Title:Optimization of a Validated Liquid Chromatographic Method with
Chemometric Approach for Estimation of Eugenol from Seed Powder
Extract and Perform Anti-oxidant Activity
Volume: 10
Author(s): Makhmur Ahmad, Jasmeet Kaur, Shamshir Khan, Patil Pallavi Mangesh, Sana Hashmi, Roshan S. and Mohamad Taleuzzaman*
Affiliation:
- Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Maulana Azad University,
Village Bujhawar, Tehsil Luni, Jodhpur 342008, Rajashthan, India
Keywords:
Eugenol, chemometric, quality by design, face-centered cubic design (FCCD), method validation, antioxidant activity.
Abstract:
Background: Developed and validated a new reverse phase high-performance liquid
chromatographic (RP-HPLC) method, and it is prompt, precise, sensitive and robust for the estimation
of eugenol in seed powder extract of Myristica fragrans.
Objective: The chemometric approach was utilized to obtain a rugged and definitive chromatographic
method for the purpose.
Methods: Method variables such as acetonitrile (%) and flow rate were investigated for robustness
and optimization by using a face-centered cubic design (FCCD). The Design Expert 12.0.1.0
software has been employed for this optimization. Further, the effects of factors were monitored
on the concentrations of eugenol recovered from seed powder extract. Chromatograms have been
developed by using an optimized mobile phase mixture containing methanol-water-acetonitrile
(10:40:50, v/v/v) and Symmetry® C18 column (5 μm, 3.9 ×150 mm). The mobile phase was derived
at a flow rate of 1 mL/min, and estimation of eugenol was performed at ʎmax 272 nm.
Results: Validation of the method has been carried out to reveal its selectivity, linearity, precision,
accuracy, LOD, and LOQ. Linear calibration plot for eugenol was held over the concentration
across 6.25 and 100 μg/mL (R2 = 0.999). The coefficient of variation was less than 1%, and
accurate recovery of eugenol was observed between 96.80 and 99.56%. The LOD and LOQ have
been established to be 1.97 and 6.25 μg/mL, respectively. Intraday and Inter-day coefficients of
variation have 1.81-1.91 and 1.92-1.57, respectively. Antioxidant activity (AA) by DPPH assay
of seed powder extract in five different solvents was performed, and % AA activity was calculated
against ascorbic acid.
Conclusion: The validated method has founded to be highly robust and will be applied for the
analysis of eugenol's formulation. The highest % AA has reported in hexane solvent.
