Osimertinib Mesylate (OSM) active and formulation samples obtained from Cipla Laboratories Ltd. (Mumbai, India). HPLC grade methanol (MeOH) was obtained from Thermo fisher Scientific India, (Mumbai, India) and HPLC and LC-MS grade Acetonitrile (ACN) obtained from Honeywell International India (New Delhi, India). Analytical reagent (AR) grade lab reagents such as formic acid was procured from S.D. Fine, ammonia solution (NH₄OH) and Hydrochloric acid (HCl) were procured from Rankem, (Avantor Thane, India) sodium hydroxide (NaOH) were procured from (Merck Life Science Pvt Ltd, Mumbai, India). To prepare all solutions and mobile phase HPLC grade water was obtained from a Milli-Q Gradient system (Millipore, Bedford, MA, USA).

Weigh equivalent to 500 mg of OSM and transferred in to 100 ml volumetric flask and added 20 ml methanol, sonicate to dissolve, further added 20 ml diluent, swirl the solution and added 10.0 mL 1.0 N NaOH solution and refluxed for 2 hrs at 100˚C temperature then cooled the solution at room temperature and makeup the volume with diluent, then filter and injected on HPLC-PDA and analyzed using analytical method illustrated in Section 2.2.1

Note: Methanol is critical for solubilizing the Osimertinib Mesylate completely, in other solvents OSM is less soluble comparative to methanol.

The OSM Mesylate drug substance and drug products are a non-compendial product and no literature was found on the public domain for Degradation products. Thus, a novel analytical method developed to separate DP, and the same method adopted for further identification with the aid of UPLC-ESI-Q-TOF-MS. Initially, samples were analyzed by available literature [17] [18] by using the reported gradient as time/solvent-B (%): 0.0/25, 5.0/25, 15/40, 30.0/80, 60.0/80, 70.0/25, 80.0/25 however interference was observed with oxidative degradants for the base DP. Thus, a new method developed by optimizing the gradient as outlined in section 2.2.1 for OSM using XBridge BEH (Waters) HPLC column (dimension 25 cm × 4.6 mm i.e., 5 µm particle size) with 1 ml of formic acid in 1000 ml water (adjusted to pH 5.5 with ammonia solution) was used as mobile phase A and acetonitrile was used as mobile phase B. The column temperature was maintained at ambient and the diluents used for the sample solution were 50:50 v/v of mobile phase A and B respectively. The DP was monitored at a wavelength of 268 nm. The analytical method development trails were conducted to optimize the mobile phase, column selection, and column temperature. The waters XBridge BEH column found suitable for stress samples. Therefore, waters XBridge BEH was chosen as the best suitable column for this analytical method and the analytical procedure as described in Section 2.2.1, The typical chromatogram of base degradation shown in Figure 2.

The newly developed chromatographic method for impurity has several advantages over the conventional HPLC methods such as this method can be directly applied for preparative/semi-preparative HPLC scale isolation for degradation products and as well as compatible with mass spectrometry characterization as the mobile phase of this method comprises mass compatible solvents, buffers (without phosphate, sulfate etc., buffers, which in turn could interfere

with mass ionization) with gradient elution. The retention time (RT), relative retention time (RRT), % DP, and peak purity are tabulated in Table 1.

The developed HPLC method was directly transferred to semi-preparative HPLC and separation was achieved by optimizing the flow rate to accommodate more quantity of samples, to collect a higher amount of degradation product with less fractions using Phenomenex Luna PREP C18 (10.0 × 250 mm), 10 micron particle size mobile phase consists of A: Buffer (0.1% formic acid with pH 5.5 with ammonia solution) and B: Acetonitrile. The gradient program was set as follows: time (min)/solvent-B (%): 0.01/20, 20.0/30, 50.0/40, 80.0/50, 85.0/90, 86.0/20, 90.0/20 with flow a rate of 2.3 ml/min in order to collect the maximum amount of degradation product within the optimal time period, several portions of impurity fractions were collected on semi-prep HPLC. The collected degradant fractions were further lyophilized. The purity of the collected degradant was tested on HPLC as per method outline in Section 2.2.1, and was found about 90%. Further, the isolated degradant analyzed on NMR spectrometry and ascertain the structure which already deduced with the help of mass spectroscopic fragmentation pathways.

The Identification of active drug and its novel degradation product was studied on Waters Xevo Q-TOF of Mass spectrometric (MS) technique with mass lynx software (ver. No. 4.1). The protonated molecule and its further fragmentation pathways obtained from ESI-Q-TOF-MS further utilized to explore to elucidate structural arrangements of OSM and its degradation product. The ESI-Q-TOF-MS condition with ion spray voltage is optimized to +3.0 kV condition for better protonated and fragments ions. The base degradant was identified along with

the target OSM drug and its RRT was found at 0.97 (DP, m/z 532.2606). The accurate mass data and its fragments are reported in Table 2 as below.

Based on molecular weight and fragmentation pattern, the proposed structure of degradation product is presented in Figure 3. The probable structure was proposed for the individual fragments are showed in Figure 4. The base degradant comprises C-C double bond of acrylamide converts to ether (by addition of methoxy group) the fragments m/z 487.2863 and m/z 459.2525 are the characteristic fragments which confirms the presence methoxy (-OCH₃).

This degradant was observed in Base hydrolysis and eluted at RRT about 0.97 (in HPLC) and shows molecular weight m/z 532.2606 Da [Figure 5(a)] which is 32 mass unit higher than the OSM API (500.2738 [M + H]+). The RDB value for DP is observed as 15.5 which decreased form the 16 RDB value of OSM API even though increase in molecular weight. It reveals that a double bond reduction in the DP. The molecular weight m/z 532.2606 further subjected to fragmentation and confirmed the structure after fragmentation Figure 5(b). Based on fragmentation pathways it is evident that the DP contains methoxy group (-OCH₃) attached to reduced C-C double bond of acrylamide. Further the structure was ascertained by 1H, and 13C NMR spectrometry, The Ethylene proton signal of acrylamide (-CH=CH₂) group is absent in NMR which was seen in OSM refer Figure 6 and Figure 7. The proton H-35 (6.54 δ ppm) and H-36’ and H-36” (5.9 and 6.52 δ ppm respectively) of OSM protons were found absent in DP. Whereas new -CH₃ and two -CH₂ proton signals observed at H-38 (3.16 δ ppm) and H-35, H-36 (at 2.92 δ ppm, 3.65 δ ppm) respectively. Additionally, DP contains -OCH₃ group is also further ascertained by 13C, DEPT-135 and 2DNMR including, HSQC, COSY and HMBC technique. The PDA spectral scan evident no change in the aromatic region. The detail interpretation of 1D and 2D NMR for DP and OSM explained in Table 3 in the HMBC experiments the main key correlations, C-38 of -OCH₃ (at 58.47 δ ppm) correlates with the proton of H-36 and C-36 of -CH₂ (at 68.68 δ ppm) correlates with proton of H-38 of -OCH₃. Additionally, the correlation of C-29 with H-36 evident there is reduction of carbon-carbon double bond of acrylamide refer Figure 8. Rest of the correlations are comparable with OSM. The explained correlation is seen in COSY experiment as well, The H-36 is correlated with H-35 Hence, The structure of base degradant unambiguously named as N-(2-((2-(dimethylamino)

^aS, singlet; d, doublet; t, triplet; dd, doublet of doublet, refer the structural formula given in Figure 3 for numbering. *protons exchanged due to solvents.

ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl) amino)phenyl)-3-methoxypropanamide. The plausible synthesis scheme is outlined in Figure 9.