hplc method development for separation of racemic … · international journal of advancements in...

International Journal of Advancements in Research & Technology, Volume 3, Issue 5, May-2014 212 ISSN 2278-7763

Copyright © 2014 SciResPub. IJOART

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.

6 References

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