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International Journal of Advancements in Research & Technology, Volume 3, Issue 5, May-2014 212 ISSN 2278-7763
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HPLC method development for separation of racemic APIs/API intermediates for scale-up in
Varicol process
Sangeeta Sangwan,*1 Satyananda Misra, 1 Ram Thaimattam, 1 Rajesh K. Thaper.*1, S. K. Dewan2
1Ranbaxy Laboratories Limited, Chemical Research Department, Research & Development Centre, Sarhaul, Sector-18, Gurgaon – 122015,
Haryana, India. Tel: +91-124-4194808, +91-9911697700, 2Department of Chemistry, Maharshi Dayanand University, Rohtak – 124001,
Haryana, India.
Email: [email protected]
ABSTRACT
HPLC methods were developed for separation of enantiomers of racemic clopidogrel, lansoprazole, omeprazole, EPB-1 and
voriconazole using chiral stationary phases (CSPs). The effect of column loadings and flow rates on the separation profiles in
terms of resolution and selectivity were investigated for developing scalable separation processes. In addition, the influence of
additives on the separation behavior has been studied. A few of these separations were also scaled up using Varicol technology. Keywords : Clopidogrel, Lansoprazole, Omeprazole, Voriconazole, Chiral stationary phase and Varicol process.
1 INTRODUCTION
Measurement and control of enantiomeric purity of chiral
active pharmaceutical ingredients (APIs) is a necessary means
to control quality of drug substances as only one enantiomer
has the desired biological and pharmacological properties.
Consequently, chiral drugs must be stereochemically pure,
which places great demands on their synthesis, analysis and
purification. Development of chiral drugs relies on four key
technologies: asymmetric synthesis,[1] chiral resolution via
crystallization[2-3] or diastereomeric salts,[4-5] enantiomeric
separation on chiral stationary phase (CSP)[6] and biocatalytic
or enzymatic synthesis.[7] Asymmetric synthesis is generally
expensive and quite often challenging. Biocatalytic or
enzymatic resolution is an attractive alternative if the enzyme
of the interest is commercially available. Chiral resolution via
crystallization is a widely used technique for production of
chirally pure drugs and chiral chromatography has become a
preferred method for continuous separation of enantiopure
compounds; both these methods are commercially viable if the
undesired enantiomer is recycled. Preparative batch
chromatography is being replaced with continuous
chromatographic processes like simulated moving bed
(SMB)[8] and its advanced variants like Varicol[9] and
Power Feed.[10]
Enantioselective chromatographic separation can also be
carried out on achiral chromatographic columns using a chiral
mobile phase or a chiral additive.[11] Strongly absorbing
additives are reported to result in a variety of band shapes in
chiral preparative chromatographic systems and additives can
be selected to address a particular separation problem by
engineering a desired band shape composition.[12]
Anti-Langmuir behavior is generally observed under
overloaded conditions, which exhibits an initial fronting
followed by a steep decay. The curvature of the anti-Langmuir
isotherm is opposite to that of the Langmuir form. Acidic or
basic additives can modify solubility, thereby improving the
solubility, but with some compromise on the selectivity. In our
continued efforts to develop HPLC methods for separation of
enantiomers of key racemic APIs in order to develop scalable
continuous separation processes, we have considered racemic
clopidogrel,[13] lansoprazole,[14] omeprazole,[14] EPB-1
[15-17] and voriconazole,[18] In this work, we present the
results of these investigations.
2 Material and Methods
All the APIs and the API intermediates were provided by
Ranbaxy Laboratories Ltd., India. Waters HPLC 2695 alliance
separation module (with customized syringe and loop volume
of 2.5 mL) with PDA 2998 and RI 2410 detectors were used for
method development. HPLC grade solvents were used as
obtained from Rankem and Qualigens. Customized chiral
columns (250 × 4.6 mm) with 5 or 20 µm particle size were
procured from Daicel Chiral Technology, Japan. Varicol
processes were carried out in Novasep VARICOL LAB
equipment using commercially available solvents.
3 Results and discussion
Chiral separation of racemic clopidogrel, lansoprazole,
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omeprazole, EPB-1 and voriconazole were studied. All these
compounds were initially run through various CSPs such as
Chiralpak AD, Chiralpak AS-V, Chiralpak IA, Chiralpak IC,
Chiralpak AZ, Chiralpak AY, Chiralcel ODI, Chiralcel OZ,
Chiralcel OD and Chiralcel OJ with a few select mobile phases
for selecting suitable column(s). The composition of the mobile
phase was then optimized based on the solubility of the
compound of interest, overall run time, resolution (Rs) and
selectivity (α) (Tables 1 through 6). For method development,
20 µm sized CSPs were considered, keeping in mind its
suitability for scale-up. All the analytical parameters were
studied at λmax but the loading studies were performed at a
higher wavelength than λmax to minimize the peak saturation
effects, except for lansoprazole wherein all the analytical
parameters were studied at a single wave length. Void volume
was calculated using 1, 3, 5-Tri-t-butylbenzene as void volume
marker.[19] Varicol simulation software was used for
designing and optimizing Varicol processes under isocratic
conditions.
3.1 Clopidogrel: S-Clopidogrel (Scheme 1) is an oral
antiplatelet agent used to inhibit blood clots in coronary artery
disease, peripheral vascular disease and cerebrovascular
disease.[13] It is marketed by Bristol-Myers Squibb and Sanofi
under the trade name Plavix. Various HPLC methods for
analysis of clopidogrel and related substances were reported,
which include the use of chiral (both protein and small-
molecule based) and achiral stationary phase with aqueous
and organic solvents as eluent. In addition, racemic
clopidogrel was separated into its enantiomers by supercritical
fluid chromatography.[20] Qualitative RP-HPLC methods
were developed for the enantiomeric separation of racemic
clopidogrel and related substances using acetonitrile-
phosphate buffer mobile phase on CHIRAL-AGP[21] and
Chromolith Performance 18e columns. [22] A quantitative
RP-HPLC method was also developed using methanol-
phosphate buffer and Sunfire C18 column.[23] In addition, a
monograph on clopidogrel API published in the US
Pharmacopoeia 29 recommends the use of RP-HPLC method
using acetonitrile-phosphate buffer as an eluent on L57
column for the detection of clopidogrel and its impurities.
Herein, we report development of scalable enantioselective
normal-phase HPLC methods for the separation of racemic
clopidogrel. The recovery of the analyte from the organic
solvent based mobile phase is much simpler compared to that
of the reported acetonitrile-phosphate buffer mixture.
Scheme 1. Chemical structures of the compounds considered
in this study.
The analytical parameters for the resolution of racemic
clopidogrel using n-hexane–isopropyl alcohol (IPA) and
n-hexane–ethanol (EtOH) solvent mixtures are shown in Table
1. The flow rate and loading plots are shown in Figure 1. It
may be noticed that even at high flow rates and sample
loadings, the peaks are well resolved. In addition, it is evident
from the chromatograms that at higher flow rates, the
resolution between the peaks decrease, and both peaks follow
linear adsorption at low column loadings and Langmuir type
adsorption under overloaded conditions. In hexane–IPA
system, the resolution and selectivity of the two enantiomers
are 0.4 and 1.38, respectively, at 400 µg loading. In contrast,
with hexane–EtOH mixture as an eluent, the resolution and
selectivity were found to be superior (1.40 and 1.72,
respectively) even at 1400 µg loading, which is consistent with
the estimated number of theoretical plates (Table 1).
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Table 1. Chromatographic data for clopidogrel enantiomers.
aNumber of theoretical plates that a real column possesses, N = 5.55 ×
tR2/w1/22
where w1/2 is the peak width at half-height. bRetention factor, k1' = (tR – tM) / tM
where tR is the retention time and tM is the time taken for the mobile phase
to pass through the column. cSelectivity factor (α) is the ratio of relative retention factors (k’); α = k2' /
k1' dResolution factor, Rs = (tR2 – tR1) / 0.5 × (tw1 + tw2)
where tR is the retention time and tw is obtained from the intersection of
the tangents with the baseline.
Figure 1. Separation of clopidogrel enantiomers using n-hexane-EtOH
(65:35, v/v) on Chiralcel OJ (20 µm, 4.6×250 mm) at different (a) flow rates
and (b) column loadings.
3.2 Lansoprazole: Dexlansoprazole (Scheme 1) is a proton
pump inhibitor that is marketed by Takeda Pharmaceuticals
with trade names, Kapidex and Dexilant for use in the
treatment of erosive oesophagitis and non-erosive
gastro-oesophageal reflux disease.[13] WO 03/051867 covers
generically the separation the lansoprazole enantiomers using
SMB technology, while disclosing the experimental details for
the separation of omeprazole enantiomers. Lansoprazole and
omeprazole is a chiral sulfoxide with the sulfur atom being the
stereogenic center.[24]
The resolution and selectivity for the lansoprazole
enantiomers were found to be reasonably good (1.5 and 1.3,
respectively) when 100 mM NaClO4 buffer was used as an
eluent at a flow rate of 3.0 mL/min and 750 µg sample loading
(Scheme 1 and Table 2a). However, the column backpressure
was found to be quite high (~195 bar) due to smaller particle
size of the CSP (Chiralcel ODR, 5.0 µm), and hence the
separations were evaluated with CSPs with 20 µm particle
size. The best separation was found on Chiralcel OZ column
using acetonitrile (ACN)–methanol (MeOH) diisopropylethyl-
amine (DIPEA) (90:10:0.1, v/v/v) mixture as the mobile phase
at a flow rate of 1 mL/min (Tables 2a and 2b). The column
back pressure was found to be 7.24 bar at 300 µg sample
loading and 5 mL/min flow rate. The peaks were well
separated ( = 1.29, Rs = 0.6) even at higher sample loadings
despite a low resolution. Both the peaks follow more or less
linear type adsorption behaviour (Figure 2). Basic additives
like DIPEA, diethylamine (DEA) and triethylamine (TEA) did
not play any detrimental role; instead they improved both the
resolution and selectivity (Table 2b). In fact, these additives
were used as stabilizers to enhance both the solution and solid
state stability of prazoles.[24] The separation of the desired
isomer, dexlansoprazole, was also scaled up in a Varicol
process[9] using MeOH-DIPEA (99.9:0.1, v/v) as the mobile
phase. Racemic lansoprazole (112 g), with a feed concentration
of 16 g/L was resolved into dexlansoprazole (52.73 g) and the
undesired enantiomer (52.03 g) with a purity of 98.80% (with
0.2% of the undesired enantiomer) and 98.46% (with 0.54% of
dexlansoprazole), respectively, using five columns with a size
distribution of 0.9 (zone 1), 1.5 (zone 2), 1.5 (zone 3) and 1.1
(zone 4) and with feed, eluent, extract, raffinate and recycling
flow rates of 18 mL/min, 167 mL/min, 130 mL/min, 55 mL/min
and 420 mL/min, respectively.
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Table 2a. Separation of lansoprazole enantiomers on Chiralcel
ODR and Chiralcel OZ columns.
Table 2b. Analytical parameters for lansoprazole enantiomers
on various columns with or without stabilizer/additive
Figure 2. Separation of lansoprazole enantiomers using ACN-MeOH-
DIPEA (90/10/0.1, v/v/v/) on Chiralcel OZ (20µm, 4.6×250 mm) at different
(a) flow rates and (b) column loadings.
3.3 Omeprazole: Esomeprazole (Scheme 1) is the S-isomer of
omeprazole, a proton pump inhibitor used for gastroesopha-
geal reflux and peptic ulcer therapy available under the brand
name, Nexium.[14] WO 03/051867 describes various HPLC
methods and scale up of the separations using SMB
chromatography using ethanol-isopropyl alcohol (30/70) and
Chiralpak AS.[24] In the present work, we present the HPLC
data using a single solvent (methanol) on Chiralcel OZ
stationary phase with or without stabilizer in the eluent.
Here racemic omeprazole was separated into stereochemically
pure esomeprazole and the undesired R-enantiomer on
Chiralcel OZ column using 100% MeOH and MeOH with 0.1%
TEA or DIPEA as mobile phase (Table 3). TEA and DIPEA
were used as the stabilizers. The chromatograms depicting the
effect of flow rate and column loading using 100% MeOH,
MeOH with 0.1% TEA, and MeOH with 0.1% DIPEA as
mobile phase are shown in Figures 3a, 3b and 3c, respectively.
With 100% MeOH as mobile phase, the eluting enantiomer
follows a linear adsorption at lower column loadings, while
Langmuir type adsorption was seen at higher loadings. The
Linear type adsorption was more pronounced in the presence
of DIPEA. However, in presence of 0.1% TEA, the peaks fol-
low Langmuir type adsorption. On Chiralcel OZ column, the
resolution factor decreased from 2.7 to 1.4 when 0.1% of TEA
or DIPEA was used, while the selectivity factor more or less
remained the same (at about 2.1). In contrast, both the
resolution and selectivity for lansoprazole enantiomers were
found to be better in presence of these additives. However, the
situation is quite opposite on Chiralpak IA column with
MeOH-ACN (90:10, v/v) as the mobile phase; TEA improved
the separation while DEA had a detrimental role (Table 3b).
Increasing the concentration of TEA from 0.1% to 0.5% did not
further improve the separation profile. The separation of
omeprazole enantiomers was further scaled up in a Varicol
process[9] using Chiralcel OZ CSP and MeOH with 0.1%
DIPEA as an additive in the mobile phase, despite the fact that
prolonged usage of such additives is known to cause memory
effect and can spoil the CSP. One kg of esomeprazole (99.8%
purity with 0.2% of the undesired enantiomer) was obtained
from 2.85 kg of omeprazole with feed, eluent, extract, raffinate
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and recycling flow rates of 22 mL/min, 137 mL/min, 94
mL/min, 65 mL/min and 429 ml/min, respectively, with an
output of about 700 g of esomeprazole/Kg CSP/day.
Table 3a. Chromatographic data for omeprazole enantiomers
on Chiralcel OZ column.
Table 3b. Chromatographic data for omeprazole enantiomers
on Chiralpak IA column.
Figure 3. Separation of omeprazole enantiomers using (a) MeOH, (b)
MeOH-TEA (99.9:0.1, v/v) and (c) MeOH-DIPEA (99.9:0.1, v/v) on Chiral-
cel OZ (20µm, 4.6×250 mm) at different flow rates shown on the left and
sample loadings on the right.
3.4 EPB-1: The R-enantiomer of EPB-1 (Scheme 1) is an in-
termediate used in the synthesis of Darunavir[15] and
Fosamprenavir,[16] while the S-enantiomer is used in the
synthesis of Atazanavir[17] Racemic EPB-1 was separated into
its enantiomers on Chiralpak AY CSP using MeOH–ACN
(90:10, v/v). The resolution and selectivity factors were found
to be in the desired range even at a high sample load of 5000
µg (Table 4). Although both the resolution and selectivity
factors were found to be acceptable even up to 9000 µg sample
loading, the number of theoretical plates is on the lower side.
The effects of flow rates and column loadings on the
chromatographic separations are shown in Figure 4. It is
apparent from the chromatograms that the individual peaks
follow different absorption isotherms. The first eluting
enantiomer follows more or less linear type adsorption, while
the second eluting enantiomer shows anti-Langmuir type
adsorption under overloaded conditions.
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Table 4. Separation of EPB-1 enantiomers on Chiralpak AY
column.
Figure 4. Separation of EPB-1 enantiomers using MeOH-ACN (90/10,
v/v/); on Chiralpak AY (20µm, 4.6×250 mm) at different (a) flow rates and
(b) column loadings.
3.5 Voriconazole: Voriconazole (Scheme 1), an antifungal
agent is used in the treatment of invasive candidiasis, invasive
aspergillosis and emerging fungal infections,[18] In this case,
only the enantioselective separation of racemic voriconazole
via diastereomeric crystallization of voriconazole salt with
R-(-)- 10-camphorsulfonic acid is reported in the literature.[25]
Herein, we report HPLC methods for developing scalable
separation of the voriconazole enantiomers.
The voriconazole enantiomers were separated on Chiralcel OZ
column using MeOH–ACN (90:10, v/v) with good selectivity
and resolution (Table 5a). The first eluting enantiomer follows
linear type adsorption; while the second eluting enantiomer
follows anti-Langmuir type adsorption at low sample loads
and Langmuir type adsorption under overloaded conditions
(Figure 5). The analytical parameters were found to be slightly
better on Chiralcel OZ column compared to that on Chiralpak
AD column, while the voriconazole enantiomers did not
separate on the other CSPs tested under similar conditions
(Table 5b). The separation was also scaled up in a Varicol
process using Chiralcel OZ CSP using MeOH–ACN (90:10,
v/v). Voriconazole (95 g), with a feed concentration of 10 g/L,
was resolved into its enantiomers – desired isomer (42 g;
99.37% purity with 0.63% of undesired enantiomer) and
undesired isomer 41.5 g; 99.73% purity with 0.27% of the
desired enantiomer) – with feed, eluent, extract, raffinate and
recycling flow rates of 15 mL/min, 240 mL/min, 142.4 mL/min,
112.6 mL/min and 287.8 mL/min, respectively.
Table 5a. Separation of voriconazole enantiomers on Chiralcel
OZ column.
Table 5b. Separation of voriconazole enantiomers on various
columns under similar conditions.
Figure 5. Separation of voriconazole enantiomers using MeOH-ACN
(90/10, v/v/); on Chiralcel OZ (20µm, 4.6×250 mm) at different (a) flow
rates and (b) column loadings.
4 Conclusions HPLC methods for separation of enantiomers of clopidogrel,
lansoprazole, omeprazole, EPB-1 and voriconazole on chiral
stationary phases (CSPs) were developed. In addition,
separation of enantiomers of lansoprazole, omeprazole and
voriconazole were scaled up in Varicol process. Various
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combinations of linear, Langmuirian and anti-Langmuirian
adsorption bands were noted for enantiomers of the
compounds studied herein. Basic additives that are primarily
used as stabilizers in the present work had both favorable and
unfavorable effects on the outcome of the enantiomeric
separations based on the type of the compound, CSP and
mobile phase used. In addition, the study revealed that the
number of theoretical plates can be a useful guide for drawing
meaningful conclusions, particularly at high sample loads.
Good solubility, high resolution and selectivity, and low
retention time are desirable for developing an efficient scalable
chromatographic separation of chirally pure compounds.
5 Acknowledgments SS thanks Ranbaxy management for their support and
encouragement.
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