Simultaneous Quantification of Precocene I and Precocene II through High‑performance Thin Layer Chromatography Validated Method in Ageratum conyzoides L. Germplasms from Western Himalayas

Background: Ageratum conyzoides L. is a traditionally used herb for various ethnoveterinary purposes. There are no earlier reports on simultaneous high‑performance thin layer chromatography (HPTLC) quantification of bioactive markers. Objective: To develop a sensitive, robust, and replicable HPTLC method for simultaneous estimation of precocene I (PI) and precocene II (PII) in A. conyzoides L. collected from the Western Himalaya region of India, with an aim to understand the level of chemotypic differences arising in the intraspecific population of this ethnobotanically important plant. Materials and Methods: A sensitive HPTLC method was developed to resolve PI and PII using toluene: ethyl acetate (9.8:0.2 v/v) as mobile phase. The method was validated for selectivity, specificity, linearity, and precision as per International Conference on Harmonization guidelines. Results: Good linearity was achieved over a five‑point concentration range with a correlation coefficient of 0.986 and 0.988 for PI and PII, respectively. The PI content varies in the range of 0.0016% (NAC‑77) to 0.0834% (NAC‑82), whereas PII was reported to be present in the range of 0.016% (NAC‑85) to 0.143% (NAC‑91) on a dry weight basis. A principal component analysis biplot of samples based on the content of PI and PII identified four elite chemotypes, namely, NAC‑81, NAC‑82, NAC‑85, and NAC‑91. Conclusion: The study identifies superior germplasms for commercial prospection and develops a validated method that can be used for the quality control of herbal drug/formulation using A. conyzoides as an ingredient.