Determination of Taxane Concentrations in Taxus canadensis Clippings Using High Performance Liquid Chromatographic Analysis with an Internal Standard Brian J. Cass, Donald S. Scott and Raymond L. Legge* Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1 Clippings of Taxus canadensis were collected and divided into needles, twigs and whole clippings samples. The biomass was extracted and analysed for taxanes using high performance liquid chromatography. The addition of n-octylbenzamide as an internal standard was found to reduce the standard deviation of calculated yields by a third. Paclitaxel was found to be highest in needles (0.0434% dry wt). The presence of paclitaxel was confirmed by photodiode array spectra. Impurities were found to co-elute with other taxanes. A compound was tentatively identified as 9-dihydro-13-acetylbaccatin III based on residence time and UV spectra. The effect of storage on paclitaxel content was investigated and it was found that paclitaxel was stable in dried needles stored at room temperature for at least 1 year. Copyright # 1999 John Wiley & Sons, Ltd. Keywords: Paclitaxel; taxanes; Taxus; internal standard; high performance liquid chromatography; n-octylbenzamide. INTRODUCTION Paclitaxel is a naturally occurring diterpenoid that has been found to be effective in treating numerous types of cancer, including ovarian cancer (Suffness, 1995). Originally, paclitaxel was produced by extraction from the bark of the Pacific yew, Taxus brevifolia. However, this production method was not sustainable owing to the low amounts of paclitaxel present in the bark (0.01– 0.03% dry wt) and to the availability of only limited amounts of this raw material. Consequently, the current focus for the production of paclitaxel is on renewable sources such as clippings, consisting of needles and small twigs. Examinations of other species of yew have demonstrated that paclitaxel and closely related taxanes are produced by all Taxus species (Suffness, 1995). The study described here was performed in order to investigate the taxane content of T. canadensis, the Eastern Canadian yew or ground hemlock. The structures of the taxanes of interest are given in Fig. 1. A comprehensive review of the analysis of paclitaxel has been published by Theodoridis and Verpoorte (1996), and it would appear that the use of an internal standard for analysing extracts of Taxus biomass is very rare. Heaton et al. (1993) used cholestenone as an internal standard for normal phase separation by supercritical fluid chromato- graphy. Xu and Liu (1991) used betamethasone as an internal standard for normal-phase high performance liquid chromatography (HPLC) of extracts of T. chinen- sis. One of the few internal standards reported for extracts with analysis by reversed-phase HPLC has been isotopically labelled 10-d3-acetylpaclitaxel with tandem mass spectrometric detection (Hoke et al., 1994). However, this internal standard is not suitable for use with the more commonly employed UV detection method. More recently, cinnamic acid has been reported as an internal standard for reversed-phase HPLC (Van Rozendaal et al., 1997). A number of researchers have reported the addition of internal standards for measuring paclitaxel in pharma- ceutical formulations and biological fluids. These internal standards include: N-nitrosodiphenylamine (El-Yazigi and Yosuf, 1995), n-octylbenzamide (Sharma et al., 1994), 2'-methylpaclitaxel (Sparreboom et al., 1995), and cephalomannine (Leslie et al., 1993; Song and Au, 1995). Cephalomannine is a naturally occurring taxane and would not be suitable for use as an internal standard in Taxus extracts; however, the other compounds could potentially be used with extracts using reversed-phase HPLC with UV detection. One of the internal standards developed for paclitaxel analysis in biological fluids, n- octylbenzamide, was chosen as an internal standard for the present study. This compound has been found to have similar chromatographic behaviour, lipophilicity and UV absorbance patterns to paclitaxel (Sharma et al., 1994). The present study extends the use of an internal standard to improve the precision and reproducibility of taxane analysis by HPLC in biomass samples. PHYTOCHEMICAL ANALYSIS Phytochem. Anal. 10, 88–92, (1999) CCC 0958–0344/99/020088–05 $17.50 Copyright # 1999 John Wiley & Sons, Ltd. * Correspondence to: Professor R. Legge, Department of Chemical Engineering, University of Waterloo, Waterloo, Ontario, Canada N2L 3G1. E-mail: rllegge@engmail.uwaterloo.lo Contract/grant sponsor: Phytogen Life Sciences, Delta, BC, Canada. Contract/grant sponsor: Natural Sciences and Engineering Research Council of Canada. Received 10 October 1997 Revised 16 February 1998 Accepted 16 February 1998