VALIDATED STABILITY- INDICATING HPTLC METHOD FOR NINTEDANIB & CHARACTERIZATION OF DEGRADANTS BY LC-MS

A simple and rapid stability-indicating method for determination of nintedanib (NTB) in bulk drug using HPTLC and LC-MS was developed and validated. Stress degradation studies were carried out by hydrolysis, oxidation, thermal and photolytic. Drug was found to be stable in thermal whereas one degradant was found in acid hydrolysis, three in basic hydrolysis, five in oxidative and two in photolytic stress. The probable structures of the degradation products were predicted & the degradation pathway was also established. Chromatography was carried out using silica gel 60 F254 TLC plate and mobile phase of Chloroform : Methanol in the ratio 7:3 v/v. The densitometric determination was done at 386 nm. The degradants were not detectable when stressed as per ICH recommended conditions but on increasing the strength of acid, base and peroxide, the degradants were very much prominent and were easily detectable in HPTLC. The LC system consisted of a Zorbax Bonus C18 (150 mm×4.6 mm, 3.5 μ). A gradient mobile phase consisting of mobile phase A: 10mM Ammonium formate (0.05% formic acid): ACN (pH 3.9) (90:10) and mobile phase B: 10mM Ammonium formate (0.05% formic acid): ACN (pH 3.9) (10:90) with a flow rate of 0.7mL/min was used to separate the degradants up to a total retention time of 15 min. Mass spectrometric detection was performed using Thermo Scientific LCQ fleet Ion Trap LC/MS.


Introduction
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Literature review suggest that various methods involving UPLC, LC-MS, UV are already reported for the estimation of NTB in bulk drug, formulation, rat plasma & human plasma. The reported methods were limited to the estimation of NTB in formulation or in plasma, but none reported about the stability profile of the drug and there by establishment of the probable degradation pathway. But, till date a validated stability-indicating HPTLC method for the estimation of NTB in bulk drug and characterization of the degradants is not reported. The current manuscript is an attempt to report a validated HPTLC method as per ICH Q2(R1) guidelines for estimation of NTB. This study was designed to develop a simple, rapid, precise & accurate HPTLC method for determination of NTB in bulk drug and to validate such as per ICH guidelines.

Degradation Studies
Degradation studies were performed as per ICH suggested stress conditions and at a drug concentration of 1600ng/band. Different stress conditions like acid & base hydrolysis, oxidative degradation, photolytic and thermal degradation. Finally, the observed results were analyzed.

Acid hydrolysis
NTB was stressed with 0.1M HCl with reflux for 8 hrs. Simultaneously control was also performed. Readings were taken at an interval of 4 hrs. After 8 hrs. also the drug was found to be stable in 0.1M HCl. Then it was again performed with 1M HCl and enough degradation (>10%) was observed within 0 hrs., hence the study was stopped. (Table 1) Base hydrolysis NTB was stressed with 0.1M NaOH with reflux for 8 hrs. Simultaneously control was also performed. Readings were taken at an interval of 4 hrs. The drug was found to be stable till 4 hours and enough degradation (>10%) was observed at the 8 th hour. (Table 2) Oxidative degradation NTB was stressed with 3% H2O2 for 6hrs at room temperature. Simultaneously control was also performed. Readings were obtained at an interval of 3hrs. (Table 3) Photolytic degradation NTB was exposed to an illumination of 1.2×10 6 lux hours as per the ICH-recommended exposure limit and the reaction was monitored periodically. (Table 4)

Thermal Degradation
Negligible degradation was observed after subjecting the drug solution to temperatures 40ºC, 60ºC & 80ºC respectively. (Table 5)      The stability profiling of the drug was carried out as per the ICH guidelines and the degradation was performed till 10%, but at this condition, the detection of the degradants was not possible due to the sensitivity of the instrument. Hence, to detect and quantify the degradants, the drug was completely degraded and then analyzed. The procedure and results are as follows: Acid Hydrolysis: Drug (4000 ng/band) was stressed with 1M HCl with reflux for 12hrs, then the solution was assayed.
Base Hydrolysis: Drug(4000ng/band) was stressed with 1M NaOH with reflux for 12hrs, then the solution was assayed.
Oxidative Degradation: Drug(4000ng/band) was stressed with 30% H2O2 at RT for 24hrs, then the solution was assayed.
Photolytic degradation: Drug(4000ng/band) was exposed to 6 × 10 6 lux hours for 24hrs, then the solution was assayed.   Based on our idea and organic synthetic disconnection approach the proposed structure and its fragmentation pattern is given below.
Proposed degradation pathway of 365.23

Summary
Summary of Validation Parameters-Summary of analytical validation parameters of NTB by HPTLC is as follows:

Summary of degradation products, probable structure, m/z & IUPAC name
Degradation products m/z IUPAC Name

Conclusion
In summary, for the first time, a simple, rapid, precise and robust stability-indicating HPTLC method has been developed and validated for the determination of Nintedanib (NTB) in bulk drug.
Furthermore, NTB was subjected to stress studies under various ICH recommended conditions.
The drug was found to degrade in acidic, alkaline, oxidative and photolytic conditions. The additional findings in this study are that the drug undergoes extensive degradation under alkaline and oxidative stress, degrades to a mild extent in acidic and photolytic conditions and is stable to thermal stress. The method was validated for parameters like linearity, precision, accuracy, robustness. The degradants were not detectable when stressed as per ICH recommended conditions but on increasing the strength of acid, base and peroxide, the degradants were very much prominent and were easily detectable in HPTLC.
However, through characterization based on only Mass Spectroscopy, structure of the degradant products was predicted, and their probable fragmentation pattern was also proposed. For further analyses and more accurate prediction, high-grade analytical tools like high resolution 1 H NMR, 13 C NMR, can be employed for the establishment of fragmentation pattern and degradation pathway.