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Cite this: DOI: 10.1039/c3ay40802g

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Supercritical uid (carbon dioxide) based ultra

performance convergence chromatography for the
separation and determination of fulvestrant
diastereomers
Ganipisetty Venkata Narasimha Rao,*a G. Gnanadev,a Bellam Ravi,a D. Dhananjaya,a
P. Manoj,a B. Indua and R. Venkata Nadhb
UltraPerformance convergence chromatography (UPC2
) is a new category of separation science which
utilizes the unrealized potential of the supercritical chromatography phenomenon. UPC2
is a standalone, viable technique that is cost eective, sustainable, and uses green technology that lowers the use
of organic solvents. Based on this advantage, we explored a simple and robust supercritical liquid-based
UPC2 method in order to increase sample throughput and productivity to quantify the diastereomers of
fulvestrant. The two isomers of fulvestrant were well separated on a chiral column (150 mm
4.6 mm,
I.D.) by applying a mixture of methanol and acetonitrile (9.5 : 0.5) as the co-solvent of the mobile phase
of carbon dioxide (75%). The detection was carried out at 280 nm. We were able to achieve a three-

Received 13th May 2013

Accepted 16th July 2013

fold reduction in retention with an isocratic mode as compared to the United States Pharmacopoeias
(USP) normal phase method. This new method was validated in accordance with the ICH guidelines; it
exhibited good intra- and inter-day accuracy, precision, and the results were linear over a range of 25%

DOI: 10.1039/c3ay40802g

to 150% of the target concentration. The method could be successfully applied for the determination of

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the diastereomeric ratio of fulvestrant as an API and in fulvestrant injectable nished products.

Introduction

Today a large amount of hazardous and-non hazardous waste in

the form of organic solvents is being generated by various
industries. The management of these waste solvents has become
a concern in the view of environmental health and safety. There
is the need to apply the principles of green chemistry in every
process at all stages of manufacturing and testing, to reduce the
resulting amounts of waste. In this context, supercritical CO2 has
emerged as a versatile solvent for various chemical separations
owing to its low toxicity and inammability. As a result, supercritical uid chromatography (SFC) has emerged as an analytical
tool for chemists for various separations of active compounds
and impurities in shorter amounts of time.1
In the pharmaceutical industry, SFC is an alternative and
complementary method to the HPLC technique. The potential
of SFC using packed columns for the analysis of impurities in
pharmaceutical compounds has been recognized for many
years.2 SFC can oer highly ecient separations in short analysis times and with a low pressure drop without compromising
a

Formulations Research and Development Centre, Mylan Laboratories Ltd, Bollaram,

Jinnaram, Medak, Hyderabad, Andhra Pradesh, 502325, India. E-mail: narasimha.
[email protected]; [email protected]; Tel: +91 9000122344

Department of Biotechnology, Vignan's Engineering College, Vadlamudi, Guntur,

Andhra Pradesh, India

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the resolution, plate counts and tailing. However, the lack of

reliable and sensitive commercial SFC systems has prevented
the extensive use of SFC in the industry. To explore the green
potential of SFC for faster separations, environmental safety
and better waste management, an attempt is made here to
develop a simple and short method for the determination of
fulvestrant diastereomers using UPC2 (UltraPerformance
convergence chromatography).3
The main aim of the current study and the future plan are to
develop and validate faster and environmentally friendly
methods to reduce the solvent consumption and analysis time
without compromising any performance parameters such as
resolution, peak tailing and plate count by utilizing the green
technology SFC. For this, we selected fulvestrant, which is a
novel endocrine therapeutic for breast cancer with a unique
structure and mode of action. Fulvestrant is the only parenteral
agent in this class which has a good side eect report and is well
tolerated. Fulvestrant is the subject of much ongoing research
regarding its novel mechanism of action and pharmacokinetic
prole, to optimize its clinical ecacy and explore new applications, including rst-line use in advanced breast cancer.47
Fulvestrant is commercially available under the name
FASLODEX from Astra Zeneca.
The chemical name of fulvestrant is 7a-[9-(4,4,5,5,5-pentauoropentylsulphinyl)-nonyl]estra-1,3,5(10)-triene-3,17b-diol. It

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Fig. 1

Chemical structure of fulvestrant.

contains six asymmetric carbon atoms, and a stereogenic

sulphoxide in the side chain. The active ingredient is a mixture
of two diastereoisomers: fulvestrant sulphoxide A and B, having
the same absolute conguration at each of the stereogenic
centers in the steroid system, but dierent absolute congurations at the sulphur atom. The chemical structure of fulvestrant
is shown in Fig. 1.8,9
Currently, the United States Pharmacopoeia monograph for
the fulvestrant drug substance prescribes an isocratic normal
phase method for the determination of the diastereomeric
ratio.9 It utilizes a mobile phase consisting of 2-methyl pentane
(an aliphatic hydrocarbon) and dehydrated alcohol 880 : 220 at
a ow rate of 1 mL min1. The run time of this method is
approximately 30 minutes. It also uses 2-methyl pentane for
sample preparation which is a costly aair and a cumbersome
process for waste management. USP and current method
chromatograms are compared in Fig. 2.
Some literature is available on the analysis of fulvestrant and
antiestrogens either by high performance liquid chromatography or HPLC coupled with a mass spectrometry technique.1021 One validated high performance liquid
chromatography method for the determination of fulvestrant in
pharmaceutical dosage forms was reported by Varanasi, which
uses normal phase separation on a cyano column using
n-hexane and isopropyl alcohol as the eluents.22 The retention
times of the two isomers are reported to be around 30 minutes
and 30.5 minutes. A dierence in retention time of 0.5 minutes
between the two peaks indicates a poor resolution. One US
patent on the separation of fulvestrant isomers by Cristian
Fazioni is available.23 The patent describes methods for separating the isomers by a reverse phase HPLC and a chiral column
using acetonitrile and n-hexane as the solvents in the mobile
phase. So far, all available literature procedures use one or more

Fig. 2

Paper
solvents described above which many laboratories would like to
reduce for health, safety, environmental, and cost reasons. No
method has been reported so far on the herein presented
detection by SFC. Hence, a green initiative was taken here to
develop a method, equivalent or superior to the existing available methods, by using the advantages of SFC to reduce the cost
of analysis, and to safeguard the environment.

Experimental

2.1

Chemicals and reagents

Samples of the fulvestrant API and injectable material used in

this study were obtained from the Mylan R&D Center (Hyderabad, India). The reference standard used was an in-house
generated standard from Mylan. The HPLC grade acetonitrile
and methanol were obtained from Merck, India. The CO2 was
purchased from Sai Padmaja Oxygen (Hyderabad, India).
2.2

Instruments and chromatographic conditions

An integrated Acquity UPC2 system from Waters Corporation,

Milford, USA, was equipped with a Waters photodiode array
detector (PDA). Data collection and analysis were performed
using the Empower soware 2pro (Waters Corporation).
Balances used for weighing the reference standards and
samples were from Mettler Toledo.
Separation was achieved on a Chiralpak AD-H (Diacel)
column with the dimensions 150 mm
4.6 mm (I.D.) and a
particle size of 5 mm. A simple mobile phase containing liquid
CO2 and mixtures of methanol and acetonitrile with the ratio
95 : 5 (v/v) as the co-solvent were used. The mobile phase ow
rate was maintained at 2.5 mL min1 throughout the run with a
column temperature of 55  C. The injection volume was 2 mL,
and the detection wavelength was 280 nm.
2.3

Standard and sample preparations

100% methanol was used as a diluent for preparing the standards and samples. A standard solution was prepared by dissolving a specic amount of fulvestrant in the diluent, which
was appropriately diluted to obtain a concentration of 1000 mg
mL1. The sample solution of the API was prepared in a similar

Typical system suitability chromatograms (a) from the USP procedure and (b) from the SFC procedure.

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Table 1

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Forced degradation studies

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Fulvestrant isomer A

Fulvestrant isomer B

Degradation conditions

Purity angle

Purity threshold

Purity
ag

Purity angle

Purity threshold

Purity
ag

Control conditions (no degradation)

Thermal degradation (at 60  C for 72 h)
Photolytic degradation (UV)
Photolytic degradation (light)

0.226
0.279
0.217

0.377
0.472
0.365

No
No
No

0.231
0.268
0.216

0.35
0.418
0.329

No
No
No

way to the standard solution (1000 mg mL1). To prepare the

injectable sample, a quantity of fulvestrant equivalent to 100 mg
was placed in a 50 mL volumetric ask, 15 mL of the diluent
were added, the mixture was sonicated for 5 minutes to dissolve
the contents, and the ask lled up to the mark with the diluent
to obtain a nal concentration of 1000 mg mL1.
2.4

Forced degradation study

Forced degradation studies were conducted on the samples and

on a plain placebo to prove the specicity of the method.
Specicity measurements were carried out by exposing test
solutions to heat (60  C for 75 hours). Photolytic studies were
carried out as per the current ICH requirements. The
percentage and peak purity of fulvestrant diastereomer peaks
was checked by using a PDA detector. The purity angle was
within the purity threshold limit for the two peaks and
demonstrated the analyte peak homogeneity. The results of the
forced degradation studies are presented in Table 1.
2.5

Solution stability and mobile phase stability

The solution stability of the standard and the sample solution were
assessed by leaving both the test solutions of the sample and the
standard at room temperature for 24 hours. The mobile phase
stability was also tested by keeping the mobile phase at room
temperature for 3 days and evaluating the system suitability.

Results and discussion

3.1

Method development and optimization

3.1.1 SFC 1st tier screening. Chiral separation of drug

substances usually involves the screening of a given set of
solvents on several columns packed with stationary phases.
Table 2

Resolution obtained from various combinations of organic modiers

S. no.

Organic modier(s)

1
2
3
4
6
7
8

Acetone
Acetonitrile

This process is oen time consuming and is generally regarded

as a bottleneck prior to the analysis. A way to speed up the
screening procedure is to improve the method development
capacity using the UPC2 multicolumn manage Aux. The rst
screening step was performed by using a generic method with a
runtime of 15 minutes in 100% CO2 and no organic modiers,
on four dierent stationary phases: BEH silica, BEH 2-ethyl
pyridine (2-EP), CSH ourophenyl and amylose tris stationary
phases with sub-2 mm particles, specically designed for UPC2
instrumentation. A sample containing a mixture of two pairs of
diastereomers was injected onto the 4 columns in sequence.
This rst screening did not result in any separation of the
two peaks. An attempt to improve the method was made by
introducing a small quantity of b-CD (beta cyclodextrin) in a
hydrophobic organic co-solvent. The results were not satisfactory, as very little separation was achieved.
3.1.2 SFC 2nd tier screening. Since fulvestrant is very
hydrophobic and CO2 is non-polar, the 2nd screening was done
by introducing organic modiers in various combinations with
CO2 on a Chiralpak AD-H column with the dimensions 150 mm

4.6 mm, and a particle size of 5 mm.
For example, dierent organic solvents such as acetone,
acetonitrile, ethanol, and methanol in various proportions were
evaluated. These methods were programmed as isocratic runs
with the same run time, temperature and back pressure settings
as those in the generic method. Trials with acetone and acetonitrile resulted in no peak elution within 10 minutes. Trials with
ethanol and methanol in dierent proportions resulted in the
separation of the two isomers. The results are shown in Table 2.
From the screening process and solubility data (see Table 3),
methanol was found to be the preferred solvent. The best
resolution was achieved by adding 5% of acetonitrile in methanol with 75% supercritical CO2 as the mobile phase.

Ethanol
Methanol
5% acetonitrile in methanol

Mobile phase A (%)

carbon dioxide

Mobile phase B
(%) organic modier

Resolution

80
70
70
75
90
75
75

20
30
30
25
10
25
25

No elution of peaks within 10 minutes

No elution of peaks within 10 minutes
1.47
1.61
No elution of peaks within 10 minutes
1.47
1.66a

Highest resolution.

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Table 3

Paper

Solubility study of fulvestrant

Solvent

Sample
weight [mg]

Solubilitya

Methanol
Methanolwater (80 : 20)
Methanolwater (70 : 30)
Acetone
Acetonewater (70 : 30)

997
1000
994.5
1000
999

Very solubleb
Not soluble
Not soluble
Freely soluble
Not soluble

a
Solubility denitions: 1000 mg of compound soluble in 1 mL very
soluble; 9 mL freely soluble; 20 mL soluble; 80 mL sparingly
soluble. b Highest solubility.

3.2

Method validation

The optimized method was validated as per the current ICH

guidelines for the determination of fulvestrant diastereomers in
the fulvestrant drug substance and formulations.24

Fig. 3

3.2.1 System suitability. The system suitability parameters

were measured to verify the system performance. The system
precision was determined in six replicate injections of standard
preparations. The USP criterion for the resolution between the
two diastereomers is not less than 1.3. In the current method a
reproducible resolution of 1.6 was obtained.
3.2.2 Linearity. The linearity of the detector response was
established by injecting the potential impurities at concentrations ranging from 25% to about 150% of the target concentration, and the correlation coecient was determined to be
0.999. A linearity plot was made for the detector response
dependent on the concentration, which is shown in Fig. 3.
3.2.3 Accuracy. The accuracy of the analytical procedure
expresses the degree of closeness of the obtained results with
the true values. The accuracy of the method was evaluated at
three dierent concentrations, namely 2.5 mg mL1, 5 mg mL1,
and 7.5 mg mL1 of the drug product, and the recovery was

(a) Linearity plot for isomer A and (b) linearity plot for isomer B.

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Table 4

Regression, precision and accuracy

Parameter

Fulvestrant A

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Regressiona equation (y)

Slope
Intercept
Correlation coecient
Residual sum of
squares
Precision (%RSD)b
Intermediate
precision (%RSD)b
Accuracy
50%
100%
150%

Fulvestrant B

67.9628
552.4918
0.9998
1 925 903.66

77.5987
19.9503
0.9999
1 265 477.63

0
0

0
0

% Recoveryc
101.4
101.7
100.2

99.4
101.1
100.6

a
The linearity range is 25150% with respect to 1000 mg mL1 of
fulvestrant. b Six determinations of each sample according to the
method described at 1000 mg mL1. c %Mean recovery.

calculated for each added amount. The average recoveries and

%RSDs were calculated. The recoveries ranged from 100.2% to
101.7%, and the %RSDs of the individual preparations were
between 0.3% and 0.4%. The results of the accuracy studies are
presented in Table 4.
3.2.4 Precision. The system precision was established by
using the fulvestrant standard (100 mg mL1) in six replicate
injections. The RSD(%) was calculated for the areas of the fulvestrant diastereomer peaks. The repeatability of the method
was established by preparing and injecting six samples at 1000
mg mL1. The RSD(%) of the results obtained for the six samples
was calculated. The intermediate precision was obtained by
having the samples analyzed by a dierent analyst on another
instrument and dierent day to evaluate the robustness of the
method. The results of the precision studies are shown in Table
4. An overlaid chromatogram of the six sample solutions is
shown in Fig. 4.
3.2.5 Robustness. The robustness of an analytical method
is a measure of its capacity to remain unaected by small but
deliberate changes in the method parameters. To determine the
robustness of the method, deliberate variations were made to
the ABPR (active back pressure regulator), ow rate, column
temperature and organic composition. The mobile phase ow

Fig. 4

Fig. 5

Graph for the method robustness.

rate was 2.5 mL min1; to study the eect of the ow rate on the
resolution, it was altered to 2.25 and 2.75 mL min1. The impact
of the column temperature on the resolution was studied at
50  C and 60  C. The variation in the organic composition in the
mobile phase was also studied. In addition to the above
parameters for UPC2, a variation in the ABPR was also studied.
This parameter helps to achieve the diusivity of CO2 to
improve the resolution. No signicant impact was observed on
the resolution with changes to the ABPR. A minor reduction in
the resolution was observed with increase in the column
temperature and ow rate to 60  C and 2.75 mL min1,
respectively. The variation in the system suitability with respect
to the robustness parameters is shown in Fig. 5.

Concluding remarks

The rapid isocratic UPC2 method developed for the quantitative

determination of fulvestrant diastereomers is specic, precise,
accurate, linear, and robust. The results obtained from the
validation studies are satisfactory. This method exhibits excellent performance in terms of sensitivity and speed, and is also
cost-eective. This technology is green and environmentally
friendly in terms of low amounts of solvent waste generated.
The method can be successfully employed for routine quality
controls of high numbers of production batches in signicantly
less time.

Overlay chromatogram of six samples from the precision experiment.

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Acknowledgements
The authors wish to thank the management of Mylan Laboratories Ltd for supporting this work. The authors wish to thank
the formulation research group for providing the samples for
the research. We would also like to thank our colleagues in the
Analytical development services for their cooperation in
carrying out this work.

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