{'fraction_non_alphanumeric': 0.040929023875263926, 'fraction_numerical': 0.04052298197173948, 'mean_word_length': 4.667280257708238, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Identification and characterization of related substances in EVT-401 by hyphenated LC–MS techniques

A sensitive and selective method was developed for the separation and characterization of related substances (RSs) in EVT-401 by hyphenated LC–MS techniques. Complete separation of the RSs was achieved with an Inertsil ODS-SP column (250 mm×4.6 mm, 5 µm) by linear gradient elution using a mobile phase consisting of 0.2% formic acid solution, methanol and acetonitrile. EVT-401 was found to be susceptible to acid, alkaline and oxidative stresses, while relatively stable under photolytic and thermal dry stress conditions. Fourteen RSs including six process-related substances and eight degradation products were detected and identified in EVT-401 with positive ESI high-resolution TOF-MS analysis of their parent ions and the corresponding product mass spectra elucidation, and some of them were further verified by chemical synthesis and NMR spectroscopy. The specific LC–MS method developed for separation, identification and characterization of RSs is valuable for EVT-401 manufacturing process optimization and quality control.


Introduction
P2X receptors, which allow non-specific passage of cations (Na + , Ca 2+ , K + ), are a kind of ligand-gated ion channels. Until now, seven kinds of receptors have been found and described [1]. P2X7 receptor, expressed on hematopoietic lineage cells including mast cells, lymphocytes, erythrocytes, fibroblasts, and peripheral macrophages, is a distinct member of the P2X subclass, which has a key role in inflammation and immunity. By inhibiting P2X7 receptor, the treatment of multiple sclerosis, inflammatory bowel disease and rheumatoid arthritis has been achieved [2][3][4][5][6]. Pfizer's CE-224535 and AstraZeneca's AZD -9056 [7] as oral small molecule P2X7 receptor antagonists have entered clinical trials for rheumatoid arthritis.
EVT-401, 2-(3-fluoro-4-(trifluoromethyl)phenyl)-N-(2-(1-hydroxypropan-2-yl)-6-methyl-1-oxo-1,2-dihydroisoquinolin-5-yl)acetamide, is a P2X7 receptor antagonist developed for the treatment of rheumatoid arthritis by preventing disease progression and symptom exacerbation [8]. As an attractive new P2X7 receptor antagonist, EVT-401 has shown efficacy of combating neuroinflammation. And the Phase I clinical trial of EVT-401 on rheumatoid arthritis has recently been completed [9]. The control of impurities by adopting quality by design (QbD) approach is very important as mentioned in the ICH guidelines for pharmaceutical development [10]. Until now, none of the methods described the identification and characterization of EVT-401 and the RSs. The identification of both process-related substances and degradation products is needed to ensure the good manufacturing of high quality EVT-401.
Hence, the purpose of present work was to determine and characterize the RSs of EVT-401 by LC-MS techniques and some of them were further synthesized and verified by NMR spectroscopy. The results obtained from the study would be of significance for quality control and the stability-indicating study of EVT-401.

Test solutions
Stock solutions were prepared accurately by dissolving EVT-401 or the synthetic reference RSs A-F in the diluent consisting of methanolacetonitrile-water (24:14:62, v/v/v) to a final concentration of 0.5 mg/ mL, respectively. 0.1% reference solution was prepared by diluting EVT-401 stock solution (0.5 mg/mL) to a final concentration of 0.5 μg/ mL. A mixed reference solution of EVT-401 and the reference RSs was prepared similarly by spiking the EVT-401 solution with a suitable volume of each of the RSs stock solution to produce a final concentration of 0.5 mg/mL for EVT-401 and 10 μg/mL for each of the RSs.

Instruments
An Agilent 1260 infinity series LC equipped with a DAD coupled to an Agilent TOF mass spectrometer (CA, USA) was used for sample analysis. Samples were separated on an Inertsil ODS-SP column (250 mm×4.6 mm, 5 µm) by using a mobile phase consisting of methanol-acetonitrile-water-formic acid (24:14:62:0.2, v/v/v/v) as solvent A and methanol-acetonitrile-water-formic acid (30:20:50:0.2, v/v/v/v) as solvent B with linear gradient elution. The program in terms of the volume ratio of solvents (A:B) was as follows: 0 min (100:0)→35 min (0:100)→50 min (0:100)→50.1 min (100:0)→60 min (100:0). The flow rate was 1.0 mL/min and the column temperature was set at 35 ℃. Analytes were monitored at 237 nm. The injection volume was 20 μL. TOF-MS was operated in high resolution mode with a dual ESI source in positive mode and protonated purine of m/z 121.0508 (C 5 H 5 N 4 + ) and protonated fluoro-phosphazine of m/z 922.0098 (HP-921, C 18 H 19 O 6 N 3 P 3 F 24 + ) were used as the internal calibration reference ions. Ionization was performed at 3.5 kV, assisted by nitrogen drying gas flow of 10 L/min at 350°C with a nebulizer pressure of 275 kPa. The m/z scanning range was from 105 to 1000 at 1 spectra/s. All the operations, acquisition and analysis were controlled by Mass Hunter Workstation.
The liquid chromatography tandem MS (LC-MS/MS) analyses were performed with a Finnigan TSQ mass spectrometer (Thermo electron corporation, San Jose, CA, USA) in positive ESI mode. The product MS spectra were collected with the following conditions: spray voltage set at 5.0 kV, heated capillary temperature at 350°C, and the nitrogen sheath gas and auxiliary gas at 350 and 50 kPa, respectively. The 0.2 Pa argon gas collision induced fragmentation was performed with the energies optimized for each impurity from 10 to 20 eV.
The NMR spectra were recorded on a Bruker AVANCE 500 MHz NMR system (Fallanden, Switzerland) with all samples dissolved in DMSO-d6 using tetramethylsilane as the chemical shift reference standard.

Method development and optimization of chromatographic conditions
To achieve good separation for active pharmaceutical ingredient (API) and potential impurities, and compatibility with LC-MS detection, a selective method should be developed. Different components of the mobile phases, proportions, buffer concentrations and column brands were tested to obtain a better separation. Finally, the mobile phases consisting of methanol-acetonitrile-water-formic acid were chosen to achieve a better separation and LC-MS compatibility with the UV detection was set at 237 nm because of a compromise between the sensitivity and the base line drifting. Typical HPLC results are shown in Fig. 1, and 14 RSs were detected in all test samples.

Chromatographic characteristics of related substances
Among the 14 RSs (Fig. 1), six process-related substances and eight degradation products were detected in all test samples. Among them, two process-related substances (RSs 1 and 14) were found in the API with the contents lower than 0.1% based on the external 0.1% selfreference method. The content of RS 1 had a significant increase in the stress conditions of acidic and alkaline hydrolysis. The content of RS 14 was not found in alkaline hydrolysis, and reduced in the other four stress conditions. Significant degradation of EVT-401 was observed under acidic (RS 1), alkaline (RSs 1 and 2) and oxidative (RSs 7-11) stress conditions, but no obvious degradation was observed in photolysis and thermal dry stress conditions. RSs 4 and 13 were produced under methanol solvation, which were not found during the storage of EVT-401. All the RSs 1-14 were well separated from EVT-401.

MS characteristics of EVT-401 and the RSs
EVT-401 and RSs 1-14 were characterized through hyphenated LC-ESI-MS techniques (Figs. 2 and 3). The chromatographic and mass spectrometric data are summarized in Table 1; the structures and the plausible fragmentation mechanisms are proposed in Figs. 4 and 5.
Six process-related substances (RSs 1, 3, 5, 6, 12 and 14) were found during the optimization research for process parameters and characterized through LC-MS techniques. According to the manufacturing processes of EVT-401 (Fig. 6), the origins of these processrelated substances were located, and the effective approaches were designed and conducted to reduce and even eliminate them as much as possible in the API. Most of the above discussed process-related substances can be eliminated by the adjustment of the mole ratios of the reaction materials, in addition to the commonly employed purity control methods of the starting materials or re-crystallization in the final steps. The modified synthesis processes were successfully applied for highquality EVT-401 manufacturing, as most of the impurities were

Characterization of the degradation products RS 2, RSs 7-11
Significant degradation of EVT-401 was observed under acidic (RS 1), alkaline (RSs 1 and 2) and oxidative (RSs 7-11) stress conditions, but no obvious degradation was observed in photolysis stress and thermal dry stress (Fig. 1). The identification and characterization of these degradants were important for the quality control of EVT-401. RS 2 was the major degradation product under alkaline stress with much decreased chromatographic retention, suggesting that the polarity of RS 2 was significantly increased compared with that of EVT-401. The accurate mass of the [M+H] + ion of RS 2 determined by ESI-TOF-  -  The product mass spectra of RSs 7-11 contained the same major product ions at m/z 435 and 395, which were 16 amu higher than those of EVT-401 (m/z 419, 379), giving further evidence of a polar hydroxyl group substitution in the structure of EVT-401. Moreover, the characteristic product ions of RSs 7-11 (Fig. 3) at m/z 231, 217 and 191 were 16 amu higher than those of EVT-401 (m/z 215, 201 and 175), confirming the presence of hydroxyl substitution in the structure of isoquinoline.
Considering the formation of intra-molecular hydrogen bond should result in the decreased polarity and the readily oxidation characteristic of the aniline ring under oxidation stress, RSs 7-11 were deduced to be 7-hydroxy derivative, 11-hydroxy derivative, 3hydroxy derivative, 8-hydroxy derivative and 5-hydroxy derivative of isoquinoline, respectively (Figs. 4 and 5).

Characterization of RSs 4 and 13
RSs 4 and 13 were produced by the action of the methanol solvation effect, and were not found during the storage of EVT-401.
Both RSs 4 and 2 were products under the alkaline stress, which were found to have similar structures. The

Characterization of the process-related substances RSs 1 and 14 by NMR
RSs 1 and 14 were found in EVT-401 and the contents of RS 1 and RS 14 in the stress conditions have a certain degree of increase or decrease. The RSs were further verified through synthesis and NMR determination (Figs. S1 and S2). The 1 H and 13 C NMR spectral data of EVT-401 and process-related RS 1 and 14 are summarized in Table 2 and Fig. 7. 1 H NMR spectra of EVT-401 and RS 1 showed extra signals of hydroxyl resonance peak at about δ 4.9 (s, 1H), and the amino resonance peak of RS 1 at about δ 5.3 (s, 1H). Due to the effect on paramagnetic shielding of carbonyl, the carbonyl carbon resonance peak lies in the bigger chemical shift region at about δ 170-160. In addition, compared with RS 1, the obvious chemical shift increases of EVT-401 and RS 14 were observed for the amino at about δ 9.9 (s, 1H).
The 13 C NMR spectra of C-3''' showed the signals for the shielding effect of F, the chemical shift generating a change at about δ160. And the split peaks of benzene indicate the connection with F. Moreover, a characteristic quartet-resonance peak was observed at δ120, confirming the structure of CF 3 .

Conclusions
The impurity profiling of EVT-401 was carried out according to ICH guidelines. EVT-401 was found to be unstable under acidic, alkaline  and oxidative stresses. Six process-related substances and eight degradation products in EVT-401 API were characterized by LC-MS and mass spectrometric techniques (Fig. 8). Most of them had much higher polarity than that of EVT-401, and the most susceptible positions for degradation in the structure of EVT-401 were found to be the amide group. The results obtained in the study will play a crucial role in providing scientific reference for the optimization of manufacturing processes and the quality assessment of EVT-401 and its formulations.

Conflicts of interest
The authors declare that there are no conflicts of interest.