424
Antitumor Agents: Taxol and Taxanes – Production by Yew Cell Culture
The
samples
are
passed
through
a
reversed phase microbore column (e.g.
Hewlett-Packard
ODS
Hypersil,
5
µ
m,
100 mm
×
2.1 mm),
using
a
mobile
phase containing methanol, water, and
acetonitrile; the linear gradient elution
profle starts with 20 : 67 : 13 (solvent A),
ends with 20 : 27 : 53 (solvent B) within
50 min, and includes a 5-min wash in
both solvents to reestablish the initial
condition. The flow rate is 0.2 mL min
1
,
and chromatograms are plotted at 227 nm,
using an HPLC system equipped with a
photodiode array detector (e.g. Hewlett-
Packard 1090A HPLC system). Typical
injection volumes are 10
µ
L, and solvents
are
all
HPLC
grade,
fltered
through
0
.
45
µ
m flters prior to use.
Microbore columns have the advantages
oF high sensitivity, low sample volume,
and low solvent consumption. ±or cell
suspension
extracts
analyzed
with
the
Foregoing solvent system, however, a better
resolution oF early eluting taxanes, such as
baccatin III and 10-deacetyl-baccatin III,
can be achieved using longer reversed
phase analytical columns; in this case,
the flow rate is increased to 1 mL min
1
,
whereas all other chromatographic and
detection parameters are not changed.
Quantifcation is done by the method
oF external standards, that is, a standard
curve is generated with authentic stan-
dard (at least fve diFFerent concentrations
covering the concentration range oF the
extract samples) and run under the same
chromatographic conditions as the sam-
ples. Taxol
and taxane identifcation is
done by retention time, UV spectra and,
cochromatography with standard. Peak
purity checks can be perFormed as addi-
tional evidence For positive identifcation
iF the HPLC equipment has a diode ar-
ray detector.
±urther prooF oF identifcation can be
obtained by collecting the corresponding
HPLC peak and perForming nuclear mag-
netic resonance (NMR) analyses. Over a
series oF injections, a concentrated sam-
ple is run through the HPLC system, the
corresponding Taxol
peak in the sam-
ple extract is collected and pooled, until
approximately 50
µ
go
FTaxo
l
has been
collected. The collected material is dried
under vacuum at 25
C and resuspended
in 150
µ
L oF HPLC grade methanol; then
a small portion oF it is injected again For
purity evaluation and fnal quantifcation.
IF a single peak with the same reten-
tion time and UV spectrum as Taxol
is present in the amount required, the
sample is dried under vacuum as beFore,
and Freeze-dried For 24 to 48 hours to
remove solvent residue. Taxol
identifca-
tion, For example, may be obtained by 1H
NMR analysis in deuterated chloroForm
(
50
µ
g oF HPLC-purifed sample Taxol
in 0.75 mL oF deuterated chloroForm with
0.05% tetramethylsilane as internal reFer-
ence) at 500 MHz and 25
ConaVar
ian
UNITY 500 spectrometer equipped with
an inverse detection 5-mm probe. Taxol
and related taxane standards may be re-
quested For nonhuman research use From
the Drug Synthesis and Chemistry Branch,
Developmental Therapeutics Program, Di-
vision oF Cancer Treatment oF the National
Cancer Institute (U.S.A.). Several taxanes
are also available From chemical sup-
ply companies.
4
Taxol
Biosynthesis
Taxol
is a highly substituted and oxy-
genated cyclic diterpenoid with a taxane
ring
system.
Its
chemical
uniqueness
among taxanes is due to the presence
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