Microsoft Word - 142-153 142 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 Spectrophotometric Determination of Amoxicillin Trihydrate in Pure and Pharmaceutical Dosage Forms Hemn A. Qader Dept. of Pharmaceutical Chemistry/College of Pharmacy/Hawler Medical University/ Erbil Nabil A. Fakhre Dept. of Chemistry/ College of Education Salahaddin University/ Erbil Received in:16/April/2015,Accepted in:23/June/2015 Abstract A rapid and sensitive method for the determination of amoxicillin trihydrate (AMXT) based on the diazo-coupling reaction was studied. Sulphanilic acid diazotizes with nitrite ion in acidic medium to produce a water soluble, colorless diazonium ion, which subsequently coupled with AMXT to form a colored azo dye in the alkaline medium, having maximum absorption at 455 nm. The calibration graph showed that Beer's law is obeyed over the concentration range of 0.3 – 30.0 μg/mL of AMXT, with the detection limit of 0.15 μg/mL and molar absorptivity was 2.3 × 104 L/mol.cm. The accuracy and the precision were acceptable depending upon the values of error percentage and relative standard deviation. The influence of common interferences was studied and the method was applied with good recovery for the determination of AMXT in pure form and different pharmaceutical formulations, which commercially available in Erbil market. Keywords: spectrophotometry, amoxicillin trihydrate, diazotization-coupling reaction. 143 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 Introduction Amoxicillin, or hydroxyl ampicillin (scheme 1), is a phenolic β-lactam antibiotic with significant activity against both Gram-positive and Gram-negative bacteria. This antibiotic is widely used to treat infectious diseases in humans and animals, and to enhance both growth and yield in agriculture (1). Amoxicillin kills bacteria by interfering with the synthesis of the bacterial cell wall. As a result, the bacterial cell wall is weakened, the cell swells and then ruptures. Amoxicillin is readily hydrolyzed by the staphylococcal penicillinase(2). In 1972, amoxicillin was introduced for the first time, which maintained broad spectrum activity of ampicillin (3), but possesses some significant advantages over ampicillin (4), include more complete gastrointestinal absorption and little or no effect on absorption of food, and other advantages over some antibiotics such as high rate of absorption and its stability under acid conditions (5). Various hydrated forms of amoxicillin, including monohydrate, dihydrate, and trihydrate, have been reported, among which, the trihydrate is the most stable hydrated form (5). The therapeutic importance of amoxicillin requires the development of a sensitive and rapid method for industrial quality control and clinical monitoring (1). From this point of view, several analytical procedures established and reported in the literature for the determination of amoxicillin in pure and pharmaceutical dosage forms, including spectrophotometric methods(6–17), derivative spectrophotometric methods (5,18,19), HPLC methods (20-22), and chemiluminescence methods (23-25). In the present work, a spectrophotometric method is established for determination of amoxicillin trihydrate in the bulk and dosage forms, with the aid of diazo-coupling reaction. Sulphanilic acid diazotizes with nitrite ion in acidic medium to produce a water soluble, colorless diazonium ion, which subsequently coupled with amoxicillin to form a colored azo dye in the alkaline medium (Scheme 2). Experimental Apparatus The spectral and absorbance measurements were carried out using 1-cm quartz cells, on a UV/Visible digital single-beam spectrophotometer of BIO-TEK Instruments (BIO-TEK Instruments manufactured in the UK for Bio-Tek instruments, model: KP-99-90283, Milan, ITALY). Reagents and solutions All chemicals were used of analytical reagent grade and all of the solutions must be prepared freshly. Amoxicillin trihydrate (AMXT) (100 μg/mL) (Fluka): prepared by dissolving 0.01 g of AMXT, in an amount of deionized water, with using ultra sonic devise for dissolving the compound and diluting to 100 mL in a volumetric flask (5). Each working standard solution was freshly prepared by suitable dilution of the stock solution with distilled water. Sulphanilic acid solution (0.5% w/v) (Hopkin and Williams, England): 0.50 g of this compound was dissolved and diluted to 100 mL with D.W. Sodium nitrite solution (0.2% w/v) (Scharlau): 0.20 g of the compound was dissolved and diluted to 100 mL with distilled water. 144 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 Hydrochloric acid, HCl (~ 0.01 N) (S D Fine-Chem Limited, Mumbai). Sodium carbonate solution (0.5 N) (Scharlau): 2.65 g of sodium carbonate, Na2CO3 was dissolved, then heated in order to dissolve and diluted to 100 mL with distilled water in a volumetric flask. Recommended Procedure Spectrophotometric determination of amoxicillin trihydrate In a series of 25 mL volumetric flasks, each one containing 0.5 mL HCl (0.01 N), 1.0 mL NaNO2 (0.2%), and 1.0 mL of sulphanilic acid (0.5%) to produce a water soluble, colorless diazonium ion. The formed diazonium ion subsequently coupled with (7.5 – 75) μg of AMXT to form an orange azo compound after the addition of 1.0 mL of Na2CO3 (0.5 N). The volume in each flask was made to the mark with distilled water, and the colored product is monitored spectrophotometrically against a reagent blank at 455 nm. The reagent blank is prepared in the same manner but without AMXT. Results and Discussions Absorption spectra When AMXT was treated according to the recommended procedure, the absorption spectra of the formed azo compound showed a maximum absorption at 455 nm. While, the blank has no significant absorbance in this region, as it is shown in the (Figure-1). Optimization of reaction conditions The effect of the type and concentration of acid with concentration of nitrite ion solution to form nitrous acid were studied. The results showed that use of 0.01 M hydrochloric acid with 1.0 mL of 0.2% NaNO2 were found to give better results (Figures 2a, 3 and 4a). The effect of volume of 0.5% sulphanilic acid solutions as diazotizing reagent was examined, 1.0 mL of this solution gave the maximum intensity (Figure 4b). The main factors for controlling azo coupling are pH value and temperature, which are to be arranged to favor coupling rather than diazo decomposition (26). Therefore, the type and the volume of different alkali solutions were examined. The results indicated that with using of 1.0 mL of 0.5 N of Na2CO3 solution, optimum condition can be obtained (Figure 2b and 4c). The order of addition of the reactants should be followed, as mentioned in the recommended procedure. The effect of using ice bath (low temperature) on the formation of the diazonium and the azo dye was examined, the results showed there was no significant difference in the absorbance measurements of the azo dye, with or without using ice, therefore, the experiments have been carried out at room temperature. Under the optimized conditions, colour stability of the azo dye was studied, the results indicated that the colour developed instantaneously and remains stable for about 15.0 minutes. Calibration graph and the statistical data Under the chosen optimum conditions, a calibration curve was constructed (Figure 5). The graph showed that the colour system is obeyed Beer’s law in the concentration range of 7.5 – 750 μg of AMXT in a 25 mL of final volume (i.e. 0.3 – 30 μg/mL of AMXT). (Table-1) shows the statistical data of the calibration curve of spectrophotometric determination of AMXT. Accuracy and precision The accuracy and the precision of the method were tested by determining five replicate of standard AMXT solution at three concentration levels. The values of the percentage of 145 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 relative error (Error %) and the percentage of the relative standard deviation (RSD %) for these replicate measurements of AMXT were calculated. The results are shown in (Table-2). Interferences Study The effects of different additives and excipients (starch, fructose, lactose, glucose, sucrose, mannitol and Mg-stearate on the determination of 15 μg/mL of AMXT via the proposed method were studied. A species considered to be interfere, when its presence caused a relative error percentage greater than ± 5.0% in the absorbance of the sample. Results, presented in Table 3, indicated that the commonly encountered excipients did not interfere in the examined method, even when they present more than 100-folds of cited drug except Mg-stearate which starts to cause interference when its concentration exceed 10-folds that of the analyte. Application of the Method The proposed method was applied successfully to the determination of AMXT in different pharmaceutical formulations, which commercially available in Erbil market. An accurately weighed amount of 10 powdered capsules or mixed content 100 mg from each one of the items under study were dissolved in minimum amount of D. W. with sonication, and transferred into a 100 mL volumetric flask then completed to the mark with distilled water. The flask with its contents was shacked well and filtered. A sample of 375 μg of AMX in a final volume of 25 mL from each sample was taken and the measurement was carried out as described earlier under the described recommended procedure. In addition, recovery experiments were performed by adding a known amount from the standard AMXT to the pre-determined dosage forms. Then total amount of AMXT, was determined with the proposed method. (Table 4) summarized composition, company, the trade name, determination of amoxicillin contained in pharmaceutical formulations with the proposed method and the recovery of the method. Conclusions The method was applied successfully for the determination of amoxicillin and amoxicillin trihydrate in the bulk and different pharmaceutical formulation samples. The proposed method offers some advantages such as; more sensitive than some of the reported methods (1, 4, 6, 9, 11, 13 – 17), rapid colour development, reproducibility, and wide applicable range compare with other published methods (14 – 16, 27, 28). In addition, the method requires neither extraction process nor heating. The application seems to be inexpensive and yielding results in good recovery (Table 5). References 1-Sueny,K. B; Valdinete, L.S; Alberto, N. A; Maria Conceição, B. S;Boaventura, F. R; and Ana Paula, S. P. (2011). A multicommuted flow analysis method for the photometric determination ofamoxicillin in pharmaceutical formulations using a diazo coupling reaction. J. Braz. Chem. Soc., 22(2):279-285. 2-Akhilesh, G; Rjkumar; Vimal, Y. and Swati, R. (2011). An analytical approach for the determination of amoxicillin and potassium clavulanate in pharmaceutical dosage form: Review. Drug Invention Today,3(4):35-37. 3-Simar, P. K; Rekha, R. and Sanju, N. (2011). Amoxicillin: a broad spectrum antibiotic. Int J Pharm PharmSci, 3(3):30-37. 146 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 4-Al-Abachi, M. Q. and Hadi, H. (2009). Flow injection spectrophotometric determination of amoxicillin in pharmaceutical samples by coupling with diazotized p-nitroaniline. Iraqi. J. Mark. Rec. Cons. Protection.1(1):105-119. 5-Al-Saidi, K. H. and Sarah, S. A. (2011). Simultaneous determination of amoxicillin and potassium clavulanate antibiotics in pharmaceutical sample using derivative spectrophotometric method. Journal of Biotechnology Research Center (Special edition). 5(3):49-60. 6-Al-Abachi, M. Q. and Hadi, H. (2007). A developed spectrophotometric determination of amoxicillin forms via charge-transfer reaction with metal. Journal of Al-Nahrain University- Science, 10(2):1-6. 7-Prakash, K; Raju, P. N; Kumari, K. S. and Narasu, M. L. (2008). Spectrophotometric estimation of amoxicillin trihydrate in bulk and pharmaceutical dosage form. E- J.Chem.5(S2):1114-1116. 8-Ünal, K; Palabiyik, İ. M; Karacan, E. and Onur, F. (2008). Spectrophotometric determination of amoxicillin in pharmaceutical formulations. Turk J. Pharm. Sci.5(1):1-16. 9-Al-Abachi, M. Q. and Hadi, H. (2009). Flow injection- spectrophotometric determination of amoxicillin based on its oxidative condensation with 4-aminoantipyrine. IJS. 50(1):8-15. 10-Rajinder, S. G. and Manirul, H. (2010). Simultaneous determination of potassium clavulanate and amoxicillin trihydrate in bulk, pharmaceutical formulations and in human urine samples by UV spectrophotometry. Int. J Biomed Sci. 6(4):335-343. 11-Patel, S. A. and Patel, N. J. (2011). Development and validation of visible spectrophotometric method for estimation of amoxicillin trihydrate in pharmaceutical dosage form. IRJP. 2(9):48-51. 12-Dhoka, M. V; Gawande, V. T. and Joshi P. P. (2011). Simultaneous estimation of amoxicillin trihydrate and bromhexine hydrochloride in oral solid dosage forms by spectrophotometric method. IRJP. 2(3):197-201. 13-Rana, S. A. (2011). Indirect spectrophotometric method for determination of amoxicillin trihydrate in aqueous solution and pharmaceutical samples. Tikrit Journal of Pure Science. 16(4):142-146. 14-Al-Uzri, W. A. (2012). Spectrophotometric determination of amoxicillin in pharmaceutical preparations through diazotization and coupling reaction. I J S. 53(4):713- 723. 15-Al-Abachi, M. Q. and Jwan A. A. (2012). Kinetic spectrophotometric methods for the determination of amoxicillin in pharmaceutical preparation. I J S. 53(1):8-16. 16-Imam, P. S; Taqui, M; Shravan K. A and Nikhila, V. (2012). New visible spectrophotometric methods for the determination ofamoxicillin trihydrate in bulk drug and their formulations. Int. Journal of Pharmacy & Industrial Research. 2(2):106-109. 17-Al-Abachi, M. Q., and Sadeem, S. (2013). Flow injection-spectrophotometric determination of phenylephrine hydrochloride and amoxicillin trihydrate in pharmaceutical preparations. Journal of Al-Nahrain University-Science, 16(1):42-52. 18-Sarmad, B. D. (2009). First- and second-order derivative spectrophotometry for individual and simultaneous determination of amoxicillin and cephalexin. National Journal of Chemistry. 34:260-269. 147 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 19-Vu, T. H. and Vu, D. H. (2009). Simultaneous determination of amoxicillin and clavulanate in combined tablets by non-derivative and derivative UV spectrophotometric techniques. Int. J. Pharm Tech Res. 1(4):1173-1181. 20-Dhiraj, S. N; Chandrakant, G. B; Surana, S. J; Venkateshwarlu, G. and Dekate, P. G. (2009). Development and validation of RP-HPLC method for simultaneous estimation of amoxicillin trihydrate and flucloxacillin sodium in capsule dosage form. Int. J. Pharm Tech Res. 1(3):935-939. 21-Madhura, D; Vandana, G. and Pranav, J. (2010). High performance liquid chromatographic method for determination of amoxicillin trihydrate and bromhexine hydrochloride in oral dosage forms. International Journal of Pharmacy and Pharmaceutical Sciences. 2(1):129-133. 22-Dorota, K. and Anna, G. (2012). HPLC analysis of amoxicillin using accq-flour reagent for pre-column derivatization. Pol. J. Environ. Stud. 21(1):139-143. 23-Xiaofeng, X. and Zhenghua, S. (2006). Ultrasensitive determination of amoxicillin using chemiluminescence with flow injection analysis. Spectroscopy: An International Journal. 20(1):37-43. 24-Hasanpour, F; Ensafi, A. A. and Khayamian, T. 2010. Simultaneous chemiluminescence determination of amoxicillin and clavulanic acid using least squares support vector regression. AnalyticaChimicaActa. 670(1-2):44-50. 25-Chivulescu, A. I; Badeadoni, M; Cheregi, M. and Danet, A. F. 2011. Determination of amoxicillin, ampicillin, and penicillin G using a flow injection analysis method with chemiluminescence detection. Rev. Roum. Chim. 56(3):247-254. 26-Hemn, A. Q. 2007. Batch and flow-injection spectrophotometric determination of nitrite in curing meat and nitrate in wastewater samples. M.Sc. Thesis. Department of Chemistry, College of Education, SalahaddinUniversity, Erbil, Iraq. Pp. 44. 27-Farhadi, K; Ghadamgahi, S; Maleki, R. and Asgari, F. S. (2002). Spectrophotometric determination of selected antibiotics using Prussian blue reaction. J. Chin. Chem. Soc.49(6):993-997. 28-Hesham, S. (2004). Selective spectrophotometric determination of phenolic β-lactam antibiotics in pure forms and in their pharmaceutical formulations. Analytica Chimica Acta. 515:333-341. 148 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 Table (1): Statistical data of the calibration curve for the determination of AMXT spectrophotometrically Value Parameter 455 λmax (nm) Brownish yellow Color 0.3-30 Linear range (μg/mL) A = 0.0602 [AMXT] + 0.0173 Regression equation 0.0602 Slop (L/mg.cm) 0.0173 Intercept 2.3×104 Molar absorptivity (L/mol.cm) 0.9999 Correlation coefficient 0.15 Detection Limit (μg/mL) 0.01823 Sandell's sensitivity (μg/cm 2) Table (2): Accuracy and precision of the proposed spectrophotometric method AMXT concentration (μg/mL) *Error % *RSD % 0.3 15 30 – 3.12 – 2.32 – 1.75 3.77 0.43 0.67 *Results for five replicates. Table (3): The effect of the presence of the most common excipients on the determination of (15 μg/mL) of AMXT. Additive and excipients Added amount (μg) Error % Starch Fructose Lactose Glucose Sucrose Mannitol Mg-stearate 2500 1500 1750 1500 1500 1500 150 – 3.76 – 3.98 – 4.16 – 3.28 – 4.13 – 4.24 + 3.26 149 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 Table (4): Determination of amoxicillin in commercial pharmaceutical formulations with the recovery result of the method. Item type Company Trade name Found amount (mg) Recovery % Capsule, 500 mg as trihydrate Hikma, Jordan Penamox 498.172 99.19 Capsule, 500 mg Global pharma, UAE Glomax 500 493.19 103.06 Capsule, 250 mg GSK, USA AmoxilTM 242.44 102.51 Capsule, 250 mg as trihydrate S.D.I, Iraq Amoxysam-250 256.28 96.98 Vial, 500 mg Panpharma S.A., France Panamoxicilline 515.34 98.64 Oral suspension, 250 mg/5 mL, as trihydrate Global pharma, UAE Glomax ® 247.98 99.63 Table (5): Comparison of the present method for the spectrophotometric determination of AMXT with some published methods. Reagents λmax nm Determination limit (μg/ml) Detection limit (μg/ml) Ref. Diazotized o-nitroaniline Diazotized p-nitroaniline Metol (N-methyl-p-hydroxy aniline) 4-aminoantipyrine Ninhydrin I3 - Diazotized p-amino benzoic acid & Diazotized procaine FeCl3 + 1,10-Phenanthroline 2,4-dinitrophenylhydrazine Fe3+ and hexacyanoferrate (III) Diazotized sulphanilic acid 435 478 620 510 578 351 435 450 510 515 700 455 25 – 400 0.5 – 100 5 – 60 1 – 60 10 – 80 2 – 40 0.4 – 10 0.4 – 14 2 – 20 1 – 40 5 – 13.5 0.3 – 30 5.1 0.104 1.494 0.173 2.41 0.1877 0.1916 0.230 0.5628 0.15 1 4 6 9 11 13 14 14 16 17 27 p. w* * p.w.= present work N S H N O O O NH2 HO CH3 CH3HO 3H2O Scheme (1): The structure of amoxicillin trihydrate (AMXT). 150 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 O S O HO NH2 Sulphanilic Acid NO2 H O S O HO N2 O S O HO N2 Diazonium Ion + Diazonium Ion N S O OH OO HO H2N HN O HH O HH O H H Amoxicillin Trihydrate Na2CO3 O S O HO N N S O OH OO HO H2N HN Azo Dye N Scheme (2): The reaction of AMXT with diazotized sulphanilic acid to produce azo dye 151 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 Fig. (1): Absorption spectra of blank against distilled water (1) and azo dye against reagent blank (2) treated according to the recommended procedure. Fig. ( 2): Effect of (a) type of acid, (b) type of base on the formation of nitrous acid. Fig. (3): Effect of concentration and volume of HCl on the reaction product. 0.1 0.2 0.3 0.4 0.5 0.6 0 0.5 1 1.5 2 2.5 3 A b so rb a n ce a) Conc. of HCl (g.equ/L) (× 3). b) Volume of 0.01 N HCl. 152 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 Fig. (4): Effect of volumes of sodium nitrite, sulphanilic acid, and sodium carbonate solution on the reaction product. Fig. (5): Calibration curve constructed under optimum conditions. 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0 0.5 1 1.5 2 2.5 3 3.5 4 A b so o rb a n ce a) volume of 0.5% sulphanilic acid. b) Volume of 0.2% NaNO2. c) volume of 0.5 N Na2CO3. y = 0.0602x + 0.0173 R² = 0.9998 0 0.4 0.8 1.2 1.6 2 0 5 10 15 20 25 30 A b so rb a n ce Concentration (µg/mL) 153 | Chemistry 2015عام ) 3العدد ( 28مجلة إبن الھيثم للعلوم الصرفة و التطبيقية المجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 28 (3) 2015 الماء في صورته النقية والمستحضر الصيدالنيلألموكسيلين ثالثي الطيفي التقدير ھيمن عبدول قادر أربيل /جامعة ھولير الطبية /كلية الصيدلة /قسم كيمياء الصيدلية نبيل عادل فخري جامعة صالح الدين /كلية التربية /قسم الكيمياء 2015/حزيران/23البحث في: ، قبل2015/نيسان/16استلم البحث في: الخالصة تم اجراء دراسة طريقة طيفية سريعة وحساسة لتقدير األموكسيلين ثالثي الماء بوساطة تفاعل األزوتة واالزدواج. تمت ازوتة حامض السلفانيليك عن طريق تفاعله مع أيون النتريت في محيط حامضي، لتكوين أيون ديازونيوم ذائب في الماء وغير زونيوم المتكون مع األموكسيلين ثالثي الماء لتكوين صبغة ازو ملونة في محيط ملون، ثم أجريت عملية ازدواج أليون الديا نانومترا. يشير الرسم البياني لالمتصاص مقابل التركيز بأن 455قاعدي التي أظھرت اعلى امتصاص عند الطول الموجي 3.2ع امتصاص مولي مقداره ميكروغرام/مل م 15.0) ميكروغرام/مل وبحد كشف 30 – 0.3قانون بير ينطبق ضمن المدى ( 410  ،وكانت قيم الدقة والتوافق للطريقة المقترحة والمحسوبة اعتمادا على قيم الخطأ النسبي واالنحراف لتر/مول.سم القياسي النسبي مقبولة. تم دراسة تأثير المتداخالت الشائعة وطبقت الطريقة المقترحة لغرض تقدير األموكسيلين ثالثي ختلفة المتوفرة في أسواق أربيل.الماء في صورته النقية وفي بعض المستحضرات الصيدالنية الم : المطباقية الضوئية، اموكسولين ثالثي الماء، تفاعل ازونة، اقتران.الكلمات المفتاحية