Analysis of Precocenes in the Essential Oil of Ageratum spp. by Reverse-phase High- performance Liquid Chromatography Kiran Sharma 1 and Om P. Sharma 2 * 1 Department of Chemistry and Biochemistry, Himachal Pradesh Krishi Vishvavidayalaya, Palampur, H.P., India 2 Biochemistry Laboratory, Indian Veterinary Research Institute, Regional Station, Kangra Valley, Palampur-176 061, Himachal Pradesh, India The chromenes precocene I (7-methoxy-2,2-dimethylchromene) and precocene II (6,7-methoxy-2,2- dimethylchromene) extracted from Ageratum houstonianum and A. conyzoides have been analysed by reverse-phase high-performance liquid chromatography. Separation was achieved using a C 18 column eluted with a linear gradient of acetonitrile and water. Precocenes I and II in the essential oils were determined to be 270  23 and 482  34 mg/mL in A. houstonianum, and 687  21 and 20  2 mg/mL in A. conyzoides. Copyright # 2001 John Wiley & Sons, Ltd. Keywords: Reverse-phase HPLC; chromenes; precocene I; precocene II; Ageratum houstonianum; Ageratum conyzoides. Introduction Precocene I and II are the bioactive chromenes in Ageratum spp. (Bowers et al., 1976; Sharma and Sharma, 1995). Both of these chromenes have insecticidal activity (Bowers et al., 1976; Polivanova, 1991); furthermore, precocenes act as anti-juvenile hormones and cause genotoxicity (Socha and Marec, 1989; Miyake and Mitsui, 1995). Both, precocene I and II may be extracted by hydrodistillation into an essential oil fraction (Kasturi et al., 1973; Ekundayo et al., 1988; Mensah et al., 1993; Menut et al., 1993; Chandra et al., 1996). Precocene I is the major component of the essential oil of Ageratum conyzoides (Ekundayo et al., 1988; Mensah et al., 1993), whilst precocene II is the predominant chromene in the essential oil of A. houstonianum (Menut et al., 1993; Chandra et al., 1996). Most of the studies reported so far have been carried out using GC systems which are limited in application to volatile compounds or to components which have been previously derivatised. HPLC analysis, however, allows the possible separation and quantification of natural products with a wide range of polarity (McMaster, 1994). Thus far, analytical systems developed for precocene I and II have shown long retention times and broad retention windows (Hsia et al., 1981; Haplin et al., 1984). An isocratic system has also been reported for the quantification of precocenes (Siebertz et al., 1990), but this system would also result in long retention times for the complete elution of complex mixtures, such as the essential oils, which contain a large number of non-polar constituents. We have, therefore, developed an alternate HPLC method which permits separation of the essential oil constituents of Ageratum spp., and we have used this procedure to analyse precocenes in the essential oils of samples of A. houstonianum and A. conyzoides collected from Palampur in the Kangra Valley (Himachal Pradesh, India), a sub-temperate area in the sub-Himalayan region. EXPERIMENTAL All solvents for HPLC analysis were of HPLC grade and purchased from Qualigens Fine Chemicals (Mumbai, India). Precocenes I and II were obtained from Sigma (St Louis, MO, USA). The aerial parts of A. houstonianum and A. conyzoides were collected from Palampur in the Kangra Valley (Himachal Pradesh, India) during the month of September. The samples were extracted by hydrodistillation using a Clevenger-type apparatus for the preparation of essential oil. Standard samples containing precocene I (1 mg/mL) and precocene II (1 mg/mL) were prepared in methanol. Extracts from A. houstonianum and A. conyzoides were prepared by dissolving 5 mL of the essential oil in 10 mL methanol. All the samples were filtered through a Whatman (Maidstone, England) stainless steel syringe assembly using a 0.22 mm Durapore (Millipore; Milford, USA) membrane filter. Analyses were performed using a Waters HPLC system consisting of model 510 and 515 pumps, a Rheodyne injector, a Novapak C 18 column PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 12, 263–265 (2001) DOI: 10.1002/pca.587 Copyright # 2001 John Wiley & Sons, Ltd. * Correspondence to: O. P. Sharma, Biochemistry Laboratory, Indian Veterinary Research Institute, Regional Station, Kangra Valley, Palampur-176 061, Himachal Pradesh, India. Email: ivriplp@nde.vsnl.net.in Received 22 February 2000 Revised 7 June 2000 Accepted 25 September 2000