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ORIGINAL ARTICLE
Simultaneous determination of hyoscine N-butyl
bromide and paracetamol in their binary mixture by
RP-HPLC method
Nouruddin W. Ali
a
, Mohammed Gamal
a,c,
*, Mohammed Abdelkawy
b
a
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Alshaheed Shehata Ahmed
Hegazy St., 62574 Beni-Suef, Egypt
b
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., 11562 Cairo, Egypt
c
Pharmacy Program, Ibn Sina National College of Medical Science, Mahgar Street 31906, Jeddah 21418, Saudi Arabia
Received 11 September 2012; accepted 15 July 2013
KEYWORDS
RP-HPLC;
Chromatography;
Hyoscine N-butyl bromide
and paracetamol
Abstract RP-HPLC chromatographic method was developed for the determination of hyoscine
N-butyl bromide (HBB) and Paracetamol (PAR). In this chromatographic method, HBB and PAR
were separated using C18 (25 cm ·4.6 mm i.d. 5 lm particle size) column as a stationary phase and
water: methanol (50:50, V/V pH adjusted to 3.9 with CF
3
COOH acid) as a mobile phase, maintaining
the flow rate at 1.0 mL min
1
with UV detection at 210 nm. The proposed method was successfully
applied for the determination of HBB and PAR in pure form over a concentration range of 2.0–
50.0 lgmL
1
for HBB with mean percentage recovery of 100.10 ± 0.475 and over a concentration
range of 5.0–200.0 lgmL
1
for PAR with mean percentage recovery of 99.87 ± 0.942 and in their
pharmaceutical formulations (Buscopan plustablets, Buscamoltablets and Buscopan plus
suppositories).
ª2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.
1. Introduction
Hyoscine N-butyl bromide is a quaternary ammonium anticho-
linergic agent. It has antispasmodic action on the smooth
muscles of the gastrointestinal, biliary and urinary tracts
(Martindale, 2007).
Paracetamol (PAR), 4-acetamidophenol, is an effective
analgesic and antipyretic for the treatment of minor, non-
inflammatory conditions in patients who are prone to gastric
symptoms (Martindale, 2007). The structural formulas of
HBB and PAR are shown in Fig. 1.
There are many reports for the determination of HBB and
PAR either separately or in combination with other drugs
including spectrophotometric methods (Mohamed et al.,
1997; Mahrous et al., 1992; Issopoulos and Pavlou-Zervou,
1994; Thomos et al., 1994; Gouda, 2010; Issa et al., 2011;
Mujahid et al., 2012; Gouda et al., 2013), chromatographic
*Corresponding author. Tel.: +20 1141618950.
E-mail address: mgamalm3000@yahoo.com (M. Gamal).
Peer review under responsibility of King Saud University.
Production and hosting by Elsevier
Arabian Journal of Chemistry (2014) xxx, xxx–xxx
King Saud University
Arabian Journal of Chemistry
www.ksu.edu.sa
www.sciencedirect.com
1878-5352 ª2013 Production and hosting by Elsevier B.V. on behalf of King Saud University.
http://dx.doi.org/10.1016/j.arabjc.2013.07.015
Please cite this article in press as: Ali, N.W. et al., Simultaneous determination of hyoscine N-butyl bromide and paracetamol in their binary mix-
ture by RP-HPLC method. Arabian Journal of Chemistry (2014), http://dx.doi.org/10.1016/j.arabjc.2013.07.015
methods (Papadoyannis et al., 1994; Lau and Mok, 1997;
Dewani et al., 2012; Favreto et al., 2012), electrochemical
methods (El-Saharty et al., 2007; Farhadi and Karimpour,
2007; Ganjali et al., 2010; Wassel and Abu-Talib, 2010), Cap-
illary electrophoresis methods (Cherkaoui et al., 1999; Chang
et al., 2000) and titrimetric methods (British Pharmacopoeia,
2009; Kumar and Letha, 1997; Vyas and Kharat, 1988).
Few methods have been mentioned for the analysis of HBB
and PAR in the binary mixture. In The first method, Erk
(1996) analyzed HBB and Paracetamol in their binary mixture
by precipitating HBB with ammonium reineckate at pH 6.0
selectively and reading the absorbance of the solution of the
precipitate in acetone at 525.0 nm for HBB and by measuring
the dA/dkvalues at 254.5 nm in the first derivative spectra of
the remaining solution for paracetamol.
In the second method (Parissi-Poulou and Panderi, 1999),
solid phase extraction procedure using strong cation exchange
cartridges followed by a reversed-phase HPLC assay was ap-
plied to the analysis of HBB, PAR and lidocaine hydrochloride
in injection forms. The chromatographic separation was per-
formed on a C-18 column. The mobile phase consisted of a
mixture of acetonitrile: ammonium acetate 0.2 M (30:70, V/
V) pumped at a flow rate 1.2 mL min
1
.This method suffers
a lot of drawbacks among which is the use of complicated pro-
cedures such as solid phase extraction procedure. This tech-
nique is expensive, time consuming and not suitable for the
routine analysis of the binary mixture of HBB and PAR in
quality control analysis so there is a great need for developing
a simple HPLC method for routine analysis of the cited drugs.
Therefore, the objective of this work is to develop sensitive,
selective and reproducible RP-HPLC method for simultaneous
determination of HBB and PAR for routine quality control
analysis of these drugs either in bulk powder or in pharmaceu-
tical formulations. Chromatographic methods are well known
for providing high selectivity and sensitivity when used for the
determination of pharmaceutical drugs.
2. Experimental
2.1. Apparatus
Shimadzu Class – LC 10 AD Liquid Chromatography sup-
plied with Shimadzu SPD – 10 A UV–vis Detector (Shimadzu
Corporation, Japan). Phenomenex C18 (25 cm ·4.6 mm i.d,
5lm particle size) column was used as a stationary phase for
HPLC determinations (USA).
Sonix TV ss-series ultrasonicator (USA).
2.2. Materials
2.2.1. Pure samples
Paracetamol (PAR) and hyoscine N-butyl bromide (HBB)
were kindly supplied by CID Co. Chemical Industries Devel-
opment, Giza, Egypt. Their purity was found to be
99.94 ± 1.537 and 99.21 ± 1.012, respectively, according to
the company analysis certificate (HPLC).
2.2.2. Market samples
1-. Buscopan plustablets (Batch No 116738T) labeled to
contain 500 mg of (PAR) and 10 mg of (HBB), CID
Co. Chemical Industries Development, Giza, Egypt.
2-. Buscamol. F.Ctablets (Batch No 12001025) labeled to
contain 500 mg of (PAR) and 10 mg of (HBB), DELTA
PHARMA, Egypt.
3-. Buscopan plusSuppositories (Batch No 105) labeled
to contain 800 mg of (PAR) and 10 mg of (HBB), CID
Co. Chemical Industries Development, Giza, Egypt.
2.2.3. Reagents
All reagents and chemicals used were of analytical grade and
were used without further purification
1-. Methanol HPLC grade (Sigma Aldrich, Germany).
2-. Deionized water (SEDICO pharmaceutical Co., 6th
October City, Egypt).
3-. Orthophosphoric acid and glacial acetic acid (EL -
NASR Pharmaceutical Chemicals Co., Abu - Zabaal,
Cairo, Egypt).
4-. Trifluoroacetic acid (Spectrochem, India).
2.3. Preparation of standard solutions
2.3.1. Paracetamol (PAR) and hyoscine N-butyl bromide
(HBB) stock standard solutions (1 mg mL
1
)
Weigh accurately 0.1 g of each drug into two separate 100-mL
volumetric flask, 50 mL methanol was added, shaken to dis-
solve then complete the volume to the mark with methanol.
2.3.2. Paracetamol (PAR) and Hyoscine N butyl bromide
(HBB) working standard solutions (100 lgmL
1
)
Transfer accurately 10 mL of the stock solution of each drug
into two separate 100-mL volumetric flasks and complete to
the volume with methanol to get 100 lgmL
1
working solu-
tion for each drug.
2.4. Procedures
2.4.1. Linearity and construction of calibration curves
Transfer accurate aliquots equivalent to (20–500) lg of HBB
and (50–2000) lg of PAR from their corresponding working
solutions (100 lgmL
1
) or stock solutions (1000 lgmL
1
)
into two separate sets of series of 10-mL volumetric flasks.
AB
Molecular formula C21 H 30 Br N O4Molecular formula C8H9NO2
Molar mass 440.4 Molar mass 151.17
Figure 1 Chemical structure of HBB (A) and PAR (B).
2 N.W. Ali et al.
Please cite this article in press as: Ali, N.W. et al., Simultaneous determination of hyoscine N-butyl bromide and paracetamol in their binary mix-
ture by RP-HPLC method. Arabian Journal of Chemistry (2014), http://dx.doi.org/10.1016/j.arabjc.2013.07.015
Complete the volume with methanol. Make triplicate 20 lL
injections for each concentration. The separation was done
on a C
18
column using water: methanol 50: 50 V/V, pH ad-
justed to 3.9 with triflouroacetic acid as a mobile phase. All
solvents were filtered through a 0.45 lm membrane filters be-
fore use and degassed in an ultrasonic bath for 20 min. Record
the chromatograms at ambient temperature maintaining the
flow rate at 1.0 mL min
1
and detect the effluent at 210 nm.
Construct the calibration curves for each compound by plot-
ting the peak area/10
4
versus the corresponding concentration
and then compute the regression equations.
2.4.2. Analysis of laboratory prepared mixtures
Prepare mixtures containing HBB and PAR in different ratios.
Proceed as mentioned under linearity and construction of cal-
ibration curves. Calculate the concentrations of the two com-
pounds from their corresponding regression equations.
2.4.3. Application of the proposed methods to pharmaceutical
formulations
2.4.3.1. For tablet dosage form. The contents of ten tablets of
Buscopan plus(also for Buscamol) were thoroughly pow-
dered and mixed then an amount of the powder equivalent
to 500 mg of PAR and 10 mg of HBB was weighed accurately
in a 250-mL beaker, 70 mL of methanol was added, stirred for
about 30 min then filtered through filter paper into a 100-mL
volumetric flask, the beaker and the funnel were washed and
the volume was completed with methanol to get a concentra-
tion of 5.0 and 0.10 mg mL
1
for PAR and HBB, respectively.
Appropriate dilutions were made to bring up a concentration
of 100.0 and 2.0 lgmL
1
for PAR and HBB, respectively.
The proposed HPLC method was applied for the analysis
and calculation of HBB and PAR concentrations.
2.4.3.2. For suppositories dosage form. The contents of five sup-
positories of Buscopan pluswere thoroughly cut into small
fragments then an amount of the fragments equivalent to
800 mg of PAR and 10 mg of HBB was weighed accurately
in a 250-mL beaker, 70 mL of methanol was added, stirred
for about 30 min, left to cool then filtered through filter paper
into a 100-mL volumetric flask, the beaker and the funnel were
washed and the volume was completed with methanol to get a
concentration of 8.0 and 0.10 mg mL
1
for PAR and HBB,
respectively. Appropriate dilutions were made to bring up a
concentration of 160.0 and 2.0 lgmL
1
for PAR and HBB,
respectively. The proposed HPLC method was applied for
the analysis and calculation of HBB and PAR concentrations.
3. Results and discussion
3.1. Method development and optimization
The aim of this work is to develop a method that can be ap-
plied successfully for separation and quantification of the stud-
ied drugs without prior separation.
A simple, selective, sensitive and accurate isocratic RP –
HPLC method was adopted for the simultaneous determina-
tion of HBB and PAR, either in bulk powder or in pharmaceu-
tical formulations.
To optimize the RP-HPLC method, it was necessary to test
the effect of different variables:
3.1.1. The choice of the stationary phase
The reversed-phase separation was preferred to the normal
phase due to the drawbacks of the normal-phase mode, for
example, hydration of the silica with water, which causes peak
Figure 2 HPLC chromatogram showing separation of mixture of HBB and PAR 50 lgmL
1
of each.
Simultaneous determination of hyoscine N-butyl bromide and paracetamol 3
Please cite this article in press as: Ali, N.W. et al., Simultaneous determination of hyoscine N-butyl bromide and paracetamol in their binary mix-
ture by RP-HPLC method. Arabian Journal of Chemistry (2014), http://dx.doi.org/10.1016/j.arabjc.2013.07.015
tailing .C
18
column was found to be more efficient than C
8
column.
3.1.2. The choice of the mobile phase
Mobile phase systems of different compositions and ratios
were tried e.g. (water: methanol) using different acids
(H
3
PO
4
,CH
3
COOH and CF
3
COOH) at different pH values.
A complete separation of HBB and PAR without interference
within a suitable time was achieved using water: methanol
(50:50, V/V pH adjusted to 3.9 with CF
3
COOH acid). This
mobile phase allows the determination of HBB and PAR in
combination without interference and within a suitable time.
3.1.3. The effect of pH
Forked peak of HBB at basic medium was a problem. Adjust-
ment of pH was done to inhibit this. It was tried using glacial
acetic acid, triflouroacetic acid and orthophosphoric acid.
Forked peak could be overcome by adjusting the pH at 3.9.
In addition, the effect of pH was studied by using acids of
different pk
a
values (H
3
PO
4
,CF
3
COOH and CH
3
COOH). It
was found that pH = 3.9 with CF
3
COOH acid was suitable
for optimum resolution and peak shape.
3.1.4. Flow rate and the scanning wavelength
Different flow rates were tried of which 1.0 mL min
1
proved
to be of choice providing good separation within 9 min.
The two drugs under investigation were dissolved in meth-
anol separately and examined by the spectrophotometer. It
was found that detection at 210 nm gave good sensitivity for
both compounds.
Finally, a satisfactory separation was obtained using C
18
(25 cm ·4.6 mm i.d. 5 lm particle size) column as a stationary
phase and water: methanol (50:50, V/V pH adjusted to 3.9 with
CF
3
COOH acid) as a mobile phase, maintaining the flow rate
at 1.0 mL min
1
with UV detection at 210 nm. The retention
times for HBB and PAR were 3.5 and 8.7 min respectively,
Fig. 2.
3.2. Method validation
Method validation was performed according to ICH guidelines
(ICH, 2005).
Linearity of the proposed method was evaluated and it was
evident in the concentration range of 2–50 lgmL
1
for HBB
and 5–200 lgmL
1
for PAR. Good linearity was evident by
the high value of the correlation coefficient and the low inter-
cept value, (Figs. 3 and 4) and (Table 4).
The regression equations were calculated and found to be:
Y1¼2:537C10s:831 r1¼0:9995
Y2¼0:651C28:794 r2¼0:9998
where Y
1
and Y
2
are the peak area/10
4
,C
1
and C
2
are HBB
and PAR concentrations in lgmL
1
respectively and r
1
and
r
2
are the correlation coefficients.
Precision of the proposed RP-HPLC method was evident as
shown in Table 4.
Accuracy of the proposed method was checked by applying
the proposed method for the determination of different blind
Figure 3 Linearity of the peak area at 210 nm to the corre-
sponding concentration of HBB (2–50 lgmL
1
) using HPLC
method.
Figure 4 Linearity of the peak area to the corresponding
concentration of PAR (5–200 lgmL
1
) using HPLC method.
Table 1 Results of accuracy for determination of pure authenticity of HBB and PAR by the proposed HPLC method.
HBB PAR
Taken (lgmL
1
) Found
*
(lgmL
1
) Recovery % Taken (lgmL
1
) Found
*
(lgmL
1
) Recovery %
2.00 2.02 101.00 5.00 4.91 98.20
10.00 10.02 100.20 10.00 10.07 100.70
20.00 19.95 99.75 50.00 49.92 99.84
30.00 29.92 99.73 100.00 100.15 100.15
40.00 40.00 100.00 150.00 151.12 100.75
50.00 49.95 99.90 200.00 199.10 99.55
Mean ± SD 100.10 ± 0.475 99.87 ± 0.942
*
Average of three determinations.
4 N.W. Ali et al.
Please cite this article in press as: Ali, N.W. et al., Simultaneous determination of hyoscine N-butyl bromide and paracetamol in their binary mix-
ture by RP-HPLC method. Arabian Journal of Chemistry (2014), http://dx.doi.org/10.1016/j.arabjc.2013.07.015
samples of HBB and PAR. The concentrations were calculated
from the corresponding regression equations. The results were
obtained as shown in Tables 1 and 2.
Accuracy of the method was assured by applying the stan-
dard addition technique on different pharmaceutical dosage
forms where good recoveries were obtained as shown in Table 3
revealing no interference from excipients and good accuracy of
the proposed method.
Specificity of the proposed method is evident from the RP-
HPLC chromatogram in Fig. 2.
Robustness of the proposed method was evaluated in the
development phase by making small changes in the composition
of the mobile phase and detection wavelength. The low value of
% RSD shows that the method is robust and that deliberate
small changes in the studied factors did not lead to a significant
change in retention values, area or symmetry of the peaks.
System suitability tests are based on the concept that the
equipment, electronics, analytical operations and samples
constitute an integral system that can be evaluated as whole.
System suitability is used to ensure system performance before
or during the analysis of the drugs. System suitability was
checked by calculating the capacity factor (K0), tailing factor
(T), column efficiency (N), the selectivity factor (c) and resolu-
tion (Rs), where the system was found to be suitable as shown
in Table 5.
Table 2 Determination of HBB and PAR in laboratory prepared mixtures by the proposed HPLC method.
Mix. No. Ratio HBB:PAR HBB PAR
Taken (lg band
1
) Found
*
(lg band
1
) Recovery % Taken (lg band
1
) Found
*
(lg band
1
) Recovery %
1 1:1 10.00 9.98 99.80 10.00 10.02 100.20
2 1: 2 10.00 10.10 101.00 20.00 19.95 99.75
3 1: 5 10.00 10.04 100.40 50.00 50.06 100.12
4 1 :10 2.00 2.00 100.00 20.00 20.04 100.20
51:50
**
2.00 2.02 101.00 100.00 99.55 99.55
6 1:80
***
2.00 1.99 99.50 160.00 159.00 99.38
Mean ± SD 100.28 ± 0.627 99.87 ± 0.357
*
Average of three determinations.
**
The ratio present in Buscopan plustablets and Buscamol tablets.
***
The ratio present in Buscopan plussuppositories.
Table 3 Application of standard addition technique to analysis of HBB and PAR in dosage forms by the HPLC method.
Dosage form Drug Taken
(lgmL
1)
Found
*
(lgmL
1
)
Found% Pure added
(lgmL
1
)
Pure found
**
(lgmL
1
)
Recovery% Mean ± SD
Buscopan plustablets
Batch No 116738T
2.00 2.00 100.00 100.15 ± 0.132
HBB 2 1.97 98.50 10.00 10.02 100.20
20.00 20.05 100.25
PAR 100 100.2 100.2 10.00 10.01 100.10 100.26 ± 0.214
20.00 20.10 100.50
30.00 30.05 100.17
Buscamol.F.Ctablets
Batch No 12001025
2.00 1.96 98.00 99.80 ± 1.637
HBB 2.00 1.97 98.5 10.00 10.12 101.20
20.00 20.04 100.20
PAR 100.00 100.45 100.45 10.00 9.91 99.10 100.07 ± 0.95
20.00 20.20 101.00
30.00 30.03 100.10
Buscopan plussuppositories
Batch No 105
2.00 1.97 98.50 99.70 ± 1.058
HBB 2.00 2.01 100.5 10.00 10.05 100.50
20.00 20.02 100.10
PAR 160.00 161.5 100.94 10.00 9.99 99.90 100.46 ± 0.764
20.00 20.03 100.15
30.00 30.40 101.33
*
Average of six determinations.
**
Average of three determinations.
Table 4 Results of assay validation parameters of HPLC for
the determination of HBB and PAR in binary mixture.
Parameters HBB PAR
Range (lgmL
1
) 2–50 (lgmL
1)
5–200 (lgmL
1)
Slope 2.537 0.651
Intercept 0.831 8.794
Correlation coefficient (r) 0.9995 0.9998
Accuracy (mean ± SD) 100.10 ± 0.475 99.87 ± 0.942
(RSD%)
a*
0.863 1.021
(RSD%)
b*
0.916 1.151
(RSD%)a
*
, (RSD%)b
*
the intra-day and inter-day relative stan-
dard deviations of the average of concentrations (20, 40 and
50 lgmL
1
for each).
Simultaneous determination of hyoscine N-butyl bromide and paracetamol 5
Please cite this article in press as: Ali, N.W. et al., Simultaneous determination of hyoscine N-butyl bromide and paracetamol in their binary mix-
ture by RP-HPLC method. Arabian Journal of Chemistry (2014), http://dx.doi.org/10.1016/j.arabjc.2013.07.015
4. Conclusion
The proposed method is efficient for providing sensitive and
accurate quantitative analysis for simultaneous determination
of HBB and PAR in bulk powder and pharmaceutical formu-
lations, without any interference from excipients. The RP-
HPLC method has the advantages of short analysis time and
the availability of the device in every quality control unit so
it is suitable for routine analysis.
The statistical analysis was performed by comparing the re-
sults of the proposed method with those of the manufacturer’s
method. No significant difference was observed regarding
accuracy and precision, as shown in Table 6.
The suggested method provides selective, accurate and sen-
sitive analytical procedure for the determination of HBB and
PAR. It is suitable for routine analysis and quality control
of HBB and PAR in their pharmaceutical formulations.
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Table 6 Statistical analysis of the results obtained by
proposed method and reference method for the determination
of HBB and PAR.
Parameter RP-HPLC method Reference method
a
HBB PAR HBB PAR
Mean% 100.10 99.87 99.21 99.94
SD 0.475 0.942 1.012 1.537
n666 6
Student’s t-test (2.23)
b
0.092 0.927
F-value (5.05)
b
4.539 2.662
a
Manufactured method personal communications.
b
The values between parenthesis are the theoretical values for t
and Fat P= 0.05.
Table 5 Statistical analysis of parameters required for system suitability testing of HPLC method.
Parameters For RP-HPLC method
Obtained value Reference value
HBB PAR
Resolution (R
s
) 10.46 >1.5
Capacity factor(K0) 2.66 0.48 1–10 acceptable
Tailing factor (T) 1.16 1.00 <1.5–2
Selectivity factor(c) 5.54 >1
Number of Theoretical plate (N) 34339600 1267360 Increases with increases efficiency
HETP (cm plate
1
) Height equivalent to theoretical plate 0.0728 0.1973 The smaller the value, the higher the efficiency
6 N.W. Ali et al.
Please cite this article in press as: Ali, N.W. et al., Simultaneous determination of hyoscine N-butyl bromide and paracetamol in their binary mix-
ture by RP-HPLC method. Arabian Journal of Chemistry (2014), http://dx.doi.org/10.1016/j.arabjc.2013.07.015
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Please cite this article in press as: Ali, N.W. et al., Simultaneous determination of hyoscine N-butyl bromide and paracetamol in their binary mix-
ture by RP-HPLC method. Arabian Journal of Chemistry (2014), http://dx.doi.org/10.1016/j.arabjc.2013.07.015
