A Simplex-Optimized Chromatographic Separation of Phytoconstituents in Cardioprotective Polyherbal Formulation and Crude Drugs

Abstract

Aim: The study aims to develop and validate accurate, precise, and robust reverse phase high performance liquid
chromatographic method for determination of phytoconstituents such as ascorbic acid, gallic acid, quercetin, and
reserpine in polyherbal formulation and crude drugs. Materials and Methods: The adequate chromatographic
separation was accomplished on C18 column, 250 × 4.6 mm, 5 μmAgilent eclipse using mobile phase 0.05 M
sodium dihydrogen phosphate buffer (pH-4 adjusted with 1% Orthophosphoric acid): Acetonitrile with properly
resolved gradient program. The flow rate was 1 ml/min, and the ultraviolet detection was monitored at 227 nm. The
retention time of ascorbic acid, gallic acid quercetin and reserpine was found to be 3.44 min, 5.26 min, 10.02 min
and 13.24 min, respectively. The method was validated as a final verification of method development concerning
precision, linearity, accuracy, sensitivity and robustness as per International Council for Harmonization (ICH)
guideline Q2 (R1). Results and Discussion: A method is considered to be linear as the correlation coefficient was
found to be within acceptance criteria. The detector response was linear in the range of 50–250 μg/ml Ascorbic
acid, 100-300 μg/ml Gallic acid, 100-300 μg/ml, Quercetin and 50–250 μg/ml Reserpine. The % RSD of peak
area response due to Ascorbic acid, Quercetin, Reserpine and Gallic acid in five replicate injections of standard
solution was to be less than 2.0%, and system suitability parameters were passed. Conclusion: The proposed
method was effectively applied for the simultaneous estimation of Ascorbic acid, Gallic acid, Quercetin and
Reserpine in Polyherbal formulation and in crude drugs.