Synthesis, isolation and characterization of process-related impurities in salbutamol sulphate

Article abstract of DOI:10.1155/2011/230231

Three known and one unknown impurities in salbutamol sulphate 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 synthesized and characterized the known and unknown imputities. Copyright E-Journal of Chemistry 2004-2011.

Full text of DOI:10.1155/2011/230231

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
2011, 8(4), 1720-1727
http://www.e-journals.net
Synthesis, Isolation and Characterization of
Process-Related Impurities in Salbutamol Sulphate
YOGESH KUMAR SHARMA*, DAU DAYAL AGARWAL, SUDESH BHURE,
RAJENDRA NATH MUKHARJEE, PRAMOD KUMAR SAHU
and SANTOSH BHARDWAJ
*Department of Industrial Chemistry, Jiwaji University
Gwalior-475011, Madhya Pradesh, India
Pharmasolve Specialities (I) Pvt. Ltd. Mumbai, India
sharmayogesh9182@rediffmail.com
Received 14 March 2011; Accepted 20 April 2011
Abstract: Three known and one unknown impurities in salbutamol sulphate
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 synthesized and characterized the
known and unknown imputities.
Keywords: Salbutamol sulphate, Impurities, Synthesis, Isolation and Characterization
Introduction
Salbutamol sulphate is a relatively selective 2-adrenergic agonist and is used as a
bronchodilator. The chemical name of salbutamol sulfate is 1-(4-hydroxy-3hydroxymeth
ylphenyl)-2-(tert-butylamino) ethanol sulfate (2:1) (salt) salbutamol sulfate is indicated for
the prevention and relief of bronchospasm with reversible obstructive airway disease
(asthma) and for the prevention of exercise induced bronchospasm. It is also indicated for
the management of acute attacks of bronchospasm. Salbutamol sulfate acts by stimulating
the adenyl cyclase enzyme, which catalyzes the formation of cyclic-3,5-adenosine
monophosphate (cyclic AMP) from adenosine triphosphate (ATP)1-2
.
There are several process impurities/related substances associated with the
manufacture of salbutamol sulfate. Different process related impurities have been
observed with various synthetic routes and/or manufacturing processes. Ten of the
known salbutamol related substances have been mentioned in Europian Pharmacopoeia
6.03. All these impurities are never present together while we are preparing bulk drug or
preparing formulation.

1721
D. AGARWAL et al.
The Europian Pharmacopoeia3 specifies that individual impurity is not more than 0.3%
and total impurity is not more than 1.0%. The British Pharmacopoeia4 specifies that the
related impurities which is determined by TLC, to be maximum of 0.5%. The united states
pharmacopoeia5 requires that the sum of the impurities in salbutamol sulphate should not be
greater than 2.0%.
During the process development of salbutamol sulphate three known and one unknown
impurities were identified in the analysis of different batches whose percent area ranged
from 0.05-0.1% by HPLC3-5. 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
authorities6 and all the impurities should be at the level of >0.1% must identify and
characterized.
A literature search revealed that only analytical procedure7-9 is available but nobody
has reported synthesis, isolation and characterization of impurities in the purified form
starting from salbutamol sulphate. The present communication involves the isolation or
preparation of impurities and characterized by chromatographic and spectroscopic
techniques10.
Experimental
The investigated samples of bulk salbutamol sulphate were received from Pharmasolve
Specialities (I) Pvt. Ltd. Mumbai.
High performance liquid chromatography (HPLC)
An in-house LC method was developed for the reaction monitoring of salbutamol impurities.
A Shimadzu model LC2010CHT equipped with a UV detector was used. The
chromatographic condition optimized were ODS, 50×4.6 mm, 3 µ column with a mobile
phase -A consisting 950:50(v/v) of 0.02 M KH2PO4 buffer and acetonitrile and mobile
phase -B consisting 600:400(v/v) of 0.02 M KH2PO4 buffer and acetonitrile with a timed
gradient programme of T/%B:0/0,3/0,8/50,15/50,16/0,20/0. Detection was carried out at 220 nm
and the flow rate 1.0 mL/min. Data was recorded by using LC solutions software.
High performance liquid chromatography (preparative)
An in-house LC method was developed for the isolation of salbutamol sulphate 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 mm i.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, 5/5, 10/10, 15/20, 20/50, 25/50, 30/80, 35/80, 36/0,
40,0. Detection was carried out at 225 nm and the flow rate was 15.0 mL/min. Data was
recorded by using empower software.
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
1
The NMR experiments were performed on Bruker avance II 400 MHz .The H chemical
shift values were reported on the δ scale in ppm, relative to TMS (δ=0.00) and the 13C chemical

Synthesis, Isolation and Characterization of Process-Related Impurities
1722
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 of impurity 1, 2 and 3
Salbutamol base (2 g) was charged into one neck round bottom flask with magnetic stirrer.
Methanol (50 mL) was added and stirring was started. NaOH (2 g) was added very slowly
and again methanol (50 mL) was introduced to dissolve the base completely. The contents
were stirred slowly with heating. The samples were checked using HPLC. The HPLC
analysis showed 3 peaks separately which confirm the presence of three different
impurities. The peaks were purified using preparative HPLC and marked as fraction-1,
2 and 3 (Scheme 1).
CH3
H
H
O
CH3
CH3
N
CH3
HO
OH
OH
H
N
CH3
CH3
H
N
H
OH
CH3
CH3
CH3
CH3OH
NaOH
H
OH
HO
H3C
H3C
CH3
N
HO
O
CH3
OH
OH
CH3
H
N
H
O
CH3
CH3
CH3
HO
CH3
Scheme 1. Synthesis of impurity 1, 2 and 3
Synthesis of impurity 4
Salbutamol base 2 g was charged with 50 mL acetone into one neck round bottom flask with
magnatic stirrer K2CO3 (1.2 g) was added and heating started with stirring. To this
benzylchloride 1.1 g was added and contents refluxed for 12 h. After 12 h the reaction mass
was checked using HPLC. The HPLC analysis showed 1 peak separately which confirm the
presence of one different impurity. This peak was purified using preparative HPLC and
marked as fraction-4 (Scheme 2).

1723
D. AGARWAL et al.
H
H
OH
CH3
CH3
N
H
OH
CH3
Acetone, C6H5CH2Cl
K2CO3,Heat
N
CH3
CH3
HO
CH3
HO
OH
OH
Scheme 2. Synthesis of impurity 4
Results and Discussion
Structure elucidation of impurity 1(Impurity-1)
Sample was analyzed by HPLC and its purity was found to be 93.14%, molecular
weight of salbutamol sulphate impurity-1 is 253.34 which is 14 mass unit higher than
that of salbutamol sulphate. The ESI mass spectrum of sample gave a protonated
molecular ion at m/z 254.1 and deprotonated molecular ion at m/z 252.6 which is the
same as salbutamol sulphate impurity-127-30, IR spectrum displayed characteristic
absorptions at 3398.89 & 2982.02,2934.06 cm-1 corresponding to >NH and aromatic
>CH stretching .The peaks at 1509.95 & 1452.92 cm-1 in IR spectrum is indicative of
1
>C=C< ring stretching, the chemical shift of the H signals showed an additional signal
at 3.15 ppm as singlet, which could be attributed to a methoxy group present in
impurity-1, 13C signals showed an additional signal at 49.84 ppm, which could be
attributed to a methoxy group present in impurity-1. HPLC purity 93.14%, mass +ve m/z
254.1 and -ve m/z 252.6 (Table 1), IR (KBr): 3398.89, 2982.02, 2934.06, 1509.66,
1452.92 (Table 1); 1H NMR: 1.09(s,9H), 1.21(d,1H), 2.58-2.80 (dd,2H), 4.17-
4.20(d,1H),6.98-7.00(dd,1H), 6.76-6.78(d,1H), 7.21-7.21(d,1H), 4.59 (s,2H), 3.15(s,3H)
(Scheme 3, Table 2); 13C NMR: 27.60, 58.85, 48.54, 82.05, 129.38, 125.21, 114.04,
153.71, 127.23, 125.24, 55.26, 49.84 (Scheme 4, Table 3).
Table 1. IR and mass spectral data of impurities
S.No.
1
Compound
Impurity-1
IR cm-1
3398.89(>N-H)
MS
+ve m/z 254.1
-ve m/z 252.6
2982.02,2934.06(Aromatic >C-H)
1509.66,1452.92(>C=C<)
3420.37 (>N-H)
+ve m/z 461.3
-ve m/z 459.5
2
3
4
Impurity-2
Impurity-3
Impurity-4
2973.72,2941.87(Aromatic >C-H)
1514.91,1478.62 (>C=C<)
3420.37 (>N-H)
+ve m/z 252.1
-ve m/z 251.6
2973.72,2941.87(Aromatic >C-H)
1514.91,1478.62 (>C=C<)
3428.93 (>N-H)
+ve m/z 330.2
2981.68,2862.63(Aromatic >C-H)
1500.88,1454.21 (>C=C<)

Synthesis, Isolation and Characterization of Process-Related Impurities
1724
11
CH3
O
11
4
H
OH
1
7
CH3
H
N
4
1
CH3
H
N
7
8
CH3
2
6
5
8
6
CH3
CH3
5
9
12
2
H
N
OH 11'
HO
4'
3
1'
CH3
3
H3C
9
9'
HO
10
6'
7'
10'
O
12
H3C
5'
2'
10
CH3
3'
OH
13
OH
12'
8'
Impurity 1
Impurity 2
1 5 '
11
CH3
O
1 6
1 5
1 4 '
H
4
1
CH3
H
7
1 1
4
1 4
N
H
O H
7
C H 3
8
6
CH3
1
N
5
8
6
C H 3
2
5
C H3
3
2
9
C H 3
3
HO
9
H O
12
1 2
10
1 0
CH3
13
O H
1 3
Impurity 3
Impurity 4
Scheme 3. 1H NMR assignment of impurities 1,2,3 & 4
Structure elucidation of impurity 2(Impurity-2)
Sample was analyzed by HPLC and its purity was found to be 87.93%, molecular weight of
salbutamol sulphate impurity-2 is 460.62 which is 221 mass unit higher than that of
salbutamol sulphate. The ESI mass spectrum of sample gave a protonated molecular ion at
m/z 461.3 and deprotonated molecular ion at m/z 459.5 which is the same as salbutamol
sulphate impurity-2, IR spectrum displayed characteristic absorptions at 3420.37 &
2973.72,2941.87 cm-1 corresponding to >NH and aromatic >CH stretching. The peaks at
1
1514.91 & 1478.62 cm-1 in IR spectrum is indicative of >C=C< ring Stretching, H signals
showed no additional signal observed but the multiplicity of signals were double this
confirm the structure of salbutamol sulphate impurity-2, 13C signals show no additional
signal but the multiplicity of signals were double. HPLC purity 87.93%, mass +ve m/z 461.3
and -ve m/z 459.5(Table 1), IR (KBr): 3420.37, 2973.72, 2941.87, 1514.91,1478.62
1
(Table 1); H NMR: 1.18-119(d,18H), 1.34(s, 2H), 2.71-2.84(dd, 4H), 3.15-3.40(m, 2H),
6.90-7.09 (m, 2H), 6.67-6.83(m, 2H), 7.28-7.56(m, 2H), 4.59-4.68(m, 4H), 10.05(s, 2H)
(Scheme 3, Table 2); 13C NMR: 26.04, 58.00, 48.98, 73.30, 132.81, 124.86, 113.92, 153.2,
124.75, 127.99 (Scheme 4, Table 3)
11
OH
6
H
N
1
14
CH3
O
CH3
8
7
H
N
1
CH3
9
CH3
H
4
8
5
2
7
CH3
12
6
9
CH3
H
N
OH
6'
10
HO
13
4
3
5
11
13
1'
H3C
2
12'
CH3
3
12
O
HO
10
13
H3C
4'
7'
8'
11'
11
5'
2'
CH3
3'
OH
10'
OH
9'
Impurity 1
Impurity 2

1725
D. AGARWAL et al.
17
18
16
15
CH3
O
H
N
1
CH3
H
17'
14
8
15
16'
C H 3
7
H
O H
6
9
CH3
8
1
4
N
5
2
10
6
C H 3
4
7
9
CH3
3
5
12
2
HO
10
13
C H 3
3
11
H O
11
12
CH3
14
13
O H
Impurity 3
Impurity 4
Scheme 4. 13C NMR assignment of impurities 1,2,3 & 4
Structure elucidation of impurity 3(Unknown impurity)
Sample was analyzed by HPLC and its purity was found to be 96.72%, molecular weight of
salbutamol sulphate impurity-3 is 251.37 which is 12 mass unit higher than that of salbutamol
sulphate. The ESI mass spectrum of sample gave a protonated molecular ion at m/z 252.1 and
deprotonated molecular ion at m/z 250.6.which confirm that molecular weight is 251. Of
salbutamol sulphate impurity-3, IR spectrum displayed characteristic absorptions at 3433.68 &
2972.11,2929.11 cm-1 corresponding to >NH and aromatic >CH stretching. The peaks at
1
1514.60 & 1478.83 cm-1 in IR spectrum is indicative of >C=C< ring stretching, H signals
showed additional signals at 2.56 & 1.10-1.13 ppm as singlet & triplate, which could be
attributed to a methoxy & methyl group present in impurity-3, 13C signals showed additional
signal at 15.05 & 78.52 ppm, which could be attributed to a methoxy & methyl group present
in impurity-3. HPLC purity 97.72%, mass +ve m/z 252.1 and -ve m/z 250.6 (Table 1), IR
(KBr): 3420.37, 2973.72, 2941.87, 1514.91, 1478.62(Table 1); 1H NMR: 1.07(s, 9H),
1.19(s,1H), 2.73-2.78(dd, 2H), 4.20-4.23(dd,1H), 6.89-6.92(dd, 1H), 6.73-6.75(d, 1H), 6.96-
6.97(d, 1H), 3.24-3.36(m, 2H), 2.56(s, 3H), 9.06(s, 1H), 1.10-1.13(t, 3H) (Scheme 3, Table
2); 13C NMR: 28.45, 63.04, 49.28, 80.92, 130.81, 124.58, 114.18, 154.71, 123.60, 128.57,
15.05, 78.52 (Scheme 4, Table 3).
Table 2. 1H NMR spectral data of impurities 1, 2, 3 & 4
Impurity 1
Impurity 2
Impurity 3
δ, ppm
Impurity 4
δ, ppm
1.21
4.66
S.No.
1,2,3
4
δ, ppm
1.09
1.21
H
δ, ppm
1.18-119
1.34
H
18H
2H
4H
2H
2H
2H
2H
4H
H
H
9H
1H
1.07
1.19
9H
1H
2H
1H
1H
1H
1H
2H
3H
1H
3H
9H
2H
2H
1H
1H
1H
1H
2H
5
2.58-2.80 2H
4.17-4.20 1H
6.98-7.00 1H
6.76-6.78 1H
7.21-7.21 1H
2.71-284
3.15-3.40
6.90-7.09
6.67-6.83
7.28-7.56
4.59-4.68
2.73-2.78
4.20-4.23
6.89-6.92
6.73-6.75
6.96-6.97
3.24-3.36
2.56
2.74-2.83
4.77-4.78
7.22-7.24
6.88-6.90
7.29-7.30
5.09
6
7
8
9
10
11
12
13
4.59
3.15
2H
3H
10.05
2H
9.06
1.10-1.13
7.31-7.36
7.38-7.43
7.47
2H
2H
1H
14,14
15,15
16
̍
̍

Synthesis, Isolation and Characterization of Process-Related Impurities
1726
Table 3. 13C NMR spectral data of impurities 1,2,3 & 4
Carbon assignment
Impurity 1
Impurity 2
Impurity 3
Impurity 4
δ, in ppm
26.04
27.60
58.85
48.54
82.05
129.38
125.21
114.04
28.45
63.04
27.04
52.60
1,2,3
58.00
4
5
6
7
8
9
48.98
49.28
69.45
73.30
80.92
70.26
132.81
124.86
113.92
130.81
124.58
114.18
135.03
125.03
125.19
10
11
12
13
14
15
153.71
127.23
125.24
55.26
153.2
124.75
127.99
58.00
154.71
123.60
128.57
63.04
110.99
154.49
130.40
58.78
49.84
15.05
78.52
49.64
137.09
128.31
127.02
127.61
16,16
17,17
18
̍
̍
Structure elucidation of impurity 4(Impurity-4)
Sample was analyzed by HPLC and its purity was found to be 99.45%, molecular weight of
salbutamol sulphate impurity-4 is 329.44 which is 90 mass unit higher than that of
salbutamol sulphate. The ESI mass spectrum of sample gave a protonated molecular ion at
m/z 330.2 which is the same as salbutamol sulphate impurity-4, IR spectrum displayed
characteristic absorptions at 3428.93 & 2981.68,2862.63 cm-1 corresponding to >NH and
aromatic >CH stretching. The peaks at 1500.88 & 1454.21 cm-1 in IR spectrum is indicative
1
of >C=C< ring Stretching, H signals showed additional signals at 7.31-7.36,7.38-7.43
& 7.47 ppm as 2 multiplet & singlet which could be attributed to a aromatic ring as reported
in impurity E, 13C signals showed additional signal at 49.64,137.09,128.31,127.02& 127.61 ppm,
which could be attributed to a additional aromatic ring present in impurity-4. HPLC purity
99.45%, mass +ve m/z 330.2 (Table 1), IR (KBr): 3428.93, 2981.68, 2862.63, 1500.88,
1
1454.21 (Table 1); H NMR: 1.21(s, 9H), 4.66(s, 2H), 2.74-2.83(dd, 2H), 4.77-4.78(d, 1H),
7.22-7.24(d, 1H), 6.88-6.90(d, 1H), 7.29-7.30(d, 1H), 5.09(s, 2H), 7.31-7.36(m, 2H), 7.38-
7.43(m, 2H), 7.47(s, 1H) (Scheme 3, Table 2) ; 13C NMR: 27.04, 52.60, 69.45, 70.26,
135.03, 125.03, 125.19, 110.99, 154.49, 130.40, 58.78, 49.64, 137.09, 128.31, 127.02,
127.61 (Scheme 4, Table 3).
Conclusion
This research paper describes the synthesis, isolation and structure elucidation of process
related impurities in salbutamol sulphate. The impurities were separated by reverse phase
chromatographic technique, further isolated by semipreparative liquid chromatography. The
isolated impurities were characterized using spectroscopic techniques. This isolated
impurities were used for mass balance of salbutamol sulphate technical material. The
synthesis of impurities was also discussed in brief.

1727
D. AGARWAL et al.
Acknowledgment
We are grateful to Mr.Sanjay Singh Rathore, R.ganga Prasad, Praveen Sharma and Mr.
Avtar Singh, Shophisticated Analytical Instrumentation Facility, Chandigarh and Punjab
University, Chandigarh for spectral analytical data.
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