Risk based HPLC method development and validation for isoniazid and ethambutol using analytical QbD approach


  • Bhagyashri Raghunath Mogarkar Department of Quality Assurance, Indira College of Pharmacy, Nanded 431606, India
  • Prakash Katakam Department of Quality Assurance, Indira College of Pharmacy, Nanded 431606, India


AQbD, Box-behnken, Experimental Design, DoE, Risk based analytical method development, quality risk management


Introduction: Analytical quality by design (AQbD), is a systematic approach to development that starts with established objectives
and places an emphasis on understanding and control of the analytical method, based on sound science and quality risk management.
The use of DoE reduces number of runs in a planned experiment and also allows the collection of higher quality data. Implementation
of QbD developes the rugged and robust method which helps to comply with ICH guideline and also facilitates continuous
improvement in the method.
Method: The experimental design is based on Box-behnken design of three key components of the HPLC method (mobile phase
composition, flow rate and wavelength) is presented. The Zorbax Eclipse XBD-C18 (4.6mm×250mm×5µm) and mobile phase
composition (90:10), flow rate (0.8 ml/min) and wavelength (212) were optimized chromatographic conditions. The approach was
created according to ICH guidelines.
Result: Isoniazid and ethambutol were found to have a retention time of 4.558 min and 6.761 min respectively. The developed
method had a linear range of 3-15 µg/ml and 8-40 µg/ml with a correlation coefficient of 0.9993 and 0.999 for isoniazid and
ethambotol respectively. The optimized approach was accurate and precise, as evidenced by less than 2% RSD for repeatability,
intraday and interday precision. The percentage recovery of spiked samples ranged from 99.45 to 100.01 as per the acceptance
requirements of ICH guidelines. The validated parameters of developed method were found in the prescribed limit as per ICH
Conclusion: Using the Design Expert version, the Box-behnken experimental design explains the interaction of mobile
phase composition, flow rate and wavelength at three different levels and the responses to be observed were retention time, peak
area, resolution, theoretical plates and asymmetric factor. The analytical QbD approach used in method development has very much
helped for better understanding of method variables hence there are less chances of failure during method validation and transfer.