Journal of Taibah University for Science

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Validated RP-HPLC method for determining the

levels of bromhexine HCl, chlorpheniramine
maleate, dextromethorphan HBr and
guaiphenesin in their pharmaceutical dosage
forms

Vishal Jain & Mukesh C. Sharma

To cite this article: Vishal Jain & Mukesh C. Sharma (2016) Validated RP-HPLC method for
determining the levels of bromhexine HCl, chlorpheniramine maleate, dextromethorphan
HBr and guaiphenesin in their pharmaceutical dosage forms, Journal of Taibah University for
Science, 10:1, 38-45, DOI: 10.1016/j.jtusci.2015.02.019

To link to this article: https://doi.org/10.1016/j.jtusci.2015.02.019

© 2015 The Authors

Published online: 16 Apr 2018.

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Journal of Taibah University for Science 10 (2016) 38–45

Validated RP-HPLC method for determining the levels of

bromhexine HCl, chlorpheniramine maleate, dextromethorphan HBr
and guaiphenesin in their pharmaceutical dosage forms
Vishal Jain, Mukesh C. Sharma ∗
School of Pharmacy, Devi Ahilya Vishwavidyalaya, Takshashila Campus, Indore, M.P. 452001, India
Available online 4 April 2015

Abstract
A simple, precise and accurate reverse phase high performance liquid chromatographic method was developed for the simultaneous
estimation of bromhexine hydrochloride, chlorpheniramine maleate, dextromethorphan hydrobromide and guaiphenesin in their
tablet dosage form. The chromatographic conditions were standardised using a Chromatopak C18 (25 cm × 4.6 mm i.d. × 5 ␮m)
with UV detection at 265 nm, and the mobile phase consisted of methanol:acetonitrile:0.025 M phosphate buffer (50:25:25, v/v/v).
The retention times of bromhexine hydrochloride, chlorpheniramine maleate, dextromethorphan hydrobromide and guaiphenesin
were 16.254 min, 12.219 min, 6.156 min and 9.432 min, respectively. The calibration curves were linear with correlation coefficients
of 0.9987, 0.9988, 0.9981 and 0.9981 over a concentration range of 4.0–24.0 ␮g/ml for bromhexine hydrochloride, 5.0–30.0 ␮g/ml
for chlorpheniramine maleate, and 10.0–60.0 ␮g/ml for both dextromethorphan hydrobromide and guaiphenesin, respectively. The
proposed method has been validated according to the ICH guidelines and was successfully applied to estimate the levels of four
drugs in a combined formulation with good accuracy and precision.
© 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Taibah University. This is an open access article under
the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Keywords: RP-HPLC; Bromhexine hydrochloride; Chlorpheniramine maleate; Dextromethorphan hydrobromide; Guaiphenesin; ICH guidelines

1. Introduction benzenemethanamine hydrochloride (Fig. 1), is a

Combinations of decongestant and antihistamine
preparations are widely used for cough and cold
treatments. Bromhexine HCl (BROM), chemically
named 2-amino-3, 5-dibromo-N-cyclohexyl-N-methyl
∗ Corresponding author. Tel.: +91 9625355114.
E-mail addresses: vishaljain.sop9849@gmail.com (V. Jain),
mukeshcsharma@yahoo.com (M.C. Sharma).
Peer review under responsibility of Taibah University.
mucolytic agent used in the treatment of respiratory
disorders associated with viscid or excessive mucus
[1,2]. This agent’s mechanism is to increase the pro-
duction of serous mucus in the respiratory tract and
it makes the phlegm thinner and less viscous. BROM
is a mucous modifying drug that helps to improve
the flow properties of bronchial mucous and eases
expectoration. A literature survey reveals several HPLC
methods that were reported for their simultaneous deter-
mination along with several other active ingredients,
which exist as various combinations in cough–cold mix-
tures [3]. These methods include liquid chromatography
[4], liquid gas chromatography [5], gas chromatogra-
phy with mass detection [6], combined formulations

http://dx.doi.org/10.1016/j.jtusci.2015.02.019
1658-3655 © 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of Taibah University. This is an open access article under the
CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
 V. Jain, M.C. Sharma / Journal of Taibah University for Science 10 (2016) 38–45
Fig. 1. Chemical structure of interacting compounds of (a) bromhexine hydrochloride (b) chlorpheniramine maleate (c) dextromethorphan hydro-
bromide (d) guaiphenesin.
using HPLC [7–9] and UV spectrophotometry [10–13].
Chlorpheniramine maleate (CHL), 3-(p-chlorophenyl)-
3-(2-pyridyl)-N,N-dimethyl propylamine (Fig. 1) is a
powerful first-generation alkyl amine antihistamine that
antagonises the H1 -receptor and is widely used for symp-
tomatic relief of the common cold and allergic rhinitis
with weak sedative properties [14]. The symptoms of
allergic rhinitis include rash, watery eyes, itchy eyes
and throat, cough, and sneezing. CHL is also effec-
tive against nausea and motion sickness, and its primary
mechanism of action being is to reduce acetylcholine
levels in the brain. A literature survey shows that several
HPLC methods have been reported for chlorpheniramine
maleate alone and in combination in pharmaceuti-
cals, such as liquid chromatographic [15–17], HPTLC
[18], spectrophotometry [19] and micellar electrokinetic
chromatography [20]. Dextromethorphan hydrobro-
mide (DEX), [(+)-3-Methoxy-17-methyl-9␣, 13␣, 14␣
morphinan hydrobromide monohydrate] is a cough sup-
pressant that is used for the relief of non-productive
cough; it has a central action on the cough centre in the
medulla [21]. Dextromethorphan hydrobromide (DEX)
is an antitussive drug that is used for pain relief and
psychological applications [22]. The chemical struc-
tures of DEX are shown in Fig. 1. The combination of
these drugs is used as an antitussive and mucolytic in
bronchitis and chronic pulmonary conditions. Several
analytical techniques have been reported in the litera-
ture, most commonly liquid chromatography [23,24],
first and second-derivative UV spectrophotometric
39
techniques [25–27], capillary electrophoresis [28], and
gas chromatography [29]. Guaiphenesin (GUA; Fig. 1),
(2RS)-3-(2-methoxyphenoxy) propane-1, 2-diol [30], is
reported to increase the volume and reduce the viscos-
ity of tenacious sputum and is used as an expectorant
for productive cough. GUA is the glyceryl ether of gua-
iacol (a constituent of guaiac resin from the wood of
Guajacum officinale Linne) and acts as an expectorant
by increasing the volume and reducing the viscosity of
secretions in the trachea and bronchi. GUA is the compo-
nent of numerous cough and cold preparations available
worldwide. Additionally, GUA has been given to patients
with altered nasal mucociliary clearance associated with
HIV infection [31]. A literature survey revealed that
some techniques have been published for the determi-
nation of guaiphenesin either alone or in combinations
with other drugs by capillary gas chromatography
[32], HPLC [33], LC-MS [34], and LC-MS/MS
[35,36].
There is no method reported for the simultane-
ous estimation of bromhexine hydrochloride (BROM),
chlorpheniramine maleate (CHL), dextromethorphan
hydrobromide (DEX) and guaiphenesin (GUA) in a com-
bined dosage form. Therefore, we communicate here a
rapid and cost-effective quality-control tool and a reli-
able method for the simultaneous assay of mixtures of
these four drugs. The method should have sufficient
accuracy and precision and permit a simple and time-
saving assay for mixtures of BROM, CHL, DEX and
GUA.
40 V. Jain, M.C. Sharma / Journal of Taibah University for Science 10 (2016) 38–45
2. Materials and methods
2.1. Apparatus
To develop a suitable LC method for the analysis
of BROM, CHL, DEX and GUA in their combined
dosage form, different mobile phases were tried. The
chromatographic system consists of a pump (Shimadzu
LC 10AT VP) with a universal loop injector (Rheodyne
7725i) with an injection capacity of 20 ␮L. The detec-
tor consists of a photodiode array detector (PDA), a
SPD-10 AVP UV-Visible detector and a Chromatopak
C18 (25 cm × 4.6 mm i.d. × 5 ␮m) column. The equip-
ment was controlled by a PC work station equipped with
CLASS M 10-VP software (Shimadzu, Kyoto, Japan). A
UV/Visible double beam spectrophotometer (Shimadzu
Model 1700) was employed with a spectral bandwidth
of 1 nm and a wavelength accuracy of 0.3 nm (with
automatic wavelength correction using a pair of 1 cm
matched quartz cells).
2.2. Reagents and materials
Pure drug samples of BROM, CHL, DEX and GUA
were generously obtained as a gift from TABLIKE
and SCHON Pharmaceutical (Indore, India). The tablet
dose form, MARICOF (Label claim: 8.0 mg BROM,
2.0 mg CHL, 10.0 mg DEX and 100.0 mg GUA), was
procured from the local market (manufactured by G.S.
Pharmaceuticals Pvt. Ltd., Roorkee, India). HPLC grade
methanol and acetonitrile were obtained from Merck
(Mumbai, India).
2.3. Chromatography conditions
The solubility of the four drugs indicated that the
reverse phase chromatographic method would be best
option for the simultaneous estimation of BROM, CHL,
DEX, and GUA. The mobile phase consists of an organic
phase of methanol, acetonitrile and 0.025 M phosphate
buffer at a ratio of 50:25:25 (v/v/v adjusted to pH
5.5 using orthophosphoric acid). The mobile phase and
working solutions were filtered through a 0.2 ␮m nylon
filter and degassed using a sonicator before use. To deter-
mine the appropriate wavelength for the simultaneous
determination of BROM, CHL, DEX, and GUA, solu-
tions of these compounds were scanned on a UV–vis
spectrophotometer in the range 200–400 nm. The suit-
able wavelength to monitor these drugs was chosen from
the overlaid UV spectra (265 nm).
2.4. Preparation of standard stock solutions
Standard stock solutions of BROM, CHL, DEX and
GUA were prepared separately by accurately weigh-
ing 10.0 mg of each of BROM, CHL, DEX and GUA
(reference standard), transferring it to a 100 ml vol-
umetric flask and dissolving it in 20.0 ml of HPLC
grade methanol. The solutions were sonicated in bath
sonicator for 10 min to ensure complete solubilisation.
After sonication, the volume was brought to 100 ml with
same HPLC grade methanol at a final concentration of
0.1 mg/ml (100 ␮g/ml) of each reference standard.
A combined standard solution containing BROM,
CHL, DEX and GUA was prepared by adding 160 mg,
40 mg, 200 mg and 2000 mg of each reference standard,
respectively, transferring it to a 1000 ml volumetric flask,
and adding 200 ml of HPLC grade methanol. The solu-
tion was sonicated in bath sonicator for 10 min to ensure
complete solubilisation. After sonication, the volume
was brought to 1000 ml with same diluent, to result in
final concentrations of 160 ␮g/ml of BROM, 40 ␮g/ml of
CHL, 2 ␮g/ml of DEX and 2000 ␮g/ml of GUA, respec-
tively.
2.5. Estimation from pharmaceutical dosage form
Twenty tablets of MERICOF were weighed to cal-
culate the average weight of one tablet. They were
homogenised to a fine powder, transferred to a 1000.0 ml
volumetric flask, dissolved in 200.0 ml of diluent (HPLC
grade methanol) and sonicated in a bath sonicator for
20.0 min to ensure complete solubilisation. After son-
ication, the supernatant was transferred to a 1000.0 ml
volumetric flask by filtering through Whatman #41 filter
paper. The residue was washed three times with 10.0 ml
of methanol and the combined filtrate was brought to
1000.0 ml with the same diluent to achieve final concen-
trations of 160.0 ␮g/ml of BROM, 40.0 ␮g/ml of CHL,
2.0 ␮g/ml of DEX and 2000.0 ␮g/ml of GUA, respec-
tively.
A constant volume of the sample solution was
injected six times under the conditions described above.
The chromatogram showed that the retention times of
BROM, CHL, DEX and GUA were 16.254, 12.219,
6.156 and 9.432, respectively, with a resolution of
3.15 between DEX and GUA, 2.72 between GUA and
CHL, and 3.98 between CHL and BROM. The capacity
factor, tailing factor, theoretical plate number results are
reported in Table 1. The total run time was 20 min. The
peak areas were measured at 265 nm for BROM, CHL,
DEX and GUA, respectively, and their concentrations
in the samples were determined using a multi-level
 V. Jain, M.C. Sharma / Journal of Taibah University for Science 10 (2016) 38–45
Table 1
Data for the evaluation of the system suitability.
Property BROM
Rt 16.254
Tf 1.09
K 1.562
N 6359
Rs 3.98
Rt , retention time; Tf , tailing factor; K , capacity factor; N, number of theoretical plates; Rs , resolution.
calibration curve and linear regression equation using
the same conditions on the same HPLC system.
2.6. Preparation of solutions to determine linearity
From the standard stock solution 1, 100.0 ␮g/ml of
each (BROM, CHL, DEX and GUA), of the different
working standards were prepared at the following con-
centrations to determine linearity: 4.0, 8.0, 12.0, 16.0,
20.0 and 24.0 ␮g/ml for BROM; 5.0, 10.0, 15.0, 20.0,
25.0 and 30.0 ␮g/ml for CHL; and 10.0, 20.0, 30.0, 40.0,
50.0 and 60.0 ␮g/ml for DEX and GUA, respectively.
Six replicates of each different working standard were
prepared for each drug. The peak areas were plotted
against the corresponding concentrations to obtain the
calibration graphs.
2.7. Analytical method validation
The method was validated for analytical procedures
according to ICH guidelines to determine the linear-
ity, sensitivity, precision and accuracy for the analyte.
A system suitability test of the chromatography system
was performed before each validation run. Five repli-
cate injections of a system suitability standard and one
injection of a test standard were made. Regression char-
acteristics, validation and system suitability parameters
for BROM, CHL, DEX and GUA in their pharmaceutical
dosage form are shown in Table 3.
2.7.1. Linearity
The method was linear from 4.0 ␮g/ml to 24.0 ␮g/ml
for BROM, 5.0 to 30.0 ␮g/ml for CHL, and 10.0 to
60.0 ␮g/ml for both DEX and GUA, respectively. The
calibration curve was plotted using area vs. the concen-
tration each compound, and had an R2 value of 0.9980
or greater.
2.7.2. Accuracy
A recovery study for MERICOF was carried out per
ICH guidelines [37], where a known concentration of
41
CHL DEX GUA
12.219 6.156 9.432
1.04 1.13 1.29
1.217 1.764 4.571
1276 4925 18,743
2.72 0.00 3.15
all four standards solutions (equivalent to 80, 100, and
120% of total drug content) was added to a pre-analysed
solution of the tablet formulation and the percentage of
recovery was calculated.
2.7.3. Precision
Intra- and inter-day precision studies for MERICOF
were calculated by assaying the sample solution (mar-
keted formulation) on the same day and different days at
different time intervals, respectively. The assay was per-
formed with at least six replicates of the sample solution.
An amount of the sample powder equivalent to 100% of
the label claim of BROM, CHL, DEX and GUA was
accurately weighed and assayed. Method repeatability
was achieved by repeating the same procedure six times
on the same day for intra-day precision. The intermedi-
ate (inter-day) precision of the method was checked by
performing the same procedure on different days under
the same experimental conditions.
2.7.4. Limit of detection and limit of quantitation
(LOD and LOQ)
For LOD and LOQ, 10.0 ␮g/ml of all four standard
solutions were prepared from each of the 100.0 ␮g/ml
standard stock solutions (BROM, CHL, DEX and GUA):
0.2, 0.4, 0.6, 0.8, 1.0, 1.2 ␮g/ml working dilutions for
BROM and CHL; 0.3, 0.5, 0.7, 0.9, 1.1 and 1.3 ␮g/ml
working dilutions for DEX; and 0.3, 0.4, 0.5, 0.6, 0.7,
0.8 ␮g/ml working dilutions for GUA. LOD and LOQ
values were calculated to assess the detection limit of
the method using the following equation, per ICH guide-
lines:
LOD = 3.3 × σ/S,
LOQ = 10 × σ/S
where σ is the standard deviation of y-intercepts of
regression lines and S is the slope of the calibration curve.
42 V. Jain, M.C. Sharma / Journal of Taibah University for Science 10 (2016) 38–45
Fig. 2. RP-HPLC chromatogram of placebo.
2.7.5. Selectivity and specificity
A combination of methanol:acetonitrile:0.025 M
phosphate buffer, pH 5.5 (50:25:25, v/v) was used as
a specific mobile phase, and 265.0 nm was selected as a
specific analytical wavelength to simultaneously deter-
mine the levels of BROM, CHL, DEX and GUA in
a marketed formulation (MERICOF) using an HPLC
method. Specificity was assessed by a qualitative com-
parison between chromatograms obtained from sample,
standard, blank and placebo solutions. The diluent was
injected as a blank. A placebo (Fig. 2) interference
study was conducted by injecting a placebo solution
prepared from the excipients most commonly used in
pharmaceutical formulations, including starch, lactose
monohydrate, and magnesium stearate.
3. Results and discussion
A new, rapid, sensitive and accurate RP-HPLC
method was developed for the simultaneous esti-
mation of bromhexine hydrochloride, chlorpheni-
ramine maleate, dextromethorphan hydrobromide and
guaiphenesin in pharmaceutical formulations. After
trying different columns, the final choice for the sta-
tionary phase that gave satisfactory resolution and run
time was the reverse-phase C18 Chromatopack Peer-
less (25 cm×4.6 mm i.d.×5 ␮m) column. There were
many mobile phases that were tested to resolve all
four chromatographic peaks, including methanol:water
(80:20, v/v) and methanol:water (50:50, v/v), but the
broadness of the peaks did not produce satisfac-
tory results in these chromatograms. To improve the
sharpness of the chromatographic peaks, we worked
with slightly acidic acetonitrile and phosphate buffer.
Finally, the mobile phase methanol:acetonitrile:0.025 M
phosphate buffer (50:25:25, v/v/v) (pH: 5.5) adjusted
with O-phosphoric acid was found to be satisfac-
tory as it gave four symmetric peaks for BROM,
CHL, DEX and GUA. The total run time was
20 min at a 1.0 ml min−1 rate and ambient tempera-
ture. The retention times of bromhexine hydrochloride,
chlorpheniramine maleate, dextromethorphan hydrobro-
mide and guaiphenesin were 16.254 min, 12.219 min,
6.156 min and 9.432 min (Fig. 3), respectively. The best
fit for the calibration curve (peak area vs. respective
concentrations) could be achieved by separate lin-
ear regression equations, which were y = 9040x + 9993
(BROM), y = 9579x − 308 (CHL), y = 25,935x + 25,500
(DEX) and y = 34,255x + 14,194 (GUA). The proposed
method was evaluated for formulations containing
BROM, CHL, DEX and GUA. Four replicate determina-
tions were performed using capsules, and found 100.45%
for BROM, 100.25% for CHL, 99.88% for DEX and
100.56% for GUA. Specificity was assessed by com-
paring the chromatogram of the tablet solution with
the placebo solution and also with the chromatograms
obtained from the standard drugs. The retention time
of all four drugs was the same in the quaternary mixed
standard solutions as well as the marketed formulation
(MERICOF) solution, and there was also no interfer-
ence from the excipients. This indicates the specificity of
the method for quantitative estimation of BROM, CHL,
DEX and GUA in a marketed formulation (Table 2).
The recoveries of all of the components were between
99.0 and 102%. The LOD and LOQ values were 21.49
and 65.13 ng ml−1 , 29.17 and 88.39 ng ml−1 , 11.65 and
35.51 ng ml−1 , and 8.1 and 24.57 ng ml−1 for BROM,
CHL, DEX and GUA, respectively (Table 2). The
excipients did not interfere with the peaks of inter-
est. Hence, the proposed method is applicable for the
 V. Jain, M.C. Sharma / Journal of Taibah University for Science 10 (2016) 38–45 43

Fig. 3. HPLC-chromatogram of marketed formulation.

Table 2
Results from assay of the marketed formulation.
Drug Label claim (mg/tab) n = 6 Amount found (mg/tab) Label claim (%) S.D. S.E. % COV

BROM 8 8.0365 100.45 0.5902 0.2410 0.5875

CHL 2 2.0052 100.25 1.2891 0.5263 1.2858
DEX 10 9.9887 99.88 0.7494 0.3059 0.7502
GUA 100 100.5601 100.56 0.7991 0.3262 0.7946

S.D, standard deviation; COV, coefficient of variance; S.E, standard error; n, number of replicates.

Table 3
Summary of validation parameters.
Parameter BROM CHL DEX GUA

Linearity range 4–24 5–30 10–60 10–60

Slope 9040 9579 25,935 34,255
Interept (y) 9993 −308 25,500 14,194
R2 0.9987 0.9988 0.9981 0.9988
Accuracy (percentage of recovery)
80% 99.67 100.0521 101.2917 101.7354
100% 101.07 100.9583 101.225 101.185
120% 100.15 101.0417 100.9931 100.1035
Intraday precision
%COV 0.1755–0.6333 0.3050–0.6715 0.1806–0.6929 0.2813–0.8976
Interdays precision
%COV 0.1018–0.2804 0.0675–0.1639 0.1529–0.4252 0.1227–0.2925
L.O.D. (ng ml−1 ) 21.49 29.17 11.65 8.1
L.O.Q. (ng ml−1 ) 65.13 88.39 35.51 24.57
Specificity/selectivity No interference
Robustness Reliable results
Ruggedness Reproducible results
44 V. Jain, M.C. Sharma / Journal of Taibah University for Science 10 (2016) 38–45
routine simultaneous estimation of BROM, CHL, DEX
and GUA in pharmaceutical dosage forms.
4. Conclusions
In the present work, we successfully and simul-
taneously analysed BROM, CHL, DEX and GUA in
a marketed formulation (MERICOF) using an RP-
HPLC method based on a literature survey. All of
the critical steps for developing the method have been
summarised and prioritised. To develop an effective RP-
HPLC method, most of the effort should be spent in
method development and optimisation, as this emphasis
will improve the final method performance. The method
validation, however, should be treated as an exercise to
summarise or document the overall method performance
for its intended purpose. Thus, the present method is
rapid, easy and accurate for the simultaneous estima-
tion of BROM, CHL, DEX and GUA in a commercially
available pharmaceutical formulation.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
The authors are thankful to TABLIKE and SCHON
Pharmaceutical, Jumbu Di Hapsi Hatod Road, Indore,
M.P, for generously providing drug samples and Head,
School of Pharmacy, DAVV Indore for providing nec-
essary facilities and chemicals. VJ thanks the All India
Council for Technical Education (AICTE), New Delhi,
India for financial support for this research. We are grate-
ful to the referees for their valuable suggestions.
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