See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/259592302
Spectrophotometric Determination of Benzocaine In Pharmaceutical Formulations Via Oxidative Coupling Reaction
Article · January 2008
CITATIONS READS 8 613
1 author:
Raeed M. Qadir Helmholtz Zentrum München
13 PUBLICATIONS 140 CITATIONS
SEE PROFILE
All content following this page was uploaded by Raeed M. Qadir on 09 January 2014.
The user has requested enhancement of the downloaded file. J. Duhok Univ., Vol. 11, No.2, pp, 2008
SPECTROPHOTOMETRIC DETERMINATION OF BENZOCAINE IN PHARMACEUTICAL FORMULATIONS VIA OXIDATIVE COUPLING REACTION
RAEED MEGEED QADIR Dept. of Chemistry, College of Science, University of Dohuk, Kurdistan region, Iraq (Received:, ; accepted for publication:,)
KEYWORD: Spectrophotometric, Benzocaine, Promethazine, Oxidative coupling
ABSTRACT A simple, rapid and sensitive spectrophotometric method for the microdetermination of benzocaine is described. The method is based on the oxidative coupling reaction of benzocaine with promethazine hydrochloride reagent using N-bromo succinimide as oxidizing agent in acidic medium to form a green colored complex, which is water soluble, stable and has a maximum absorption at 611 nm. Beer’s law is obeyed in a concentration range of 5 - 300 g of benzocaine in a final volume of 25 ml (i.e. 0.2 – 12 ppm), with a molar absorptivity of 1.51104 L.mol-1.cm-1, and the sandell's sensitivity is 0.01097 g.cm-2, with a relative error of –0.51 to +1.31% and relative standard deviation of 0.31 to 1.96% depending on the concentration levels. The proposed method has been successfully applied to the determination of benzocaine in two pharmaceutical formulations.
INTRODUCTION EXPERIMENTAL
enzocaine, p-aminobenzoic (ethyl 4- Apparatus B aminobenzoate) acid ester is a local All spectral and absorbance measurements were anesthesia used as surface anesthetic, for the local performed on UNICAM Helios Beta 9423 UV- and temporal relief of pain related, among other Visible recording spectrophotometer using 1 cm silica disorders, to buccal affections for such reasons, it is a cell, pH meter type HANNA 301 pH - Ion meter is ® ® drug extensively used in odontology(1). Benzocaine used for pH readings, and OHAUS – Voyager acts as local anesthesia by preventing transmission of analytical balance was used for weighing processes. impulses along nerve fibres and at nerve endings. It is Reagents comparatively non-irritating and has low systemic All chemicals used in this investigation were of toxicity(2,3). This essential drug is found in the liquid, analytical – reagent grade and the standard gel, lozenge, lotion, ear drop and aerosol spray forms. pharmaceutical materials was provided from general It used as a temporary topical anesthetic that numbs establishment for medical appliance and drugs / the nerve endings near the surface of the skin where SDI – Samaraa / Iraq. applied. Physicians throughout the world prescribe Solutions benzocaine for people four months and older(4). Benzocaine solution, 100 g.ml-1. A considerable number of publications have This solution was prepared by dissolving 0.01 g of appeared describing different methods for the benzocaine in 2 ml of ethanol and 20 ml of distilled determination of benzocaine in pure form and in water to increase solubility and diluted to 100 ml in a pharmaceutical formulations, these methods included volumetric flask with distilled water. (5) (6,7) colorimetric , spectrophotometric , and Promethazine hydrochloride solution, 0.1 % (w/v). kinetic(8).Other methods includes the using of (9) (10) This solution was prepared by dissolving 0.1 g of chemiluminescence , and luminescence . High promethazine hydrochloride reagent in 100 ml of performance liquid chromatography (HPLC) is also distilled water in a volumetric flask. described for determination benzocaine (11,12) , beside to gas chromatography method (13). N-Bromo succinimide solution, 0.1 % (w/v). The oxidative coupling reaction is one type of This solution was prepared by dissolving 0.1 g of organic reactions that have a big importance in N-bromo succinimide in 25 ml of distilled water with analytical chemistry because of the high sensitivity of heating and the volume completed to 100 ml in a such reactions. Oxidative coupling reactions depend volumetric flask. on the reaction of two or more of organic compounds Hydrochloric acid solution, 0.1 N. in presence of an oxidizing agent to yield a colored This solution is prepared by diluting 2.13 ml of product in most cases(14). the concentrated hydrochloric acid (36%) solution to The purpose of the investigation reported in this 250 ml with distilled water in a volumetric flask. paper is to evaluate a simple spectrophotometric Eardrop (OTIC) solution. method for the microdetermination of benzocaine A suitable volume (1 ml) of the pharmaceutical based on it is oxidative coupling reaction with solution containing exactly 0.05 g of benzocaine was promethazine hydrochloride in the presence of N- treated with 2 ml of ethanol and diluted to 100 ml bromo succinimide as oxidizing agent yielding highly with distilled water to give 500 g.ml-1 solution of colored dye in acidic medium. Application part benzocaine, different volumes of this solution latter included determination of benzocaine in two was used to be determinates by proposed method. pharmaceutical formulations. Sore lozenge solution. One lozenge which contains exactly 0.01g of benzocaine was allowed to be dissolved in a solution
J. Duhok Univ., Vol. 11, No.2, pp, 2008
of 20 ml distilled water and 2 ml ethanol with reagent, 2 ml of 0.1% N-bromo succinimide, and 1 heating, then the volume completed to 100 ml by ml of 0.1N hydrochloric acid to 200 g of benzocaine distilled water in a volumetric flask to give 100 in a final volume of 25 ml. g.ml-1 solution of benzocaine, different volumes of Optimization of Conditions this solution latter was used to be determinates by The effect of various variables on the color proposed method. development of the product is tested to establish the Interferences solutions, 1000 g.ml-1. optimum conditions. These solutions were prepared by dissolving 0.1 g Effect of pH of each interferent in minimum amount of a suitable The preliminary experiments have shown that the solvent (distilled water or methanol) and then diluted formation of the colored dye occurs in acidic medium to 100 ml by distilled water in a volumetric flask. .The effect of amounts of different widely used acids Different volumes of these solutions were use to had been studied (Table 1). check the selectivity of the proposed method.
RESULTS AND DISCUSSION
The preliminary Study In the preliminary study a green dye that have a maximum absorption at 611 nm was obtained by the addition of 3 ml of 0.1% promethazine hydrochloride
Table (1):- Effect of pH on absorbance
ML ADDED H2SO4 HCL CH3COOH H3PO4 OF 0.1 N Abs. pH Abs. pH Abs. pH Abs. pH ACID 0 0.014 4.91 0.014 4.91 0.014 4.91 0.014 4.91 1 0.657 2.97 0.653 2.98 0.565 3.87 0.664 3.15 2 0.673 2.78 0.692 2.75 0.571 3.77 0.702 2.96 3 0.686 2.61 0.709 2.60 0.571 3.68 0.698 2.84 4 0.685 2.50 0.713 2.45 0.572 3.63 0.687 2.65 5 0.688 2.39 0.719 2.36 0.578 3.56 0.677 2.57 6 0.689 2.33 0.714 2.29 0.579 3.53 0.673 2.51 7 0.688 2.30 0.715 2.23 0.578 3.51 0.676 2.42
The results indicates that addition of 5 ml of coupling reaction with benzocaine in presence of hydrochloric acid (0.1 N) solution give the highest N-bromo succinimide (Table 3). intensity of the colored dye at pH 2.36 and therefore Table( 3):- Effect of coupling reagent it is recommended for subsequent experiments. * COUPLING REAGENT TESTED , ABSORBAN MAX , Selection of oxidizing agent (0.1%) CE NM Different types of oxidizing agents (0.1%, w/v) 4-Amino antipyrine 0.091 481 were investigated in acidic medium (pH 2.36), the 3-Amino phenol 0.181 449 results are shown in table 2. Promethazine. HCl 0.721 611 Orcinol 0.101 597 Table( 2):- Effect of oxidizing agent Resorcinol 0.100 570 * OXIDIZING AGENT , * ABSORBANCE MAX, NM (0.1%) 3 ml added N-Bromo succinimide 0.717 611 The results in table 3 indicate that the K2Cr2O7 0.159 602 promethazine hydrochloride reagent gives highly K2CrO4 0.246 602 colored (green) dye comparing with the other KIO3 0.207 608 coupling reagents and therefore promethazine NH4VO3 Turbid --- hydrochloride reagent is recommended for * 2 ml added subsequent experiments. Effect of the concentration of oxidizing agent The results illustrated in table 2 indicated that The effect of different volumes (0.5 - 3 ml) of using of N-bromo succinimide as oxidizing agent N-bromo succinimide (0.1%) solution on the color give the more sensitive reaction (highest intensity) intensity has been studied and it was observed that and therefore it is recommended for subsequent 1 ml of N-bromo succinimide (0.1%) solution is the experiments. more suitable amount since it gives the highest value Selection of coupling reagent of correlation coefficient of measurements (Table 4). Various organic compounds (0.1%, w/v) have been tested as coupling reagent for the oxidative
J. Duhok Univ., Vol. 11, No.2, pp, 2008
Table( 4):- Effect of oxidizing agent amount ABSORBANCE G OF BENZOCAINE ADDED CORRELATI * ON ML OF 0.1% NBS COEFFICIEN SOLUTION 10 25 50 100 200 250 T (R) 0.5 0.045 0.117 0.209 0.410 0.554 0.552 0.95183 1.0 0.045 0.100 0.206 0.400 0.791 0.978 0.99994 1.5 0.054 0.094 0.210 0.386 0.719 0.973 0.99807 2.0 0.049 0.069 0.164 0.350 0.715 0.969 0.99777 2.5 0.033 0.069 0.159 0.344 0.686 0.971 0.99642 3.0 0.001 0.058 0.154 0.309 0.606 0.802 0.99908 * NBS = N-bromo succinimide
Therefore, the volume of 1 ml of N-bromo that using 2 ml of promethazine hydrochloride (0.1%) succinimide (0.1%) solution is recommended for the solution give the highest value of correlation subsequent experiments. coefficient (Table 5). Effect of the concentration of the coupling reagent Various volumes of promethazine hydrochloride (0.1%) solution were tested and the results indicated
Table( 5):- Effect of coupling reagent amount CORRELATIO ML OF 0.1% ABSORBANCE G OF BENZOCAINE ADDED N PROMETHAZINE. COEFFICIENT HCL SOLUTION 20 40 80 160 200 260 (R) 0.5 0.003 0.011 0.033 0.099 0.148 0.271 0.96704 1.0 0.020 0.050 0.243 0.504 0.650 0.961 0.99594 2.0 0.096 0.150 0.318 0.639 0.796 1.015 0.99966 3.0 0.079 0.149 0.320 0.621 0.796 0.997 0.99952 4.0 0.095 0.159 0.320 0.629 0.797 0.965 0.99856 5.0 0.087 0.163 0.322 0.644 0.801 0.939 0.99631
The volume of 2 ml of promethazine surfactant (cationic, anionic and neutral) to the hydrochloride (0.1%) solution is recommended for medium of reaction. The results are shown in table 6. the subsequent experiments The selected surfactants are: Effect of Surfactants Cetyltrimethylammonium bromide (CTAB) (cationic) The effect of surfactant was studied by the Sodium dodecyl sulphate (SDS) (anionic) addition of different volumes of various types of iso-Octylphenoxypolyethoxyethanol (Triton X-100) (neutral)
Table( 6):- Effect of surfactants and the order of additions
ABSORBANCE /VOLUME OF SURFACTANTS ADDED SURFACTANTS ADDED 0.0 ml 1.0 ml 2.0 ml 4.0 ml CTAB, 110-3M 0.673 0.654 0.606
SDS, 110-3M 0.387 0.301 0.219 0.797 Triton X-100, 1% (v/v) 0.644 0.642 0.638
The results in table 6 indicate that all types of Order of addition of reagents surfactant had a negative effect on the intensity of the The order of additions of reagents was examined colored dye absorption and therefore, the addition of in a final volume of 25 ml and the results are shown surfactant not recommended. in table 7.
Table( 7):- Effect of order of addition NO. ORDER OF ADDITIONS OF REAGENTS ABSORBANCE I 200g Benzocaine+ 2ml 0.1% Promethazine+ 5ml 0.1N HCl+ 1ml 0.1% NBS 0.310 II 200g Benzocaine+ 2ml 0.1% Promethazine+ 1ml 0.1% NBS + 5ml 0.1N HCl 0.795 III 200g Benzocaine+ 1ml 0.1% NBS + 2ml 0.1% Promethazine+ 5ml 0.1N HCl 0.565 IV 200g Benzocaine+ 1ml 0.1% NBS+ 5ml 0.1N HCl + 2ml 0.1% Promethazine 0.416 V 200g Benzocaine+ 5ml 0.1N HCl + 2ml 0.1% Promethazine + 1ml 0.1% NBS 0.657 VI 200g Benzocaine+ 5ml 0.1N HCl+ 1ml 0.1% NBS + 2ml 0.1% Promethazine 0.614
J. Duhok Univ., Vol. 11, No.2, pp, 2008
The results in the above table indicate that the The effect of time on the development and stability order (II) (given under present procedure) of addition period of the colored dye was investigated under the optimum conditions of the reaction. The results of reagents is the optimum order that gives the indicate that the absorbance of the colored dye highest intensity of the colored dye. remained constant for at least 30 minutes (Table 8).
Effect of reaction time Table( 8):- Effect of time on absorbance
G OF *ABSORBANCE / MINUTES BENZOCAINE IN 5 10 15 20 30 40 50 60 25ML 10 0.075 0.073 0.072 0.072 0.071 0.069 0.069 0.068 50 0.225 0.225 0.224 0.223 0.223 0.221 0.221 0.220 100 0.382 0.381 0.381 0.380 0.378 0.377 0.375 0.373 150 0.577 0.574 0.572 0.572 0.571 0.567 0.565 0.564 200 0.798 0.796 0.796 0.794 0.794 0.791 0.788 0.786 * Absorbance readings after 10 minutes of developing time.
The results shown in table 8 indicate that the promethazine hydrochloride reagent in presence of stability period is sufficient to allow several N-bromo succinimide in an acidic medium shows measurements to perform sequentially. maximum absorption at 611 nm in contrast to the reagent blank, which shows a weak absorption at the Final Absorption spectra same wavelength (Fig.1) The absorption spectra of the colored complex formed from the reaction between benzocaine and
0.95
0.75 B
0.55 A
Absorbance 0.35
0.15
-0.05 400 450 500 550 600 650 700 750 800 Wavelength, nm
Fig (1):- Absorption spectra of 100 g of benzocaine / 25 ml treated according to the recommended procedure and measured (A) against reagent blank, (B) reagent blank measured against distilled water.
Procedure and calibration graph flasks, the volumes are completed to the mark with To a series of 25 ml calibrated flasks, an distilled water, after 10 minutes the absorbances are increasing volumes (0.05- 5 ml) of (100 g.ml-1) of read at 611 nm against reagent blank. The calibration benzocaine are transferred followed by addition of 2 graph is linear over the range 5 - 300 g of ml of (0.1% ,w/v) promethazine hydrochloride benzocaine in a final volume of 25 ml (i.e. 0.2 – 12 reagent solution, with shaking, followed by 1 ml of ppm) (Fig. 2). The apparent molar absorptivity has (0.1% ,w/v) N-bromo succinimide solution and then a been found to be 1.51104 L.mol-1.cm-1. 5 ml of 0.1N of hydrochloric acid is added to the
J. Duhok Univ., Vol. 11, No.2, pp, 2008
1.6
1.4 y = 0.0036x + 0.037 r = 0.9996 1.2 1
0.8
Absorbance 0.6
0.4
0.2
0 0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600
g of Benzocaine in 25 ml
Fig (2):- The calibration graph for benzocaine determination by oxidative coupling reaction with promethazine and N-bromo succinimide.
Accuracy and precision To check the accuracy and precision of the Table( 10):-Effect of interferences on the determination of 100 g of benzocaine method, benzocaine has been determined for five replicates at four different concentrations. The results Relative error, (%) / g of interference illustrated in table 9 indicate that satisfactory Interference added precision and accuracy could be obtained. 100 200 300 Glucose +2.50 -2.40 -3.60 Table( 9):- Accuracy and precision of the proposed method Dextrose +0.76 -2.60 -3.30 Amount of * Relative standard Lactose +1.30 -2.90 -4.50 benzocaine taken Relative error , (%) * deviation , (%) Starch -1.40 -1.80 -4.80 (g / 25ml) Acacia -0.78 +2.33 +3.32 20 +0.56 1.96 Salicylic acid -0.62 +0.71 +1.40 50 +1.31 0.60 Benzoic acid -0.32 -0.76 -1.10 100 -0.42 0.49 200 -0.51 0.31 Nature of the dye * Average of five determinations The composition of the colored dye yielded from the oxidative coupling reaction between benzocaine Interferences and promethazine hydrochloride reagent has been To demonstrate the selectivity of the proposed established using continuous variation (Job’s) method, the interfering effect of various method(15) ( Fig. 3 ) indicate that the colored dye has pharmaceutical additives were examined by a composition of 1:2, benzocaine to promethazine determining 100g of benzocaine in presence of each hydrochloride reagent at 611 nm. of the interferences in final volume of 25 ml using the recommended procedure. The results obtained are summarized in table 10.
J. Duhok Univ., Vol. 11, No.2, pp, 2008
0.5
0.4
0.3
0.2 Absorbance 0.1
0 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 [S] / [S]+[R]
Fig (3) :- Job’s plot for benzocaine (S)- promethazine hydrochloride(R)
The apparent stability constant has been calculated (16) and is found to be 1.50108 L2.mol-2, indicate that the formed dye is stable. Therefore, the structure of the formed dye may be written as follows.
CH3 CH3 CH CH 3 3 N CH H2C CH 3 H2C CH N CH3
N N
S N S
C OCH2CH3 O
Green complex
APPLICATION OF THE METHOD applying proposed procedure, good recovery is obtained as shown in table 11. The proposed method is applied to determine benzocaine in two pharmaceutical formulations. On
Table( 11):- Application of the proposed method for the determination of benzocaine
Pharmaceutical g of Benzocaine g of Benzocaine Recovery Drug Supplier Certified value preparation present /25 ml measured/25 ml (%) 50 48.95 97.90 DIAMOND ® OTIC solution 100 98.40 98.40 Tampain PHARMA 50 mg (Eardrop) (Syria) 150 148.65 99.10 200 189.80 94.90 50 49.98 99.96 Anesthetic sore BAYER 100 102.30 102.30 Cepacol® 10 mg lozenges (Germany) 150 149.85 99.60 200 200.2 100.10
1.6 J. Duhok Univ., Vol. 11, No.2, pp, 2008 1.4 y = 0.0036x + 0.037 r = 0.9996 1.2 Comparison of the method 1 Table 12 shows the comparison between some of analytical variables obtained from the present 0.8 method with that of a recent analytical method.
Table (12):-Comparison of the methods
Absorbance 0.6 ANALYTICAL PARAMETERS PRESENT METHOD LITERATURE METHOD (5)
0.4 pH Acidic Acidic Temperature (○C ) At room temperature At room temperature 0.2 Development time( minutes) 10 10
λ max ( nm ) 166 525 0 Medium of reaction Aqueous Aqueous 0 40 80 120 160 200 240 280 320 360 400 440 480 520 560 600 Reagent Promethazine. HCl p-Benzoquinone Type of analytical reaction Oxidative coupling Charge-transfer g of Benzocaine in 25 ml Beer’s law range (ppm) 0.2 – 12 5 – 70
Molar absorptivity 4 3 -1 -1 1.5110 1.710 ( L.mol .cm ) Relative error (%) –0.51 to +1.31 ±1.5 RSD ( % ) 0.31 to 1.96 ±0.83 Color of the dye Green Red Sandell's sensitivity (g.cm-2) 0.01097 0.0972 Nature of the dye 1 : 2 1 : 1 Determination of benzocaine in two Determination of benzocaine in one Application of the method pharmaceutical formulations pharmaceutical formulations
The results indicate that the proposed method has a 8- M. Carmona, M. Silva, and D. Pere-Bendito, (1992), A selective good sensitivity compared with the above literature and Sensitive Kinetic Method for the Determination of Procaine method. and Benzocaine in Pharmaceuticals, J. Pharm. Biomed. Anal., CONCLUSION 10(2-3), 145-152.
9- X. R. Zhang, W. G. Baeyens, G. V. Derweken, A. C. A simple, rapid and sensitive method has been Calokerinos and K. Imai, (1995), Chemiluminescence proposed for the microdetermination of benzocaine in Determination of Some Local Anesthetics, Anal. Chem. Acta, aqueous solution. The method based on the oxidative 303(1), 137-142. coupling reaction of benzocaine with promethazine 10- A. M. Casas-Hernandez, M. P. Aguilar-Caballos and A. hydrochloride reagent using N-bromo succinimide as Gomez-Hens, (2002), Application of Time-Resolved oxidizing agent in acidic medium. The proposed Luminescence to Dry Reagent Chemical Technology, Anal. Chem. method requires neither temperature control nor Acta, 452(2), 169-175. solvent extraction and provides accurate and precise 11- B. Gigant, A. M. Barros, A. Teixeira and M. J. Marcelo- results when applied successfully to the determination Curto, (1991), Separation and Simultaneous High Performance of benzocaine in two types of pharmaceutical Liquid Chromatographic Determination of Benzocaine and Benzyl formulations. Benzoate in Pharmaceutical Preparation, J. Chro. A., 549, 217-220.
12- G. S. Sadana and A. B. Ghogar, (1991), Simultaneous REFERENCES Determination of Chloramphenical and Benzocaine in Topical 1- S.C. Sweetman , (2002), Martindale The Complete Drug Formulation by High Performance Liquid Chromatographic, J. Reference, 33rd edition, Pharmaceutical Press, UK, p. 1302–1306. Chro. A., 542, 515-520. 2- British Pharmacopeia on CD-ROM, (2000). 3rd edition, System 13- T. A. Biemer, N. Asral and J. A. Albanese, (1992), Simulation Ltd, Stationary Office, London. Simultaneous Stability Indicating Capillary Gas Chromatographic 3- http://medical-dictionary.thefreedictionary.com/benzocaine. Assay for Benzocaine and the Two Principal Benzyl Esters of 4- http://www.drugs.com/cons/Benzocaine.html Balsam Peru Formulated in a Topical Ointment, 5- Alaa S. Amin and Akram M. El-Didamony, (2003), Colorimetric J. Chro. A., 623(2), 395-398. Determination of Benzocaine, Lignocaine and Procaine 14- C. S. Sastery, B. G. Rao, B. S. Reddy and S. S. Murty, Hydrochlorides in Pure Form and in Pharmaceutical Formulations (1981), Spectrophotometric Determination of Pharmaceutically Using p-Benzoquinone, Anal. Sci., 19, 1457-1459. Important Arylamines with Metol and NBS, J. Indian Chem. Soc., 6- R. A. Zakaria, (2004), Spectrophotometric Determination of 58, 655-658. Benzocaine and Salbutamol Sulphate using Diazotization Coupling 15- R. Delevie, (1997), Principles of Quantitative Chemical Method- Application to Some Drugs Preparation, M.Sc. Thesis, Analysis, McGraw-Hill, International edition, Singapore, p. 498. University of Mosul, College of Science, pp. 5- 34. 16- L.G. Hargis, (1988), Analytical Chemistry, Principles and 7- N. D. Dinesh, P. Nagaraja, and K. S. Rangappa, (2002), Techniques, Prentice-Hall International, London, p. 424-427. Sensitive Spectrophotometric Method for the Analysis of Some Anesthetic Drugs, Indian J. Pharm. Sci., 64(5), 485-488.
View publication stats