Separation, Identification, Isolation and Characterization of Degradation Product of Osimertinib Tablets by UPLC-QTOF-MS/MS and NMR: Evaluation of In-Silico Safety Assessment for Osimertinib (OSM) and Degradation Product (DP)

The present work encompasses identification and characterization of major degradation product (DP) of OSM observed in base hydrolytic stress study. The separation of DP was carried out on a non-polar stationary phase by using high-performance liquid chromatography system (HPLC). Using waters X-bridge (250 mm × 4.6 mm, 5 µm) C18 column with gradient elution program. For the characterization study, stress samples were subjected to HPLC and UPLC-QTOF-MS/MS and based on mass fragmentation pattern, plausible structure was deduced. Further, the DP was isolated using semi-prepara-tive liquid chromatography and concentrated the fractions using lyophiliza-tion. The isolated DP was subjected to extensive 1D (1H, 13C, and DEPT- 135) and 2D (COSY, HSQC and HMBC) nuclear magnetic resonance (NMR) studies to authenticate the structure. The impurity was unambiguously named as N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-metho-xy-5-((4-(1-methyl -1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-methoxypropanamide. Add-itionally, the In-Silico structure activity relation (QSAR) assessed through statistical based software’s DEREK Nexus TM , and MultiCASE, Case Ultra TM widely accepted and respected software’s for DP and OSM.


Introduction
Osimertinib Mesylate (OSM) (Figure 1; IUPAC name: "N-(2-{2 dimethylaminoethyl-methylamino}-4-methoxy-5-{ [4-(1-methylindol-3-yl)pyrimidin-2yl]am-ino}phenyl)prop-2-enamide mesylate salt" molecular formula: C 28 H 33 N 7 O 2 •CH 4 O 3 S; molar mass: 596 g/mol [1]. Osimertinib is an oral, third-generation, irreversible EGFR-TKI that is proved to selectively inhibit both EGFR-TKI-sensitizing and EGFR T790M resistance mutations [2] [3] [4]. It was also approved to be one of the first-line treatment options for EGFR-mutant NSCLC patients owing to the positive results from the FLAURA study [5], which demonstrated significant survival benefits in both PFS and OS [6] [7]. In April 2014 based on phase I clinical trial results, the OSM was designated as a Breakthrough Therapy [8]. The tablet formulation of OSM Mesylate (Tagrisso TM ) developed by Astra-Zeneca has been granted accelerated approval by the United States Food and Drug Administration (US-FDA) in November 2015, for the therapy of patients with metastatic EGFR T790M mutation-positive NSCLC who have progressed on or after the first and second generation of EGFR TKI therapy [9]. Now a day's impurity profiling considers critically by all regulatory agencies to ensure the safety and efficacy of all pharmaceutical preparations. Certainly, out of specification (OOS) or out of trend (OOT) results are mostly observed when dealing with a relatively high number of degradants or impurities in finished product or drug substance during product storage [10]. The impurity profiling is the one of the critical quality attributes for the pharmaceutical preparations which ensures the pure and safe drug to the patient [11]. Therefore, chromatography plays a crucial role in separation of process/degradation impurities, based on the chemistry of molecule suitable column was used to develop a unique chromatography to ensure the purity of OSM drug. As per the ICH guidance, the stability studies of drug product or drug substance are one of the crucial quality control testing in the pharmaceutical industry [12], which assist to the drug manufacturer to maintain the quality of finished formulations or raw materials. The quality of the drug may alter with several environmental variables such as light, humidity, temperature, storage time, or physical parameters [13].
For the finished product or raw materials self-life would be assigned based on the stability studies and trend data of degradation products on various intervals of testing. Thus, degradation studies of drugs are paramount importance to forecast

High-Performance Liquid Chromatography (HPLC)
The method development for degradation product was done on Agilent 1220 HPLC system, photodiode array detector and automatic liquid sampler injector. Chromatographic data were recorded and processed using chromeleon software (Thermofisher Scientific ver. No 6.80). The test sample solutions were made with diluent (Mobile phase A: B 50:50 v/v). The sample solutions for analysis were injected on a newly developed reverse-phase chromatographic method with Waters X-bridge C18 column (25 cm × 4.6 mm internal diameter, 5-micron particle size) used for the impurity separation and identification. Mobile phase A consists of buffer (0.1% formic acid with pH 5.5 with ammonia solution) was filtered through 0.45 m PTFE filter and degassed, Acetonitrile as mobile phase B.
The analysis was carried out using the liner gradient program was set for separation of components as follows: The analytes were monitored at a wavelength of 268 nm. The column temperature was maintained at ambient. 10 µL injection volume was used for sample analysis.

Semi-Preparative High-Performance Liquid Chromatography
The semipreparative HPLC separation and fraction collection of degradation product was carried out on Agilent HPLC 1200 with 900 µl loop size with semi preparative column Luna C18 column (250 cm × 10 mm internal diameter; 10 micron particle size) using Agilent quaternary pump, the sample concentration was optimized to 10 mg/ml, prepared in diluent. The mobile phase consists of A: 3 ml/min. The analytes were monitored at 268 nm, with injection volume was used as 400 µL for faction collection. The fractions were, pooled together and lyophilized using lyophilizer (Make: BUCHI, Lyovapor, L-300).

Electrospray Ionization Mass Spectrometry (ESI-Q-TOF-MS)
The isolated and purified degradation product was further subjected for mass and its further fragmentation using an electrospray ionization-mass spectrometry system (Waters Xevo Q-tof Waters USA) with an electrospray ionization source at positive ion full scan mode mass spectrometry. Ion source temperature was set at 120˚C, Desolvation Temperature was set to 350˚C and the capillary cone voltage was +3 kV. Nitrogen gas (99.99% purity) was used as curtain gas and CAD gas was used as Helium at a pressure of 15 psi and 10 psi respectively with gas flow-rate of 6 mL•min −1 and nebulizer gas flow-rate was at 10 mL•min −1 . Zero air at a pressure of 45 psi was used as heater gas. The mass spectra were acquired in positive ion mode over the mass range of 50 -4000 Da.
All the chromatographic conditions used for UPLC-Q-TOF-MS are same as chromatographic conditions of HPLC, outlined in section 2.2.1.

Nuclear Magnetic Resonance Spectroscopy (NMR)
1D and 2D NMR spectra of the isolated DP and OSM Drug was recorded on varian TM 500 MHz (Advance Neo) using best suitable solvent as methanol-d4 for OSM and DMSO-d6 for DP and Tetramethylsilane (TMS) as internal standard.

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

Isolation of Novel Degradation Product
The developed HPLC method was directly transferred to semi-preparative Further, the isolated degradant analyzed on NMR spectrometry and ascertain the structure which already deduced with the help of mass spectroscopic fragmentation pathways.

Mass Spectrometric Characterization of Novel Degradation Product
The Identification of active drug and its novel degradation product was studied  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 (-OCH 3 ).

Characterization of the Degradation Product [(m/z 532.2606), M + H]
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 (-OCH 3 ) 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=CH 2 ) 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 -CH 3 and two -CH 2 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 -OCH 3 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         H-10 C-10 C-2, C-5  Figure 3 for numbering. *protons exchanged due to solvents.

In-Silico Safety Assessment of Degradation Product
The safety assessment for impurities is crucial for pharmaceuticals, in this context the application of computational methods e.g., Quantitative Structure-Activity Relationships (QSARs), Structure-Activity Relationships (SARs), and/or expert systems for the evaluation of genotoxicity are needed, especially when very limited information on impurities is available [19]. The SAR was predicted through widely accepted and respected predication software tools e.g., DEREK, Lhasa ltd and MCASE, CASE Ultra, the endpoints of the predictions are based on molecular structure and reactivity. DEREK yields semi-quantitative assessments of a DNA reactive functional groups (e.g., Genotoxicity, or chromosome damage, Mutagenic,) of the input chemical structure. DEREK is a knowledge and rule-based semi-quantitative estimations tools, the learning set was created using both bacterial mutagenicity and all other available genotoxicity data [20].
MCASE dissociates the input molecule into multiple fragments and evaluates statistically for bacterial mutagenicity by using two models GT1_BMUT (OECD 471 Bacterial Mutagenicity) and GT_EXPERT (Expert Rules for Bacterial Mutagenicity) from the available database for mutagenicity and generates quantitative data from various reputed sources the outcomes are tabulated in Table 4.
Based on the safety assessment done on the above-mentioned software's, the OSM and DP are observed as Non-genotoxic.

Conclusions
In contrast to our previously developed method for oxidative degradants, the newly developed method is considered to be more appropriate to separate all the plausible OSM degradants. The OSM was studied under base hydrolytic conditions and yielded one major degradant. Further, it is well characterized by using UPLC-Q-TOF-MS and NMR studies (1H, 13C, DEPT-135, COSY, HMBC and HSQC) and the structure is proposed as N-(2-((2-(dimethylamino)ethyl)(methyl)amino)-4-methoxy-5-((4-(1-methyl-1H-indol-3-yl)pyrimidin-2-yl)amino)p henyl)-3-methoxypropanamide. This impurity can be controlled in finished formulations or drug substance by adequate control of residual methanol and diluent for sample preparation. Further, in silico safety assessment was predicted for DP with OSM using widely-respected and accepted software's i.e., MCASE and DEREK Nexus. The results showed that the DP was observed as non-genotoxic. The shreds of evidence in this study are expected to be useful for generic drug manufacturer during product development.