International Journal of Chemical and Analytical Science ISSN: 0976-1206 Research Article www.ijcas.info Simultaneous Determination of some anticholinergic drugs (Biperiden and Chlorphenoxamine) in Various Dosage forms by Spectrophotometry
Issa FAA1, EL-Helbawy SM2 and Abed EL- Magied AM2* 1Chemistry Department, faculty of science, Menoufia University 2Medico-Legal organization, Ministry of Justice, Cairo, Egypt
Simple and rapid spectrophotometric procedures were established for quantization of biperiden hydrochloride (BPN) and chlorphenoxamine hydrochloride (CPA). The procedures are based on the reaction between the examined drugs BPN and CPA and picric acid (I), alizarin (II), bromothymol blue (III) and chlorophenol red (IV) producing ion-associates which can be measured at the optimum wavelength. The optimization of the reaction conditions was investigated. Beer’s law is obeyed in the concentration ranges 58.8 –166.4 µgml−1. The molar absorptivity, Sandell sensitivity are also calculated. The correlation coefficient was ≥0.9996 (n = 6) with a relative standard deviation (R.S.D.) ≤1.35 for four determinations. The methods are successfully applied to determine of BPN and CPA in pharmaceutical formulations.
Keywords: Spectrophotometry, Ion-Associates, Drugs, Biperiden, Chlorphenoxamine
INTRODUCTION MATERIALS AND METHODS
Biperiden hydrochloride (BPN) is α-Bicyclo [2.2.1] hept–5–en– Apparatus: The spectral measurements were carried out by 2–yl-α-phenyl–1–piperidinepropanol. Chlorphenoxamine using U.V-visible spectrophotometer (Helios Alpha) with a hydrochloride (CPA) is 2-[1-(4-Chlorophenyl)-1–phenyl- 10mm quartz cell, optical path length was used. phenylethoxy]-N, N-dimethylethanamine. They are a weak peripheral anticholinergic[1,2] Agent. It has, therefore, some Reagents: Picric acid 2,4,6 tri nitro phenol (I), Alizarin 1,2 di antisecretory, antispasmodic, mydriatic and antihistaminic hydroxy anthraquinone (II), bromothymol blue 3,3 effects. The mechanism of action of centrally active dibromothymol sulphonaphthalein (III) and chlorophenol anticholinergic drugs such as BPN, CPA are considered to red 5,5'- dichlorophenol sulphonaphthalein (IV) were Merck relate to competitive antagonism of acetylcholine at products. A stock solution (1× 10-2 M ) was prepared by cholinergic receptors in the corpus striatum, which then dissolving the appropriate weights of I and II in doubly restores the balance [3,4]. Several methods have been applied distilled water, while III and IV were dissolved in the least for the determination of biperiden hydrochloride and volume alcohol then completed by distilled water. Biperiden chlorphenoxamine hydrochloride in dosage forms and in hydrochloride (provided from Kahira Pharm Company biological fluids. The different techniques used in this action Egypt), chlorphenoxamine hydrochloride (provided from include Spectrophotometry [5,6], High Pereformance Liquid Eipico Company Egypt) and their pharmaceutical Chromatography [7], Gas Chromatography [8], Polarography formulation (i.e. Akiniton 2mg and alleregex tablets). [9], and cyclic voltammetery [10]. General procedure: Into 50 ml separating funnel, 5ml of (1 × The chemical structure of the studied drugs 10-2 M ) of I, II, III and IV were added by pipette, different α-Bicyclo[2.2.1]hept–5–en–2–yl-α-phenyl–1–piperidinepropanol volumes of solution containing (1 ×10-2 M ) of drugs of BPN and CPA were added and the volume was made up to 10ml with distilled water. The formed ion–associates was extracted with 20 ml chloroform by shaking for two minutes and allowed to separates into two phases. The organic layer was collected and dried with anhydrous sodium sulphate then complete to 25 ml chloroform. The absorbance of the extract BIPERIDEN was measured at the recommended wavelength as recorded in table.1 against reagent blank prepared in the same way
2-[1-(4-Chlorophenyl)-1–phenylphenylethoxy]-N,N-dimethylethanamine without addition of the examined drugs. All measurements were carried out at room temperature (25 ± 5 ◦C).
Application to various dosage forms: At least 5 tablets of the drug were weighed into a small dish, powdered and mixed well. A portion equivalent to 100 mg was weighed and dissolved in 100 ml water, shaken well and filtered through a filter paper to give clear solution. The clear solution was diluted to 250 ml with water in a 250 ml calibrated flask. The drug content of this solution was obtained by applying the general procedure to aliquot containing different volumes of CHLORPHENOXAMINE solution containing of (1 × 10-2 M) drugs as described above.
Corresponding Author: Abed EL- Magied AM, Medico-Legal organization, Ministry of Justice, Cairo, Egypt. Received 13-09-2011; Accepted 21-12-2011
January, 2012 International Journal of Chemical and Analytical Science, 2012, 3(1), 1296-1300 1296 Issa FAA, et al.: Simultaneous Determination of some anticholinergic drugs (Biperiden and Chlorphenoxamine) in Various Dosage forms by Spectrophotometry
Stoichiometric relationship: Job’s method of continuous used (methylene chloride, chloroform, benzene, n-hexane, variation was employed of (1 × 10-2 M) standard solution of cyclohexane and diethyl ether). The most convenient solvent BPN, CPA and of (1 × 10-2 M) solution of reagents (I–IV) were found to give the maximum colour intensity and extracting used. A series of solutions were prepared in which the total power of ion –associates was chloroform for BPN and CPA. volume of drug and reagent was kept constant at 5.0 ml. The The study revealed that a volume ratio of 1-1 (aqueous – reagents were mixed in various proportions and diluted to organic) was the most suitable for the ion–associates volume in 10 ml calibrated flask with the appropriate solvent extraction. following the above mentioned procedures. Effect of the reagent concentration: The effect of reagent RESULTS AND DISCUSSION concentration was tested by using varying amounts 1-5 ml of (1 × 10-2 M) solution of BPN and CPA. The result shows that Several parameters such as reagent concentration, sequence of 2.5 ml of (1 × 10-2 M) solution of the reagent (I – IV) were addition, effect of extracting solvent, effect of pH, effect of sufficient to achieve the maximum absorbance value. time, were optimized to achieve high sensitivity, stability and reproducible results. Effect of pH: The effect of pH on the formation of ion– associates was studied using stock solution of sodium Optimization: Most favorable conditions were examined to hydroxide and hydrochloric acid (0.5 M). 5 ml of (5 × 10-3 M ) achieve maximum colour intensity in the quantitative of reagent was mixed with 1ml (2 × 10-3 M ) of drug solution determination of the examined drug (BPN and CPA). The and adding drops of HCl or NaOH to adjust pH then the absorption spectra of BPN and CPA and their ion–associates volume was made up to 10 ml with distilled water. The with picric acid, alizarin, bromothymol blue and optimum pH range for BPN and CPA was (2 – 6) pH for (I – chlorophenol red under the optimum conditions are recorded IV) reagents, respectively. Fig (3, 4). in table 1. The absorption band of BPN and CPA ion– associates are located at 426, 410, 400, and 413 nm, 426, 407, 1.2 420 and 418 nm with reagents (I–IV), respectively. However, AR in all instances, the absorbance was measured at those λmax 1 PA CPR against reagent blank; the influence of each of the following BTB variables on the reaction was tested. Fig (1 ,2). 0.8
0.6
1.2 Absorbance 0.4 BTB CPR 1 0.2 PA 0.8 AR 0 0 2 4 6 8 10 12 14 0.6 pH Absorbance 0.4 Fig (3): Effect of pH on BPN ion-associates with AR, PA, CPR 0.2 and BTB.
0 300 340 380 420 460 500 540 580 0.6 AR wavelength nm 0.5 PA CPR Fig (1): Absorption spectra of BPN ion-associates with AR, 0.4 BTB PA, CPR, and BTB 0.3
0.2 BTB Absorbance 1.2 CPR PA 0.1 1 AR 0 0.8 0 2 4 6 8 10 12 14 pH 0.6
absorbance 0.4 Fig (4): Effect of pH on CPA ion-associates with AR, PA, CPR
0.2 and BTB
0 Effect of temperature: The effect of temperature on the 300 340 380 420 460 500 540 580 formation and stability of ion–associates was studied by wave length nm measuring the absorbance of the extracted ion–associates at Fig (2): Absorption spectra of CPA ion-associates with AR, temperature range form 25-70°C.The results show that the PA, CPR and BTB. ion-associates were formed almost instantaneously at room temperature (25 ± 5 ◦C) and remain constant up to 45°C for Effect of the extracting solvent: The polarity of the solvent reagents I-IV. Fig (5, 6). affects both extracting efficiency and absorptivity of the ion– associates. Various water immiscible organic solvents were
January, 2012 International Journal of Chemical and Analytical Science, 2012, 3(1), 1296-1300 1297 Issa FAA, et al.: Simultaneous Determination of some anticholinergic drugs (Biperiden and Chlorphenoxamine) in Various Dosage forms by Spectrophotometry
PA 1.2 1.4 AR AR PA 1.2 CPR 1 CPR BTB BTB 1 0.8 0.8
0.6 0.6 Absorbance Absorbance 0.4 0.4 0.2 0.2 0 0 0 5 10 15 20 25 30 35 40 45 50 0 20 40 60 80 100 Time, hours Temperature, °C
Fig (5): Effect of temperature on the stability of BPN ion- Fig (8): Effect of time on the stability of CPA ion – associates associates with AR, PA, CPR, and BTB. with AR, PA, CPR, BTB.
1.4 Effect of Sequence of mixing: The optimum sequence of AR mixing was (reagent-drug–solvent). For production the 1.2 PA CPR highest colour intensity and shortest time for maximum 1 BTB absorbance, while other sequences needed longer time in addition to lower stability. 0.8
0.6 The stoichiometry of the ion–associates Absorbance The stoichiometry of the ion–associates formed between 0.4 drugs under investigation and reagents (I–IV) was [11] 0.2 investigated by applying the continuous variation method and molar ratio method[12]. Fig (9, 10, 11, 12, 13 and 14) the 0 0 20 40 60 80 100 result indicates the existence of [1:1] charge transfer complex Temperature, °C at a definite λmax recorded in Table 1.
Fig (6): Effect of temperature on the stability of CPA ion- Table 1: Characteristics and analytical data of BPN and associates with AR, PA, CPR and BTB. CPA ion-associates with (I – IV) reagents.
CPA BPN CPA Effect of time: The effect of time on the formation and Parameters IV III II I IV III II I II III IV I II III IV stability of ion–associates was studied by measuring the absorbance of the extracted ion–associates at increasing time λ max 426 410 400 413 426 407 420 418 intervals. The results show that the developed color remained Beer’s law 166.4 59.6 138.7 98.8 78.95 58.8 118.65 138.45 stable for 30 hr. with reagents from I - IV after these intervals, Molar 0.19 0.52 0.24 0.44 0.42 0.52 0.32 0.22 slight decrease in color intensity occurred. Fig (7, 8). absorpitity x104 x104 x104 x104 x104 x104 x104 x104 Sandell 0.183 0.067 0.145 0.079 0.081 0.065 0.106 0.155 1.2 PA sensitivity AR Regressin equation* 1 CPR BTB Intercept 0.0014 0.0035 0.0012 0..0025 0.0261 0.0344 0.0016 0.0014 0.8 Slope 0.0055 0.015 0.0068 0.0125 0.0119 0.0145 0.0094 0.0064 Correl. 0.9998 0.9997 0.9998 0.9996 0.9998 0.9999 0.9998 0.9997 0.6 coefficien 0.4 Absorbance *A = a +bc where c is the concentration µg/mL 0.2
0 1 PA 0.9 0 5 10 15 20 25 30 35 40 45 50 AR 0.8 Time, hours BTB 0.7 CPR Fig (7): Effect of time on the stability of BPN ion – associates 0.6 0.5 with AR, PA, CPR, BTB. 0.4 Absorbance 0.3 0.2 0.1 0 0 0.2 0.4 0.6 0.8 1
Mole-fraction of drug
Fig (9): Continuous variation of (BPN) ion-associates with AR, PA, CPR and BTB.
January, 2012 International Journal of Chemical and Analytical Science, 2012, 3(1), 1296-1300 1298 Issa FAA, et al.: Simultaneous Determination of some anticholinergic drugs (Biperiden and Chlorphenoxamine) in Various Dosage forms by Spectrophotometry
1.4 AR PA PA 0.9 1.2 CPR AR BTB 0.8 CPR 1 0.7 BTB 0.6 0.8 0.5 0.6 0.4 Absorbance
Absorbance 0.3 0.4 0.2 0.2 0.1 0 0 0 20 40 60 80 100 120 140 0 0.2 0.4 0.6 0.8 1 Chlorphenoxamine concentration,ug/ml Mole-fraction of drug
Fig (10): Continuous variation of CPA ion-associates with AR, Fig. 14: Standard curves of CPA ion-associates with AR, PA, PA, CPR and BTB. CPR and BTB
0.9 AR Interference: No interference was observed in the 0.8 PA determination of BPN and CPA with different reagents (I–IV) CPR 0.7 in the presence of different additives such as lactose, glycerol, BTB 0.6 propylene glycol, sugar and starch. Which are present in its 0.5 pharmaceutical preparations. 0.4 Absorbance 0.3 Validation of the method: Results obtained were compared
0.2 with those of the official methods. The statistical treatment of
0.1 obtained results revealed that there is no significant difference between both as shown in Table 3. Six replicate determination 0 0 0.5 1 1.5 2 at different concentration levels were carried out to test the molar ratio [D]/[R] precision of the method. The recoveries in the range (99.3 – 100.1%) as in Table 2, reflecting high accuracy of the results in Fig (11): Molar ratio of BPN ion-associates with AR, PA, CPR addition to high precision indicated by very low values of and BTB standard deviation. The performance of the proposed method was assessed by calculation of t and f tests compared with the [13, 14] 1.2 Pharamacopial method . Mean values were obtained PA AR with student’s t and f testes at 95% confidence level for five 1 CPR degrees of freedom were in the accepted values.
0.8 BTB Table 2: Evaluation of accuracy of the proposed method for 0.6 determination of BPN and CPA
Absorbance 0.4 Pure solution (µg/mL) Tablets (µg/mL) Ion-associates 0.2 Taken Found Recovery % Taken Found Recovery % BPN-I 20 19.95 99.75 20 20.06 100.30 0 40 40.08 100.20 40 38.95 97.37 0 0.5 1 1.5 2 80 79.15 98.80 80 80.05 100.06 Molar ratio D / R 120 120.10 100.08 120 119.08 99.20 140 139.07 99.30 140 139.99 99.99 BPN-II 20 20.08 100.04 20 19.99 99.95 25 24.68 98.72 25 25.05 100.20 Fig (12): Molar ratio of CPA ion-associates with AR, PA, CPR 30 29.95 99.85 30 30.10 100.35 and BTB. 40 40.09 100.22 40 39.10 97.75 50 49.97 99.96 50 50.04 100.08 BPN-III 20 19.95 99.75 20 20.15 100.75 40 39.95 99.88 40 39.99 99.98 AR 80 79.08 98.85 80 80.09 100.15 CPR 100 100.05 100.05 100 100.10 100.10 BTB 1.4 120 119.96 99.97 120 119.05 99,20 PA BPN-IV 20 19.85 99.35 20 19.95 99.75 1.2 30 29.79 99.25 30 29.85 99.55 1 50 50.09 100.18 50 50.02 100.04 70 69.70 99.60 70 70.10 100.15 0.8 80 79.98 99.98 80 80.06 100.08 CPA-I 20 19.96 99.90 20 20.04 100.02 0.6 30 30.04 100.14 30 29.99 99.97 Absorbance 0.4 40 39.15 97.75 40 40.08 100.20 50 50.10 100.20 50 49.15 98.30 0.2 60 59.30 98.80 60 60.05 100.08 CPA-II 20 19.96 99.80 20 19.98 99.94 0 30 30.10 100.30 30 30.05 100.18 0 20 40 60 80 100 120 140 160 180 40 39.55 98.97 40 39.25 98.12 Biperiden concentration, ug/ml 50 50.85 101.65 50 50.02 100.04 55 54.94 99.89 55 55.15 100.28 Fig. 13: Standard curves of BPN ion-associates with AR, PA, CPA-III 20 20.05 100.25 20 20.05 100.25 40 39.95 99.88 40 40.22 100.55 CPR and BTB 60 59.75 99.60 60 59.99 99.98 80 79.90 99.88 80 80.30 100.08 100 98.98 98.98 100 99.35 99.35
January, 2012 International Journal of Chemical and Analytical Science, 2012, 3(1), 1296-1300 1299 Issa FAA, et al.: Simultaneous Determination of some anticholinergic drugs (Biperiden and Chlorphenoxamine) in Various Dosage forms by Spectrophotometry
CPA -IV 20 20.02 100.10 20 19.85 99.25 [15]. This finding indicates that the proposed methods are 40 39.85 99.64 40 39.95 99.88 80 80.04 100.05 80 80.02 100.03 successful. Also this method is applicable to wide range of 100 99.27 99.27 100 99.98 99.98 concentration, less time consuming and needs simple reagents 120 119.80 99.84 120 120.02 100.02 which are available, thus offering an economic method for routine determination of the cited drugs. Table 3: Statistical treatment of data obtained for BPN and CPA applying the proposed methods in comparison with the Pharamacopial method REFERENCES Pharm. Parameters I II III IV method Pure solution (BPN) 1. Moffat AC, Ed Clarks, Isolation and Identification of Drugs, X±SD 99.98±0.97 99.6±0.9 99.8±0.8 99.70±0.9 99.65±0.9 Pharmaceutical Press, London, 1994. n 3 6 6 6 6 t value* 1.80 1.65 2.05 1.35 2. Parfitt K, Ed Martindale, The Extra Pharmacopoeia, the Pharmaceutical F value 1.45 1.75 1.84 1.60 Press, London, 32nd ed., 1999.
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Source of support: Nil Conflict of interest: None Declared
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