Stereoselective HPLC separation of alvimopan on cellulose-based immobilized polysaccharide as a chiral stationary phase

Article abstract of DOI:10.1002/chir.22859

Chiral separation by normal phase high performance liquid chromatography is one of the most powerful technique to quantify the chiral purity of the compounds. In this study, a novel, simple, and specific analytical method was proposed to ascertain the chi

Full text of DOI:10.1002/chir.22859

Received: 21 December 2017
DOI: 10.1002/chir.22859
Revised: 30 January 2018
Accepted: 17 February 2018
R E G U L A R A R T I C L E
Stereoselective HPLC separation of alvimopan on cellulose‐
based immobilized polysaccharide as a chiral stationary
phase
1,2
1
1,3
Nitin H. Dhekale
| Dattatray B. Gunjal | Anil H. Gore
|
2
2
1
Yagnakirankumar Komaravolu
| K. Hima Bindu | Govind B. Kolekar
1
Fluorescence Spectroscopy Research
Abstract
Laboratory, Department of Chemistry,
Shivaji University, Kolhapur, 416 004
Maharashtra, India
Chiral separation by normal phase high performance liquid chromatography
is one of the most powerful technique to quantify the chiral purity of the
compounds. In this study, a novel, simple, and specific analytical method
was proposed to ascertain the chiral purity of alvimopan (ALV). The normal
phase HPLC method was developed based on cellulose tris (3,5‐
dichlorophenylcarbamate) stationary phase. The separation of ALV isomers
achieved by using column CHIRALPAK IC (250 × 4.6 mm, 5 μm), mobile
phase n‐hexane: isopropyl alcohol: ethanol: diethylamine (650:200:150:5
2
Analytical Research and Development,
Dr. Reddy's Laboratories Ltd. Hyderabad,
Hyderabad, 500 090 Telangana, India
3
Rajarshi Chhatrapati Shahu College
Kolhapur, 416 004 Maharashtra, India
Correspondence
Govind B. Kolekar, Fluorescence
Spectroscopy Research Laboratory,
Department of Chemistry, Shivaji
University, Kolhapur 416 004,
Maharashtra, India.
−1
v/v), column oven temperature 30°C, flow rate 1.0 mL min , injection vol-
ume was 10 μL, chromatographic response monitored at 273 nm. The devel-
oped method was validated as per the ICH guidelines and found precise,
accurate, and linear. The advantage of the method is a good separation of
ALV isomers within 35 minutes of the analysis time. Therefore, this method
is suitable for routine determination of chiral purity of ALV active pharma-
ceutical ingredient.
Email: gbkolekar@yahoo.co.in
KEYWORDS
alvimopan, HPLC, method development, method validation, stereoselective
2-4
1
| INTRODUCTION
the blood‐brain barrier.
Preclinical and small‐scale
clinical studies demonstrated that ALV administered
orally can selectively antagonize the gastrointestinal
effects of opioid agonists such as morphine without
Alvimopan is a potent selective peripherally acting
1-4
antagonist of the human μ‐opioid receptor
that is
2-5
being developed for the management of acute postoper-
ative ileus and for the reversal of the delayed gastroin-
testinal and colonic transit that result in symptoms
such as constipation, nausea, and motility disorders in
affecting analgesia. The adverse effects of opioids on
the gastrointestinal tract can be reversed by μ‐opioid‐
receptor antagonists such as naloxone and nalmefene,
but these drugs are also active in the central nervous
system, and so inhibit the analgesic effects of systemic
opioids. To overcome this issue, efforts have been
directed at identifying μ‐opioid receptor antagonists
1
patients treated with opiate analgesics. It is orally avail-
able, but its activity is peripherally restricted because its
moderately large, zwitterions form and polarity limit
gastrointestinal absorption and prevent passage through
6-8
with peripherally restricted activity.
Chirality. 2018;1–6.
wileyonlinelibrary.com/journal/chir
© 2018 Wiley Periodicals, Inc.
1

2
DHEKALE ET AL.
Chirality infuses the natural world and is of particu-
(A)
(B)
(D)
lar importance to life, as the molecules that comprise
living things are chiral and their chirality is crucial to
9-11
their biological function.
Recent trend of pharmaceu-
tical research and development is most inclined to the
developing the selective isomer and testing of individual
enantiomers become a growing priority. So far, no
method has been published for the determination of
the ALV chiral purity. By considering an inherent criti-
cality of reversed phase mode for chiral separation, nor-
mal phase mode preferred for separation of ALV chiral
(C)
12-14
isomers.
The objective of the present study was to
develop a stereoselective HPLC‐UV method with cellu-
lose tris‐(3,5‐dichlorophenylcarbamate) as a stationary
phase. This method allows to determine the chiral
purity of ALV without any derivatization. In this study,
the developed method was validated as per the ICH
guidelines. The proposed method is novel, simple, and
accurate for quantification of ALV isomeric impurities.
The advantage of the method is a good separation of
ALV isomers within 35 minutes of analysis time.
Therefore, this method is suitable for routine determina-
tion of chiral purity of ALV active pharmaceutical
ingredient.
FIGURE 1 Structures of ALV and stereoisomers. A, 2‐((S)‐2‐
benzyl‐3‐((3R, 4R)‐4‐(3‐hydroxyphenyl)‐3,4‐dimethylpiperidin‐1‐yl)
propanamido) acetic acid. (2S,3R,4R). B, 2‐((R)‐2‐benzyl‐3‐((3R, 4R)‐
4‐(3‐hydroxyphenyl)‐3,4‐dimethylpiperidin‐1‐yl) propanamido)
acetic acid. (2R,3R,4R). C, 2‐((S)‐2‐benzyl‐3‐((3S, 4S)‐4‐(3‐
hydroxyphenyl)‐3,4‐ dimethylpiperidin‐1‐yl) propanamido) acetic
acid. (2S,3S,4S). D, 2‐((R)‐2‐benzyl‐3‐((3S, 4S)‐4‐(3‐hydroxyphenyl)‐
2
2
| MATERIALS AND METHODS
.1 | Reagents and chemicals
3,4‐dimethylpiperidin‐1‐yl) propanamido) acetic acid. (2R,3S,4S)
All the chemical reagents are of analytical reagent grade
and used as received without further purification
throughout the experiments. n‐Hexane and isopropyl
alcohol were purchased from Rankem (Mumbai), etha-
nol was purchased from (Hyman), and diethylamine
was purchased from Merck (India). ALV and isomeric
impurities were supplied by Dr. Reddy's Laboratories
3
| EXPERIMENTAL DETAILS
.1 | Preparation of solutions
3
3.1.1 | Selection of diluents
Special attention should be paid on the solubility of the
enantiomers as well as racemate while preparing of
sample, because the separation shall be decided based on
(Figure 1)
1
5-
the solubility of enantiomers into the particular solvents.
As part of solubility study, different solvents were investi-
gated. It was found that the enantiomers are soluble in
the methanol and mixture of methanol and ethanol,
whereas it is practically insoluble in n‐hexane, isopropyl
alcohol, and ethyl acetate.
The mode of separation was normal phase (non‐polar);
therefore, the n‐hexane and alcohols are selected as sol-
vents for the mobile phase. Initially, the sample was dis-
solved in methanol. Due to the difference in polarity of
mobile phase and diluent, the peak splitting of ALV was
observed. Typical chromatogram of ALV isomers using
methanol as diluent is shown in Figure 2A,C. Further
ALV isomer was dissolved in methanol: isopropyl alcohol
(20:80 v/v). The isomers were dissolved in methanol and
2
.2 | Instrumentation and
chromatographic conditions
A Waters HPLC model 2695 equipped with pump, auto-
mated injector, column oven, and photo diode array
detector was set at 273 nm. Experiment was carried out
by
250 mm × 4.6 mm, 5 μm). The mobile phase consists of
n‐hexane: isopropyl alcohol, ethanol, and diethylamine
650:200:150:5 v/v) which is operated at 30°C column oven
temperature at isocratic mode. The mobile phase flow of
using
Diacel
CHIRALPAK‐IC
column
(
(
−
1
the method was 1.0 mL min . Injection volume was
0 μL. Data acquisition and interpretation were per-
formed by using empower‐2 software.
1

DHEKALE ET AL.
3
FIGURE 2 A, The chromatogram of ALV stereoisomer 2S 3R 4R using methanol (100%) diluent. B, The chromatogram of ALV
stereoisomer 2S 3R 4R using methanol and isopropyl alcohol (20:80 v/v). C, The chromatogram of ALV stereoisomers 2S 3S 4S, 2R 3R 4R,
2
4
S 3R 4R, and 2R 3S 4S using methanol (100%) diluent. D, The chromatogram of ALV stereoisomers 2S 3S 4S, 2R 3R 4R, 2S 3R 4R, and 2R 3S
S using methanol and isopropyl alcohol (20:80 v/v)
diluted volume with isopropyl alcohol. The symmetric
peak shape of ALV was found. Typical chromatogram of
ALV isomers using methanol and isopropyl alcohol as dil-
uent shown in Figure 2B,D. Therefore, the methanol and
isopropyl alcohol combination (20:80 v/v) was finalized
as diluent in ALV chiral analysis.
4 | RESULTS AND DISCUSSION
4.1 | Method development and
optimization
Chiral separation and estimation are a very complex phe-
nomenon. Normal phase chromatography with chiral sta-
tionary phase considered for separation of ALV and
17
isomeric impurities.
3.1.2 | Sample and standard solution
preparation
4
.2 | Theory of chiral separation
The details of ALV and isomeric impurities are summarized
in Figure 1. The ALV chiral isomers were dissolved in the
diluent (methanol: isopropanol 20:80 v/v) as stock solutions.
Initially, samples are dissolved in methanol and diluted
volume in isopropanol and sonicated for 3 minutes.
There are several articles available in literature describing
about the separation mechanism of enantiomers on poly-
saccharide phases.
14,17-21
In this chiral separation study,
specific interactions are most important like, π‐π interac-
tion between the aromatic rings of the ALV enantiomers
and chiral stationary phase, hydrogen bond interaction
between carbonyl group of chiral stationary phase and
acidic proton of ALV isomers, dipole‐dipole interaction
and the steric effect when nonpolar group of ALV isomer
comes contact with chiral stationary phase. Wainer et al
reported that the analyte‐containing aromatic functional-
ities could provide additional stabilizing effect to the iso-
mer‐chiral stationary phase complex by insertion of the
−1
ALV isomers were spiked in ALV sample (0.5 mg mL ).
3.1.3 | Evaluation of system suitability and
method validation
Based on the criticality of the method, peak tailing and
peak resolution (Rs) were considered as system suitability
criteria for this method. The developed method was vali-
15,16
dated as per ICH guidelines.

4
DHEKALE ET AL.
1
4,18,21
aromatic portion into the chiral cavity.
This type of
of the method, the effect of column oven temperature on
stereoisomers was studied in the range of 20 to 50°C. The
retention of chiral isomers can be expressed by using Van't
Hoff's equation. The temperature dependence of retention
stabilization effect may also exist due to the presence of
aromatic function in Alvimopan molecule.
9,10
can be expressed using Van't Hoff's equation.
Multiple
4
4
.3 | Experiments
type of interaction making contributions on the chiral
recognition. At lower temperature, the viscosity of the
mobile phase was high compared with higher
temperature; therefore, slow elution of the ALV isomer
observed at lower temperature and increased the
resolution and decreased USP plate count. Figure 3 shows
the dependence of resolution on the column oven temper-
ature, and Figure 4 shows the dependence of USP plate
count on the column oven temperature.
.3.1 | Experiment no. 1
During development, the initial experiment was per-
formed on Chiracel OD‐H with the combination of
0:50 v/v n‐hexane and isopropyl alcohol. Spiked chiral
isomers in ALV were injected. ALV and isomers peak
was eluted with 3 minutes and no resolution was found
between the ALV and isomers.
5
4.3.2 | Experiment no.2
4.5 | Elution order of ALV isomers
To resolve the ALV isomers, mobile phase combination
was changed to n‐hexane, ethanol, and isopropyl alcohol
and performed the experiment using CHIRALPAK‐IC col-
umn and methanol used as diluent. The ALV isomers were
not resolved well and asymmetric peak shape was found.
During chiral method development, order of elution of
ALV isomers was monitored by injecting individual pure
4.3.3 | Experiment no.3
This experiment was performed using mobile phase con-
sist of n‐hexane: isopropyl alcohol, ethanol, and
diethylamine (650:200:150:1 v/v) and CHIRALPAK‐IC
(250 x 4.6 mm, 5 μm) column. Resolution was found
between the ALV and isomers peak but the peak shape
was asymmetric (split peak). Upon literature survey, it
was understood that the cause of peak splitting was differ-
ence in the polarity of the diluent (methanol) and mobile
phase solvents. Hence, the diluent was changed from
methanol to combinaion of methanol and isopropyl alco-
hol (20:80 v/v). To improve the peak symmetry/peak
shape and resolution between isomers, further experi-
ments were carried out with modifying the camposition
of mobile phase. The resolution between the peaks
improved with increasing the volume of diethylamine in
the mobile phase because it improves the resolution, peak
FIGURE 3 Graphical presentation of effect of the column oven
temperature on resolution of alvimopan stereoisomers
10
symmetry, and plate number.
The mobile phase combination, n‐hexane: isopropyl
alcohol, ethanol, and diethylamine (650:200:150:5 v/v)
and keeping chromatographic conditions same as per
the above, obtained a good resolution between ALV and
isomers and peak symmetry.
4.4 | Study of column oven temperature
Thorough literature survey reveals that the improvement
of enantiomeric resolution at sub‐ambient temperature
by using polysaccharide based CSPs. During optimization
FIGURE 4 Graphical presentation of effect of the column oven
temperature on plate count of alvimopan stereoisomers
9

DHEKALE ET AL.
5
isomer (Chiral purity mentioned in Table 1) because the
elution order of isomers are most crucial for chiral
method development. A typical system suitability chro-
matogram of ALV chiral separation is shown in Figure 5.
The system suitability of the method was monitored by
using mobile phase as n‐hexane, isopropyl alcohol, etha-
nol, and diethylamine in the ratio of 650:250:100:5 (v/v)
TABLE 2 Summarized data of system suitability
Peak
Name of Isomer
Resolution
Tailing Factor
Peak‐1
Peak‐2
Peak‐3
Peak‐4
2S,3S,4S
2R,3R,4R
2S,3R,4R
2R,3S,4S
NA
1.23
1.03
1.33
1.25
2.97
1.42
2.56
−
1
with flow rate of 1.0 mL min and column oven tempera-
ture 30°C on CHIRALPK IC column (250 × 4.6 mm, 5 μm).
2S,3S,4S, 2R,3R,4R, 2R,3S,4S isomer in the precision study
at 50% level obtained was 0.97%, 1.29%, and 0.65%
whereas at 100% level obtained was 1.23%, 0.96%, and
4.6 | Method validation
0
.71%, respectively, which confirms the good precision
Blank solution (20:80 methanol: isopropyl alcohol v/v)
was injected to ensure the absence of any peak at reten-
tion time of ALV‐2S,3R,4R, ALV‐2R,3R,4R, ALV‐
of the method. The resolution between ALV‐2R,3R,4R
and ALV‐2S,3R,4R (n = 6) obtained was 1.4.
2
S,3S,4S, ALV‐2R,3S,4S ALV isomer peak. Straight base-
line indicated that there was no carryover or any other
peak at retention time of analytes/isomers peak.
4
.6.3 | Limit of detection and limit of
quantification
Limit of detection and limit of quantification of the
method was calculated based on the signal to noise ratio
The retention time region of ALV isomers in blank was
considered for baseline noise measurement by using
empower 2. For 2S,3S,4S, 2R,3R,4R, 2R,3S,4S isomers,
limit of quantification were found 0.136, 0.285, and
4
.6.1 | System suitability
System suitability criteria were evaluated to ensure the
satisfactory performance of the method as well as ensure
the analytical method can be used within established
chromatographic conditions. System suitability results
are summarized in Table 2.
0
.153 μg/mL, respectively, and limit of detection were
found 0.048, 0.109, and 0.061 μg/mL, respectively. The
RSD for the area of the 2S,3S,4S, 2R,3R,4R, 2R,3S,4S iso-
%
4.6.2 | Precision study
mer in the precision study at LOQ level was 1.86, 1.41,
and 1.56%, respectively.
Precision study of the method was performed. The per-
centage relative standard deviation of the area of the
4.6.4 | Linearity study
TABLE 1 Chiral purity of alvimopan isomers
All the 3 isomers of the ALV were prepared in the range
from LOQ to 150% of the target concentration 0.15% was
prepared. Linear curve was plotted with a peak area of
ALV‐2R,3R,4R, ALV‐2S,3S,4S, ALV‐2R,3S,4S isomer
against its concentration. The correlation coefficient
obtained was 0.9991, 0.9995, and 0.9999, respectively.
Sr. No.
Isomer Name
Chiral Purity (% area)
1
2
3
4
2S,3R,4R
2R,3R,4R
2S,3S,4S
2R,3S,4S
99.2
68.5
99.2
99.6
4.6.5 | Accuracy
Accuracy study for ALV isomers was evaluated in tripli-
cate at 3 concentration levels (0.10%, 0.15% and 0.20%
with respect to ALV). The percentage recovery of
2S,3S,4S, 2R,3R,4R, 2R,3S,4S isomers in the samples at
LOQ level was 100.6, 98.0 and 100.9%, respectively, at
5
9
0% level 99.1, 98.8, 98.7%, respectively, at 100% level
8.7%, 99.3%, and 99.9%, respectively, and at 150% level
FIGURE
alvimopan chiral separation
5
Typical system suitability chromatogram of
99.6, 98.4, and 101.2%, respectively. From the above
results, the method was found accurate.

6
DHEKALE ET AL.
1
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| CONCLUSION
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A novel, simple chiral analysis HPLC method was
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50:250:100:5 (v/v) as mobile phase within 35 minutes
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ORCID
Nitin H. Dhekale http://orcid.org/0000-0003-3976-3307
6. ICH harmonized tripartite guideline, validation of analytical
procedures: text and methodology, Q2 (R1). Current step 4 ver-
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rated in November 2005). http://www.ich.org/fileadmin/
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Yagnakirankumar Komaravolu
002-8914-7265
http://orcid.org/0000-
0
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DB, Gore AH, Komaravolu Y, Hima Bindu K,
Kolekar GB. Stereoselective HPLC separation of
alvimopan on cellulose‐based immobilized
polysaccharide as a chiral stationary phase.
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