anes were obtained from the National Cancer Institute (NCI),
Drug Synthesis and Chemistry Branch, Developmental Thera-
peutics Program (Bethesda, MD), with the exceptions of 7-epi-
taxol, which was received from Bristol-Myers Squibb (Wallingford,
CT), and the paclitaxel side chain methyl ester, 3′-(benzoylamino)-
2′-hydroxybenzenepropanoate, which was purchased from Hauser
Chemical Research (Boulder, CO). The water used was distilled,
followed by deionization (DI-DI) in a NANOpure II four-cartridge
system (Barnstead/ Thermolyne Corp., Dubuque, IA). The fungal
medium was prepared from a modified version of Schenk and
Hildebrandt’s (SH) and autoclaved prior to use.21
Apparatus. High-performance liquid chromatography (HPLC)
analysis was performed on a Perkin-Elmer (Norwalk, CT) LC250
binary system with modifications to our published HPLC method.20
Quantitation was performed with PE Nelson 1020 software (v4.71)
by the external standard calibration method. The membrane SPE
filtration device was either a 47 mm all-glass 1 L filtration apparatus
(XX15 047 00, Millipore, Bedford, MA) or a six-position vacuum
manifold equipped at each position with a 47 mm glass reservoir
and tabulated glass base (98050300809, 3M Corp., St. Paul, MN).
Each filtration apparatus was fitted with a Kel-F (3M Corp.) 47
mm support disk. A 47 mm Empore C18 membrane (7460-06, J.
T. Baker) was placed on the Kel-F support disk, and a 47 mm
poly(propylene) (PP) separator of 10 µm pore size (61757, Gelman
Sciences, Ann Arbor, MI) was set on top of the Empore
membrane. Kinetics experiments were performed in sealed
vacules or ampules (Wheaton Glass, Millville, NJ).
P rocedure. Analytical HPLC. The mobile phase tubing was
maintained at 30-31 °C, while the MetaChem Taxsil SAFE-
GUARD guard cartridge (2.0 mm i.d. × 13 mm) and MetaChem
Taxsil analytical column (2.0 mm i.d. × 150 mm, 5 µm) were kept
at 30 °C. A 5 µL injection of a six-taxane standard, prepared in
Omnisolv methanol from the authentic compounds, was made at
low and high concentration levels. The low and high level
calibration standards contained 0.015-0.050 and 0.65-2.5 µg/ µL
of each taxane, respectively. The mobile phase consisted of
acetonitrile in reservoir A and a 90:10 (v/ v) solution of water/
methanol in reservoir B. HPLC operating conditions were as
follows: a 0.28 mL/ min flow rate at 36% A was maintained for 20
min, followed by a linear gradient over 2 min to 65% A, held for
12 min, and a linear gradient to 36% A over 5 min. The final
composition was held for 20 min to reequilibrate the system prior
to the next injection. The structures of the taxanes of interest,
baccatin III, 10-deacetyltaxol, cephalomannine, 7-epi-10-deacetyl-
taxol, paclitaxel, and 7-epi-taxol, are provided in Figure 1. These
six taxanes are resolved under the specified conditions (Figure
2).
SPE Isolation of Taxanes and Fungal Mycelia. A modification
of our SPE method for the large-scale isolation of taxanes from
Taxus crude extracts20 was used for collection of the fungal mycelia
and taxane/ metabolite extraction from the SH medium. The 47
mm PP separator/ Empore membrane combination was condi-
tioned prior to sample loading, using ethyl acetate, methanol, and
water, successively, to remove contaminants and to solvate the
C18 chains in the disk. Following conditioning, 25 mL of water
was delivered into the reservoir and passed through at 1 mL/
min to ensure that all of the methanol was displaced from the
membrane. When =10 mL of water remained in the reservoir,
the vacuum was released, and the medium/ fungal sample,
typically =15 mL, was added to the reservoir. The vacuum was
reestablished and the liquid drawn through the membrane until
it was just dry. Water (20 mL) was delivered into the reservoir
to wash off any polar compounds from the fungal mat and from
the Empore membrane. The vacuum was maintained for 5 min
to remove thoroughly residual water from the SPE membrane
and the support disk. The mycelium and PP separator were
removed. Methanol (15 mL) was then delivered into the reservoir
above the Empore membrane, and, with the vacuum in place, the
methanol fraction was collected, transferred to a round-bottom
flask, and concentrated according to one of the two procedures
detailed below. Data from the fungal growth studies will be
published elsewhere.
Concentration Procedure 1. The methanol fraction was con-
centrated to dryness in a water bath at 41-43 °C by rotary
evaporation at reduced vacuum (85 kPag), established with water
aspiration for 20 min. The resulting residue was reconstituted
using sonication in methanol to a final volume of 2 mL for analysis
by HPLC.
Concentration Procedure 2. The methanol fraction was con-
centrated to ∼0.5 mL on a rotary evaporator at high vacuum,
established with a Duo Seal vacuum pump (Welch Manufacturing
Co., Chicago, IL). The sample was kept in a water bath at ambient
temperature (∼21 °C) during evaporation. Solvent reduction
required 6-7 min, after which the sample was reconstituted with
methanol to 2 mL and analyzed by HPLC.
Kinetics of Paclitaxel Degradation in Solution. We investigated
paclitaxel degradation kinetics in DMSO, SH medium, Omnisolv
methanol, chloroform, 1:1 (v/ v) SH medium/ DMSO, and 1:1 (v/
v) SH medium/ methanol. Stock solutions of paclitaxel in DMSO,
methanol, or chloroform were prepared as needed, typically at
1.5 µg/ µL. The ampule or vacule was charged with 10 µL of
appropriate paclitaxel stock solution and 490 µL of the solvent
under study.
Sealed ampules or vacules containing the paclitaxel solution
were placed in a constant temperature bath and removed at
selected times for HPLC analysis. During the course of the
experiments, the DMSO and SH solutions generally changed from
colorless to orange/ dark brown.
RESULTS AND DISCUSSION
Kinetics Studies. It has been reported that paclitaxel
converts primarily to 7-epi-taxol, the thermodynamically more
stable isomer,22 upon heating in the dry state,23 in organic
solvents,22-25 and in cell culture medium.26 It is reasonable that
7-epi-taxol is the thermodynamically more stable isomer due to
hydrogen bonding between the C7R-OH and C4R-acetate acyl
oxygen.25 Epimerization at C7 is reversible and proceeds via a
proposed24 retroaldol/ aldol intramolecular mechanism. The pro-
posed pathway requires transfer of the hydroxyl hydrogen from
C7 to the C9 carbonyl with concomitant aldehyde formation at
C7, ring cleavage between C7 and C8, double-bond formation
between C8 and C9, and hence enol formation at C9. Free rotation
(22) Huang, C. H. O.; Kingston, D. G. I.; Magri, N. F.; Samaranayake, G.;
Boettner, F. E. J. Nat. Prod. 1 9 8 6 , 49, 665-669.
(23) Richheimer, S. L.; Tinnermeier, D. M.; Timmons, D. W. Anal. Chem. 1 9 9 2 ,
64, 2323-2326.
(24) McLaughlin, J. L.; Miller, R. W.; Powell, R. G.; Smith, C. R., Jr. J. Nat. Prod.
1 9 8 1 , 44, 312-319.
(25) Miller, R. W.; Powell, R. G.; Smith, C. R., Jr.; Arnold, E.; Clardy, J. J. Org.
Chem. 1 9 8 1 , 46, 1469-1473.
(21) DiCosmo, F.; Norton, R.; Towers, G. H. N. Naturwiss 1 9 8 2 , 69, 550-551.
(26) Ringel, I.; Horwitz, S. B. J. Pharm. Exper. Ther. 1 9 8 7 , 242, 692-698.
Analytical Chemistry, Vol. 69, No. 1, January 1, 1997 73