Implementation of green analytical principles to develop and validate the HPLC method for the separation and identification of degradation products of Panobinostat, and its characterization by using LC-QTOF-MS/MS and its in-silico toxicity prediction using ADMET software

Abstract

Panobinostat (PAN) is an inhibitor of histone deacetylase (HDAC) that has been granted approval by the US Regulator for the purpose of treating chronic lymphocytic leukemia. Stress studies were conducted on PAN to assess its inherent stability under physical (thermal and photolytic) and chemical conditions (acidic, basic, neutral, and oxidative) in diluent (water: ethanol 50:50 v/v). The developed HPLC method exhibits both selectivity and specificity towards PAN and its degradation products (DPs). PAN and DPs were resolved using a Waters Xbridge C18 3.0 μm (50 × 4.6 mm) column. The mobile phase consisted of a gradient program (0/15, 2/15, 6/25, 8/25, 10/70, 12/70, 14/90, 16/90, 18/15, and 20/15 (T min/%B)). using mobile phase A as 10 mM ammonium formate buffer (pH 3.0) and mobile phase B as Ethanol with 0.5 mL/min as flow rate, 3 µL as injection volume and at 277 nm as UV wavelength. The PAN exhibited instability under solution state conditions characterized by acidic, basic, and oxidative conditions. The developed chromatographic method has been expanded to include QTOF-MS/MS to characterize the DPs in positive ionization mode. The developed HPLC method has undergone validation in compliance with the ICH guideline Q2 (R1). The method found linear from 12 µg/mL (LOQ) to 300 µg/mL with r2 ≥ 0.99. The specificity of the method was assessed through the peak purity of analyte and DPs. The% recovery for analyte was fall in the range of 99.28 to 100.36 with 0.60 % RSD. The%RSD for the analyte in Method precision and intermediate precision is below 1.0. The sample and standard solutions are stable upto 48 h at room temperature. The Green Analytical Principles (GAP) applied to the analytical method using GAPI, AGREE and Eco-Scale tools to assess the Greenness of the analytical method. The PAN and DPs were applied with ADMET prediction software indicated the carcinogenicity, mutagenicity for PAN and only carcinogenicity for DPs. The reproductive toxicity for shown for PAN, DP-2 and DP-3. The DP-3 has shown the penetration of the blood-brain barrier. The method is suitable for the quantification of PAN and its DPs as both active pharmaceutical ingredient and formulations in quality control and stability studies for its regular use.