Synthesis, isolation and characterization of process-related impurities in oseltamivir phosphate

Article abstract of DOI:10.1155/2012/327351

Three known impurities in oseltamivir phosphate bulk drug at level 0.1% (ranging from 0.05-0.1%) were detected by gradient reverse phase high performance liquid chromatography. These impurities were preliminarily identified by the mass number of the impur

Full text of DOI:10.1155/2012/327351

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
2012, 9(1), 113-120
http://www.e-journals.net
Synthesis, Isolation and Characterization of Process-
Related Impurities in Oseltamivir Phosphate
YOGESH KUMAR* SHARMA, DAU DAYAL AGARWAL, SUDESH BHURE, SANJAY
SINGH RATHORE, CHAKRAVIR RAWAT and RAJENDRANATH MUKHARJEE
*Department of Industrial Chemistry, Jiwaji University
Gwalior-475011, Madhya Pradesh, India
Pharmasolve Specialities (I) Pvt. Ltd. Mumbai, India
sharma_yogesh9182@rediffmail.com
Received 28 May 2011; Accepted 21 July 2011
Abstract: Three known impurities in oseltamivir phosphate bulk drug at level
0.1% (ranging from 0.05-0.1%) were detected by gradient reverse phase high
performance liquid chromatography. These impurities were preliminarily
identified by the mass number of the impurities. Different experiments were
conducted and finally the known impurities were synthesized and
characterized.
Keywords: Oseltamivir phosphate, Impurities, Synthesis, Isolation and Characterization
Introduction
Oseltamivir is an ester prodrug, which is rapidly and extensively hydrolysed in vivo to its
active metabolite oseltamivir carboxylate, a potent and selective inhibitor of influenza virus
neuraminidase1 is considered the leading currently available antiviral to counter a serious
epidemic or pandemic outbreak of influenza2,3. The current concerns over avian influenza A
(H5N1) have created an increased demand for this drug. Pharmaceutical counterfeiting is a
well recognized global health problem with a particular impact in developing countries
where drug-regulatory systems are weak or ineffective4. There have been many alarming
reports lasting recent years of counterfeit antimalarials, antibiotics, hormones and steroids,
analgesics and antipyretics, anti-asthma and anti-allergy drugs5-11
.
Oseltamivir phosphate is a white crystalline solid with the chemical name (3R,4R,5S)-
4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylic acid, ethyl ester,
phosphate (1:1). The chemical formula is C16H28N2O4 (free base). The molecular weight of
oseltamivir is 312.4 and 410.4 for oseltamivir phosphate salt12. The structural formula is as
follows:

114
Y. K. SHARMA et al.
CH3
O
O
O
CH3
CH3
HN
NH2
O
CH3
There are several process impurities/related substances associated with the manufacture
of oseltamivir phosphate. Different process related impurities have been observed with
various synthetic routes and/or manufacturing processes. Six of the known oseltamivir
phosphate related substances have been mentioned in International Pharmacopoeia. All these
impurities are never present together while we are preparing bulk drug or preparing
formulation.
The International Pharmacopoeia12 specifies that individual impurity is not more than
0.1% and total impurity is not more than 0.7%. During the process development of
oseltamivir phosphate three known impurities were identified in the analysis of different
batches whose percent area ranged from 0.05-0.1% by HPLC12. A comprehensive study has
been carried out to isolate or to prepare and characterize these impurities. To the best of our
knowledge, synthesis rout of impurities is novel and not reported in literature. Due to the
importance of regulatory authorities13 all the impurities which are at the level of >0.1% must
be identified and characterized.
A literature search revealed that only analytical procedure14-18 is available but nobody has
reported synthesis, isolation and characterization of impurities in the purified form starting
from oseltamivir phosphate. The present communication involves the isolation or preparation
of impurities and characterization by chromatographic and spectroscopic techniques19.
Experimental
The investigated samples of bulk oseltamivir phosphate were received from pharmasolve
specialities (I) Pvt. Ltd. Mumbai.
High performance liquid chromatography (HPLC)
An LC method was used for monitoring the reaction of oseltamivir impurities. A shimadzu
model LC2010CHT equipped with a UV detector was used. The chromatographic conditions
optimized were ODS, 50×4.6 mm, 3 µ column with a mobile phase -A consisting 900:100(v/v)
of 0.05 M KH2PO4 buffer pH 6.0 and acetonitrile and mobile phase-B consisting
100:900(v/v) of 0.05 M KH2PO4 buffer pH 6.0 and acetonitrile with a timed gradient
programme of T/% B:0/20,3/50, 8/100, 15/100, 16/20, 20/20. Detection was carried out at
220 nm and the flow rate 1.5 mL/min. Data was recorded by using LC solutions software.
High performance liquid chromatography (preparative)
An inhouse LC method was developed for the isolation of oseltamivir impurities. A waters
LC controller equipped with a UV detector was used. The Prep-HPLC method uses a C18
column (YMC Pack Pro.C-18,250 mm* 20 mmi.d., 10 um particle size) with a mobile phase
A consisting of 0.02 M ammonium acetate and mobile phase B consisting acetonitrile timed
gradient programme of T/%B: 0/0, 5/0, 8/80, 15/80, 16/0, 20/0. Detection was carried out at
207 nm and the flow rate was 15.0 mL/min. Data was recorded by using Empower software.

Synthesis, Isolation and Characterization of Process-Related Impurities
115
Mass spectrometry
Electrospray ionization mass spectroscopy was performed using an ion trap mass
spectrometer (Model 6310 agilent). The positive and negative electrospray MS data was
obtained by switching the capillary voltage between n+ 5000 and -4500V respectively.
NMR spectroscopy
The NMR experiments were performed on Bruker avance II 400 MHz. The 1H chemical shift
values were reported on the δ scale in ppm, relative to TMS (δ=0.00) and the 13C chemical
shift values were reported relative to CDCl3 (δ=77.00 ppm) and DMSO, d6 (δ=39.50 ppm) as
internal standards. 1H and 13C experiment was run using a mixing time of 1000 ns.
FT-IR spectroscopy
The IR spectra were recorded in the solid state as KBr dispersion medium using Perkin
Elmer spectrum 100 FT-IR spectrophotometer.
Synthesis of impurities
Synthesis if impurity 1 and 2
Oseltamivir base (1 g) was charged with 100 mL methanol into a double necked round
bottom flask with magnetic stirrer. Methanolic sodium hydroxide was added drop wise and
the reaction mass was heated very carefully and the reaction mass was refluxed at methanol
boiling point. The reaction was monitored after every 1 h by HPLC. After completion of
reaction the reaction mass was neutralised with dilute HCl. The HPLC analysis shown 2
peaks separately which confirm the presence of two different impurities. Both peaks were
purified using preparative HPLC and marked as fraction-1 and 2 (Scheme 1). The salt was
removed by preparative HPLC after desalting and the sample was kept for freeze drying.
CH3
O
O
OH
CH3
H2N
HN
O
CH3
CH3
O
Impurity-1(A)
O
CH3OH
Heat + NaoH
O
CH3
CH3
HN
NH2
CH3
O
CH3
O
Oseltamivir
O
OH
CH3
HN
NH2
O
CH3
Impurity-2(B)
Scheme 1. Synthesis if impurity 1 and 2

116
Y. K. SHARMA et al.
Synthesis of impurity 3
Oseltaivir base (1 g) was charged with 100 mL methanol into a double necked round bottom
flask with megnatic stirrer. To this methanolic sodium hydroxide was added and the reaction
mass was stirred at room temperature. The reaction was monitored after every 1 h by HPLC.
The HPLC analysis had shown 1 peak which confirms the presence of one different
impurity. This peak was purified using preparative HPLC and marked as fraction-3 (Scheme 2).
After completion of reaction the reaction mass was neutralised with dilute HCl. The salt was
removed by preprative HPLC after desalting the sample was kept for freeze drying.
CH3
CH3
O
O
O
CH3OH
O
CH3
O
CH3
O
CH3
NaoH
CH3
HN
HN
NH2
O
CH3
NH2
O
CH3
Oseltamivir
Impurity-3(D)
Scheme 2. Synthesis if impurity 3
Results and Discussion
Structure elucidation of impurity 1(Impurity-A)
Sample was analyzed by HPLC and its purity was found to be 94.13%, molecular weight of
oseltamivir phosphate impurity-A is 284.36,28 mass unit less than that of oseltamivir
phosphate. The ESI mass spectrum of sample gave a protonated molecular ion at m/z 285.1,
which is the same as oseltamivir phosphate impurity-A, IR spectrum displayed characteristic
absorptions at 3430.01 & 2968.79,2939.87 cm-1 corresponding to >NH and aliphatic >CH
stretching. The peaks at 1642.69 & 1200.32 cm-1 in IR spectrum are indicative of >C=O
1
stretching & O-C stretching, H signals showed the absence of signals at 4.11-4.19 &
1.20-1.24 ppm was observed as multiplet & triplet, which could be attributed to a ethyl
group absent in impurity-A, but a significant change observed in the position of 1C & 5C
protons which is due to change double bond position in impurity-A, 13C signals showed
absence of signals at 60.52 & 14.02 ppm, which could be attributed to a ethyl group absent in
impurity-A, but a significant change observed in the position of 1C & 5C carbons which is
due to change double bond position in impurity-A. HPLC purity 94.13%, mass +ve m/z
1
285.1 (Table 1), IR (KBr): 3430.01, 2968.79,2939.87, 1642.69 & 1200.32 (Table 1); H
NMR: 2.03-2.69 (dd,2H), 3.30 (s,1H), 3.54-3.56 (d,1H), 3.95 (m*,1H), 6.30 (s,1H), 8.11
(s, 1H), 1.84 (s, 3H), 2.91 (s,1H), 1.39 (s,4H), 0.78-0.83 (d,6H) (Scheme 3, Table 2); 13C
NMR: 32.72, 75.85, 55.55, 49.70, 134.94, 131.19, 169.97, 23.09, 80.58, 25.11, 25.74, 8.91,
9.46 (Scheme 4, Table 3).
Structure elucidation of impurity 2(Impurity-B)
Sample was analyzed by HPLC and its purity was found to be 92.61%, molecular weight of
oseltamivir phosphate impurity-B is 284.36,28 mass unit less than that of oseltamivir
phosphate. The ESI mass spectrum of sample gave a protonated molecular ion at m/z 285.0,
which is the same as oseltamivir phosphate impurity-B, IR spectrum displayed characteristic
absorptions at 3419.69 & 2967.75,2939.35 cm-1 corresponding to >NH and aliphatic >CH

Synthesis, Isolation and Characterization of Process-Related Impurities
117
stretching. The peaks at 1646.68 & 1200.14 cm-1 in IR spectrum are indicative of >C=O
1
stretching & O-C stretching, H signals showed the absence of signals at 4.11-4.19 &
1.20-1.24 ppm was observed as multiplet & triplet, which could be attributed to a ethyl group
absent in Impurity-B, 13C signals showed absence of signals at 60.52&14.02 ppm, which could
be attributed to a ethyl group absent in Impurity-B. HPLC purity 92.61%, mass +ve m/z
285.0 (Table 1), IR (KBr): 3419.69, 2967.75,2939.35, 1646.68 & 1200.14 (Table 1); 1H NMR:
6.19 (s, 1H), 3.38 (s, 1H), 3.83-3.85 (d,1H), 3.34-3.36 (m,1H), 2.53-2.60 (dd,2H), 1.83
(s, 3H), 3.26-3.29 (m,1H), 1.33-1.42 (m, 4H), 0.76-0.85 (m, 6H) (Scheme 3,Table 2); 13C
NMR: 137.41, 76.46, 57.97, 50.35, 35.38, 129.20, 169.49, 170.98, 22.99, 80.26, 25.19,
25.82, 8.97, 9.51 (Scheme 4, Table 3).
Table 1. IR and mass spectral data of impurities 1, 2 & 3
IR cm-1
3430.01 (>N-H)
S.No. Compound
MS
1
2
3
Impurity-1 2968.79,2939.87 (aliphatic >CH stretching)
1642.69 & 1200.32 (>C=O & O-C stretching)
3419.69 (>N-H)
Impurity-2 2967.75,2939.35 (aliphatic >CH stretching)
1646.68 & 1200.14 (>C=O & O-C stretching)
3434.02 (>N-H)
+ve m/z 285.1
+ve m/z 285.0
+ve m/z 299.1
Impurity-3 2966.54,2939.46 (aliphatic >CH stretching)
1648.06, 1249.80 (>C=O & O-C stretching)
12'
1 2 '
C H3
11
C H
3
1 1
O
O
1
4
1
11'
1 1 '
1 2
O
O
N
1 0
10
O H
9
OH
2
2
C H
3
C H3
4
9
5
12
5
3
6
H
2
3
H N
6
8
H N
O
N H2
O
8
C H3
7
C H
3
7
Impurity 1
Impurity 2
12'
CH3
11
O
1
11'
O
10
CH3
9
O
2
CH3
4
5
3
HN
6
NH2
O
8
CH3
7
Impurity 3
Scheme 3. 1H NMR assignment of impurities 1,2 & 3

118
Y. K. SHARMA et al.
Structure elucidation of impurity 3(Impurity-D)
Sample was analyzed by HPLC and its purity was found to be 97.37%, molecular weight
of oseltamivir phosphate impurity-D is 298.39,14 mass unit less than that of oseltamivir
phosphate. The ESI mass spectrum of sample gave a protonated molecular ion at m/z
299.1, which is the same as oseltamivir phosphate impurity-D, IR spectrum displayed
characteristic absorptions at 3434.02 & 2966.54,2939.46 cm-1 corresponding to >NH and
aliphatic >CH stretching. The peaks at 1648.06 & 1249.80 cm-1 in IR spectrum are
indicative of >C=O stretching & O-C stretching, 1H signals showed the absence of signals
at 4.11-4.19 & 1.20-1.24 ppm was observed as multiplet & triplet, which could be
attributed to a ethyl group absent in impurity-D, but an singlet observed at 3.70 ppm
which could be attributed to a methoxy group present in Impurity-D, 13C signals showed
absence of signals at 60.52 & 14.02 ppm, which could be attributed to a ethyl group
absent in Impurity-D, there was a new 13C signal at 48.95 ppm was observed which could
be attributed to a methoxy group present in impurity-D. HPLC purity 97.37%, mass +ve
m/z 299.1 (Table 1), IR (KBr): 3434.02, 2966.54,2939.46, 1648.06, 1249.80 (Table 1);
1H NMR: 6.65 (s, 1H), 3.63-3.68 (m, 1H), 4.15-4.17 (m,1H), 3.37-3.40 (m,1H), 2.21-2.78
(dd, 2H), 8.16-8.18 (d, 1H), 1.87 (s, 3H), 5.49 (b, 2H), 3.70 (s, 3H), 3.09-3.16 (m,1H),
1.34-1.48 (m, 4H), 0.76-0.85 (m, 6H) (Scheme 3, Table 2); 13C NMR: 138.60, 74.37,
53.74, 51.97, 29.87, 127.35, 170.28, 165.80, 23.19, 81.01, 24.97, 25.58, 8.88, 9.42, 48.95
(Scheme 4, Table 3).
12'
CH3
12'
11'
CH3
O
7
11'
1
4
O
7
11
12
O
10
1
OH
11
12
O
2
10
6
CH3
OH
2
6
5
CH3
3
H2N
4
5
3
HN
HN
O
8
8
NH2
O
CH3
9
CH3
9
Impurity 1
Impurity 2
12'
C H3
11'
O
7
1
11
O
C H3
10
O
13
2
6
5
C H3
12
4
3
H N
8
N H2
O
C H3
9
Impurity 3
Scheme 4. 13C NMR assignment of impurities 1,2 & 3

Synthesis, Isolation and Characterization of Process-Related Impurities
119
Table 2. 1H NMR spectral data of impurities 1, 2 & 3
Impurity 2
δ, ppm
6.19
Impurity 3
δ, ppm
6.65
Impurity 1
δ, ppm
2.03-2.69
3.30
S.No.
H
H
H
1H
1H
1H
1H
2H
1H
1H
1H
1H
2H
1H
3H
2H
3H
1H
4H
6H
1
2
2H
1H
1H
1H
1H
1H
3H
3.38
3.63-3.68
4.15-4.17
3.37-3.40
2.21-2.78
8.16-8.18
1.87
3.83-3.85
3.34-3.36
2.53-2.60
3
3.54-3.56
3.95
6.30
4
5
6
8.11
1.84
7
1.83
3H
8
9
10
5.49
3.70
2.91
1.39
1H
4H
6H
3.26-3.29
1.33-1.42
0.76-0.85
1H
4H
6H
3.09-3.16
1.34-1.48
0.76-0.85
11,11̍
12,12̍
0.78-0.83
Table 3. 13C NMR spectral data of impurities 1, 2 & 3
Impurity 3
Carbon assignment
Impurity 1
Impurity 2
δ, in ppm
138.60
74.37
1
2
32.72
75.85
137.41
76.46
53.74
3
55.55
57.97
51.97
4
49.70
50.35
29.87
5
134.94
131.19
169.97
169.97
23.09
35.38
127.35
170.28
165.80
23.19
6
129.20
169.49
170.98
22.99
7
8
9
80.26
81.01
10
80.58
25.1125.74
8.919.46
25.1925.82
8.979.51
24.9725.58
8.889.42
48.95
1111̍
1212'
13
Conclusion
This research paper describes the synthesis, isolation and structure elucidation of process
related impurities in oseltamivir phosphate. The impurities was separated by reverse phase
chromatographic technique, further isolated these impurities by semi preparative liquid
chromatography. The isolated impurities was characterized using spectroscopic techniques.
This isolated impurities was used for mass balance of oseltamivir phosphate technical
material. The synthesis of impurities was also discussed in brief.
Acknowledgment
We are grateful thanks to Mr. R. Ganga Prasad, Praveen Sharma and Mr. Avtar Singh,
Shophisticated Analytical Instrumentation Facility, Chandigarh and Punjab University,
Chandigarh for spectral analytical data.

120
Y. K. SHARMA et al.
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