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Supercritical fluid (CO2) chromatography for
quantitative determination of selected cancer
therapeutic drugs in the prescence of potential
impurities in injection formulations
Venkata Narasimha Rao G., *a
Ravi B.,a
Jalandhar D.,a
Manoj P.a
and R. Venkata Nadhb
In the present study, two cancer therapeutic drugs (docetaxel and bortezomib) were separated from their
potential impurities on a chromatographic platform by utilizing CO2 gas (supercritical state) and quantified.
The chromatographic separations were achieved on two short columns BEH-2EP (100 mm  3 mm, 1.7 mm)
and CHIRALPAK AD-3 (100 mm  4.6 mm, 3 mm) for docetaxel and bortezomib, respectively. The present
work describes the role of organic modifiers in the separation of polar compounds by supercritical fluid
chromatography. The two new methods were fully validated in accordance with the current ICH
(International Council for Harmonization of technical requirements for pharmaceuticals for human use)
guidelines. The stability indicating power of the methods was demonstrated from the stress studies
conducted on the injection formulations of the two compounds. The methods are precise with % RSD of
0.4, linear with the correlation coefficient of r2
$ 0.999 and accurate in the range of 50–150% of the
target assay concentration. The two methods can be equally employed for the assay determination of
docetaxel and bortezomib APIs as well.
1 Introduction
Supercritical uid carbon dioxide has emerged as a versatile
solvent for scientists to use in various chemical operations
owing to its sustainable (green) properties, relative chemical
inertness, greater applications in manufacturing, and abun-
dance,.1–5
This resulted in supercritical uid chromatography
(SFC) as an alternative and complementary method to the HPLC
technique. The potential of SFC using packed columns for
analysis in the pharmaceutical industry has been recognized for
many years.7
SFC can offer highly efficient separations in
shorter analysis times and at a low-pressure drop without
compromising the resolution, plate counts and tailing factors.
However, the lack of reliable and sensitive commercial SFC
systems has prevented the extensive use of SFC in the industry.
With newly available instruments, several SFC methods have
been reported for a variety of compounds.6–9
The present study is an application of SFC for the assay
determination of docetaxel and bortezomib in injection
formulations.
Docetaxel is an antineoplastic agent belonging to the taxane
compound family and used in the treatment of several cancers
(ovarian, prostate, esophageal, etc.) by either monotherapy or in
combination with several other drugs like gemcitabine, irinote-
can, cisplatin and carboplatin.10–14
The chemical name of doce-
taxel is (2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester,
13-ester with 5b-20epoxy-1,2a,4,7b,10b,13a-hexahydroxytax-11-
en-9-one 4-acetate 2-benzoate, trihydrate. Its formulation is
available as Taxotere Injection Concentrate15,16
in the market.
Bortezomib is available in the market as VALCADE® from
Millennium Pharmaceuticals, Inc., and Janssen-Cilag and is
used for the treatment of patients with multiple myeloma.17
Multiple myeloma (MM) is an incurable disease with a poor
survival, which has not been affected even by high-dose
chemotherapy. Bortezomib appears to improve survival and
response rates of patients with previously untreated MM in
spite of higher risk of peripheral neuropathy.18
It is one of the
novel agents that has improved rate of remission during ASCT
(autologous stem cell transplantation) without increasing
toxicity.19,20
Bortezomib is a modied dipeptidyl boronic acid. The
chemical name of bortezomib is [(1R)-3-methyl-1-[[(2S)-1-oxo-3-
phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl] boronic
acid.21
The molecular weight is 384.24, and the molecular
formula is C19H25BN4O4. The chemical structures of docetaxel
and bortezomib are shown in Fig. 1.
Currently docetaxel is official in the United States Pharma-
copoeia (USP), European Pharmacopoeia (EP), and Indian
a
Formulations Research and Development Centre, Mylan Laboratories Ltd, Jinnaram,
Medak, 502325, India. E-mail: venkata.nr@gmail.com
b
GITAM University, Bengaluru, Karnataka-562163, India
Cite this: DOI: 10.1039/c7ay00779e
Received 23rd March 2017
Accepted 7th April 2017
DOI: 10.1039/c7ay00779e
rsc.li/methods
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Pharmacopoeia (IP), while bortezomib is not official in any
pharmacopoeia.21–23
Each pharmacopeia has published mono-
graphs of docetaxel and docetaxel injection. The USP mono-
graph prescribes a reversed phase, and a 60 minute gradient
method for the assay and RS determination using a 150 mm Â
4.6 mm, 3.5 mm, L1 column. The mobile phase contains water,
acetonitrile and acetic acid mixed in the ratio of 1 : 1 : 0.1,
respectively. The detection was carried out at 232 nm, with
a sample load of 20 mL. Ph. EP and IP prescribe a common
method (72 minutes gradient) for assay and RS, similar to USP.
A few HPLC and UPLC coupled with mass spectrometry (HPLC-
MS/MS, UPLC-MS/MS) methods for plasma docetaxel are avail-
able in the literature.24–30
Few analytical methods are available on bortezomib esti-
mation in bulk and formulations.
Chandra Sekhar K et al. reported one ultraviolet method for
the determination of bortezomib in bulk and dosage form.31
A
few RP-HPLC and HPLC coupled with mass spectrophotometer
methods were reported for the estimation of bortezomib and
impurities in bulk and injection formulations.32–40
All these
methods use octadecylsilyl columns, and the mobile phase
consists of acetonitrile in major proportions, 60%,35
65%,36
80%,37
50% (ref. 38) combined with buffers.
All the above reported methods on docetaxel and bortezomib
are reverse phase methods and use a variety of organic solvents in
their mobile phases. Until today, there are no reported literature
studies available on the utilization of CO2 as a mobile phase for
carrying out chromatographic separations of cancer drugs from
their potential impurities. Hence, an attempt is made here and
novel methods were developed, which are ecofriendly.
The biggest challenge in developing these methods was the
selection of organic modiers and their quantity in the sepa-
ration of polar impurities from the main active compound.
2 Materials and methods
2.1 Chemicals and reagents
Isopropyl alcohol was obtained from Rankem (India). HPLC
grade methanol and diethylamine (DEA) were procured from
Merck, India. Triouro acetic acid (TFA) was from Acros
organics. Ethanol used in the study was obtained from
Commercial alcohols. Carbon dioxide gas was purchased from
Sai Padmaja Oxygen at Hyderabad, India. The samples of
docetaxel injection and bortezomib for injection used in the
study were obtained in-house from Mylan (Hyderabad, India).
Docetaxel CRS and docetaxel identication CRS were procured
from US Pharmacopoeia. Bortezomib standard and its impuri-
ties were obtained from an API supplier.
2.2 Instruments and chromatographic conditions
An integrated acquity UPC2
system was from Waters Corpora-
tion, Milford, USA and equipped with a Waters photodiode
array detector (PDA). Data collection and analysis were per-
formed using Empower soware 2pro (Waters Corporation).
Balances used for weighing the reference standards and
samples were from Metler and Sartorius. The two columns,
namely BEH-2EP and CHIRALPAK AD-3 were procured from
Waters India and Diacel respectively.
2.2.1 Chromatographic conditions for docetaxel injection.
A BEH-2EP (100 mm  3.0 mm, I.D. and 1.7 mm) column was
used for separating docetaxel and its potential impurities. The
column oven temperature was maintained at 45 
C. The mobile
phase consisting of liquid CO2 and a co-solvent (a mixture of
ethanol and methanol (1 : 1) containing 0.1% diethylamine)
was pumped in an isocratic mode (85 : 15) at a constant ow
rate of 1.5 mL minÀ1
throughout the run. 1 mL of the sample was
injected and detected at a wavelength of 228 nm. During the
analysis, the active backpressure of the system was maintained
at 1900 psi. The sample cooler temperature was maintained at
10 
C.
2.2.2 Chromatographic conditions for bortezomib dry
powder for injection. For bortezomib the mobile phase consists
of liquid CO2 (% A) and a mixture of 0.5% TFA in an ethanol and
IPA mixture (1 : 1) (% B). The mobile phase was pumped into
the chromatograph at a ow rate of 1.3 mL minÀ1
using a linear
gradient program. The gradient programme at time T
(minutes), ramp of % B (organic modier) is T/% B, 0/10, 1/10,
3/20, 8/20, 8.5/10, 10/10.
Fig. 1 Chemical structures of (a) docetaxel and (b) bortezomib.
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A CHIRALPAK AD-3 column (100 mm  4.6 mm I.D., 3
mm particle size) from Diacel was used for the separation of
bortezomib and its impurities. The column oven tempera-
ture was kept at 45 
C throughout the experiments. A
sample volume of 1 mL was injected into the chromato-
graph and detected at 270 nm. The active backpressure of
the system was maintained at 1900 psi throughout the
analysis. The sample cooler temperature was maintained at
10 
C.
2.3 Standard and sample preparations
2.3.1 Diluent preparation. HPLC grade methanol was used
as the diluent, for the preparation of standards, system suit-
ability solutions, and samples for both the methods. This
selection was based on the solubility studies conducted on the
two active drug substances.
2.3.2 Docetaxel standard, USP identication solution
(system suitability) and sample preparation. An amount of
docetaxel USP CRS was weighed accurately in a suitable volu-
metric ask. 10 mL of the diluent was added, sonicated to
Table 1 Results of forced degradation studiesa
S. no. Stress condition
Assay of degraded
sample
%
degradation
Purity
angle
Purity
threshold
Assay of
degraded sample
%
degradation
Purity
angle
Purity
threshold
Docetaxel Bortezomib
1 Acid hydrolysis 78.6 20.5 0.121 0.313 92.2 7.1 1.259 1.870
2 Base hydrolysis 66.9 32.2 0.135 0.456 91.4 7.9 1.647 2.625
3 Thermal degradation 96.6 2.5 0.090 0.323 99.9 0.6 0.985 1.529
4 Oxidation 90.5 8.6 0.106 0.290 81.8 17.5 0.721 1.453
5 Photolytic degradation (UV) 96.7 2.4 0.231 0.421 96.7 2.6 0.978 1.752
6 Photolytic degradation (light) 95.3 3.8 0.100 0.370 93.7 5.6 1.054 1.494
a
Control sample results for calculating the % degradation, docetaxel assay ¼ 99.1 and bortezomib assay ¼ 99.3.
Fig. 2 Ultraviolet-Visible spectra of (a) docetaxel and (b) bortezomib.
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dissolve the contents and diluted suitably to obtain a concen-
tration of 1000 mg mLÀ1
. USP docetaxel identication RS
contains docetaxel and small amounts of listed impurities
2-debenzoxyl 2-pentenoyl docetaxel, 6-oxodocetaxel, 4-epi-
docetaxel, and 4-epi-6-oxodocetaxel. It was used as a system
suitability and spiked sample solution. The sample solution was
prepared similar to the USP CRS solution by diluting an
appropriate volume of the sample with the diluent to get the
concentration of 1000 mg mLÀ1
.
2.3.3 Bortezomib standard and sample preparation. The
standard solution was prepared by dissolving an amount of
bortezomib in the diluent and diluting suitably to obtain
a concentration of 180 mg mLÀ1
. The sample solution was
prepared similar to that of the standard to get the concentration
of 175 mg mLÀ1
. Bortezomib has seven impurities namely, (S,S)-
diastereomers, (R,R)-diastereomers, R,S enantiomer, impurity
A, impurity B, impurity C and impurity D. The impurities of
bortezomib were prepared by dissolving a suitable quantity in
the diluent and spiked into the sample preparation.
2.4 Forced degradation study
2.4.1 For docetaxel injection. Forced degradation studies
were performed to assess the stability indicating power of the
method. Docetaxel injection samples were deliberately sub-
jected to various stress conditions such as acidic (1 mL of 1 N
HCl for 12 h at room temperature), basic (1 mL of 1 N NaOH for
12 h room temperature), oxidative (1 mL of 1% H2O2 for 30
minutes), humidity (90% RH for 12 hours) UV light (200 watt
hours per square meter), and light (1.2 m lux hours). The
stressed samples were further neutralized and diluted suitably
to get nal concentrations of 1000 mg mLÀ1
. All samples were
then chromatographed and the peak purity plots were obtained
for the docetaxel peak. Peak purity testing determines the peak
homogeneity by comparing each spectrum within the peak
against the peak apex. The chromatograms of the stressed
samples were evaluated for peak purity of docetaxel using
Waters Empower Networking Soware. The values of the purity
angle and purity threshold were used to estimate the spectrum
homogeneity and the peak purity. Table 2 shows the
Fig. 3 Chromatograms obtained from primary screenings (Trial-1).
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degradation data with purity angle and purity threshold. The
criterion for peak homogeneity is that the purity angle should
be less than the peak threshold as per the waters empower
soware. The purity angle and purity threshold from Table 2
show that the docetaxel peak is homogenous in the presence of
degradation products as obtained from the stress samples.
2.4.2 For bortezomib injection. Bortezomib for injection
samples were subjected to the following stress conditions.
Acid hydrolysis (1 mL of 0.5 N HCl for 6 hours), base hydrolysis
(1 mL of 1 N NaOH for 4 hours), heat exposure (at 60 
C for 24
hours), oxidative (3 mL of 10% H2O2), photolytic exposure (UV-
200 watt hours per square meter, light-1.2 million lux hours)
studies were carried out as per the current ICH guidelines
(international council for harmonisation of technical
requirements for pharmaceuticals for human use). The
exposed samples were neutralized and diluted to get
a concentration of 175 mg mLÀ1
. The bortezomib peak was
evaluated for homogeneity by measuring the purity angle and
purity threshold. The results are tabulated in Table 1. The
purity angle of bortezomib obtained from various stress
studies was found less than the purity threshold. This indi-
cates that the bortezomib peak is homogenous in the presence
of its degradants.
Fig. 4 Chromatograms obtained from Trial 2.
Fig. 5 Development chromatograms obtained from Trial 3.
Fig. 6 Purity plot of the docetaxel peak obtained from development Trial 3.
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3 Results and discussion
3.1 Method development and optimization
A systematic approach was adopted for developing methods
using the SFC principle. The two compounds were studied for
ultra-violet absorption to determine the detection lambda
maxima. For this, solutions of docetaxel and bortezomib were
prepared having appropriate concentrations within the UV-
Visible range and absorbance study was conducted. The two UV
spectra are shown in Fig. 2. Docetaxel exhibited two maxima,
228 nm, and 272 nm. Bortezomib exhibited three maxima, 209
nm, 266 nm, and 318 nm.
Fig. 7 Typical chromatogram of optimized chromatographic conditions.
Fig. 8 Chromatograms obtained from primary screenings on (a) BEH-2EP and (b) CHIRALPAK AD-3 (Trial-1).
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3.1.1 Determination of docetaxel in docetaxel injection
3.1.1.1 SFC primary screening and optimization of docetaxel
SFC primary screening. Trial-1. In primary screening three
different stationary phases, namely BEH (ethylene bridged
hybrid) silica, BEH-2EP (ethylene pyridine), and CSH (charged
surface hybrid)-ourophenyl with sub-2 mm particles which are
specically designed for SFC, were employed to have predict-
able separations of sample components. This selection of
columns was based on their chemistries, polarities, pressure
tolerability (up to 18 000 psi or 241 bar), operable temperature
(up to 60 
C) and compatibility with acidic and basic additives
like TFA, DEA, etc.
In view of the study objective, primary screenings were done
with 100% CO2 mobile phase with a ow rate of 10 mL minÀ1
.
The system pressure was maintained at 1900 units through an
active back pressure regulator. The initial column temperature
was kept at 40 
C. With these set of chromatographic condi-
tions, the rst trial was made by injecting USP identication
Fig. 9 Development chromatogram obtained from Trial 2.
Fig. 10 Development chromatogram obtained from Trial 3.
Fig. 11 Development chromatogram obtained from Trial 4.
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solution (1 mL) into the chromatographic system. The chro-
matograms are shown in Fig. 3.
The inference from above chromatograms was that no
detectable peaks were observed in BEH silica and CSH our-
ophenyl columns. BEH-2EP showed some promising retention
of peaks with very low response. Hence, further development
strategy was continued with the BEH-2EP column. Supercritical
CO2 is a nonpolar solvent with poor solvating power. This is one
of the reasons for poor retention of polar compounds. Berger
and Deye et al., suggest that the addition of modiers to the
mobile phase can increase the extraction efficiency of CO2.41–43
Trial-2. To increase the mobile phase strength, a few experi-
ments were conducted with a small quantity of ethanol (0–5%)
Fig. 12 Typical chromatogram of optimized chromatographic conditions.
Fig. 13 System suitability chromatogram obtained from docetaxel identification solution.
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along with a gradient programme. The increase in the polarity
of the mobile phase has increased the sample partition and 5%
ethanol has resulted in the elution of docetaxel at a RT of about
5.6 minutes. All the other components eluted closely before the
docetaxel peak. The chromatogram is shown in Fig. 4.
Trial 3. The separation and elution of majority of compo-
nents occurred at the isocratic portion (95 : 5 CO2 : ethanol) of
the gradient programme and hence the isocratic composition
was nalized to even avoid the gradient hump. With the new
chromatographic conditions, the identication solution was
injected and the chromatogram was recorded. This trial resul-
ted in a better baseline and improved separations among the
components. The specimen chromatogram is shown in Fig. 5.
The corresponding purity plot of the DCTX peak is shown in
Fig. 6. The purity angle (PA ¼ 0.529) and threshold (TH ¼ 0.385)
indicate that the DCTX peak is not homogenous (criteria: PA 
TH). Hence further separation is required.
Trial 4. From the literature, the addition of small amounts,
typically 0.1–1% of additives, to the mobile phase has been
shown to have a benecial effect on the peak shape and sensi-
tivity in SFC. Based on this 0.1% of DEA was added to the
organic composition. This has further improved the peak shape
and resolution of closely eluting peaks. The nal optimized
chromatogram is shown in Fig. 7.
3.1.1.2 SFC primary screening and optimization for
bortezomib
SFC primary screenings. The structural evaluation shows that
bortezomib possesses chiral centers and may give rise to chiral
related substances. Having this information, the entire method
development was strategized combining both chiral and achiral
screening and optimization.
Fig. 14 System suitability chromatogram obtained for bortezomib.
Fig. 15 Acid stressed sample chromatogram.
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Trial-1. In primary screening, four columns having different
stationary phases namely HSS T3, BEH-2EP (ethylene pyridine),
and CSH (charged surface hybrid)-ourophenyl with sub-2 mm
particles which are specically designed for SFC, and amylose
tris (3,5-dimethylphenylcarbamate) were employed to have
predictable separations of sample components. This selection
of columns was based on their chemistries, polarities, pressure
tolerability (up to 18 000 psi or 241 bar), operable temperature
(up to 60 
C) and compatibility with acidic and basic additives
like TFA, DEA, etc. The mobile phase was kept 100% CO2 in view
of study objective at a ow rate of 1.0 mL minÀ1
. The system
pressure was maintained at 1900 psi through the active
backpressure regulator. The initial column to temperature was
kept at 45 
C to maintain the supercritical state of CO2. With
these set of chromatographic conditions the bortezomib
impurity mixture (1 mL) was injected into the chromatographic
system in each of the stationary phase. No peaks were detected
on the CSH, and HSS T3 column. BEH-2EP and CHIRAL-AD 3
columns showed few promising peaks. Fig. 8 shows the chro-
matogram obtained on the two columns, respectively.
Trial-2. Further development strategy was continued with
CHIRALPAK-AD 3 (100 Â 4.6, 3 mm). According to Berger and
Deye et al., addition of organic modier to the mobile phase
increases the extraction efficiency of CO2. Based on this, ethanol
Fig. 16 Base stressed sample chromatogram.
Fig. 17 Peroxide stressed sample chromatogram.
Fig. 18 Thermal stressed sample chromatogram.
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was introduced into the mobile phase, and several experiments
were conducted with varied percentages using a gradient pro-
gramme. The increase in polarity of the mobile phase has
increased the sample partition. The column temperature and
ow rate were reduced to 45 
C and 1.0 mL minÀ1
respectively to
check their impact on the separation. With these changes,
bortezomib standard solution and individual impurities were
injected into the chromatograph. Due to these changes, borte-
zomib was slightly retained and separation among the four
peaks was improved a little. The other three peaks were iden-
tied as isomers of bortezomib. The chromatogram obtained
from Trial 2 is shown in Fig. 9.
Trial 3 and 4. In Trial 3 IPA was introduced along with
ethanol in a 1 : 1 ratio. The gradient programme was slightly
modied to improve separation among the closely eluting
isomers and elute late-eluting impurities.
This trial resulted in better separation among the isomer
peaks, and the other peaks were not eluted within 10 minutes.
The obtained chromatogram with Trial 3 is shown in Fig. 10.
In Trial 4 the ow rate was increased from 1.0 to 1.2 mL
minÀ1
to check for the other two impurities (impurity C and
impurity D). Both impurities were eluted within 10 minutes
with good separation. All peaks were eluted and are well
Fig. 19 Sample chromatogram UV degradation.
Fig. 20 Sample chromatogram light degradation.
Fig. 21 Acid stressed sample chromatogram.
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resolved from the bortezomib peak. The development chro-
matogram obtained from Trial 4 is shown in Fig. 11.
3.2 Method optimization
Furthermore, the method included optimization studies which
were conducted to ensure method repeatability and reproduc-
ibility. The optimization studies were conducted by changing
the column temperature (40 
C, and 50 
C) and ow rate (1.0,
1.1, 1.2, 1.5 mL minÀ1
),and by adding TFA (0.1%, 0.2%, 0.5%) to
organic modiers for sharp peaks. The optimized chromato-
gram obtained is shown in Fig. 12.
3.3 Method validation
The optimized methods were fully validated as per the
requirements of current ICH guidelines for validation of
analytical procedures, i.e., Q2 (R1).44
The detailed validation
experiments and results are discussed below.
Fig. 22 Base stressed sample chromatogram.
Fig. 23 Peroxide stressed sample chromatogram.
Fig. 24 Thermal stressed sample chromatogram.
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3.3.1 System suitability. System suitability parameters were
measured to verify the system performance for the intended
analysis. For this purpose the USP identication reference
standard (CRS) was prepared and injected into the chromato-
graph (for docetaxel). The USP system suitability parameters
i.e., USP plate count, USP tailing, and USP resolution were
evaluated. The chromatogram obtained from the system suit-
ability solution, and the peak purity data are shown in Fig. 3.
The retention time of docetaxel was 2.679 minutes. Docetaxel
was separated from its closest impurity (RT ¼ 2.513) with
a resolution of 1.7 (minimum criteria for resolution as per USP
is 1.5). The USP plate count was 11 772 and the USP tailing
factor was 1.1 for the docetaxel peak.
For bortezomib the system suitability was determined from
six replicate injections of bortezomib standard preparation and
spiked sample preparation. The resolution between bortezomib
Fig. 25 Sample chromatogram UV degradation.
Fig. 26 Sample chromatogram light degradation.
Table 2 Precision data for docetaxel and bortezomib
S. no. Docetaxel Bortezomib
Sample number Method precision
Intermediate
precision Method precision
Intermediate
precision
1 99.1 99.7 99.4 98.7
2 99.7 100.7 98.6 98.9
3 98.9 99.3 99.3 98.9
4 99.0 100.2 99.9 99.3
5 99.5 100.8 99.0 99.0
6 98.5 98.3 99.4 98.4
Mean 99.1 99.8 99.3 98.9
SD 0.43 0.95 0.44 0.30
% RSD 0.4 0.9 0.4 0.3
Overall % RSD
(% RSD of 12 measurements)
0.80 0.42
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and its closely eluting peak was achieved to be 2.4. USP tailing
and plate counts for the bortezomib peak were 1.2 and 5662
respectively. The % RSD for six replicate standard injections was
1.3.
Typical system suitability solutions for docetaxel and borte-
zomib are shown in Fig. 13 and 14 respectively.
3.3.2 Specicity. The specicity of the method was deter-
mined to evaluate the interference due to diluent, placebo, and
specied impurities, by injecting the diluent (blank), placebo,
spiked sample and stress samples. There was no interference
found at the retention time of docetaxel and bortezomib due to
diluent, placebo, specied impurities and other degradants
from the stress samples at the retention time of docetaxel and
bortezomib. Furthermore, the homogeneity of the peaks was
conrmed from the peak purity data. The content of docetaxel
and bortezomib in the presence of their potential impurities
was found to be 99.0 and 100.3, respectively. The forced
degradation chromatograms are shown in Fig. 15–26.
3.3.3 Precision (repeatability). Precision of a method is
a measure of repeatability. It is performed in two ways, method
precision and intermediate precision. Method precision was
performed by preparing six replicate samples and injecting one
aer the other, under the conditions as similar as possible, and
two injections were taken from each sample. Intermediate
precision was performed similar to the method precision on
a different day, using different chemicals and columns. The
response of docetaxel and bortezomib was measured and the
relative standard deviation was calculated. Table 2 shows the
precision data for docetaxel and bortezomib. The overall % RSD
Table 3 Linearity data for docetaxel and bortezomib
Statistics Docetaxel Bortezomib
Linearity Correlation 0.9999 0.9999
Slope 666.24 1133.4
Intercept À4478.0264 À962.03
Table 4 Accuracy data for docetaxel and bortezomib
Accuracy Docetaxel Bortezomib
Levels Level-1 (50%) Level-2 (100%) Level-3 (150%) Level-1 (50%) Level-2 (100%) Level-3 (150%)
Sample-1 99.1 99.8 99.6 98.3 100.3 99.6
Sample-2 98.7 99.8 99.4 98.9 99.7 99.8
Sample-3 98.7 99.6 98.9 98.3 100.0 99.6
Mean recovery 98.8 99.7 99.3 98.5 100.0 99.7
% RSD 0.2 0.1 0.4 0.4 0.3 0.1
Table 5 Robustness and method sensitivity data for method 1 (docetaxel injection)
S. no. Condition
RT of docetaxel
(minutes)
Assay of docetaxel
in spiked sample
USP
resolution
USP
tailing
% RSD
of standard
Method sensitivity
yes/no
1 Control (no change) 2.679 99.1 1.7 1.1 0.5 NAa
2 Flow (+)1.65 mL minÀ1
2.415 98.9 1.5 1.1 0.3 Yes
3 Flow (À)1.35 mL minÀ1
3.250 99.0 1.8 1.1 0.7 Yes
4 Temperature (+)50 
C 2.558 99.0 1.7 1.1 0.4 No
5 Active back pressure regulator (+)2000 psi 2.600 99.0 1.7 1.1 0.4 No
6 Active back pressure regulator (À)1900
psi
2.55 98.9 1.6 1.1 0.4 Yes
a
Not applicable.
Table 6 Robustness and method sensitivity data for method 2 (bortezomib for injection)
S. no. Condition
RT of bortezomib
(minutes)
Assay of bortezomib
in spiked sample
USP
resolution
USP
tailing
% RSD
of standard
Method sensitivity
yes/no
1 Control (no change) 3.847 99.3 2.4 1.2 0.8 NAa
2 Flow (+)1.43 mL minÀ1
3.058 99.1 1.8 1.2 1.0 Yes
3 Flow (À)1.17 mL minÀ1
4.215 99.1 2.5 1.2 1.0 No
4 Column oven temp (+)50 
C 3.812 99.1 2.5 1.2 1.0 Yes
5 Active back pressure regulator (+)2000 3.999 99.0 2.5 1.2 0.8 No
6 Active back pressure regulator (+)1900 psi 3.999 99.0 2.5 1.2 0.8 No
a
Not applicable.
Anal. Methods This journal is © The Royal Society of Chemistry 2017
Analytical Methods Paper
Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41.
View Article Online
of 0.80 for docetaxel and 0.42 for bortezomib shows that the
methods are precise.
3.3.4 Linearity. The linearity of the method was estab-
lished in the concentration range of 246.5–1479.18 mg mLÀ1
(i.e., 25% to 150% of the target concentration) for docetaxel
and 29.0–290.1 mg mLÀ1
(15% to 160% of the target concen-
tration) for bortezomib. The linear regression data from the
calibration plot were indicative of an excellent linear rela-
tionship between the peak area and concentration over the
range specied above and the data are provided in Table 3. A
correlation of 0.9999 was obtained for docetaxel and bortezo-
mib, establishing a linear relationship between the concen-
tration and detector response.
3.3.5 Accuracy. The accuracy of an analytical procedure
expresses the degree of the closeness of the obtained results
with the true values. The accuracy of the method was tested by
the recovery procedure. A known amount of the active drug
substance was spiked to the placebo and the recovery was
estimated. The accuracy of the method was evaluated from 50%
to 150% of target sample concentrations for docetaxel (500 mg
mLÀ1
, 1000 mg mLÀ1
, and 1500 mg mLÀ1
), and bortezomib (87 mg
mLÀ1
, 175 mg mLÀ1
, and 350 mg mLÀ1
). The average assays were
in the range of 98.7 to 99.7 with % RSD 0.1–0.4 for docetaxel and
98.5 to 100.0 with 0.1–0.4% RSD for bortezomib. Table 4
summarizes the accuracy results.
3.3.6 Robustness. The robustness of an analytical method
is its capacity to remain unaffected by small but deliberate
changes in the method parameters. The robustness of the
method is determined by making deliberate variations in the
ow rate, column temperature and ABPR (active backpressure
regulator). The various altered conditions and the measured
system suitability are shown in Tables 5 and 6.
The above robustness data state that an increase in the ow
rate resulted in signicant variations in the USP resolution and
could inuence the system suitability results. The column
temperature and ABPR have no signicant impact on the
results.
4 Conclusions
The newly developed CO2 based assay methods for docetaxel
and bortezomib are specic, precise, accurate, linear, and
robust. The results obtained from the validation studies were
satisfactory and they exhibit excellent performance in terms of
sensitivity, speed and cost-effective. This technology being
a green technology is environmentally friendly in terms of the
waste generated from the system. The method can successfully
be employed for high accuracy routine assay testing and release
production batches.
Acknowledgements
The authors wish to thank the management of Mylan Labora-
tories Ltd for supporting this work. The authors also like to
thank colleagues in the Analytical development services for
their co-operation in carrying out this work.
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Supercritical fluid (CO2) chromatography for quantitative determination of selected cancer therapeutic drugs in the prescence of potential impurities in injection formulations

  • 1. Supercritical fluid (CO2) chromatography for quantitative determination of selected cancer therapeutic drugs in the prescence of potential impurities in injection formulations Venkata Narasimha Rao G., *a Ravi B.,a Jalandhar D.,a Manoj P.a and R. Venkata Nadhb In the present study, two cancer therapeutic drugs (docetaxel and bortezomib) were separated from their potential impurities on a chromatographic platform by utilizing CO2 gas (supercritical state) and quantified. The chromatographic separations were achieved on two short columns BEH-2EP (100 mm  3 mm, 1.7 mm) and CHIRALPAK AD-3 (100 mm  4.6 mm, 3 mm) for docetaxel and bortezomib, respectively. The present work describes the role of organic modifiers in the separation of polar compounds by supercritical fluid chromatography. The two new methods were fully validated in accordance with the current ICH (International Council for Harmonization of technical requirements for pharmaceuticals for human use) guidelines. The stability indicating power of the methods was demonstrated from the stress studies conducted on the injection formulations of the two compounds. The methods are precise with % RSD of 0.4, linear with the correlation coefficient of r2 $ 0.999 and accurate in the range of 50–150% of the target assay concentration. The two methods can be equally employed for the assay determination of docetaxel and bortezomib APIs as well. 1 Introduction Supercritical uid carbon dioxide has emerged as a versatile solvent for scientists to use in various chemical operations owing to its sustainable (green) properties, relative chemical inertness, greater applications in manufacturing, and abun- dance,.1–5 This resulted in supercritical uid chromatography (SFC) as an alternative and complementary method to the HPLC technique. The potential of SFC using packed columns for analysis in the pharmaceutical industry has been recognized for many years.7 SFC can offer highly efficient separations in shorter analysis times and at a low-pressure drop without compromising the resolution, plate counts and tailing factors. However, the lack of reliable and sensitive commercial SFC systems has prevented the extensive use of SFC in the industry. With newly available instruments, several SFC methods have been reported for a variety of compounds.6–9 The present study is an application of SFC for the assay determination of docetaxel and bortezomib in injection formulations. Docetaxel is an antineoplastic agent belonging to the taxane compound family and used in the treatment of several cancers (ovarian, prostate, esophageal, etc.) by either monotherapy or in combination with several other drugs like gemcitabine, irinote- can, cisplatin and carboplatin.10–14 The chemical name of doce- taxel is (2R,3S)-N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester with 5b-20epoxy-1,2a,4,7b,10b,13a-hexahydroxytax-11- en-9-one 4-acetate 2-benzoate, trihydrate. Its formulation is available as Taxotere Injection Concentrate15,16 in the market. Bortezomib is available in the market as VALCADE® from Millennium Pharmaceuticals, Inc., and Janssen-Cilag and is used for the treatment of patients with multiple myeloma.17 Multiple myeloma (MM) is an incurable disease with a poor survival, which has not been affected even by high-dose chemotherapy. Bortezomib appears to improve survival and response rates of patients with previously untreated MM in spite of higher risk of peripheral neuropathy.18 It is one of the novel agents that has improved rate of remission during ASCT (autologous stem cell transplantation) without increasing toxicity.19,20 Bortezomib is a modied dipeptidyl boronic acid. The chemical name of bortezomib is [(1R)-3-methyl-1-[[(2S)-1-oxo-3- phenyl-2-[(pyrazinylcarbonyl)amino]propyl]amino]butyl] boronic acid.21 The molecular weight is 384.24, and the molecular formula is C19H25BN4O4. The chemical structures of docetaxel and bortezomib are shown in Fig. 1. Currently docetaxel is official in the United States Pharma- copoeia (USP), European Pharmacopoeia (EP), and Indian a Formulations Research and Development Centre, Mylan Laboratories Ltd, Jinnaram, Medak, 502325, India. E-mail: venkata.nr@gmail.com b GITAM University, Bengaluru, Karnataka-562163, India Cite this: DOI: 10.1039/c7ay00779e Received 23rd March 2017 Accepted 7th April 2017 DOI: 10.1039/c7ay00779e rsc.li/methods This journal is © The Royal Society of Chemistry 2017 Anal. Methods Analytical Methods PAPER Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online View Journal
  • 2. Pharmacopoeia (IP), while bortezomib is not official in any pharmacopoeia.21–23 Each pharmacopeia has published mono- graphs of docetaxel and docetaxel injection. The USP mono- graph prescribes a reversed phase, and a 60 minute gradient method for the assay and RS determination using a 150 mm  4.6 mm, 3.5 mm, L1 column. The mobile phase contains water, acetonitrile and acetic acid mixed in the ratio of 1 : 1 : 0.1, respectively. The detection was carried out at 232 nm, with a sample load of 20 mL. Ph. EP and IP prescribe a common method (72 minutes gradient) for assay and RS, similar to USP. A few HPLC and UPLC coupled with mass spectrometry (HPLC- MS/MS, UPLC-MS/MS) methods for plasma docetaxel are avail- able in the literature.24–30 Few analytical methods are available on bortezomib esti- mation in bulk and formulations. Chandra Sekhar K et al. reported one ultraviolet method for the determination of bortezomib in bulk and dosage form.31 A few RP-HPLC and HPLC coupled with mass spectrophotometer methods were reported for the estimation of bortezomib and impurities in bulk and injection formulations.32–40 All these methods use octadecylsilyl columns, and the mobile phase consists of acetonitrile in major proportions, 60%,35 65%,36 80%,37 50% (ref. 38) combined with buffers. All the above reported methods on docetaxel and bortezomib are reverse phase methods and use a variety of organic solvents in their mobile phases. Until today, there are no reported literature studies available on the utilization of CO2 as a mobile phase for carrying out chromatographic separations of cancer drugs from their potential impurities. Hence, an attempt is made here and novel methods were developed, which are ecofriendly. The biggest challenge in developing these methods was the selection of organic modiers and their quantity in the sepa- ration of polar impurities from the main active compound. 2 Materials and methods 2.1 Chemicals and reagents Isopropyl alcohol was obtained from Rankem (India). HPLC grade methanol and diethylamine (DEA) were procured from Merck, India. Triouro acetic acid (TFA) was from Acros organics. Ethanol used in the study was obtained from Commercial alcohols. Carbon dioxide gas was purchased from Sai Padmaja Oxygen at Hyderabad, India. The samples of docetaxel injection and bortezomib for injection used in the study were obtained in-house from Mylan (Hyderabad, India). Docetaxel CRS and docetaxel identication CRS were procured from US Pharmacopoeia. Bortezomib standard and its impuri- ties were obtained from an API supplier. 2.2 Instruments and chromatographic conditions An integrated acquity UPC2 system was from Waters Corpora- tion, Milford, USA and equipped with a Waters photodiode array detector (PDA). Data collection and analysis were per- formed using Empower soware 2pro (Waters Corporation). Balances used for weighing the reference standards and samples were from Metler and Sartorius. The two columns, namely BEH-2EP and CHIRALPAK AD-3 were procured from Waters India and Diacel respectively. 2.2.1 Chromatographic conditions for docetaxel injection. A BEH-2EP (100 mm  3.0 mm, I.D. and 1.7 mm) column was used for separating docetaxel and its potential impurities. The column oven temperature was maintained at 45 C. The mobile phase consisting of liquid CO2 and a co-solvent (a mixture of ethanol and methanol (1 : 1) containing 0.1% diethylamine) was pumped in an isocratic mode (85 : 15) at a constant ow rate of 1.5 mL minÀ1 throughout the run. 1 mL of the sample was injected and detected at a wavelength of 228 nm. During the analysis, the active backpressure of the system was maintained at 1900 psi. The sample cooler temperature was maintained at 10 C. 2.2.2 Chromatographic conditions for bortezomib dry powder for injection. For bortezomib the mobile phase consists of liquid CO2 (% A) and a mixture of 0.5% TFA in an ethanol and IPA mixture (1 : 1) (% B). The mobile phase was pumped into the chromatograph at a ow rate of 1.3 mL minÀ1 using a linear gradient program. The gradient programme at time T (minutes), ramp of % B (organic modier) is T/% B, 0/10, 1/10, 3/20, 8/20, 8.5/10, 10/10. Fig. 1 Chemical structures of (a) docetaxel and (b) bortezomib. Anal. Methods This journal is © The Royal Society of Chemistry 2017 Analytical Methods Paper Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 3. A CHIRALPAK AD-3 column (100 mm  4.6 mm I.D., 3 mm particle size) from Diacel was used for the separation of bortezomib and its impurities. The column oven tempera- ture was kept at 45 C throughout the experiments. A sample volume of 1 mL was injected into the chromato- graph and detected at 270 nm. The active backpressure of the system was maintained at 1900 psi throughout the analysis. The sample cooler temperature was maintained at 10 C. 2.3 Standard and sample preparations 2.3.1 Diluent preparation. HPLC grade methanol was used as the diluent, for the preparation of standards, system suit- ability solutions, and samples for both the methods. This selection was based on the solubility studies conducted on the two active drug substances. 2.3.2 Docetaxel standard, USP identication solution (system suitability) and sample preparation. An amount of docetaxel USP CRS was weighed accurately in a suitable volu- metric ask. 10 mL of the diluent was added, sonicated to Table 1 Results of forced degradation studiesa S. no. Stress condition Assay of degraded sample % degradation Purity angle Purity threshold Assay of degraded sample % degradation Purity angle Purity threshold Docetaxel Bortezomib 1 Acid hydrolysis 78.6 20.5 0.121 0.313 92.2 7.1 1.259 1.870 2 Base hydrolysis 66.9 32.2 0.135 0.456 91.4 7.9 1.647 2.625 3 Thermal degradation 96.6 2.5 0.090 0.323 99.9 0.6 0.985 1.529 4 Oxidation 90.5 8.6 0.106 0.290 81.8 17.5 0.721 1.453 5 Photolytic degradation (UV) 96.7 2.4 0.231 0.421 96.7 2.6 0.978 1.752 6 Photolytic degradation (light) 95.3 3.8 0.100 0.370 93.7 5.6 1.054 1.494 a Control sample results for calculating the % degradation, docetaxel assay ¼ 99.1 and bortezomib assay ¼ 99.3. Fig. 2 Ultraviolet-Visible spectra of (a) docetaxel and (b) bortezomib. This journal is © The Royal Society of Chemistry 2017 Anal. Methods Paper Analytical Methods Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 4. dissolve the contents and diluted suitably to obtain a concen- tration of 1000 mg mLÀ1 . USP docetaxel identication RS contains docetaxel and small amounts of listed impurities 2-debenzoxyl 2-pentenoyl docetaxel, 6-oxodocetaxel, 4-epi- docetaxel, and 4-epi-6-oxodocetaxel. It was used as a system suitability and spiked sample solution. The sample solution was prepared similar to the USP CRS solution by diluting an appropriate volume of the sample with the diluent to get the concentration of 1000 mg mLÀ1 . 2.3.3 Bortezomib standard and sample preparation. The standard solution was prepared by dissolving an amount of bortezomib in the diluent and diluting suitably to obtain a concentration of 180 mg mLÀ1 . The sample solution was prepared similar to that of the standard to get the concentration of 175 mg mLÀ1 . Bortezomib has seven impurities namely, (S,S)- diastereomers, (R,R)-diastereomers, R,S enantiomer, impurity A, impurity B, impurity C and impurity D. The impurities of bortezomib were prepared by dissolving a suitable quantity in the diluent and spiked into the sample preparation. 2.4 Forced degradation study 2.4.1 For docetaxel injection. Forced degradation studies were performed to assess the stability indicating power of the method. Docetaxel injection samples were deliberately sub- jected to various stress conditions such as acidic (1 mL of 1 N HCl for 12 h at room temperature), basic (1 mL of 1 N NaOH for 12 h room temperature), oxidative (1 mL of 1% H2O2 for 30 minutes), humidity (90% RH for 12 hours) UV light (200 watt hours per square meter), and light (1.2 m lux hours). The stressed samples were further neutralized and diluted suitably to get nal concentrations of 1000 mg mLÀ1 . All samples were then chromatographed and the peak purity plots were obtained for the docetaxel peak. Peak purity testing determines the peak homogeneity by comparing each spectrum within the peak against the peak apex. The chromatograms of the stressed samples were evaluated for peak purity of docetaxel using Waters Empower Networking Soware. The values of the purity angle and purity threshold were used to estimate the spectrum homogeneity and the peak purity. Table 2 shows the Fig. 3 Chromatograms obtained from primary screenings (Trial-1). Anal. Methods This journal is © The Royal Society of Chemistry 2017 Analytical Methods Paper Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 5. degradation data with purity angle and purity threshold. The criterion for peak homogeneity is that the purity angle should be less than the peak threshold as per the waters empower soware. The purity angle and purity threshold from Table 2 show that the docetaxel peak is homogenous in the presence of degradation products as obtained from the stress samples. 2.4.2 For bortezomib injection. Bortezomib for injection samples were subjected to the following stress conditions. Acid hydrolysis (1 mL of 0.5 N HCl for 6 hours), base hydrolysis (1 mL of 1 N NaOH for 4 hours), heat exposure (at 60 C for 24 hours), oxidative (3 mL of 10% H2O2), photolytic exposure (UV- 200 watt hours per square meter, light-1.2 million lux hours) studies were carried out as per the current ICH guidelines (international council for harmonisation of technical requirements for pharmaceuticals for human use). The exposed samples were neutralized and diluted to get a concentration of 175 mg mLÀ1 . The bortezomib peak was evaluated for homogeneity by measuring the purity angle and purity threshold. The results are tabulated in Table 1. The purity angle of bortezomib obtained from various stress studies was found less than the purity threshold. This indi- cates that the bortezomib peak is homogenous in the presence of its degradants. Fig. 4 Chromatograms obtained from Trial 2. Fig. 5 Development chromatograms obtained from Trial 3. Fig. 6 Purity plot of the docetaxel peak obtained from development Trial 3. This journal is © The Royal Society of Chemistry 2017 Anal. Methods Paper Analytical Methods Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 6. 3 Results and discussion 3.1 Method development and optimization A systematic approach was adopted for developing methods using the SFC principle. The two compounds were studied for ultra-violet absorption to determine the detection lambda maxima. For this, solutions of docetaxel and bortezomib were prepared having appropriate concentrations within the UV- Visible range and absorbance study was conducted. The two UV spectra are shown in Fig. 2. Docetaxel exhibited two maxima, 228 nm, and 272 nm. Bortezomib exhibited three maxima, 209 nm, 266 nm, and 318 nm. Fig. 7 Typical chromatogram of optimized chromatographic conditions. Fig. 8 Chromatograms obtained from primary screenings on (a) BEH-2EP and (b) CHIRALPAK AD-3 (Trial-1). Anal. Methods This journal is © The Royal Society of Chemistry 2017 Analytical Methods Paper Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 7. 3.1.1 Determination of docetaxel in docetaxel injection 3.1.1.1 SFC primary screening and optimization of docetaxel SFC primary screening. Trial-1. In primary screening three different stationary phases, namely BEH (ethylene bridged hybrid) silica, BEH-2EP (ethylene pyridine), and CSH (charged surface hybrid)-ourophenyl with sub-2 mm particles which are specically designed for SFC, were employed to have predict- able separations of sample components. This selection of columns was based on their chemistries, polarities, pressure tolerability (up to 18 000 psi or 241 bar), operable temperature (up to 60 C) and compatibility with acidic and basic additives like TFA, DEA, etc. In view of the study objective, primary screenings were done with 100% CO2 mobile phase with a ow rate of 10 mL minÀ1 . The system pressure was maintained at 1900 units through an active back pressure regulator. The initial column temperature was kept at 40 C. With these set of chromatographic condi- tions, the rst trial was made by injecting USP identication Fig. 9 Development chromatogram obtained from Trial 2. Fig. 10 Development chromatogram obtained from Trial 3. Fig. 11 Development chromatogram obtained from Trial 4. This journal is © The Royal Society of Chemistry 2017 Anal. Methods Paper Analytical Methods Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 8. solution (1 mL) into the chromatographic system. The chro- matograms are shown in Fig. 3. The inference from above chromatograms was that no detectable peaks were observed in BEH silica and CSH our- ophenyl columns. BEH-2EP showed some promising retention of peaks with very low response. Hence, further development strategy was continued with the BEH-2EP column. Supercritical CO2 is a nonpolar solvent with poor solvating power. This is one of the reasons for poor retention of polar compounds. Berger and Deye et al., suggest that the addition of modiers to the mobile phase can increase the extraction efficiency of CO2.41–43 Trial-2. To increase the mobile phase strength, a few experi- ments were conducted with a small quantity of ethanol (0–5%) Fig. 12 Typical chromatogram of optimized chromatographic conditions. Fig. 13 System suitability chromatogram obtained from docetaxel identification solution. Anal. Methods This journal is © The Royal Society of Chemistry 2017 Analytical Methods Paper Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 9. along with a gradient programme. The increase in the polarity of the mobile phase has increased the sample partition and 5% ethanol has resulted in the elution of docetaxel at a RT of about 5.6 minutes. All the other components eluted closely before the docetaxel peak. The chromatogram is shown in Fig. 4. Trial 3. The separation and elution of majority of compo- nents occurred at the isocratic portion (95 : 5 CO2 : ethanol) of the gradient programme and hence the isocratic composition was nalized to even avoid the gradient hump. With the new chromatographic conditions, the identication solution was injected and the chromatogram was recorded. This trial resul- ted in a better baseline and improved separations among the components. The specimen chromatogram is shown in Fig. 5. The corresponding purity plot of the DCTX peak is shown in Fig. 6. The purity angle (PA ¼ 0.529) and threshold (TH ¼ 0.385) indicate that the DCTX peak is not homogenous (criteria: PA TH). Hence further separation is required. Trial 4. From the literature, the addition of small amounts, typically 0.1–1% of additives, to the mobile phase has been shown to have a benecial effect on the peak shape and sensi- tivity in SFC. Based on this 0.1% of DEA was added to the organic composition. This has further improved the peak shape and resolution of closely eluting peaks. The nal optimized chromatogram is shown in Fig. 7. 3.1.1.2 SFC primary screening and optimization for bortezomib SFC primary screenings. The structural evaluation shows that bortezomib possesses chiral centers and may give rise to chiral related substances. Having this information, the entire method development was strategized combining both chiral and achiral screening and optimization. Fig. 14 System suitability chromatogram obtained for bortezomib. Fig. 15 Acid stressed sample chromatogram. This journal is © The Royal Society of Chemistry 2017 Anal. Methods Paper Analytical Methods Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 10. Trial-1. In primary screening, four columns having different stationary phases namely HSS T3, BEH-2EP (ethylene pyridine), and CSH (charged surface hybrid)-ourophenyl with sub-2 mm particles which are specically designed for SFC, and amylose tris (3,5-dimethylphenylcarbamate) were employed to have predictable separations of sample components. This selection of columns was based on their chemistries, polarities, pressure tolerability (up to 18 000 psi or 241 bar), operable temperature (up to 60 C) and compatibility with acidic and basic additives like TFA, DEA, etc. The mobile phase was kept 100% CO2 in view of study objective at a ow rate of 1.0 mL minÀ1 . The system pressure was maintained at 1900 psi through the active backpressure regulator. The initial column to temperature was kept at 45 C to maintain the supercritical state of CO2. With these set of chromatographic conditions the bortezomib impurity mixture (1 mL) was injected into the chromatographic system in each of the stationary phase. No peaks were detected on the CSH, and HSS T3 column. BEH-2EP and CHIRAL-AD 3 columns showed few promising peaks. Fig. 8 shows the chro- matogram obtained on the two columns, respectively. Trial-2. Further development strategy was continued with CHIRALPAK-AD 3 (100 Â 4.6, 3 mm). According to Berger and Deye et al., addition of organic modier to the mobile phase increases the extraction efficiency of CO2. Based on this, ethanol Fig. 16 Base stressed sample chromatogram. Fig. 17 Peroxide stressed sample chromatogram. Fig. 18 Thermal stressed sample chromatogram. Anal. Methods This journal is © The Royal Society of Chemistry 2017 Analytical Methods Paper Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 11. was introduced into the mobile phase, and several experiments were conducted with varied percentages using a gradient pro- gramme. The increase in polarity of the mobile phase has increased the sample partition. The column temperature and ow rate were reduced to 45 C and 1.0 mL minÀ1 respectively to check their impact on the separation. With these changes, bortezomib standard solution and individual impurities were injected into the chromatograph. Due to these changes, borte- zomib was slightly retained and separation among the four peaks was improved a little. The other three peaks were iden- tied as isomers of bortezomib. The chromatogram obtained from Trial 2 is shown in Fig. 9. Trial 3 and 4. In Trial 3 IPA was introduced along with ethanol in a 1 : 1 ratio. The gradient programme was slightly modied to improve separation among the closely eluting isomers and elute late-eluting impurities. This trial resulted in better separation among the isomer peaks, and the other peaks were not eluted within 10 minutes. The obtained chromatogram with Trial 3 is shown in Fig. 10. In Trial 4 the ow rate was increased from 1.0 to 1.2 mL minÀ1 to check for the other two impurities (impurity C and impurity D). Both impurities were eluted within 10 minutes with good separation. All peaks were eluted and are well Fig. 19 Sample chromatogram UV degradation. Fig. 20 Sample chromatogram light degradation. Fig. 21 Acid stressed sample chromatogram. This journal is © The Royal Society of Chemistry 2017 Anal. Methods Paper Analytical Methods Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 12. resolved from the bortezomib peak. The development chro- matogram obtained from Trial 4 is shown in Fig. 11. 3.2 Method optimization Furthermore, the method included optimization studies which were conducted to ensure method repeatability and reproduc- ibility. The optimization studies were conducted by changing the column temperature (40 C, and 50 C) and ow rate (1.0, 1.1, 1.2, 1.5 mL minÀ1 ),and by adding TFA (0.1%, 0.2%, 0.5%) to organic modiers for sharp peaks. The optimized chromato- gram obtained is shown in Fig. 12. 3.3 Method validation The optimized methods were fully validated as per the requirements of current ICH guidelines for validation of analytical procedures, i.e., Q2 (R1).44 The detailed validation experiments and results are discussed below. Fig. 22 Base stressed sample chromatogram. Fig. 23 Peroxide stressed sample chromatogram. Fig. 24 Thermal stressed sample chromatogram. Anal. Methods This journal is © The Royal Society of Chemistry 2017 Analytical Methods Paper Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 13. 3.3.1 System suitability. System suitability parameters were measured to verify the system performance for the intended analysis. For this purpose the USP identication reference standard (CRS) was prepared and injected into the chromato- graph (for docetaxel). The USP system suitability parameters i.e., USP plate count, USP tailing, and USP resolution were evaluated. The chromatogram obtained from the system suit- ability solution, and the peak purity data are shown in Fig. 3. The retention time of docetaxel was 2.679 minutes. Docetaxel was separated from its closest impurity (RT ¼ 2.513) with a resolution of 1.7 (minimum criteria for resolution as per USP is 1.5). The USP plate count was 11 772 and the USP tailing factor was 1.1 for the docetaxel peak. For bortezomib the system suitability was determined from six replicate injections of bortezomib standard preparation and spiked sample preparation. The resolution between bortezomib Fig. 25 Sample chromatogram UV degradation. Fig. 26 Sample chromatogram light degradation. Table 2 Precision data for docetaxel and bortezomib S. no. Docetaxel Bortezomib Sample number Method precision Intermediate precision Method precision Intermediate precision 1 99.1 99.7 99.4 98.7 2 99.7 100.7 98.6 98.9 3 98.9 99.3 99.3 98.9 4 99.0 100.2 99.9 99.3 5 99.5 100.8 99.0 99.0 6 98.5 98.3 99.4 98.4 Mean 99.1 99.8 99.3 98.9 SD 0.43 0.95 0.44 0.30 % RSD 0.4 0.9 0.4 0.3 Overall % RSD (% RSD of 12 measurements) 0.80 0.42 This journal is © The Royal Society of Chemistry 2017 Anal. Methods Paper Analytical Methods Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 14. and its closely eluting peak was achieved to be 2.4. USP tailing and plate counts for the bortezomib peak were 1.2 and 5662 respectively. The % RSD for six replicate standard injections was 1.3. Typical system suitability solutions for docetaxel and borte- zomib are shown in Fig. 13 and 14 respectively. 3.3.2 Specicity. The specicity of the method was deter- mined to evaluate the interference due to diluent, placebo, and specied impurities, by injecting the diluent (blank), placebo, spiked sample and stress samples. There was no interference found at the retention time of docetaxel and bortezomib due to diluent, placebo, specied impurities and other degradants from the stress samples at the retention time of docetaxel and bortezomib. Furthermore, the homogeneity of the peaks was conrmed from the peak purity data. The content of docetaxel and bortezomib in the presence of their potential impurities was found to be 99.0 and 100.3, respectively. The forced degradation chromatograms are shown in Fig. 15–26. 3.3.3 Precision (repeatability). Precision of a method is a measure of repeatability. It is performed in two ways, method precision and intermediate precision. Method precision was performed by preparing six replicate samples and injecting one aer the other, under the conditions as similar as possible, and two injections were taken from each sample. Intermediate precision was performed similar to the method precision on a different day, using different chemicals and columns. The response of docetaxel and bortezomib was measured and the relative standard deviation was calculated. Table 2 shows the precision data for docetaxel and bortezomib. The overall % RSD Table 3 Linearity data for docetaxel and bortezomib Statistics Docetaxel Bortezomib Linearity Correlation 0.9999 0.9999 Slope 666.24 1133.4 Intercept À4478.0264 À962.03 Table 4 Accuracy data for docetaxel and bortezomib Accuracy Docetaxel Bortezomib Levels Level-1 (50%) Level-2 (100%) Level-3 (150%) Level-1 (50%) Level-2 (100%) Level-3 (150%) Sample-1 99.1 99.8 99.6 98.3 100.3 99.6 Sample-2 98.7 99.8 99.4 98.9 99.7 99.8 Sample-3 98.7 99.6 98.9 98.3 100.0 99.6 Mean recovery 98.8 99.7 99.3 98.5 100.0 99.7 % RSD 0.2 0.1 0.4 0.4 0.3 0.1 Table 5 Robustness and method sensitivity data for method 1 (docetaxel injection) S. no. Condition RT of docetaxel (minutes) Assay of docetaxel in spiked sample USP resolution USP tailing % RSD of standard Method sensitivity yes/no 1 Control (no change) 2.679 99.1 1.7 1.1 0.5 NAa 2 Flow (+)1.65 mL minÀ1 2.415 98.9 1.5 1.1 0.3 Yes 3 Flow (À)1.35 mL minÀ1 3.250 99.0 1.8 1.1 0.7 Yes 4 Temperature (+)50 C 2.558 99.0 1.7 1.1 0.4 No 5 Active back pressure regulator (+)2000 psi 2.600 99.0 1.7 1.1 0.4 No 6 Active back pressure regulator (À)1900 psi 2.55 98.9 1.6 1.1 0.4 Yes a Not applicable. Table 6 Robustness and method sensitivity data for method 2 (bortezomib for injection) S. no. Condition RT of bortezomib (minutes) Assay of bortezomib in spiked sample USP resolution USP tailing % RSD of standard Method sensitivity yes/no 1 Control (no change) 3.847 99.3 2.4 1.2 0.8 NAa 2 Flow (+)1.43 mL minÀ1 3.058 99.1 1.8 1.2 1.0 Yes 3 Flow (À)1.17 mL minÀ1 4.215 99.1 2.5 1.2 1.0 No 4 Column oven temp (+)50 C 3.812 99.1 2.5 1.2 1.0 Yes 5 Active back pressure regulator (+)2000 3.999 99.0 2.5 1.2 0.8 No 6 Active back pressure regulator (+)1900 psi 3.999 99.0 2.5 1.2 0.8 No a Not applicable. Anal. Methods This journal is © The Royal Society of Chemistry 2017 Analytical Methods Paper Publishedon11May2017.DownloadedbyUniversityofCalifornia-SanDiegoon11/05/201716:24:41. View Article Online
  • 15. of 0.80 for docetaxel and 0.42 for bortezomib shows that the methods are precise. 3.3.4 Linearity. The linearity of the method was estab- lished in the concentration range of 246.5–1479.18 mg mLÀ1 (i.e., 25% to 150% of the target concentration) for docetaxel and 29.0–290.1 mg mLÀ1 (15% to 160% of the target concen- tration) for bortezomib. The linear regression data from the calibration plot were indicative of an excellent linear rela- tionship between the peak area and concentration over the range specied above and the data are provided in Table 3. A correlation of 0.9999 was obtained for docetaxel and bortezo- mib, establishing a linear relationship between the concen- tration and detector response. 3.3.5 Accuracy. The accuracy of an analytical procedure expresses the degree of the closeness of the obtained results with the true values. The accuracy of the method was tested by the recovery procedure. A known amount of the active drug substance was spiked to the placebo and the recovery was estimated. The accuracy of the method was evaluated from 50% to 150% of target sample concentrations for docetaxel (500 mg mLÀ1 , 1000 mg mLÀ1 , and 1500 mg mLÀ1 ), and bortezomib (87 mg mLÀ1 , 175 mg mLÀ1 , and 350 mg mLÀ1 ). The average assays were in the range of 98.7 to 99.7 with % RSD 0.1–0.4 for docetaxel and 98.5 to 100.0 with 0.1–0.4% RSD for bortezomib. Table 4 summarizes the accuracy results. 3.3.6 Robustness. The robustness of an analytical method is its capacity to remain unaffected by small but deliberate changes in the method parameters. The robustness of the method is determined by making deliberate variations in the ow rate, column temperature and ABPR (active backpressure regulator). The various altered conditions and the measured system suitability are shown in Tables 5 and 6. The above robustness data state that an increase in the ow rate resulted in signicant variations in the USP resolution and could inuence the system suitability results. The column temperature and ABPR have no signicant impact on the results. 4 Conclusions The newly developed CO2 based assay methods for docetaxel and bortezomib are specic, precise, accurate, linear, and robust. The results obtained from the validation studies were satisfactory and they exhibit excellent performance in terms of sensitivity, speed and cost-effective. This technology being a green technology is environmentally friendly in terms of the waste generated from the system. The method can successfully be employed for high accuracy routine assay testing and release production batches. Acknowledgements The authors wish to thank the management of Mylan Labora- tories Ltd for supporting this work. 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