Research J. Pharm. and Tech. 13(8): August 2020 1 ISSN 0974-3618 (Print) www.rjptonline.org 0974-360X (Online) RESEARCH ARTICLE Development and Validated Stability indicating RP-HPLC method for quantitative determination of Ascorbic acid and piperine in Dhathryaristam Dr Paramita Das, Dr Padmavathi. P. Prabhu, Jithender Reddy 1 Dept of Pharmaceutical Chemistry, Krupanidhi College of Pharmacy, Bengaluru, Karnataka 2 Srinivas College of Pharmacy, Mangalore, Karnataka 3 Vaageswari College of Pharmacy, Telangana *Corresponding Author E-mail: paramitadas04@gmail.com ABSTRACT: Dhathryaristam is an ayurvedic medicine described in Charaka samhita chikitsasthana. The preparation consists of Phyllanthus emblica (amlaki, Indian goose berry) and Piper longam (Indian long pepper). The aim of this research was to determine a finger print methods for ascorbic acid and piperine using simple mobile phase((buffer: acetonitrile) in raw material as well as formulation. The efficient separation of chemical marker was carried out using reverse phase C18 eluted with gradient mobile phase. The separation occurred at 4.1min ascorbic acid and 5.6min piperine. Method validation was demonstrated by system suitability, accuracy, precision, repeatability and recovery. The calibration curve was found to be within the tested ranges. The recoveries were between 98-102%. The photo degradation studies exhibit the formation of various degraded product. The quantification of ascorbic acid in raw material was 1.917μg/ml in Phyllanthus emblica and marketed formulation D-I and D-II are 1.877μg/ml and 1.871μg/ml respectively. The quantification of piperine in raw material was found to be 10.106μg/ml in Piper longam in two marketed formulation D-I and D-II are 10.845 μg/ml and 10.106μg/ml respectively. The developed and validated method can be effectively applied to the quantitative determination of herbal formulation containing ascorbic acid and piperine. KEYWORDS: Dhathryaristam, Phyllanthus emblica, Piper longam, marker, Quantification. INTRODUCTION: Ayurveda is one of the world’s oldest holistic medicine systems also known as “mother of all healing”. In India, plant based products are commonly used while; poly- herbals have existed worldwide with a long history and are used in various ayurvedic therapies 1 . A key challenge for scientist in developing a polyherbal formulation is to develop a well-designed methodology and quantitative analysis of marker for standardization of herbal medicine. Quantitative estimation of chemical marker in polyherbal formulation is always difficult task 2 . Standardization of herbal drug is challenging in terms of modern parameter. Received on 25.04.2020 Modified on 11.06.2020 Accepted on 05.08.2020 © RJPT All right reserved Research J. Pharm. and Tech. 2020; 13(8):. DOI: The specific guidelines for the same have set by WHO for the evaluation of herbal drugs. Lot of advancement has taken place in chromatographic techniques; mostly RP-HPLC with gradient elution is most commonly used for polyherbal formulations. Markers can be separated by different separation techniques with good resolution and less interference from each other. Dhathryaristam also known as Amalakarishtam is an herbal medicine consists of Phyllanthus emblica (ascorbic acid) and Piper longam (piperine). Ascorbic acid{R-3,4- dihydroxy-5-((S)-1,2-dihydroxyethyl) furan-2(5H)- one}is hydro soluble compound and plays an essential role in defence against radical oxygen species 3 . Piperine {(2E,4E)-5-(2H-1,3-Benzodioxol-5-yl)-1-(piperidin-1- yl) penta-2,4-dien-1-one} having potent bronchodilator properties. Dhathryaristam contains 5-10% of self generated alcohol. Ascorbic acid and piperine are excellent herbs that have been considered for various formulations. It has been proven that ascorbic acid extract has strong antioxidant 4 properties and protect against immune deficiencies 5 . Piperine has got