*Author for correspondence JOURNAL OF NATURAL REMEDIES DOI: 10.18311/jnr/2023/31612 Article Received on: 19.10.2022 Accepted on: 11.02.2023 Revised on: 10.02.2023 1. Introduction Ayurvedic medicines are polyherbal formulations that contain a wide variety of chemical constituents in each herb. Plants, which are regarded as a traditional source, are the source of a signifcant number of phytochemicals. Ayurvedic medicines are polyherbal formulations with a variety of chemical constituents in each herb. Te efectiveness of herbal remedies must be evaluated to support their inclusion in the current medical system. Manufacturing and primary processing of herbal substances have an impact on the quality of the active medicinal constituent 1 . It is challenging to quantify markers in any polyherbal composition and to standardise polyherbal formulations. Herbal products can be standardised using advanced techniques like UV, visible, infrared, thin-layer chromatography, High-Performance Liquid Chromatography (HPLC), high-performance thin-layer chromatography, gas chromatography with mass spectrometry, liquid chromatography with a mass spectrometer, atomic absorption spectrometry, spectrofuorimetric, and other techniques. Various methods for estimating p-Cymene and aloe-emodin alone or in conjunction with other markers have been developed, but no HPLC analysis method for aloe-emodin and p-Cymene has been measured simultaneously. More than 100 diferent plant species contain the monoterpene p-Cymene, which is used in both medicine and food. Its chemical name is 1-methyl-4-(1- methyl ethyl)-benzene. It has antibacterial, anticancer, calming, painkilling, and anti-infammatory properties. RESEARCH ARTICLE Development and Validation of Novel High-Performance Liquid Chromatography Method for Simultaneous Estimation of p-Cymene and Aloe-emodin Jyotanshi Bisht and Avani Khristi* Department of Pharmaceutical Quality Assurance, Parul Institute of Pharmacy, Parul University, Waghodiya, Vadodara – 391760, Gujarat, India; avani.khristi@paruluniversity.ac.in Abstract The objective of the present investigation was to develop a novel, accurate, precise, and linear High-Performance Liquid Chromatographic (HPLC) method for the simultaneous estimation of p-Cymene and aloe-emodin in the novel topical herbal formulation and validated as per ICH guidelines. In the current study, good chromatographic separation was achieved in isocratic mode using an HPLC C18 column (250mm × 4.6), 5 μm, and a mobile phase consisting of acetonitrile:water in the ratio of 80:20, at a flow rate of 1.0 mL/min and column temperature maintained at 25°C. The response obtained was monitored at 225 nm wavelength with a UV-Visible detector. The retention times of Aloe-emodin and p-Cymene were found to be 4.3 min and 9.0 min respectively. Linearity was established for both p-Cymene and aloe-emodin in the range of 10-90 µg/mL, respectively. For the method, % Recovery was found in the range of 99.67-100.51 % for p-Cymene and 98.68-100.4 % for aloe-emodin respectively. The LOD and LOQ were found to be 0.01 and 0.04 for p-Cymene and 0.12 and 0.36 for aloe- emodin respectively. This method can be successfully employed for simultaneous quantitative analysis of p-Cymene and Aloe-emodin in the novel topical herbal formulation. Keywords: Aloe-emodin, HPLC, p-Cymene, Simultaneous Method, Validation