Rapid titrimetric and spectrophotometric methods for salbutamol sulphate in pharmaceuticals using N-bromosuccinimide.

Abstract

One titrimetric and two spectrophotometric methods which are simple, sensitive and rapid are described for the assay of salbutamol sulphate (SBS) in bulk drug and in tablet dosage forms using N-bromosuccinimide (NBS) and two dyes, rhodamine-B and methylene blue, as reagents. In titrimetry, aqueous solution of salbutamol sulphate is treated with a measured excess of NBS in acetic acid medium and after the oxidation of SBS is complete, the unreacted oxidant is determined iodometrically. Spectrophotometric methods entail addition of a known excess of NBS in acid medium followed by the determination of residual oxidant by reacting with a fixed amount of either rhodamine B and measuring the absorbance at 555 nm (method A) or methylene blue and measuring the absorbance at 665 nm (method B). In all methods, the amount of NBS reacting corresponds to the amount of SBS content. Titrimetric method is applicable over 1.74 x 10(-4) - 8.68 x 10(-4) mol L(-1) range and the reaction stoichiometry is found to be 1:6 (SBS:NBS). In spectrophotometric methods, the absorbance is found to increase linearly with the concentration of SBS, which is corroborated by the correlation of coefficients of 0.9993 and 0.9988 for method A and method B, respectively. The systems obey Beer's law for 0.25-1.75 microg mL(-1) (method A) and 0.5-5.0 microg mL(-1) (method B). The calculated apparent molar absorptivity values were found to be 2.10 x 10(5) and 6.16 x 10(4) L mol(-1) cm(-1), for method A and method B, respectively. The limits of detection and quantification are also reported for both spectrophotometric methods. Intra-day and inter-day precision and accuracy for the developed methods were evaluated. The methods were successfully applied to the assay of SBS in tablet and capsule formulations and the results were statistically compared with those of a reference method. No interference was observed from common tablet adjuvants. The accuracy and reliability of the methods were further ascertained by recovery experiments via the standard-addition technique.

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87
Acta Pharm. 57 (2007) 87–98
10.2478/v10007-007-0007-7
Original research paper
Rapid titrimetric and spectrophotometric methods
for salbutamol sulphate in pharmaceuticals using
N-bromosuccinimide
KANAKAPURA BASAVAIAH1*
BANKAVADI CHIKKASWAMY SOMASHEKAR1
VEERAIAH RAMAKRISHNA2
1Department of Chemistry, University
of Mysore, Manasagangotri
Mysore-570 006, India
2Department of Pharmaceutical
Chemistry, Government College
of Pharmacy, Bangalore-27, India
Accepted December 4, 2006
One titrimetric and two spectrophotometric methods which
are simple, sensitive and rapid are described for the assay
of salbutamol sulphate (SBS) in bulk drug and in tablet
dosage forms using N-bromosuccinimide (NBS) and two
dyes, rhodamine-B and methylene blue, as reagents. In ti-
trimetry, aqueous solution of salbutamol sulphate is treat-
ed with a measured excess of NBS in acetic acid medium
and after the oxidation of SBS is complete, the unreacted
oxidant is determined iodometrically. Spectrophotometric
methods entail addition of a known excess of NBS in acid
medium followed by the determination of residual oxidant
by reacting with a fixed amount of either rhodamine B and
measuring the absorbance at 555 nm (method A) or methy-
lene blue and measuring the absorbance at 665 nm (me-
thod B). In all methods, the amount of NBS reacting corre-
sponds to the amount of SBS content. Titrimetric method is
applicable over 1.74 x 10–4– 8.68 ´ 10–4mol L–1range and
the reaction stoichiometry is found to be 1:6 (SBS:NBS). In
spectrophotometric methods, the absorbance is found to
increase linearly with the concentration of SBS, which is
corroborated by the correlation of coefficients of 0.9993 and
0.9988 for method A and method B, respectively. The sys-
tems obey Beer’s law for 0.25–1.75 mg mL–1(method A)
and 0.5–5.0 mg mL–1(method B). The calculated apparent
molar absorptivity values were found to be 2.10 x 105and
6.16 x 104L mol–1cm–1, for method A and method B, re-
spectively. The limits of detection and quantification are
also reported for both spectrophotometric methods. Intra-
day and inter-day precision and accuracy for the develo-
ped methods were evaluated. The methods were success-
fully applied to the assay of SBS in tablet and capsule for-
mulations and the results were statistically compared with
those of a reference method. No interference was ob-
served from common tablet adjuvants. The accuracy and
reliability of the methods were further ascertained by re-
covery experiments via the standard-addition technique.
Keywords: salbutamol sulphate, assay, titrimetry, spectro-
photometry, N-bromosuccinimide
* Correspondence, e-mail: basavaiahk@yahoo.co.in

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Salbutamol sulphate (SBS) whose structure is given in Fig. 1 is a selective b-2-ago-
nist antiasthmatic. Its primary action is to stimulate adenyl cyclase which catalyzes the
formation of cyclic adenosin monophosphate. The drug is official in European Pharmaco-
poeia (1), which describes a potentiometric titration in non-aqueous medium, British Phar-
macoeia (2) and Indian Pharmacopoeia (3). Some different methods of analysis have been
reported for the determination of SBS, including HPLC (4–6) and UV-spectrophotometry
(7, 8), but most of them require extensive sample preparation prior to the measurement
step, some are less sensitive and some other are relatively complicated in terms of assay
procedure or equipment required for analysis.
SBS in pharmaceuticals has been assayed by visible spectrophotometric methods ba-
sed on reactions such as redox (9, 10), reduction followed by chelation (11), oxidative co-
upling (12, 13), diazotization and coupling (14, 15), nitrosation (16), nitration (17), nitra-
tion followed by Meisenheiner complex formation (18) and charge-transfer complex for-
mation (19). However, many of these procedures suffer from some disadvantage, such as
poor sensitivity, heating or extraction step, critical working conditions or the use of or-
ganic solvents, and are hence unsatisfactory for routine analysis. The only visual titrime-
tric method (20) reported employs NBS as the oxidimetric titrant in the presence of po-
tassium bromide and using methyl red as indicator. However, the method is applicable
over a macro scale. Recently, Issa et al. (21) have reported a conductometric titration met-
hod using phosphotungstic and phosphomolybdic acids as titrants. Even these proce-
dures are time consuming and less sensitive.
This paper describes three assay methods for SBS in tablets and capsules. The meth-
ods employ N-bromosuccinimide as an oxidizing agent, rhodamine-B and methylene blue
dyes as reagents. The proposed methods have the advantages of being rapid and simple
88
K. Basavaiah et al.: Rapid titrimetric and spectrophotometric methods for salbutamol sulphate in pharmaceuticals using N-bromosucci-
nimide, Acta Pharm. 57 (2007) 87–98.
OH
NH
S
O
O
2
+
N
NH
O
O
Br
CH COOH
3
2
+ 6 HBr
6
O
O
6
+
+
2 C(CH ) NH
3 32
+
H SO
24
OH
OH
H C
3H C
3
CH3
OH
OH
OH
CHO
O
CHO
Fig. 1. Tentative reaction pathway.

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and are free from interferences from common tablet and capsule excipients. The results
obtained were closely comparable to those of a reported method, and recovery tests we-
re also found to be satisfactory.
EXPERIMENTAL
Apparatus
A Systronics Model 106 digital spectrophotometer (Systronics India Ltd., India) pro-
vided with 1-cm matched quartz cells was used for absorbance measurements.
Reagents and standard solutions. – All chemicals used were of analytical reagent grade
and solutions were made in distilled water.
NBS solution (0.01 mol L–1) was prepared by dissolving N-bromosuccinimide (SRL
Research Chemicals, India) in water with the aid of heat and standardized (22). The so-
lution was kept in an amber coloured bottle stored in a refrigerator and used for titri-
metry. It was diluted with water to get 70 and 150 mg mL–1NBS for use in spectrophoto-
metric methods.
Sodium thiosulphate solution (0.01 mol L–1, Sisco Chem. Industries, India) was pre-
pared in water and standardized. Potassium iodide (10%) and starch (1%) were prepa-
red in the usual way. A stock solution of rhodamine B (500 mg mL–1) was prepared by
dissolving the dye (s. d. Fine Chem. Ltd., India, dye content 80%) in water and filtered
using glass wool. The dye solution was diluted to 50 mg mL–1for method A. 400 mg mL–1
methylene blue was prepared by dissolving the dye (s. d. Fine Chem., dye content 70%)
in water and filtered. It was diluted to 40 mg mL–1with water and used in method B.
Pharmaceutical grade SBS (certified to be 99.7% pure) was procured from Cipla In-
dia Ltd. (India) and was used as received. A stock standard solution equivalent to 1 mg
mL–1SBS was prepared in water and was used in titrimetric work. This solution was ap-
propriately diluted with water to yield working concentrations of 5 and 10 mg mL–1for
use in method A and method B, respectively.
Assay procedures
Titrimetry. – A 10-mL aliquot of pure drug solution containing 1.0–5.0 mg of SBS
was accurately measured and transferred into a 100-mL titration flask. The solution was
acidified by adding 5 mL of 5 mol L–1acetic acid followed by the addition of 10 mL of
0.01 mol L–1NBS. The content was mixed well and the flask was kept aside for 15 min
under occasional swirling. Then, 5 mL of 10% potassium iodide was added to the flask
and the liberated iodine was titrated with 0.01 mol L–1sodium thiosulphate to a starch
end point. A blank titration was run under the same conditions.
Spectrophotometric method A. – Aliquots of pure SBS solution (0.5 to 3.5 mL, 5 mg
mL–1) were transferred into a series of 10-mL calibrated flask and the total volume was
adjusted to 4 mL with water. To each flask, 1 mL of 1 mol L–1hydrochloric acid, was
added, followed by 1 mL of NBS solution (70 mg mL–1). The contents were mixed and
89
K. Basavaiah et al.: Rapid titrimetric and spectrophotometric methods for salbutamol sulphate in pharmaceuticals using N-bromosucci-
nimide, Acta Pharm. 57 (2007) 87–98.

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the flasks were set aside for 10 min under occasional shaking. Finally, 1 mL of 50 mg
mL–1rhodamine B solution was added to each flask, diluted to the mark with water and
the absorbance of solution was measured at 555 nm against a reagent blank after 10 min.
Spectrophotometric method B. – Varying aliquots (0.5–5.0 mL) of standard 10 mg mL–1SBS
solution were accurately measured and delivered into a series of 10-mL calibrated flasks,
and the total volume was made up to 5.0 mL with water. To each flask, 1 mL each of 5
mol L–1hydrochloric acid and 150 mg mL–1NBS solution were added successively; the
flasks were let stand for 10 min under occasional shaking. Then, 1 mL of 40 mg mL–1
methylene blue solution was added to each flask, the volume was adjusted to the mark
with water and mixed. The absorbance of each solution was measured at 665 nm against
a reagent blank after 5 min.
In each spectrophotometric method, the concentration of the unknown was read from
the calibration graph or computed from the regression equation derived from Beer’s law
data.
The limits of detection (LOD) and quantification (LOQ) were calculated according
to the current ICH guidelines (23) using the following formulae:
LOD
SD
a
B
=
3.3
and
LOQ
SD
a
B
=10
where SDBis the standard deviation of seven reagent blank determinations and a is the
slope of the calibration curve.
Procedure for tablets
The following formulations containing SBS were purchased from local commercial
sources and used in the investigation: A, Asthalin tablets, (Cipla India Ltd.) each con-
taining 2/4 mg of SBS, B, Asmanil tablets, (Inga Pharmaceuticals, India) containing 2/4
mg of SBS per tablet, C. Bronkotab tablets, (GSK, India) containing 2/4 mg of SBS and D.
Salbu capsules (Upjohn Pharm., India) containing 4/8 mg of SBS per capsule.
Fifty to 100 tablets or the contents of 50 capsules were accurately weighed and pow-
dered. An amount of tablet/capsule powder equivalent to 100 mg of SBS was accurately
weighed into a 100-mL calibrated flask, 60-mL of water was added and shaken for 20
min. Then, the volume was diluted to the mark with water, the content was mixed well
and filtered using a Whatman No. 42 filter paper. The first 10-mL portion of the filtrate
was discarded and a convenient aliquot (e.g., 3 mL) of the subsequent portion was ana-
lyzed by titrimetry as described earlier. The filtrate (1 mg mL–1in SBS) was diluted with
water to obtain 5 and 10 mg mL–1concentrations and subjected to spectrophotometric
analysis as described above.
Recovery experiment. – To a fixed and known amount of drug in the tablet/capsule
powder (pre-analysed), pure SBS was added at three different levels, and the total was
found by the proposed methods, from which the percent recovery of pure drug added
was calculated.
90
K. Basavaiah et al.: Rapid titrimetric and spectrophotometric methods for salbutamol sulphate in pharmaceuticals using N-bromosucci-
nimide, Acta Pharm. 57 (2007) 87–98.

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Selectivity testing
A separate selectivity test was performed by applying the proposed methods to the
determination of SBS in a synthetic mixture consisting of SBS (100 mg), talc (250 mg),
starch (300 mg), lactose (30 mg), calcium gluconate (50 mg), calcium dihydrogenortho-
phosphate (20 mg), sodium alginate (70 mg) and magnesium stearate (100 mg), in the ratio
of 1:2.5:3.0:0.3:0.5:0.2:0.7:1. SBS was extracted with three 20 mL portions of water and fil-
tered. The filtrate was washed with water; the filtrate and washings were collected in a
100-mL calibrated flask and diluted to volume with water and mixed well. A convenient
aliquot of the extract was subjected to analysis by titrimetry. The extract was suitably dilut-
ed to get 5 and 10 µg mL–1solutions and was analyzed by spectrophotometric methods.
RESULTS AND DISCUSSION
The proposed methods are indirect and are based on the determination of surplus
NBS after allowing the reaction between SBS and NBS as oxidant to occur. In titrimetry,
the unreacted NBS is determined iodometrically, and in spectrophotometric methods, the
same is determined by reacting with a fixed amount of either rhodamine-B or methylene
blue. The latter methods make use of the bleaching action of NBS on either dye, the dis-
colouration being caused by the oxidative destruction of the dye.
Titrimetry
The reaction stoichiometry that was used for all calculations was found to be 1:6.
The relation between the amount of the drug and titration end point was examined. The
linearity is apparent from the calculated correlation coefficient of –0.9954 and suggests
that the reaction between SBS and NBS proceeds stoichiometrically in the ratio 1:6. Non-
stoichiometric results were obtained in a hydrochloric acid medium as well as sulphuric
acid medium. Quantitative results were obtained in an acetic acid medium and a 1.0 mol L–1
acetic acid concentration was found optimal although the reaction stoichiometry was un-
affected in the range 0.2–2.0 mol L–1acetic acid. The oxidation reaction was found to be
complete and quantitative in 15 min and contact times up to 25 min had no effect on the
stoichiometry and the results. Beyond 25 min and up to 60 min, a small amount of NBS
was consumed but without producing any definite stoichiometry. Hence, it is necessary
to terminate the oxidation step at the end of the 15thmin to obtain accurate and precise
results. A 10-mL aliquot of 0.01 mol L–1NBS (0.1 mmol) solution was found adequate for
quantitative oxidation of SBS in the range determined 0.1–0.5 mg mL–1.
Spectrophotometry
In the spectrophotometric methods, SBS was added to a fixed and known amount
of NBS, and after the reaction was judged to be complete, residual NBS was determined
by reacting with a fixed amount of either rhodamine B or methylene blue. SBS, when
added in increasing amounts to a fixed amount of NBS, consumed the latter, and a con-
91
K. Basavaiah et al.: Rapid titrimetric and spectrophotometric methods for salbutamol sulphate in pharmaceuticals using N-bromosucci-
nimide, Acta Pharm. 57 (2007) 87–98.