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Simultaneous determination of hyoscine N-butyl bromide and paracetamol in their binary mixture by RP-HPLC method
⁎Corresponding author. Tel.: +20 1141618950. mgamalm3000@yahoo.com (Mohammed Gamal)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
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 μm particle size) column as a stationary phase and water: methanol (50:50, V/V pH adjusted to 3.9 with CF3COOH 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 μg mL−1 for HBB with mean percentage recovery of 100.10 ± 0.475 and over a concentration range of 5.0–200.0 μg mL−1 for PAR with mean percentage recovery of 99.87 ± 0.942 and in their pharmaceutical formulations (Buscopan plus® tablets, Buscamol® tablets and Buscopan plus® suppositories).
Keywords
RP-HPLC
Chromatography
Hyoscine N-butyl bromide and paracetamol
1 Introduction
Hyoscine N-butyl bromide is a quaternary ammonium anticholinergic 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.
Chemical structure of HBB (A) and PAR (B).
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., 2014; Gouda et al., 2013), chromatographic methods (Papadoyannis et al., 1994; Lau and Mok, 1997; Dewani et al., 2014; Favreto et al., 2012), electrochemical methods (El-Saharty et al., 2007; Farhadi and Karimpour, 2007; Ganjali et al., 2010; Wassel and Abu-Talib, 2010), Capillary 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/dλ values 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 applied to the analysis of HBB, PAR and lidocaine hydrochloride in injection forms. The chromatographic separation was performed 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 procedures such as solid phase extraction procedure. This technique 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 pharmaceutical 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 supplied with Shimadzu SPD – 10 A UV–vis Detector (Shimadzu Corporation, Japan). Phenomenex C18 (25 cm × 4.6 mm i.d, 5 μm 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 Development, 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
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Buscopan plus® tablets (Batch No 116738T) labeled to contain 500 mg of (PAR) and 10 mg of (HBB), CID Co. Chemical Industries Development, Giza, Egypt.
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Buscamol. F.C® tablets (Batch No 12001025) labeled to contain 500 mg of (PAR) and 10 mg of (HBB), DELTA PHARMA, Egypt.
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Buscopan plus® Suppositories (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
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Methanol HPLC grade (Sigma Aldrich, Germany).
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Deionized water (SEDICO pharmaceutical Co., 6th October City, Egypt).
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Orthophosphoric acid and glacial acetic acid (EL - NASR Pharmaceutical Chemicals Co., Abu - Zabaal, Cairo, Egypt).
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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 dissolve then complete the volume to the mark with methanol.
2.3.2 Paracetamol (PAR) and Hyoscine N butyl bromide (HBB) working standard solutions (100 μg mL−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 μg mL−1 working solution for each drug.
2.4 Procedures
2.4.1 Linearity and construction of calibration curves
Transfer accurate aliquots equivalent to (20–500) μg of HBB and (50–2000) μg of PAR from their corresponding working solutions (100 μg mL−1) or stock solutions (1000 μg mL−1) into two separate sets of series of 10-mL volumetric flasks. Complete the volume with methanol. Make triplicate 20 μL injections for each concentration. The separation was done on a C18 column using water: methanol 50: 50 V/V, pH adjusted to 3.9 with triflouroacetic acid as a mobile phase. All solvents were filtered through a 0.45 μm membrane filters before 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 plotting the peak area/104 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 calibration curves. Calculate the concentrations of the two compounds 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 powdered 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 concentration 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 μg mL−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 suppositories of Buscopan plus® were 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 μg mL−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 applied successfully for separation and quantification of the studied drugs without prior separation.
A simple, selective, sensitive and accurate isocratic RP – HPLC method was adopted for the simultaneous determination of HBB and PAR, either in bulk powder or in pharmaceutical 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 tailing .C18 column was found to be more efficient than C8 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 (H3PO4, CH3COOH and CF3COOH) 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 CF3COOH 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. Adjustment 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 pka values (H3PO4, CF3COOH and CH3COOH). It was found that pH = 3.9 with CF3COOH 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 methanol 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 C18 (25 cm × 4.6 mm i.d. 5 μm particle size) column as a stationary phase and water: methanol (50:50, V/V pH adjusted to 3.9 with CF3COOH 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.
HPLC chromatogram showing separation of mixture of HBB and PAR 50 μg mL−1 of each.
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 μg mL−1 for HBB and 5–200 μg mL−1 for PAR. Good linearity was evident by the high value of the correlation coefficient and the low intercept value, (Figs. 3 and 4) and (Table 4). (RSD%)a∗, (RSD%)b∗ the intra-day and inter-day relative standard deviations of the average of concentrations (20, 40 and 50 μg mL−1 for each).
Linearity of the peak area at 210 nm to the corresponding concentration of HBB (2–50 μg mL−1) using HPLC method.
Linearity of the peak area to the corresponding concentration of PAR (5–200 μg mL−1) using HPLC method.
Parameters
HBB
PAR
Range (μg mL−1)
2–50 (μg mL−1)
5–200 (μg mL−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
The regression equations were calculated and found to be: where Y1 and Y2 are the peak area/104, C1 and C2 are HBB and PAR concentrations in μg mL−1 respectively and r1 and r2 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 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.
HBB
PAR
Taken (μg mL−1)
Found⁎ (μg mL−1)
Recovery %
Taken (μg mL−1)
Found⁎ (μg mL−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
Mix. No.
Ratio HBB:PAR
HBB
PAR
Taken (μg band−1)
Found⁎ (μg band−1)
Recovery %
Taken (μg band−1)
Found⁎ (μg 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
5
1: 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
Accuracy of the method was assured by applying the standard 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.
Dosage form
Drug
Taken (μg mL−1)
Found⁎ (μg mL−1)
Found%
Pure added (μg mL−1)
Pure found⁎⁎ (μg mL−1)
Recovery%
Mean ± SD
Buscopan plus® tablets 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.C® tablets 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 plus® suppositories 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
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 (K′), tailing factor (T), column efficiency (N), the selectivity factor (γ) and resolution (Rs), where the system was found to be suitable as shown in Table 5.
Parameters
For RP-HPLC method
Obtained value
Reference value
HBB
PAR
Resolution (Rs)
10.46
>1.5
Capacity factor(K′)
2.66
0.48
1–10 acceptable
Tailing factor (T)
1.16
1.00
<1.5–2
Selectivity factor(γ)
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
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 formulations, 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 results 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.
Parameter
RP-HPLC method
Reference methoda
HBB
PAR
HBB
PAR
Mean%
100.10
99.87
99.21
99.94
SD
0.475
0.942
1.012
1.537
n
6
6
6
6
Student’s t-test (2.23)b
0.092
0.927
F-value (5.05)b
4.539
2.662
The suggested method provides selective, accurate and sensitive 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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