Iraqi J Pharm Sci, Vol.25(1) 2016 Spectrophotometric determination of olanzapine 42 The Spectrophotometric Determination of Olanzapine via Coupling with Diazotized p-Nitroaniline Sahar R. Fadhel *,1 , Najwa I. Abdulla ** and Intidhar D. Sulaiman ** * Department of Chemistry, College of Science, University of Diyala, Diyala, Iraq. ** Department of Chemistry, College of Education for Pure Sciences (Ibn Al-Haitham), University of Baghdad, Baghdad, Iraq. Abstract A new spectrophotometric method has been developed for the assay of olanzapine (OLN.) in pure and dosage forms. The method is based on the diazocoupling of (OLN.) with diazotized p-nitroaniline in alkaline medium to form a stable brown colored water-soluble azo dye with a maximum absorption at 405 nm. The variables that affect the completion of reaction have been carefully optimized. Beer’s law is obeyed over the concentration range of (0.5-45.0 μg.mL -1 ) with a molar absorptivity of 1.5777×10 4 L.mol -1 .cm -1 . The limit of detection was 0.3148 μg.mL -1 and Sandell’s sensitivity value was 0.0198 μg.cm -2 . The proposed method has been applied successfully to the determination of olanzapine in tablet pharmaceutical preparations. Keywords: Spectrophotometry, Olanzapine, Diazotization, p-nitroaniline, tablet dosage form. ويترواويليه-ت باراووالوسابيه باقتراوه مع العامل المؤزالتقدير الطيفي لال سحر ريحان فاضل *،1 وجوى اسحق عبد هللا ، ** اوتظار داّود سليمان، ** * انعراق. ،دٍاني ،جايعت دٍاني ،لسى انكًََاء, كهَت انعهوو ** انعراق،بغداد ،جايعت بغداد ،انھَثى( ابٍ) أنصرفت نهعهوو انخربَت كهَت،انكًََاء لسى الخالصة ٌ الخرا عهي انطرٍمت حعخًد. انصَدالََت انًسخحضراث فٌ و انُمَت بصورحه طَفَت جدٍدة نخمدٍر االوالَسابٍَ طرٍمت حطوٍر ىح فٌ ذائبه بٌُ نوٌ ذاث يسخمرة صبغه نخكوٍٍ لاعدً فٌ وسظ َاٍخرواَهٍَ انداٍازوحاٍسً-انباراانداٍازو نعمار االوالَسابٍَ يع كاشف بدلت انخفاعم اكخًال فٌ حؤثر انخٌ انعوايم يٍ درسج انعدٍد. َاَويخر 405 انًوجٌ انطول عُد ايخصاص اعظى اعطج انًاء انخٌ يم.ياٍكروغراو ( 45,0 – 0,5)انخراكَس يا بٍَ يٍ يدى ضًٍ بَر نمد اَطبك لاَوٌ .انفضهي نهخفاعم انظروف عهي نهحصول -1 . 10يوالرٍت حمدر حَث اعطج ايخصاصَه 4 يول.نخر 1.5111× -1 سى. -1 يم.ياٍكروغراو 0.3140انكشف يساوٍا نـ نمد كاٌ حد . -1 ايا سى.ياٍكروغراو 0.01.0 حساوً ساَدل فمد كاَج حساسَت -2 فٌ حمدٍر االوالَسابٍَ فٌ بُجاح خرحتمانً انطرٍمت حطبَك حى . يسخحضراث انحبوب انصَدالََت. ويترواويليه, مستحضرات الحبوب الصيدالوية.-, اوالوسابيه, االزوته, باراالطيفي التقديرالكلمات المفتاحية: Introduction Olanzapine (OLN.) )C17H20N4S( is known chemically as 2-methyl-4-(4-methyl - 1piperazinyl) 10H-thieno [2,3-b] [1,5] benzodiazepine (Scheme 1), has molar mass of 312.439 g.mol -1 and it is a yellow crystalline solid substance with a melting point of 195ºC. (1,2) It is an atypical antipsychotic drug used in the treatment of schizophrenia and other psychotic syndromes (3) . Scheme (1):- The structural formula of olanzapine. (1) The mode of action of olanzapines’ of antipsychotic activity having efficacy in schizophrenia is unknown, (4) it has been proposed that drug achieve this seemingly highly selective approach to treatment is the antagonism of a specific serotonin receptor, 5- HT2A .This receptor can be found on the axon terminal of neurons that produce dopamine. When serotonin activates those receptors, the release of dopamine decreases. By building into the typical antipsychotics a mechanism to block the 5-HT2A receptors from serotonin, the dopamine release is increased. (3,5) Since its introduction in 1996 in over 84 countries (6) (OLN.) was determined by using several methods that have been reported for the analysis in pure form, dosage forms or in combination with other drugs. These methods include spectrophotometry, (7-10) HPLC, (11,12) flow injection analysis, (13) electro-analytical method, (14,15) and capillary electrophoresis (16) . 1 Corresponding author E-mail: saharchemst2011@yahoo.com Received: 19 /1/2016 Accepted: 12/4/2016 Iraqi J Pharm Sci, Vol.25(1) 2016 Spectrophotometric determination of olanzapine 43 The aim of the present study is to develop an accurate, simple and sensitive spectrophotometric procedure for the determination of olanzapine in pure and tablet dosage forms. The method is based on the coupling of olanzapine with diazotized p-nitroaniline in basic medium to form a colored complex. In addition, the reaction conditions were studied univariatly one-factor-a time to provide an optimized analytical response. Experimental Apparatus A PG instrument, UV- visible spectrophotometer model T80 (U.K) with 1 cm matched quartz cells was used for the absorbance measurements. Sartorius BL 210S electronic balance was used for weighing the samples. Hot plate with magnetic stirrer, Ijlassco, India. Materials and methods All chemicals used were of analytical reagent grade and were obtained from BDH and Panreac. Olanzapine standard powder was kindly provided by the State Company for Drug Industries and Medical Appliances (SDI), Samara-Iraq. The pharmaceutical preparation; Zyprexa  tablet (5 mg) Lilly (Spain), Zyprexa  tablet (10 mg) Lilly (Spain) and Olan  tablet (5 mg) Micro (India) were purchased from local markets. Olanzapine stock solution [1000 µg.mL -1 ] The stock solution of (OLN.) was prepared by dissolving an accurately weighed 0.1000 g of pure drug in 5 ml of 0.1 M HCl and the volume was made up to the mark in a 100 mL volumetric flask with distilled water. The stock solution was protected from light and stored at 5 ºC. Olanzapine working solution [100 μg.mL -1 ]: prepared by diluting 10 mL of the stock solution to 100 mL in a volumetric flask with distilled water. Diazotized p-Nitroaniline reagent solution, (DPNA), [0.01 M] p-Nitroaniline (PNA) (0.2762) g was dissolved in 50 ml distilled water and 6.7 ml of concentrated HCl was then added to this solution with stirring. The mixture was heated to obtain a clear solution, transferred to 200 ml volumetric flask, and cooled to (0-5) C in an ice-bath. NaNO2 (0.1380) g was then added and the mixture was stirred vigorously. Five minutes later, the solution was made up to the mark with cold distilled water. The solution is kept in a brown bottle into refrigerator and used after three hours of preparation. It is stable for at least 72 hours. Sodium hydroxide solution (~2 M): prepared by dissolving (8.000) g of NaOH in a suitable volume of distilled water and the volume was made up to the mark in 100 mL volumetric flask. Olanzapine tablets solution [1000 µg.ml -1 ] Seven tablets of both strengths of Zyprexa and ten tablets of Oln-5 were accurately weighed and separately, grinded into fine powder and mixed well then the average weight was calculated for each brand. An amount of the powder equivalent to (0.5928) g, (0.2972) g and (0.5848) g (containing 0.0200 g of the drug olanzapine) of Zyprexa-5 mg, Zyprexa- 10 mg and Oln-5 mg respectively was accurately weighed, dissolved in 5 ml of 0.1 M HCl and stirred for 10 min to ensure complete dissolution of the drug, then transferred into 20 mL volumetric flask and diluted to the mark with distilled water to get 1000 µg.mL -1 of (OLN.). The solution was filtered by using Whatman filter paper No.41 to avoid any suspended or un-dissolved material before use. Working solution 100 µg.mL -1 was freshly prepared and analyzed by the recommended procedure. General recommended procedure for calibration In a series of 10 mL volumetric flasks, 1 mL of 0.01 M of the diazotized p- nitroaniline solution, aliquots of working drug solution (100 µg.mL -1 ) in the range (0.05, 0.1, 0.3, 0.5……4.5) mL were added to each flask followed by 1 ml of 2 M sodium hydroxide with shaking and allowed to stand for 5 min. The contents were diluted to the mark with distilled water and mixed well. After 5 min, the absorbance of the brown colored azo-dye was measured at 405 nm against the reagent blank prepared in the same manner without the analyte. Results and Discussion Absorption Spectra for primary test The primary test for the present method involved diazotization of p-nitroaniline followed by coupling with olanzapine (OLN.). The test was done by adding 1 ml of 0.005 M diazotized p-nitroaniline in 10 mL volumetric flask, followed by the addition of 1 ml of 100 µg.ml -1 (OLN.) with shaking. 1 ml of 1 M NaOH was then added to the above mixture. The contents were diluted to the mark with distilled water. The absorbance and λmax of the colored azo-dye was measured against the reagent blank prepared in the same manner without the analyte. (Figure 1) shows that the maximum absorption was obtained at a wavelength of 405 nm. Iraqi J Pharm Sci, Vol.25(1) 2016 Spectrophotometric determination of olanzapine 44 Figure (1): Absorption spectra of: (a) the complex of 10 µg.mL -1 (OLN.) with diazotized p-nitroaniline against reagent blank, (b) blank solution against distilled water under the primary test conditions. Optimization of reaction variables The various parameters related to the colored product formation have been studied by varying the parameters one at a time and controlling all others fixed and the optimum conditions have been selected 1. Effect of the diazotized p-nitroaniline concentration The optimum diazotized p-nitroaniline concentration was estimated by adding 1mL of various concentrations [0.005- 0.030] M of diazotized p-nitroaniline reagent solution, The results showed that 0.01 M of the reagent solution is sufficient for production of maximum and reproducible color intensity (Figure 2). Therefore, the recommended concentration of diazotized p-nitroaniline was chosen to be 1 mL of 0.01 M and used for all subsequent measurements. Figure (2): Effect of diazotized p- nitroaniline concentration on the color development in the determination of 10 µg.mL -1 (OLN). 2. Effect of type of the base The effect of different alkaline solutions with concentration of 1 M on the absorption intensity of the colored azo-dye formed was investigated. It was found that sodium hydroxide gave the maximum absorption intensity of the colored product which is used for the subsequent work, (Table 1). Table (1): Effect of different bases. Alkaline medium [1M] Absorbance NaOH 0.216 KOH 0.188 Na2CO3 0.104 NH4OH 0.025 2. Effect of sodium hydroxide concentration The stability of the formed azo dye product depends upon the nature of reaction medium. (17) The formed azo dye was found to have a reasonable stability when the reaction medium was rendered alkaline via the addition of 1ml of 2 M sodium hydroxide solution which was optimum and recommended for the subsequent work, (Figure 3). Figure (3): Effect of sodium hydroxide concentration on the color development in the determination of 10 µg.mL -1 (OLN.). 4. Effect of coupling reaction time The optimum time of coupling reaction was determined by choosing different time periods (0-20) min for development the color of azo-dye at room temperature, it was found that 5 minutes period was required for full color development as shown in (Table 2). Table (2): Effect of coupling reaction time. Time (min) Absorbance 0 0.305 5 0.392 10 0.362 15 0.314 20 0.288 5. Effect of order of mixin The effect of different orders of component addition on chromogen formation was studied by changing the order for three times as Iraqi J Pharm Sci, Vol.25(1) 2016 Spectrophotometric determination of olanzapine 45 shown in (Table 3). Results shows that mixing order number one was recommended and thus was followed in the subsequent experiments, since it resulted in obtaining maximum absorbance. Table (3): Variation of absorbance with reactants addition order on the determination of 10 µg.mL -1 (OLN.). No Sequence Absorbance 1 Diazotized reagent + Drug + Base 0.392 2 Diazotized reagent + Base+ Drug 0.330 3 Drug + Base + Diazotized reagent 0.327 6. Stability Under the aforementioned optimum condition, the effect of time on the formation of the azo-dye product was investigated by allowing the reaction to proceed by varying periods. It was found that the absorbance reach to a maximum constant value after 5 min, and the color of the azo product was nearly stable for at least 60 min as shown in (Figure 4). Figure (4): The stability of colored reaction product with time. Final absorption spectra When 10 µg. mL -1 of (OLN.) is treated with diazotized p- nitroaniline reagent, under the aforementioned optimum conditions, an absorption peak is obtained showing intense brown azo - dye absorption at 405 nm, the reagent blank showed almost nil absorption at this maximum wavelength as shown in (Figure 5). Figure (5): Absorption spectra of: (a) the complex of 10 µg.mL -1 (OLN.) with iazotized p-nitroaniline against reagent blank, (b) blank solution against distilled water under the optimum conditions. Calibration curve and analytical data According to the optimum experimental conditions, linear calibration graph for (OLN.) was obtained (Figure 6), which shows that Beerʼs law was obeyed in the concentration range of (0.5- 45.0) µg.mL -1 . The regression equation, correlation coefficient, molar absorptivity, Sandell's sensitivity, limit of detection (LOD) and limit of quantification (LOQ) were given in (Table 4). Table (4): Optical characteristics and statistical data for the determination of (OLN.). Parameter Value λmax (nm) 405 Color Brown Regression equation Y=0.0505[(OLN.) µg.mL -1 ]-0.0119 Linearity range (µg.mL -1 ) 0.5 - 45 Calibration sensitivity (mL.µg -1 ) 0.0505 Correlation coefficient (r) 0.9994 Correlation of linearity (R 2 ) 0.9989 Molar absorptivity (L.mol -1 .cm -1 ) 1.5777x10 4 Sandellʼs sensitivity (µg.cm -2 ) 0.0198 L.O.D. (µg.mL -1 ) 0.3148 L.O.Q. (µg.mL -1 ) 1.0495 Iraqi J Pharm Sci, Vol.25(1) 2016 Spectrophotometric determination of olanzapine 46 Figure(6): Calibration curve for the determination of (OLN.) under optimum conditions. Nature of the dye product Job's method (18) and mole ratio method (19) have been used in the determination of the reaction ratio of (OLN.) with p-nitroaniline reagent. The obtained results in (Figures 7 and 8) showed that 1:1 (OLN.) to diazotized p- nitroaniline reagent ratio is obtained. Hence the azo-dye may have the proposed mec hanism illustrated in (Scheme 2). Figure (7): Continuous variation method for reaction (OLN.) with diazotized p- nitroaniline Figure (8): Mole ratio method for (OLN.) with diazotized p-nitroaniline. Scheme (2):- The suggested reaction mechanism between DPNA and (OLN.). Iraqi J Pharm Sci, Vol.25(1) 2016 Spectrophotometric determination of olanzapine 47 Comparison of the methods (Table 5), shows the comparison between some analytical variables of the present method with another spectrophotometric methods in literature. Precision and accuracy The precision and accuracy for the determination of (OLN.) via the proposed method were studied by calculating the values of coefficient of variation (C.V%) and percentage of relative error (Er %), for three replicates at three different concentration levels of (OLN.) drug. The results in (Table 6) show acceptable values for accuracy and precision. Interference study The effect of various excipients, which may be present in pharmaceutical products and affecting the reaction between (OLN.) and diazotized p- nitroaniline, was studied. Optimum experimental conditions, were employed to determine 10μg.mL -1 concentration of (OLN.). (Table 7) shows that the studied excipients did not interfere in the present method. Application in pharmaceutical preparation The application of the method for the assay of olanzapine in drugs has been applied successfully, and the results obtained were listed in (Table 8) for each sample in three replicates Table (5): Analytical parameters for the analysis of olanzapine by the proposed ethodcomparing to the methods. Methods Linear range μg.mL -1 (ε) L.mol -1 .cm -1 Correlation Coefficient ( R) C.V% range Ref. Proposed method 0.5-45.0 1.5777x10 4 0.9994 0.033-0.080 … Spectrophotometric 4.0-20.0 1.74×104 0.9998 0.355-0.822 6 Spectrophotometric 5.0-160.0 0.60 x 10 3 0.9999 0.820–0.910 7 Spectrophotometric 5.0 -40.0 ---- 0.9980 0.120-0.590 9 Spectrophotometric 0.4 -8.0 2.08 × 10 4 0.9994 0.669-2.278 20 Spectrophotometric 40.0 -0.4 4.375 × 10 3 0.9999 0.270-0.700 21 RP-UPLC 25.0-150 ---- 0.9990 0.210-0.330 10 HPLC 2.5-20.0 ---- 0.9999 0.150-0.460 11 Table (6): Evaluation of accuracy and precision for the determination of (OLN.) by proposed method. Conc. of (OLN.) μg.mL -1 Er% C.V% Taken Found* 10.000 9.951 -0.490 0.458 18.000 18.013 0.072 0.013 32.000 32.046 0.143 0.033 *Average of three measurements. Table (7): Recovery values for 10 µg.mL -1 of (OLN.) in the presence of different excipients. Excipients Olanzapine Conc. Recovery (%) Name Conc. (µg.mL - 1 ) Taken (µg.mL -1 ) Found (µg.mL -1 ) Lactose 500 10.000 9.981 99.810 Glucose 500 9.839 98.390 Sucrose 500 9.990 99.900 Starch 1000 9.971 99.710 Magnesium Stearate 1000 9.952 99.520 Iraqi J Pharm Sci, Vol.25(1) 2016 Spectrophotometric determination of olanzapine 48 Table (8): Application to the olanzapine concentration measurement in tablets. Sample Weight Found* (mg) Concentration (µg.mL -1 ) Recovery % C.V % Taken Found* Zyprexa- 5 mg 5.026 10.000 10.052 100.523 0.469 5.027 30.000 30.166 100.553 0.285 Zyprexa- 10 mg 10.145 10.000 10.145 101.450 0.514 10.031 30.000 30.093 100.310 0.107 Olan- 5 mg 4.882 10.000 9.947 99.473 0.280 4.986 30.000 29.917 99.725 0.185 *Average of three measurements. Conclusion The proposed method permits rapid, precise, and accurate determination of olanzapine. It makes use of simple reagents, which an ordinary analytical laboratory can afford. The method was found to be free from interference by the excipients. The wide applicability of the new procedure for routine quality control was well established by the assay of olanzapine in pure form and in pharmaceutical preparations. References 1. British harmacopoeia on - , er ajesty s Stationary office, London, 2013. 2. Andrew, W.; Pharmaceutical manufacturing encyclopedia, 3 rd edition, Elsevier Saunders, 2013; 5-6. 3. Kassahun, K.; Mattiuz, E. 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