Application of chemometric approach for QbD-Enabled development and validation of an RP-HPLC method for estimation of methotrexate

Abstract

The present work describes Quality by design (QbD)-oriented development of a reverse phase high performance liquid chromatographic (RP-HPLC) method for quantification of methotrexate (MTX). Initially, for performing HPLC studies with UV detector, an appropriate wavelength was selected using a novel technique of Principal Component Analysis (PCA). Risk assessment study was performed, followed by factor screening study using a fractional factorial design (FFD). Subsequently, factor optimization was conducted using a central composite design (CCD), with flow rate and mobile phase ratio as the critical method parameters (CMPs), and tailing factor (TF), theoretical plate count (TPC) and % assay selected as the critical analytical attributes (CAAs). Polynomial modeling was performed to embark upon an optimal solution, followed by numerical and graphical optimization. Chromatographic separation was conducted using the mobile phase composition of acetonitrile (ACN) and ammonium acetate buffer (10 mM, pH 6) in the ratio of 25:75 v/v, flow rate of 0.8 mL/min, column oven temperature of 40 °C, and UV detection at 257 nm. Further, the analytical method was validated as per ICH Q2(R1) guidelines for linearity, accuracy, precision, sensitivity and robustness, ratifying that all the parameters were within the acceptable limits for quantification of MTX.

GRAPHICAL ABSTRACT

KEYWORDS:

• Analytical quality by design
• analytical validation
• design of experiments
• principal component analysis
• method robustness
• risk assessment

Acknowledgements

Prof Bhupinder Singh gratefully acknowledges the generosity of M/s Stat-Ease Inc., Minneapolis, USA, for providing the perpetual license of Design Expert^® software, while conferring upon him with the “Stat-Ease QbD Performance Award 2014” and “Premier Academic Status” for his stellar contributions in the area of QbD-based pharmaceutical research work. Financial support granted by the University Grants Commission (UGC), New Delhi, India, and generous supply of drug sample by M/s IPCA Laboratories Ltd., Mumbai, India, are gratefully acknowlegded. Atul Jain expresses his gratitude to M/s Oniosome Healthcare Pvt. Ltd., Mohali, Punjab, India for extending vital analytical support to him.

Conflict of interest

The authors report no conflict of interest.