A Validated Quantitative HPLC Method for Proanthocyanidin A2 in Pometia pinnata Leaves

Abstract

A reverse-phase high-performance liquid chromatographic method was described for the determination of proanthocyanidin A2 in Pometia pinnata leaf extracts. The method utilized a Phenominex® Luna 5u Hilic column with a mixture of 2% acetic acid and acetonitrile (step gradient elution as follows: 0-4 min, 5:95; 5-9 min, 10:90; 10-14 min, 80:20 v/v) as the mobile phase at a flow rate of 1 mL/min, and UV detection at 280 nm. The parameters of linearity, precision, accuracy, specificity and sensitivity of the method were evaluated. The extraction methods for proanthocyanidin A2 were also examined. Proanthocyanidin A2 was eluted within 7 min with a satisfactory peak resolution. The recovery of the HPLC method was 96-98% with a good linearity (r2≥ 0.9999) for proanthocyanidin A2 in the concentration range of 7.7 - 250 µg/mL. A high degree of specificity as well as repeatability and reproducibility (RSD values less than 5%) were also achieved. The limits of detection and quantification were 1.25 and 2.50 µg/mL, respectively. This eatablished specific, precise, accurate, rapid and reproducible HPLC method was successfully used to quantify the active principle, proanthocyanidin A2 in P. pinnata leaf extracts. Proanthocyanidin A2 was found to be a major constituent in the crude methanol extract of P. pinnata leaves, at 33.8 ± 0.4 mg/g dried extract. Microwave-assisted extraction (MAE) was selected as the best extraction method for proanthocyanidin A2. The optimized MAE method increased the amount of proanthocyanidin A2 extracted from the dried leaf powder up to 36.6 %w/w.