Detection of Paclitaxel in a Taxus brevifolia sample

Kayla SteevesChem 4101 Dec 5, 2010

What is Paclitaxel?[10]

• Paclitaxel is a natural product originally isolated from the bark of the tree Taxus brevifolia.

• Paclitaxel is a chemotherapeutic agent, and is used to treat advanced cancer of the ovaries, breast, lung, and Kaposi sarcoma.

• Taxus brevifolia is native to the pacific northwest region of North America

• Hypothesis-A compound analogous to the diterpenoid paclitaxel, will be isolated from a sample collected from Taxus brevifolia.

Significance- As a valuable anti-cancer drug, It should be established quantitatively how much Pacitaxel can be isolated from the root of the Taxus brevifolia.

This information can then be used to determine if other methods of production should be explored.

Appropriate Technique: Liquid Chromatography/Mass Spectrometry/Mass

spectrometry[6,7,8,9]

• Suitable for complex plant matrices• Provides molecular mass, structural, and relative

concentrations information• Suitable for samples of limited quantities• Taxane database containing characteristic

chromatographic and mass spectrometric data assessable • LC columns specific for taxane analysis• Direct sample introduction

Instrumentation[6,7]

• Varian 1200L LC/MS/MS (used $69,900)

• Liquid Chromatography• Curosil-PFP, 5 μm, 150 × 4.6 mm, column ($645)

• Flow rate-1mL/min

• Gradient elution-Acetonitrile/Water (25:75) to Acetonitrile/Water (65:35)

• 40 Minutes

• 20°C

• Tandem Mass Spectrometry• Triple Quadrupole

• Electrospray ionization, operated at a 350°C and 5500 V

• Multiple-reaction monitoring mode- enhance the selectivity and specificity of low content compounds in Taxus species

• Full-scan spectra acquired over the m/z range of 10–1000 amu, step size 0.1 amu and dwell time of 1.5 s.

Sample PreparationStep 1:•Ground with a mortar•Extract with 25 mL methanol, sonicate for 1 hour, sit over night 20°CStep 2:•Vacuum filter, dispose of plant residue•Dry solution with rotary evaporatorStep 3:•In separatory funnel combine with 100 mL ethyl acetate and 100 mL water•Dry out ethyl acetate fraction with rotary evaporatorStep 4:•In separatory funnel combine with 90 mL methanol, 90 mL hexane, and 20 mL water•Retain aqueous solution, dry using rotary evaporator• Dilute in methanol, centrifuge,collect supernatant for analysis

Figures of Merit[6]

• High selectivity• High sensitivity• Good reproducibility• LOD 8 ng/mL• LOQ 26 ng/mL• Linearity range 0.060-38.8 μg/mL• Correlation factor (R2) 0.9937

• Ma-mass

Data Analysis[6]

Using the external standard method, perform linear regression, construct a calibration curve

Generate curve from 8 concentrations of standards

Three replica injections of each concentration standard, use average value for the peak area values

Following HPLC separation, measure the peak area corresponding to the transition of m/z 854.4→ 286.1, retention time 31.56 minutes

Peak areas obtained from the MRM used to quantify

Sample Spectrum

Mass Spectrum[7]

LC-MS Mass Chromatogram[6]

Alternative MethodsMethod Advantages DisadvantagesUltraviolet-Visible Absorption[3,8]

Inexpensive, all organic samples suitable, low LOD

Does not detect all taxanes

Infrared Spectroscopy[8] Quick, some structural information, FTIR possess high accuracy and precision

Many functional groups present in paclitaxel

Micellar Electrokinetic Chromotography[8,10]

Superior resolution, neutral compounds, complex matrices allowed, quick, chiral separation capabilities

Developed for low molecular mass compounds

Gas Chromatography/Mass Spectrometry[8]

Quick, ideal technique for separating and identifying species in mixtures, longer columns

Limited to volatile species

Conclusions• LC/MS/MS allows for sensitivity, precise, and repeatable quantitative

analysis of Taxus brevifolia samples.

• Multiple-reaction monitoring mode helpful for optimizing sensitivity and accuracy.

• Curosil-PFP, 5 μm, 150 × 4.6 mm, column, selective and optimum for this study.

• Quadrupoles are great for MS/MS. Despite the presence of a poor vacuum quadrupoles can funtion, since the ions are produced under atmospheric pressure conditions; this makes them well suited to electrospray ionization. Quadrupole mass spectrometers have a relatively low cost which also makes them attractive as electrospray analyzer.

• Future studies could be completed on the sap produced by the Taxus brevifolia.

• Further studies could also be explored for other trees in the Taxus genus.

References1 Auriola O.K.; Lepisto M.A.; Naaranlahti T. Determination of Taxol by High-Performance

Liquid Chromatography-Thermospray Mass Spectrometry. Journal of Chromatography. 1992, 594, 153-158.

2 Bhat, S.V.; Nagasampagi, B.A.; Sivakumar, M. Chemistry of Natural Products; Springer: India, 2005.

3 Bitsch, F.; Wenwen, M.; Macdonald, F; Nieder M.; Analysis of Taxol and Related Diterpenoids From Cell Cultures by Liquid Chromatography-Electrospray Mass Spectrometry. Journal of Chromatography. 1993, 615, 273-280.

4 Gross, H.J. Mass Spectrometry, Springer-Verlag: Germany, 2004.5 Hanson R. James, Natural Products the Secondary Metabolites. The Royal Society of

Chemistry, 2003. 6 Kerns, H. E.; Volk, J.K.; Hill, E. S.; Profiling Taxanes in Taxus Extracts Using LC/MS and

LC/MS/MS Techniques. Jounral of Natural Products. Voi.57, No. I0,pp. 1391-1403, 1994.7 Li, Shuangming.; Fu, Yujie.; Yuangang, Zu.; Determination of Paclitaxel and Other six

Taxoids in Taxus Species by High-Performance Liquid Chromatography–Tandem Mass Spectrometry. Journal of Pharmaceutical and Biomedical Analysis 49. 2009. 81–89

8 Skoog, Douglas A., F. James Holler, and Stanley R. Crouch. Principles of Instrumental Analysis. Thomson Brooks/Cole. 2007. 6th Ed.

9 Steven L. Richheimer, David M. Tinnermeier, Daniel W. Timmons. High-Performance Liquid Chromatographic Assay of Taxol. Anal. Chem., 1992, 64 (20), pp 2323–2326.

10 Suffness, M. Taxol Science and Applications. CRC press: Boca Raton. 1995.