STABILITY INDICATING VALIDATED HPLC METHOD FOR THE DETERMINATION OF ZANUBRUTINIB IN BULK AND PHARMACEUTICAL DOSAGE FORM

Objective: An accurate, rapid economical and straight forward, reliable assay technique was evolved and showed for the evaluation of zanubrutinib using reversed-phase high-performance liquid chromatography. Methods: In the proposed method, efficient chromatographic separation was achieved applying acetonitrile and 0.1% orthophosphoric acid (50:50 v/v) as a mobile phase with a flow of 1 ml/min and the wavelength was observed at 220 nm. Chromatography was administered isocratically at ambient temperature and run time was approximately 6 min and the retention time (Rt) was observed as 4.358 min. Results: The method was justified as per ICH guidelines. System suitability parameters were studied by injecting the quality six fold and results were well under acceptance criteria. Linearity study was administered between 10% and 150% levels, regression coefficient value was observed as 0.999. Limit of detection and limit of quantification were observed as 0.02 µ g/ml and 0.2 µ g/ml, respectively. Precision was found to be 0.74 for repeatability and 0.68 for intermediate precision. Recovery of the drug was found to be 98–102%, indicates that the recovery is in the acceptable limit. Validation results were found to be satisfactory and the method applicable for bulk and formulation analysis. Hence, it was evident that the proposed method was said to be suitable for regular analysis and quality control of pharmaceutical preparations. Conclusion: The validation results were in good agreement with the acceptable limit. Relative standard deviation values which are <2.0% indicating the accuracy and precision of this method. Assay of retail formulation was administered and found to be 100.24% was present using the above method. Stress conditions of degradation in acidic, alkaline, peroxide, and thermal were studied. This developed method showed reliable, precise, accurate results under optimized conditions.


INTRODUCTION
The brand name of zanubrutinib was Brukinsa is used to cure adult patients who were suffering from the disease of mantle cell lymphoma (MCL) [1][2][3] who have received a minimum of one prior therapy. Zanubrutinib is judged as a Bruton's tyrosine kinase (BTK) inhibitor [4][5][6]. It is taken orally. Efficacy [7,8] was evaluated in BGB-3111-206 (NCT03206970), a phase II clinical trial open-label, multi-center, singlearm trial of 86 patients with MCL who received a minimum of one prior therapy. Zanubrutinib was given orally at 160 mg 2 times daily until disease [9][10][11] passage or intolerable toxicity [12]. Effectiveness was also estimated in BGB-3111-AU-003 (NCT 02343120), a phase I/II open-label, growth, global, multi-center, single-arm trial of B cell malignancies [13,14], including 32 previously treated MCL patients treated with zanubrutinib administered orally at 160 mg twice daily or 320 mg once daily.

Equipment
Chromatographic system of e-2695 with a quaternary pump and a PDA detector of 2996 was used. The chromatographic data were analyzed with Empower Software of version 2.0.

Chromatographic conditions
Using chromatographic conditions, separation was administered in isocratic mode at temperature employing a Luna C 18 (250 × 4.6 mm, 5 µ) column. The combination of 0.1% OPA and acetonitrile 50:50 v/v with a flow 1 ml/min was used as a mobile phase. The volume of injection was 10 µl and eluent was observed at 220 nm, so this was selected. The spectrum was shown in Fig. 1.

Preparation of standard solution
Weigh 20 mg of zanubrutinib working standard and transferred into a flask volume of 100 ml and diluted to volume with diluents. Further, dilute 5 ml of the prepared solution to 50 ml with diluents.

Preparation of sample solution
Transfer 38.5 mg of zanubrutinib sample into a flask of 100 ml and add 70 ml of diluents, sonicate to dissolve it, and makeup to the mark. Further, dilute 5 ml of the prepared sample solution to 50 ml with diluents.

Method validation
In this method validation parameter [15] (system precision, linearity, precision, accuracy, limit of detection (LOD), limit of quantification (LOQ), robustness, forced degradation, and stability), studies were validated for the chosen drug of Zanubrutinib. Sample and standard chromatograms of the proposed technique were shown in Figs. 2 and 3.

System suitability
The HPLC system was stabilized for 60 min to urge a stable bottom line. Six replicate injections of the quality solution containing 20 µg/ml of zanubrutinib were assessed to see the system suitability. The amount of the theoretical plate counts was 6527 and tailing factor was 1.04, respectively. These parameters were found to be within the acceptable limit.

Linearity
Linearity of the tactic was evaluated by preparing a typical solution containing 20 µg/ml of zanubrutinib (100% of the targeted level of the assay concentration). Sequential dilutions were performed to the given solutions at 10%, 25%, 50%, 100%, 125%, and 150% of the target concentrations. These were injected, and therefore, the peak areas are to plot calibration curves against the concentration. The correlation coefficient value of those analytes was 0.999. The results were shown in Table 1 and Fig. 4.

LOD and LOQ
LOD and LOQ minimum concentration level at which the analyte is often reliably detected, quantified using the quality formulas (3.3 σ/s and 10 σ/s for LOD and LOQ, respectively). LOD value of zanubrutinib was 0.02 µg/ml and s/n value is 5. LOQ value of zanubrutinib was 0.2 µg/ml and its s/n value is 26.

Precision
Method precision was investigated by the analysis of six separately prepared samples of an equivalent batch. From these six separate samples, solution was injected, and therefore, the peak responses obtained wont to calculate mean and percentage relative standard  Table 2.

Accuracy
Accuracy decided by recovery studies which were administered in three individual concentration levels (50%, 100%, and 150%). APIs with concentrations 10, 20, and 30 µg/ml were prepared. As per the test method, the test solution was injected to three preparations at each spike level and therefore the assay was performed. The percentage recovery values were observed in between 99.3 and 100.65 of zanubrutinib. The share recovery values were observed as 2%. The results were given in Table 3. Table 4 shows the assay results of zanubrutinib.

Ruggedness
Ruggedness of the tactic was studied and showed that the chromatographic patterns did not significantly change when different HPLC system, analyst, and column. The worth of the percentage of RSD was below 2% exhibits the ruggedness of the developed method.

Robustness
Robustness of the tactic was found to attract RSD should be but 2%. Slightly variations were wiped out the optimized method parameters like flow (±0.2 ml/min), organic content in the mobile phase (±10%). The results were shown in Table 5.

Stability
By observing the stability techniques of Shyamal et al. [16], Kalpana et al. [17], stability of ordinary and sample solutions is studied from initial to 24 h stored at RT. They were injected at different time intervals and difference between initial to 24 h percentage of the assay was not quite 2%. There is no effect in storage conditions for zanubrutinib drug. The results are shown in Table 6.

Forced degradation
Forced degradation conditions containing acidic, basic, peroxide, hydrolysis, reduction, and thermal stress were studied in 0.1 N and 1 N concentration levels (Table 7).

CONCLUSION
This method describes the quantification of zanubrutinib in bulk and pharmaceutical formulation according to ICH guidelines. The evolved technique was observed to be accurate, precise, linear, and reliable. The advantage lies within the simplicity of sample preparation and therefore, the less costly reagents were used. The proposed HPLC conditions ensure sufficient resolution and, therefore, the precise quantification of the compounds. Analysis of the testing results indicates that the precision and reproducibility data are satisfactory. The developed chromatographic technique was often successfully applied for routine analysis in drug research.

ACKNOWLEDGMENT
The authors are thankful to Professor Dr. Ch. Balasekhar Reddy for giving his valuable guidance and suggestions. I heartily thank full to Brilliant Group of Technical Institutions, Abdullapurmet, Rangareddy (Dt.) Hyderabad and Shree Icon Pharmaceutical Laboratories, Vijayawada, India, for providing research facilities for this work.

AUTHORS' CONTRIBUTIONS
Dr. Ch. Balasekhar Reddy has provided the design intellectual content, innovations, protocol for conducting the experiment along with