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Anal. Chem. 1992, 64, 2323-2328
High-Performance Liquid Chromatographic Assay of Taxol Steven L. Richheimer,' David M. Tinnermeier, and Daniel W. Timmone Hauser Chemical Research, Inc., Boulder, Colorado 80301
A roverubphaso hlgh-porformance llquld chromatographic (HPLC) mothodfor assaylngtaxol buk drug or promssampbs k doscrlbod. The mothod ut1Uz.r a commerclally avallabh ponta~oroph.nyl(PFP) packing makrlal that has groater wkctlvlty for taxol and relatod taxanm than othot rovomdpham modla tortd. Tho mothod has boon shown to bo accurate, Ilnoar, prock, rpeclflc, and ruggod, and can bo us8dto assay tho bulk drug and doterminoits chromatographic purtty as w d l as to assay taxd In process and biomasssampbs.
ANH
Ph
I: Taxol
INTRODUCTION Taxol (I) is isolated in large quantities from the bark of the Pacific yew tree, Taxus breuifolia. It is a unique antitumor drug that appears to exert ita activity as a result of interference with microtubular structure and function.' Several other HPLC assaye for taxol using reversed-phase media have appeared in the literature.2-9 However, these methods have focused on the separation of taxol (I) from the closely eluting analog cephalomannine (111) and tend to be long and tedious. None of the published methods separate taxol from another closely eluting taxane: 7-epi-10-deacetyltaxol (II), which tends to elute between I and I11 on reversed-phase columns. This report describes a new reversed-phase liquid chromatographic (HPLC) method that adequately separates I from I1 and I11 and from other closely eluting compounds that occur naturally in the bark and leaves of Taxus species.
OH
II: 7-Epl-1O-deacetyltaxol
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:
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EXPERIMENTAL SECTION Reagents and Materials. Acetonitrile and water (HPLC grade) and methanol (reagentgrade) were purchased from Fisher Scientific Co. (Fair Lawn, NJ). Reagent-grade phosphoric acid (ca. 85%),hydrochloric acid, glacial acetic acid, and methylene chloridewere purchased from B&r ScientificProducts (McGraw Park, IL). I and I1 were isolated in-housefrom bark of the Pacific yew tree. Taxol was shown to be over 99% pure by chromatography. 7-Epitaxol (XII)was prepared by heating taxol at 140 "C for 70 h. Cephalomannine (III), baccatin I11 (VII), and 10deacetylbaccatin I11 (X) were obtained from the National Cancer Institute (Bethesda, MD). Phenyl HPLC columns (4.6 mm X 250 mm), packed with 5-pm diphenyl material, were purchased from Supelco,Inc. (Bellefonte,PA) and Metachem Technologies, Inc. (Redondo Beach CA). Pentafluorophenyl (PFP) HPLC columns (4.6 mm X 250 mm, 5 pm, 60 A) were purchased from (1)Schiff, P. B.; Faut, J.; Horwitz, S. B. Nature 1979,277,665. (2)Harvey, S. D.; Campbell, J. A.; Kelsey, R. G.; Vance, N. C. J. Chromatogr. 1991,587,300. (3)Witherup, K. M.; Look, S. A.; Staeko, M. W.; McCloud, T. G.; Isaaq, H. J.; Muechik, G. M. J. Liq. Chromatog. 1989,12 (ll),2117. (4) Longnecker, S.M.; Donehower, R. C.; Cabs, A. E.; Chen, T. L.; Brundrett, R. B.; Grochow, L. B.; Ettinger, D. S.; Colvin, M. Cancer Treat. Rep. 1987,71,53. (5)Magri, N.F.; Kingston, D. G. J. Org. Chem. 1986,51,797. (6) Senih, V.; Blechert, S.;Colin, M.; Guenard, D.; Picot, F.; Potier, P.; Varenne, P. J. Not. Prod. 1984,47,131. (7)Hamel, E.; Lin,C. M.; Johns,D. G. Cancer Treat. Rep. 1982,66, 1381. (8) Miller, R. W.; Powell, R. G.; Smith, C. R., Jr.; Arnold, E.; Clardy, J. J. Org. Chem. 1981,46,1469. (9) Wani, M. C.; Taylor, H. L.;Wall, M. E.; Coggon, P.; McPhail, A. T. J. Am. Chem. SOC.1971,93,2325.
111:
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Cephalomannine
ES Industries (Marlton, NJ) and Metachem Technologies. A 4.6-mm X 20-mm phenyl guard column (Jones Chromatography, Lakewood, CO) was used for all analyses. Crude extracts were filtered through 0.2-pm, 13-mm PVDF filters (Baxter Scientific Products). Apparatus. The HPLC system consisted of a Model L-6200 pump, Model AS-4000autosampler equipped with a 100-pLloop, and a Model L-4000 or L-3000 UV/VIS/DAD detector (Hitachi Instruments, Inc., Fremont, CA). The system was equipped with an NEC 286 computer with 40M hard drive (Boxborough,MA) and Lab Manager HPLC software (Hitachi Instruments, Inc.). Chromatographicreports were printed on a Star MicronicsModel NX-1000 dot matrix printer (New York, NY) or a Panasonic Model KX-P4450i laser printer (Chicago, IL). Partial loop fill method of injection was used. Phenyl Column Conditions. The mobile phase for elution of taxanes on diphenyl columns was a linear gradient beginning with 25:75 MeCN/water at a flow rate of 1 mL/min, reaching 6040 after 35 min. PFP Column Conditions. The mobile phase for isocratic elution on PFP columns consisted of a 4555 (v/v) mixture of acetonitrile (MeCN) and water or 0.1% phosphoric acid solution (1mL of phosphoric acid/L of solution). The flow rate was 1.5 mL/min. Under these conditions, I eluted in about 10 min. For chromatographic purity testing with the PFP column, the initial isocraticconditionswere 36-39 7% MeCN with the remainder water or 0.1% phosphoric acid solution; this was held for 35 min at a flow of 2 mL/min. This was followed by a linear gradient to
0003-2700/92/0364-2323$03.00/0 0 1992 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
6040 MeCN/water at 50 min. Under these conditions, I eluted in about 30 min. Detection was at 227 nm throughout, and 10 pL of sample was injected for all samples except for chromatographic purity testing, where 15 pL was injected. Sample and Standard Preparation. Pure I (100 mg) was dissolved in 100.0 mL of methanol containing approximately 100 pL (0.1% ) of acetic acid. Acetic acid was used to neutralizetraces of alkali present in the methanol and increase the shelf-lifeof the standard solution. Crude extracts of T.breuifolia biomass were obtained by exhaustivesoxhlet extraction with methanol followed by dilution to known volume with methanol. Assay Procedure. Standard and sample preparations (1mg/ mL) were injected on the PFP column under isocratic conditions (4555, 1.5 mL/min). Either peak areas or peak heights were measured. Tasol was quantitated by comparing the average peak response of the sample to that of the standard. Ultraviolet Spectra. UV spectra of taxol and related taxanes were obtained in the range of 200-360 nm with the Model L-3OOO diode array detector and DAD Manager software (Hitachi Instruments, Inc.). All spectra were normalized. NMR Spectra. Proton NMR spectra were obtained on a Varian EM-390 operating at 90 Mhz for or on a Bruker ACP-300 operating at 300.13 MHz. Spectra were acquired at ambient temperature using a 5-mm probe in deuterated chloroform at approximately 20 mg/mL. All chemical shifts are reported relative to tetramethylsilane.
RESULTS AND DISCUSSION Identification of 7-Epi-10-deacetyltaxol (11). I1 was first described byMcLaughlin.10 I1was isolated from a crude methanolic extract of T. breuifolia bark by silica column chromatography followed by crystallization from ethyl acetate. The UV spectrum of I1 was identical to that of 10deacetyltaxol (IV), and heating IV at 120 "C converted it partially to 11. It has been previously reported that epimerization a t C-7 decreases the polarity of the epimer, probably due to intramolecular hydrogen bonding between the 7ahydroxyl and the carbonyl oxygen of the 4a-acetoxy group.10 Hence, 7-epitaxol (XII) eluted after I by reversed-phase chromatography, and similarly, I1 eluted after IV. The structure of I1 was confirmed by two-dimensional proton NMR. Preparation and Identification of Taxol Acid Side. Taxol acid side chain [B(S)-(benzoylamino)-a(R)-hydroxybenzenepropanoic acid, XI] was prepared by treating I, dissolved in a 1:l mixture of methanol and water, a t room temperature with concentrated ammonium hydroxide. XI was extracted from the acidified solution with methylene chloride. The methyl ester of the taxol acid side chain (1x1 was prepared similarly in the absence of water. NMR, UV, and HPLC retention times were consistent with the assigned structures. Chromatography and Comparison to Other Methods. Witherup et al. reported good separation of I and I11 using a phenyl column and a mobile-phase mixture of acetonitrile, methanol, and water.3 We found that when methanol was eliminated from the mobile phase, the chromatographic separation of I and I11 improved on a phenyl column. However, I1 still coeluted with I under these conditions. Several brands of phenyl columns were tried with mixed results. However, we found that a high carbon load diphenyl column (Supelco or Metachem Technologies), using a methanol-free gradient, could achieve baseline separation of these closely eluting taxanes (see Figure 1). In comparison, a pentafluorophenyl column was found to better separate these closely eluting taxanes (see Figure 2). The PFP column appeared to be more selective for I, 11, and I11 and could achieve baseline separation of I, 11,and I11 under isocratic conditions (45% MeCN and 55% HzO at 1.5 mL/min) in less (10)McLaughlin, J. L.; Miller, R. W.; Powell, R. G.; Smith Jr., C. R.
J. Not. Prod. 1981,44, 312.
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Fl#m2. Chromatogram of a "reof taxol( I), 7epClWeecetyltaXol (II), and cephekmannlm, (111) on PFP column. Chromatographic conditions: isocratlc for 35 mln at 2 W m l n with MeCN/HzO (37:83), followed by a linear gradient to MeCN/H20 (6040) at 50 mln.
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naUn3. Chromatogramofa mlxhreof taxol (I), 7+1 Weecetyltaxol (111, and cephalomannine (111) on a PFP column. Chromatographic conditkns: isocratlc for 1.5 mumin with MeCN/H20 (4555).
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Flgurr 4. Chromatogram of a sampk of degraded taxol standard on a PFP column. Chromatcgraphlc condltkns: isocratk for 35 min at 2 W m i n with MeCN/H20 (37:83), followed by a linear gradknt to MeCN/Hfl(8040)at 50 mln. I =taxaI 1 = 7-epCiO-deacetyttaxoI; I V = 1OdeacetylteXol; V = 7-epibaccatin 111; V I = methylbenzoate; VI1 = baccatin 111; I X = methyl ~ ~ b e n z o y l a m i n o ~ ~ ( i + h y d r o x ybenzenepropanoate; X = lodeacetylbaccatin 111; XI1 = 7-epltaxol.
than 10min (seeFigure 3). Thew chromatographic conditions were found suitable for assaying both I bulk drug, and I in crude Taxus extracts. Figure 4 shows the chromatogram obtained on a sample of alkali-degraded I using the PFP column. Figure 5 illustrates the chromatogram obtained on the same sample using the diphenyl column. The phenyl
ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
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11K Fbum 1. Chromatogram of a sample of degraded taxol standard on Flgure 7. Arrhenius plots for the degradation of taxol: ( 0 )In boaMetechemInterdl5-wphenyicolumn. chrome to graph kc^ butanol; (0)dry state. linear gradient at 1 ml/mln starting with MeCN/H20(25:75) and going to MeCNIH20 (60:40) after 35 mln. I = taxol; I 1 = 7-epi-10deecetyltaxol; I V = 1Odeacetyitaxol; V = 7-eplbaccatln 111; V I = methyl benzoate; VI1 = baccatln 111; VI11 = 7-epClO-deacetylio'oo baccatln 111; I X = methyla(SHbenroylamlno)-~(~y~o~~nzenrena propanoate; X = 1Odeacetylbaccatln 111; XI1 = 7-epitaxol.
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Flgure 8. Chromatogram of a sample of taxol bulk drug. Peak assignments are the same as In Figure 1. The closely elutlng unknown compound (U) Is separated from I by the method. Chromatographic condltions: lsocratlc for 35 mln at 2 mL/mln with MeCN/H20 (37:63), followed by a linear gradlent to MeCN/H20 (6040) at 50 mln. I = taxol; II = 7-epC 1O-deacetyttaxol; III = cephalomannlne; I V = 10. deacetyttaxol; VI1 = baccatln 111.
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column was superior in separating naturally occurring taxanes from one another, while the PFP column was more selective for I and was superior in separating I from closely eluting taxanes. M a t of the base-catalyzed degradation products have been describedpreviously10 and were identified by their relative retention time and UV spectra, and in some cases by spiking with known standards. I1 tailed on new PFP columns. This tailing could be eliminated by adding phosphoric acid (1:lOOO) to the mobile phase. The addition of phosphoric acid had no effect on the chromatography of other taxanes, and after such treatment, water could be substituted for the dilute phosphoric acid and the tailing did not return. Linearity. The linear peak response for I was determined over the range of 0.25-1.5 mg/mL. The standard curve was h e a r (r = 0,9999)for both peak area and peak height. The standard curves are shown in Figure 6. Precision. The injection precision was measured by performing 10 replicate injections of both 5 and 10 pL of a standard preparation (1mg/mL). The standard error (2 u %) was 0.8% for a 5-rL injection and 0.6% for a 10-pL injection. The overall assay variability was determined by performing five separate assays on a sample of bulk I (concentration 0.9-1.1 mg/mL). The ratio of milligrams of I to peak area was calculated; the standard error was 2.0%. Stability of the Bulk Drug. Heating pure, previously dried I at 80 OC or higher converted it primarily to XII. An Arrhenius plot (seeFigure 7) was generated for the conversion of I to XII. Extrapolation of the curve to 25 "C indicated that about 0.02% would degrade per year. An Arrhenius plot was also generated for the degradation of I in solution.
The plot has approximately the same slope, but the rate of degradation was approximately 5-6 times faster in solution than in the dry state (see also Figure 7). Stability of Standard and Sample Solutions. Taxol underwent hydrolysis and transesterification in methanolic solutions. A standard consisting of reagent- or HPLC-grade methanol typically lost about 30% of the taxol peak area after storage for 2 weeks at room temperature (see alsoFigure 1). A sample with 0.1% acetic acid added to the methanol showed no sign of degradation. The preservation effect of acetic acid appeared to be due to its ability to neutralize traces of alkali (probably ammonia) present in methanol. Experiments indicated that taxol standards containing 0.1 % acetic acid showed no detectable degradation when stored 7 weeks at room temperature or 3 months at 4 O C . Chromatographic Purity Testing. The PFP column run under isocratic conditions with less MeCN achieved separation of I from other closely eluting compounds such as the closely eluting taxane U (see also Figure 8). No peak corresponding to U was seen in the chromatogram of the same sample using a phenyl column, and presumably U coelutes with I under these conditions. Isocratic conditions of approximately 37:63 MeCN/water on the PFP colurnn were found to separate I from impurities and degradation products that might normally be encountered in process samples and bulk drug. Less polar compounds such as XII, which elute after I, were eluted by increasing the MeCN to 60% after the completion of the isocratic phase. This chromatographic method is suitable for performing chromatographic purity testing of the bulk drug. Degradation of Taxol by Alkali. In aqueous or methanolic alkaline solution, I was destroyed rapidly and totally. In aqueous solution, the acid side chain (XI) and benzoic acid were isolated. In methanolic solution, IX was produced in addition to VI1 and X. Other deacetylated and debenzoylated derivatives of VI1 were probably also produced by alkaline hydrolysis, but debenzoylated derivatives of VI1
ANALYTICAL CHEMISTRY, VOL. 64, NO. 20, OCTOBER 15, 1992
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Fburr 10. iog/log plot of the rate of degradation of taxol In methanolic HCI solutlon vs the formality of the solution.
would be poorly retained on the column and have weak UV absorbance at 227 nm, and none were observed. Degradation of Taxol by Acid. Taxol was degraded rapidly at room temperature in a 1:l mixture of methanol and concentrated HCl. Several polar degradation products were produced, including 11, IV, VI, and IX (see also Figure 9). However, unlike base-catalyzed degradation of I, the chromatographicand UV data indicated that no derivatives of VI1 formed. In contrast to weakly alkaline solutions, I was stable in methanol containing0.1 7% acetic acid. In dilute methanolic HC1 solutions, the rate of degradation of I was dependent on the concentration of acid (see Figure 10). Usefulness of the Method for Analyzing Crude Biomass Extracts. The phenyl column method was superior to the P F P method for separating polar b a n e s such as baccatin I11 (VII) and 10-deacetylbaccatin I11 (X) from other
Figure 11. Chromatogram of a crude ~ n o "3 k of Taxw brevffdle leaf. Chromatographic conditions: bcratk at 2 Wmln at 111 MeCNIH20 (35~65). I 3 taxol; I1 = 7-epl-lOd~cetyltaxd;
cephalomannlne.
polar material occurring in methanolextractaof Taxus.Methanolic extracts of T.brevifolia bark were found to contain only traces of I1 and could be analyzed by either the phenyl column method or the PFP column method. On the other hand, some Taxus leaf samples had several non-taxane compounds that eluted between I and 111, and near I1 (see also Figure 11). Using the PFP column, these unknown compounds were separated from I, while they interfered with the analysis of I using the phenyl column method. Even after multiple analyses of crude methanolic Taxus extracts neither the PFP columns nor the phenyl columns developed high back pressure. Both columns showedlonglifetimeswhen protected with a standard phenyl guard column. Therefore, preliminary cleanup steps appear to be unnecessary when using these columns, and crude methanolic extracta of Talcus can be analyzed directly.
ACKNOWLEDGMENT We thank Thomas H. Warden for suggesting that we try using the PFP column for analyzingTaxus extracts. We thank
the National Cancer Institute (NCI) taxane standards;Bernard J. Floor of Bristol-Myers Squibb Co., Pharmaceutical Research and Development Division, Syracuse, NY, for his contribution toward developing conditions for the chromatographic purity test; Jeff T. Beckvermit of Hauser Chemical Research for obtainingcrystalline7-epi-10-deacetyltaxol;and Chris Rithner of Colorado State University for running the 300-MHZ NMR spectra. RECEIVEDfor review February 18, 1992. Accepted June 24, 1992.
