100.1273± 0.200213   0.199959 80% 8 10 18 17.986 99.922 80% 8 10 18 18.058 100.322 100% 10 10 20 20.064 100.320 100.165 ± 0.195768       0.195445 100% 10 10 20 19.989 99.945 100% 10 10 20 20.046 100.230 120% 12 10 22 22.075 100.340 100.210 ± 0.253385       0.252854 120% 12 10 22 21.982 99.918 120% 12 10 22 22.082 100.372

37. 3 . Linearity Method: As per Test Assessed under Above Linearity (Plotting a Calibration Curve) Preparation Dilutions of Olaparib for Linearity Study Standard solutions of Olaparib in the concentration range of 10  g/ml to 60  g/ml were obtained by transferring (1,2,3 and 4,5,6, ml) of Olaparib stock solution (100ppm) to the series of clean & dry 10 ml volumetric flasks. The volumes in each volumetric flask were made up with the solvent system and mixed The absorbances of the solutions were measured at 278 nm against the solvent system as blank and calibration curve is plotted. The Lambert-Beer’s Law is linear in concentration range of 10 to 60  g/ml at 278 nm for Olaparib

38. . Results of calibration curve of olparib 4.PRECISION: The precision of developed analytical method said to be the closeness of agreement between a series of measurement obtained from the multiple sampling of the homogenous sample solution under the prescribed experimental conditions. The precision of the developed method can be analysed by the 6 different homogenous solutions. The precision can be divided into following types. 1. Repeatability and 2. Intermediate precision. In this first one is Repeatability or Intra-day precision was determined on six replicates of same sample solutions on the same day. Concentration (µg/ml) Absorbance (n=6) 10 0.214 20 0.415 30 0.631 40 0.844 50 1.035 60 1.236

39. Inter-day precision was estimated by analyzing newly prepared sample solutions in triplicate over the 3 consecutive days. Both inter day and intraday precision was expressed as % RSD. The % RSD values for intraday precision for Method A was found to be within the limits. Repeatability Repeatability was assessed using: Six time repetition of target concentration 100 % that is (10µg/ml). Intermediate precision can be assessed by intra-day and inter day analysis. Method: In the study of the repeatability precision which was conducted on the solution which has the concentration value 100 % of the target concentration (n  6).

40. S.No . Conc. (  g/ml) Wavelength (nm) Absorbance 1 10 278 0.214 2 10 278 0.217 3 10 278 0.216 4 10 278 0.219 5 10 278 0.215 6 10 278 0.218 Mean  S.D. 0.2165 Standard Deviation 0.001871 % RSD 0.864124 Data of Repeatability of Absorbances Intermediate Precision: Intra-Day & Inter-Day: The intra & inter day variation of the method was carried out & the high values of mean assay & low values of standard deviation & % RSD (% RSD < 2%) within a day & day to day variations for Olaparib revealed that the proposed method is precise

41.   Con. taken ( μg /mL) Observed Conc. of Olaparib (µg/ml) by the proposed method Intra-Day Inter-Day Con. found ( μg /mL) % RSD Con. found (μg/mL) % RSD 8 7.983 0.986 8.068 0.598 10 10.014 0.752 10.054 0.964 12 12.136 0.685 12.365 1.083 Intra-Day and Inter-Day Precision for Method 5.ROBUSTNESS: Robustness of the method was determined by carrying out the analysis under different temperature condition i.e. at 23°C, 25°C and at 28°C. The respective absorbances of 10µg/ml.

42. RESULTS SHOWING ROBUSTNESS OF OLAPARIB TEMPERATURE 23 ° C TEMPERATURE 25 ° C Concentration( μg /ml) Absorbance Statistical Analysis 10 0.215   Mean =0.216 SD =0.001789 % RSD =0.828173 10 0.219 10 0.214 10 0.217 10 0.216 10 0.215 Concentration( μg /ml) Absorbance Statistical Analysis 10 0.228 Mean = 0.225833 SD = 0.002787 % RSD = 1.23404 10 0.229 10 0.227 10 0.226 10 0.222 10 0.223

43. 6.RUGGEDNESS : In the ruggedness study, the influence of small, deliberate variations of the analytical parameters on the absorbance of the drug was examined. The factor selected was a change in the analyst. The Ruggedness of the method was determined by carrying out the analysis by different analyst and the respective absorbance of 10µg/m. Concentration( μg /ml) Absorbance Statistical Analysis 10 0.236  Mean =0.236833 SD =0.001835 % RSD =0.774742 10 0.238 10 0.234 10 0.239 10 0.238 10 0.236 TEMPERATURE 28 ° C

44. Concentration( μg /ml) Absorbance Statistical Analysis 10 0.219  Mean =0.215833 SD =0.002787 % RSD =1.291216 10 0.212 10 0.213 10 0.218 10 0.216 10 0.217 Results showing ruggedness of Olaparib. ANALYST :1 ANALYST:2 Concentration( μg /ml) Absorbance Statistical Analysis 10 0.256   Mean =0.255333 SD =0.002338 % RSD =0.915701 10 0.252 10 0.254 10 0.259 10 0.256 10 0.255

45. Concentration( μg /ml) Absorbance Statistical Analysis 10 0.262   Mean =0.265333 SD =0.002805 % RSD =1.05707 10 0.268 10 0.266 10 0.264 10 0.269 10 0.263 ANALYST :3

46. 7.LIMIT OF DETECTION AND LIMIT OF QUANTIFICATION: The limit of detection (LOD) and the limit of quantification limit (LOQ) are measured by using the following equations: L.O.D. =3.3 (SD/S) L.O.Q. = 10 (SD/S) Where, SD = Standard deviation of the response S = Slope of the calibration curve The slope S and the SD may be estimated from the calibration curve of the analyte/sample. The LOD was found to be 0.369  g/ml and LOQ was found to be 1.107  g/ml for Olaparib respectively which represents that sensitivity of the method is high.

47. RESULTS AND DISCUSSION   The standard solutions of Olaparib in Methanol (10μg/ml) subjected to a scan individually at the series of wavelengths of 200 nm to 400 nm. Absorption maximum of Olaparib was found to be at 278 nm. Therefore, 278nm was selected as λ max of Olaparib for the present study. The calibration curve of Olaparib was found to be linear in the range of 10-60 μg /ml at 278 nm. Therefore, it was clear that Olaparib can be determined without interference of any irrelevant substance in single component pharmaceutical products.

48. The used technique was initially attempted on bulk drugs in their synthetic sample and concentrations were estimated.   The % recovery was carried out at 3 levels, 80%, 100% and 120% of Olaparib standard concentration. Three samples were prepared for each recovery level. Level of Recovery % Recovery   % RSD 80% 100.138   0.199959 80% 99.922 80% 100.322 100% 100.320     0.195445 100% 99.945 100% 100.230 120% 100.340     0.252854 120% 99.918 120% 100.372

49. The solutions were then analysed, and the percentage recoveries were found to be satisfactory within the acceptable limits as per the content of the label claim for marketed tablet dosage form. The newly developed method was validated according to the ICH guidelines and the method validation parameters. The developed method was subjected to do the various method validation parameters such as specificity, accuracy, precision, linearity and range, limit of detection and limit of quantification, robustness and ruggedness etc.

50. CONCLUSION As per the result & discussion, it was concluded that most method developments and validation are successfully done on anticancer drugs. Which starts with the solvent selection according to particular drugs solubility within it. Because when method development starts at the first stage searching for various solvents as per solubility parameter then its easy availability and showing off the intense absorption peaks. Then go for the proposed analytical method that is simple, sensitive, easy, and cost-effective. The proposed method is exact and accurate. The UV spectrophotometric method offers adequate data quickly and plays a significant role in method development and validation. After that Methanol, absolute ethanol, acetonitrile, solvents were used which showed the same criteria that are required to show better results invalidation process. Next λ max also plays important role in the validation of the method and the maximum anticancer drugs recorded in this project shows λ max are between 210 nm to 278 nm. As a result, the UV methods developed in this study offer simplicity, precision, and accuracy of chemistry.

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