Conseguences of soil crude oil pollution on some wood properties of olive trees Chemistry |112 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Modified Simplex - Spectrophotometric Determination of Clonazepam via Charge-Transfer Complexation Faeza Hazim Zankanah Sarmad Bahjat Dikran Dept. of Chemistry/College of Education for Pure Science (Ibn Al-Haitham) /University of Baghdad Received in:28/September/2016,Accepted in:2/November/2016 Abstract A simple, sensitive, accurate, and precise spectrophotometric method for the determination of clonazepam (CLNZ) was developed. The method is based on charge transfer reaction between CLNZ and p-Bromanil (p-Br) to form a colored complex. The optimum conditions of complex formation were investigated by (1). Unvariable method, for the optimization of reagent concentration, base concentration, temperature, and time. (2). Multivariable simplex method including the effect of three experimental factors via; reagent concentration, concentration of NaOH and time. The linearity range of CLNZ was (1-30) μg.mL -1 at 378 nm under condition established via simplex method with molar absorptivity (1.9069x10 4 ) L.mol -1 .cm -1 , Sandell's sensitivity index (0.0165) μg.cm -2 , detection limit of 0.2957μg.mL -1 , quantification limit 0.9858µg.mL -1 and association constant of the formed complex (2333.3). The proposed method has been successfully applied for the determination of CLNZ in pure form and pharmaceutical preparations. Keywords: Clonazepam, charge transfer complexation, p-Bromanil, spectrophotometry. Chemistry |113 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Introduction Clonazepam (CLNZ) is a benzodiazepine, has the IUPAC name (5-(2-Chlorophenyl)-7- nitro-1, 3-dihydro-2H-1, 4-benzodiazepin-2-one) [1] (Figure 1), medically considered as an anticonvulsant drug which is broadly used in the controlling of epilepsy. It affects chemicals in the brain that may be unbalanced [2, 3]. For the importance of CLNZ compound, several researches have been conducted to deal with its estimation of that compound. In this respect, different methods for CLNZ identification have been reported in all dependent pharmacopeia, such as IR and TLC [1]; potentiometric and HPLC methods [4,5]. Different analytical methods have been used for the determination of CLNZ such as (HPLC) [6, 7], (TLC) [8, 9], (LC) [10, 11], capillary electrophoresis [12] and spectrophotometry [13-15]. Simplex optimization of experimental parameters was first introduced by Spendley [16] and then modified by Nelder [17] and Aberg [18]. A simplex is a geometric figure in which there are n +1 vertices, where (n) represents the number of variables [19]. The method found a lot of applications in field of analytical chemistry [20-23], because it offers the capability of optimizing several factors simultaneously depending on a statistical design search to find out the maxima or minima of response, by rejecting the point producing the worst response and a replacement of it by the new point which is obtained statistically. The aim of this work is to develop an inexpensive, simple and fast spectrophotometric method based on charge transfer (CT) reaction between CLNZ as donor and Bromanil (p-Br) as acceptor in alkaline medium. Experimental Materials All the chemicals and reagents used in the present study were of analytical grade. CLNZ was provided by the State Company for Drug Industries and Medical Appliances, Samara-Iraq (SDI). Absolute ethanol and acetonitrile were supplied from Aldrich while p-Br was supplied from Merck. Rivotril tablets (Switzerland) labeled to contain 0.5 mg of CLNZ per tablet was purchased from commercial source. Preparation of stock and working standard solutions: A stock solution of CLNZ (250 µg.mL -1 ) was prepared by dissolving accurately 0.0625g of CLNZ in 250 mL ethanol. The stock solution was further diluted to get working concentrations of (1.0, 5.0, 10.0, 15.0, 20.0, 25.0 and 30.0) µg.mL -1 . p -Bromanil (1x10 -2 M) solution: Prepared by dissolving 0.4237g of p-Br in 40 mL acetonitrile and the solution was diluted to final volume of 100 mL with acetonitrile. Sodium hydroxide (~ 0.4M) solution: Prepared by dissolving 1.6000 g of NaOH in 30 mL distilled water and diluted to 50 mL in volumetric flask with distilled water. Potassium hydroxide (~0.4 M) solution: Prepared by dissolving 2.2444 g of KOH in 50 mL distilled water and diluted to 100 mL in volumetric flask with distilled water. Preparation of sample solution: Fifteen of Rivotril tablets were weighed and finely powdered. A quantity of powder equivalent to 0.7545g of CLNZ (an equivalent amount of five tablets) was dissolved in 25 mL ethanol, left to stand for 5 min and transferred to a 50.0 mL volumetric flask and diluted with ethanol to obtain 50µg .mL -1 CLNZ. The solution was filtered by using Whatman filter paper No.41 to avoid any suspended or undissolved material before use. Chemistry |114 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Apparatus All spectrophotometric measurements were performed using Shimadzu 1800 UV-Vis; with match silica cells, a Sartorius BL 210S balance and water bath memmert W-200 RING were used throughout the work. General procedure for the determination of clonazepam i. Under condition established by univariate method: To a series of 25mL volumetric flasks, 5.0 mL of standard solution containing (25.0-750.0) μg of CLNZ were transfered. Then, 5.0mL of 5.0x10 -3 M p-Br solution was added to each flask with shaking followed by the addition of 5.0mL of 0.2M NaOH. The flasks were allowed to stand in water bath for 25 minutes at 70 o C, and completed with the ethanol .The absorbance was measured at 384.0 nm against the reagent blank. ii. Under condition established by simplex method: A series of calibrated 25 mL volumetric flasks, 5.0 mL aliquots of standard solutions containing (25.0-750.0) μg of CLNZ and 5.0 mL of 1.0x10 -3 M p-Br solution different amounts and 5.0 mL of 0.12M KOH were shaking, The solution were then allowed to stand in water bath for 15 minutes at 70 o C . The volume of each flask was then completed to the mark with ethanol and the absorption value of the resulted product was measured against the corresponding reagent blank at 378.0 nm. Results and discussion Selection of wavelength The absorption spectra of the product solution versus reagent blank and for reagent blank versus ethanol were recorded (Figure 2-I). The product shows a maximum absorption at 384.0 nm under primary test. Optimization studies Optimization of reagent concentration The effect of p-Br concentration on the colour intensity of the product was examined in the range of 1.0x10 -3 to 9.0x10 -3 M. However, 1 mL of 5.0 x 10 -3 M p-Br is found to be suitable for quantitative determination of CLNZ as well as for attainment of maximum and reproducible colour intensity (Figure 3-a). Optimization of KOH concentration The effect of different concentrations in the range of (0.04 - 0.40M) of KOH on the absorbance has been investigated. Figure 3-b shows that 1.0 mL of 0.2M KOH solution was optimum and it was recommended for the subsequent experiment. Optimization of the base type The effect of 0.2 M solution of different bases (NaOH, KOH and Na 2 CO3) was investigated (Table 1(. It was found that NaOH solution gives the maximum absorption intensity of the colored complex, which is used for the following experiments. Optimization of heating time The absorbance of the developed colored complex with respect to different time intervals (1-35 minutes) was investigated (Figure 4-a). Constant absorbance value was obtained of the 25 min of heating, which is used in the subsequent experiments. Chemistry |115 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Optimization of temperature The effect of different heating temperatures (10 to 100 °C) was examined and the results are depicted (figure 4-b). The absorbance attains maximum colour intensity at temperature 70 °C, while higher temperatures gave no satisfactory results because the product solution starts to show a slight turbidity. Optimization of solvent type Different organic solvents (ethanol, methanol, acetone, DMSO, benzene, toluene and acetonitrile) have been tried for dissolving the reagent by studies on the absorbance. Acetonitrile was found to be the most suitable solvent and also gives optimum stability of the absorbance values of the formed charge transfer complexes (Table 2). Optimization of diluting solvent The effect of solvent on the absorptivity of the CLNZ-Br complexes was studied by using different solvents (absolute ethanol, ethanol: H2O (1:1), H2O and acetonitrile) for diluting the reaction mixture. Ethanol was proved to be the most suitable diluting solvent (Table 3). Sequence of addition Different orders of addition of reagents were experimented (Figure 5-a). It was found that the order of addition: CLNZ + p-Br + Base was efficient in producing the obtained results. Stability The effect of time on formed charge transfer product was investigated by allowing standing for varying times. The results showed that the complex remains stable at least for 60 minutes (Figure 5-b). Simplex method Simplex method is used to confirm the optimum conditions, which were obtained by the univariate procedure. The simplex procedure (Table 4) optimized three major parameters: reagent concentration, concentration of NaOH and heating time. After setting the boundary conditions for each variables (Table 5), four (n + 1) arbitrary experimental conditions were chosen, within specified boundaries for each, at which they affected the measured absorption signal of the colored product (experiments 1 - 4 in Table 4). The absorbance of these four experiments was fed into the modified multisimplex program, which starts to reflect the worst point through the centroid of other points to obtain a new point 5. An experiment was then performed utilizing the variable setting as a reflected point; because this value was better than that at point 3, the latter was rejected and replaced by point 5. A measured absorption signal was fed again to the program and the process was repeated successively until optimum conditions were obtained similarly to those obtained by the univariate method. Final absorption spectra Figure 2-II shows the final spectra of charge transfer product which exhibits maximum at 384 nm, 378nm under the univariate conditions and simplex method respectively. Validation of Beer’s law (Linearity, accuracy and precision) According to the optimum conditions, linear calibration graph was obtained by plotting absorbance versus varying concentration of CLNZ and Beer’s law is valid over the concentration range of 1.0-30.0 μg.mL -1 for both univariate and simplex methods (Figures 6-a b) at 384nm and 378 nm respectively. Table (6) shows the different analytical parameters obtained such as slope, intercept, correlation coefficient, Sandell’s sensitivity, molar Chemistry |116 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 absorptivity (ε), standard deviation, limit of quantification, limit of detection and relative standard deviation. The precision and accuracy of the univariate and simplex methods were evaluated by performing three replicate analyses on pure drug solutions at three different concentration levels within the Beer’s law limits. The percent error (RE %) and relative standard deviation (RSD) values presented in Table (7) reveal the high accuracy and precision of the methods. Stoichiometric ratio The stoichiometric ratio of the reactants was determined by employing Job’s method of continuous variation [24] and molar ratio method [25]. The results indicated that the interaction occurs between equimolar solutions of CLNZ and p-Br and the complex was formed in the ratio of 1:1 as illustrated in Figures (7 –a  b) and Scheme 1. Association constant (Benesi–Hildebrand equation) The association constants of CLNZ- p-Br complex has been calculated via Benesi- Hildebrand equation [26]: Where [Aº] is the initial concentration of acceptor, [Dº] is the initial concentration of donor. A CT is the absorbance of the charge transfer complex, ε CT and K CT are the molar absorptivity and association constant of the complex respectively. A straight line was obtained by plotting [A o ]/A CT versus 1/ [D o ] (Figure8) and the calculated parameters are presented in Table (9). Interferences study The results showed that no interferences were found in the presence of 10 μg.mL -1 of the studied excipients (lactose, sucrose and glucose) in the determination of clonazepam. (Table 10). Application of the method to pharmaceutical preparation (commercial tablet) The proposed method was applied successfully to determine CLNZ in the commercial dosage form as tablets (0.5 mg/tablet) and the obtained results are given in Table (11). The recommended method was statistically compared with other methods, no significant differences were found between the calculated and theoretical values of t- test at 95% and 90% and F- test at 95% confidence limit (Table 12). Analytical application by standard additions method (SAM) Standard addition method has been followed to check the validity of the proposed method. Good recoveries suggesting non-interference were obtained as presented in Figure (9- a and b). Conclusion A charge-transfer complexation between CLNZ with p-Br reagent occurred with a 1:1 stoichiometry and maximum wavelength of absorption at 378 nm. The proposed method is beneficial over univariate method due to its sensitivity, accuracy, low relative standard deviation and high percentage of recovery and therefore it can be used in rapid quantitative determination of CLNZ in both pure and dosage form. Chemistry |117 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 References 1. British Pharmacopeia, (2013) CD-ROM Her Majesty, s Stationary office, London. 2. Dahlin, M.G., Amark, P.E., Nergårdh, A.R.; Amark; Nergårdh (January 2003). "Reduction of seizures with low-dose clonazepam in children with epilepsy". Pediatr. Neurol. 28 (1): 48–52. Doi: 10.1016/S0887-8994(02)00468-X. PMID 12657420. 3. Martindale, (2009), The Complete Drug Reference, Pharmaceutical press, 36th Edition, 478-479. 4. The Indian Pharmacopoeia, (2010), The Indian Pharmacopoeia commission, Ghaziabad, 6th edition, II: 1111-1114. 5. United State Pharmacopoeia, (2007) Rockville, USP convention, Inc, 30th edition 1795- 1797. 6. Bhimanadhuni, C. N., Garikapati, D. O.and Usha R.,(2012)," Development and validation of an RP-HPLC method for the simultaneous determination of Escitalopram Oxalate and Clonazepam in bulk and its pharmaceutical formulations", International Current Pharmaceutical Journal, 1(8): 193-198. 7. Patil, P.M.; Wankhede, S. B. and Chaudhari, P. D., (2015)," A Validated Stability– Indicating HPLC Method estimation of Clonazepam In the bulk drug and Pharmaceutical Dosage Form", Pharm Anal Acta, 6:2. 8. Thangaduraia, S., Dhanalakshmia, A., and Kannan M. S. (2013)," Separation and Detection of certain Benzodiazepines by Thin-Layer Chromatography" Malaysian Journal of Forensic Sciences 4(1). 9. Khedkar, T. S.; Reddy, Y. R., and Mali, B. D., (2012)," A thin-layer chromatographic chromatography detection of some benzodiazepines "International Journal of Medical, Legal Medicine. 15. 10. John, C.; Ghosh, P.; Varshney, K. M. ; Kaur, S.; Shukla, S. K. and Satyanarayana S., (2014)," determination of clonazepam in human hair and nail using Liquid Chromatography tandem Mass Spectrometry (LC-MS/MS)", Journal of Liquid Chromatography & Related Technologies, 37:1917–1928. 11. Jing, D.; Yintao, S.; and Junwei W., (2014)," Qualitative and Quantitative Analysis of Clonazepam and its Metabolite 7-aminoclonazepam in Blood by LC-tandem QTOF/MS and LC-MS/MS", FORENSIC SCI SEM, 4(1): 45-52. 12. Wo´zniakiewicz, A.; Wietecha-Posłuszny, R.; Wo´zniakiewicz, M.; Bryczek, and E., Ko´scielniak P., (2015)," A quick method for determination of psychoactive agents in serum and hair by using capillary electrophoresis and mass spectrometry", Journal of Pharmaceutical and Biomedical Analysis 111, 177–185. 13. Hadi, H.,(2015) "Spectrophotometric Determination of Clonazepam in Pure and Dosage forms using Charge Transfer Reaction, Iraqi J Pharm Sci,.24(1). 14. Ibrahim, F., El-Enany, N., Shalan, S., and and Elsharaway R.,(2016)," Validated spectrofluorometric method for the determination of clonazepam in pharmaceutical preparations", Department of Analytical Chemistry, University of Mansoura, Mansoura, Egypt, 31, 3, 682–687. 15. Karajgi, S.; Patil, A.; and Kotnal, R., (2016)," Area under Curve UV Spectrophotometric Method for the Determination of Clonazepam in Tablets" Human Journals, 6 (2): 314-323. 16. Snežana, S. M.; Aleksandra, N. P.;Snežana, B. T.; Emilija, T. P.; Milan, N. M. and Milan B. S., (2012)," Development and Application of Ligand-Exchange Reaction Method for the Determination of Clonazepam", Tropical Journal of Pharmaceutical; 11 (1): 91-98. Chemistry |118 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 17. Spendley, W.; Hext, G.R.; and Himusworth, F.R.T. (1962) application of Simplex designs in optimisation and evolutionary Sequential operation, Journal of Technometrics. 4: 441- 462. 18. Nelder, J. A. and Mead, R. A. (1965) Asimplex Method for Function Minimization, Computer Journal, 7: 308-313. 19. Aberg, E. R. and Gustavsson, A.G.T. (1982) Design and Evaluation of Modified Simplex Methods, Analytica Chemica Acta, 144:39-53. 20. Walters, F. H.; Parker, L. R.; Morgan, S. L.; and Deming, S. N. (1991) SequentialSimplex Optimization, 1st, CRC Press,Inc., Boca Raton, Florida, 44. 21. Momenbeik, F.; Momeniz, Z. and Kharasani, H., J.,(2005) Separation and determination of Vitamins E and A in multivitamin sy rup using micellar liquid chromatography and simplex optimization, Journal of Pharmaceutical and Biomedical Analysis, 37(2): 383- 387. 22. Murillo Pulgarn, J.A.; Alanon Molina, A. and Alanon Pardo, M .T. (2002) the use of modified simplex method to optimize the room temperature phosphorescence variables in the determination of an antihypertensive drug, Journal of Talanta, 57, 795-805. 23. Tinoi, J.; Rakariyatham, N. and Deming, R.L. (2005) Simplex optimization of carotenoid production by Rhodotorula glutinis using hydrolyzed mung bean waste flour as substrate, Journal of Process Biochemistry 40, 7: 2551-2557. 24. Yoe, J. H., and Jones, A. L. (1944) Colorimetric determination of iron with disodium-1, 2- dihydroxybenzene-3, 5-disulfonate, Anal. Chem.16, 111-5. 25. Delvie, R. (1997). "Principles of Quantitative Chemical Analysis", International Edn. McGraw-Hill Inc., Singapore, 498 p. 26. Benesi, H. A., and Hildebrand, J. H. (1949). A spectrophotometric investigation of the interaction of iodine with aromatic hydrocarbons. Journal of the American Chemical Society, 71(8), 2703-2707. 27. Al-Abachi, M. Q. Hammoudi, M. A. (2015)," Batch and Flow Injection Spectrophotometric Methods for the Determination of Clonazepam in Pharmaceutical Preparations via Oxidative Coupling with Pyrocatechol"Iraqi Journal of Science, Vol 56, .2A, and: 898-908. 28. MouayedQ.,Hadi H. (2015) Flow Injection- Spectrophotometric Determination of Clonazepam Based On Its Oxidative Condensation with Promethazine Hydrochloride, Al- Mustansiriyah Journal of Science,. 26, 1. Table (1): Effect of type of bases. Type of bases(0.2M) Absorbance NaOH 0.5521 KOH 0.5333 LiOH.H2O 0.3748 Na2CO3 0.3509 Chemistry |119 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Table (2): Effect of different types of solvent on the absorbance of 10µg.mL -1 CLNZ. Table (3): Effect of different diluting solvents on the absorbance of 10µg.mL -1 CLNZ. Table (4): Boundary of Simplex independent variables for determination of CLNZ. Variable Minimum boundary Maximum boundary Step size Con. of p-Br (M) 1.0x10 -3 9.0x10 -3 1.0x10 -3 Con. of NaOH (M) 0.04 0.40 0.04 Time(minute) 0.0 35.0 5.0 Table (5): Simplex method for determination of 10µg.mL -1 CLNZ. Solvent Absorbance Acetonitrile 0.6327 Methanol 0.4370 Acetone 0.2142 Ethanol 0.5633 DMSO 0.6515 Benzene Immiscible Toluene Immiscible Solvent Absorbance Acetonitrile 0.6845 H2O 0.2460 Ethanol 0.6327 Ethanol: H2O 0. 4998 Exp.No Factors Absorbance at 378 nm Con.of p-Bromanil (M x 10 -3 ) Con. Of NaOH (M) Heating time (minute) 1 1.0 0.08 5.0 0.6643 2 6.0 0.32 20.0 0.5840 3 5.0 0.04 1.0 0.3294 4 2.0 0.04 11.0 0.6877 5 1.0 0.20 20.0 0.6485 6 1.0 0.04 0.0 0.6600 7 2.0 0.04 0.0 0.2826 8 1.0 0.12 15.0 0.7015 9 1.0 0.16 25.0 0.6568 10 1.0 0.08 15.0 0.6667 11 1.0 0.12 25.0 0.6544 12 1.0 0.08 10.0 0.6911 13 1.0 0.12 10.0 0.6481 Chemistry |120 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Table (6): Optical characteristics and statistical data for the determination of CLNZ by univariate and simplex methods. Parameter univariate method Simplex method λmax (nm) 384.0 378.0 Colour Purple Yellow Linearity, (μg. mL -1 ) 1.0-30.0 1.0-30.0 Regression equation Y=0.0567X +0.0329 Y=0.0604X +0.0544 Slope (mL. µg -1 ) 0.0567 0.0604 Intercept 0.0329 0.0544 Correlation coefficient (r) 0.9983 0.9986 Molar absorptivity (L.mol -1 .cm -1 ) ε =17963.967 ε =19068.956 Sandell's sensitivity(µg.cm -2 ) 0.0176 0.0165 *Detection limit (µg.mL -1 ) 0.0632 0.0594 **Quantification limit (µg.mL -1 ) 0.2108 0.1979 *LOD = 3.3 σ /S , **LOQ = 10 σ/S Table (7): Evaluation of accuracy and precision via intra-day and inter-day. Method Taken Con. (μg. mL -1 ) Intra-day accuracy and precision Inter-day accuracy and precision Found* (μg. mL -1 ) RE% RSD% Found* (μg. mL -1 ) RE% RSD% Univariate 5.00 4.975 -0.500 1.6470 4.890 -2.200 1.930 12.00 12.046 0.300 1.0242 12.110 0.900 1.545 20.00 19.880 -1.000 1.5180 19.604 -1.978 1.848 Simplex 3.00 3.020 0.670 0.3340 3.042 1.334 0.827 15.00 15.145 0.967 0.1285 15.170 1.130 0.289 25.00 24.861 0.563 0.2060 24.890 0.440 0.244 *Average of three measurements. Table (8): Analytical parameters for the analysis of CLNZ by the proposed and other methods. Methods Linearity (µg.mL -1 ) max (nm) Correlation Coefficient ( R) Recovery% RSD% Ref. Proposed method 1.0-30.0 378 0.9986 96.50 - 97.90 0.846-1.253 - Spectrophotometric 5.0-40.0 532 0.9984 99.10 - 104.11 1.07 - 4.32 [13] spectrofluorometric 0.1-0.5 383 0.9999 99.33 - 101.55 0.776 [14] Spectrophotometric 0.32 – 4.1 425 0.9985 97.28 - 103.12 1.53 - 3.39 [15] RP-HPLC 20.0-120.0 ------ 0.9992 99.41-99.95 ------- [6] HPLC 5.0 – 25.0 254 0.9993 99.00-101.00 0.2222- 0.5810 [7] Chemistry |121 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Table (9): Parameters from Benesi–Hildebrand plot for the formed complex. Parameter value Intercept 7E-05 Slope 3E-08 Correlation coefficient (r) 0.9973 *ε CT (L.mol-1.cm-1) 14285.7 ** K CT 2333.3 Log K CT 3.367 *** ΔGº, J/mol -22112.7 ΔGº,KJ/mol -22.1127 * ε CT = 1/Intercept ** K CT = Intercept/Slope *** (ΔGº=-2.303RT Log K CT ) Table (10): Percent recovery for 10 µg.mL -1 of CLNZ in the presence of 10µg.mL -1 of excipient. Recovery% Con. found µg.mL -1 Excipients 107.00 10.56 Sucrose 105.00 10.71 Glucose 106.00 10.66 Lactose 102.89 10.29 Starch Table (11): Determination of CLNZ in pharmaceutical tablet. *Average of three determinations. Table (12): T- and F- values for the analysis of 10 µg.mL -1 CLNZ in pharmaceutical compound. X N S.D t- values a F-valuesb Ref. 19.580 3 0.1874 3.8799 ---------- Proposed Method c 19.880 4 0.3698 1.266 3.8961 [13] 20.139 5 0.3242 2.6763 2.9945 [27] 19.970 4 0.1997 2.6192 1.1362 [28] a. Theoretical value for t-test: N=2(4.303), N=3(3.182) and N=4(2.776) at 95% confidence limit. b. Theoretical values for F-test : N = (3,2), at 95% is (19.16) and N= (4,2) at 95% is (19.25) c. Clonazepam 0.5mg/tablet, Switzerland. Clonazepam (0.5mg tablet) Switzerland Found amount mg Conc. taken µg.mL -1 Conc.* found µg.mL -1 Recovery * % S.D* RSD* % 0.4825 5.0 4.8250 96.50 0.0408 0.8457 0.4879 10.0 9.7593 97.65 0.1223 1.2533 0.4883 20.0 19.5802 97.90 0.1874 0.9571 Chemistry |122 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Figure (1): Chemical structure of clonazepam. Figure (2): Absorption spectra a-(II) 10 μg.mL. -1 of clonazepam only against ethanol. b. 10 μg.mL. -1 of clonazepam against reagent blank: (I)-under primary test. (II) -under the optimum conditions c. 10 μg.mL. -1 of clonazepam under simplex conditions d. The reagent blank measured against ethanol. Figure (3): (a) Effect of p-Br concentration. (b) Effect of KOH concentration on the absorbance of 10μg.mL -1 of CLNZ. 0.0 0.2 0.4 0.6 1.0 5.0 9.0 A b so rb a n ce Concentration of p-Br( M) x 10-3 a 0.36 0.41 0.46 0.51 0.56 0.04 0.13 0.22 0.31 0.4 A b so rb a n ce Concetration of KOH(M) b Chemistry |123 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Figure (4): (a) Effect of heating time. (b)Effect of temperature on the absorbance of 10μg.mL -1 of CLNZ. Figure (5): (a) Effect of order of addition (D: Drug, R: Reagent, B: Base) (b) Effect of time on the stability of CLNZ- p-Br complex (10 μg mL-1). Figure (6): Calibration graph for CLNZ by (a) univariate method (b) the simplex condition. Figure (7): (a) Job’s method of continuous variation (b) Mole ratio method ([CLZ], [p-Br] = 1.5x10 -5 M, λmax=387nm). 0.1 0.3 0.5 0.7 1 9 17 25 33 A b so rb a n ce Heating time(minute) a 0.1 0.3 0.5 0.7 0.9 10 20 30 40 50 60 70 80 90 100 A b so rb a n ce Tempreature (oC) b 0.40 0.50 0.60 0.70 1 2 3 A b so rb a n ce Number order of addition a R+B+D R+D+B D+B+R 0.55 0.59 0.63 0.67 1 11 21 31 41 51 A b so rb a n ce Time(minute) b y = 0.0567x + 0.0329 R² = 0.9969 0.0 0.4 0.8 1.2 1.6 0 10 20 30 A b so rb a n ce µg CLNZ/25 mL a y = 0.0604x + 0.0544 R² = 0.9973 0.0 0.5 1.0 1.5 2.0 0 10 20 30 A b so rb a ce µg CLNZ / 25mL b 0.0 0.2 0.4 0.6 0.0 0.3 0.5 0.8 1.0 A b so rb a n ce Mole fraction of CLNZ a 0.3 0.4 0.5 0.6 0.7 0.0 1.0 2.0 3.0 A b so rb a n ce Molar ratio p-Br to CLNZ b Chemistry |124 2017(عام 2العدد ) 30هجلة إبن الهيثن للعلىم الصرفة والتطبيقية الوجلد Ibn Al-Haitham Jour. for Pure & Appl. Sci. Vol. 30 (2) 2017 Figure (8): Benesi–Hildebrand plot for CLNZ –p-Br complex. Figure (9): Determination of CLNZ in tablet (Rivotril) by SAM (a) (0.5 mL from 50 µg.mL -1 ). (b) (1.0 mL from 50 µg.mL -1 ). Scheme (1): Charge Transfer complex formed between CLNZ and p-Br. y = 8E-09x + 2E-05 R² = 0.9948 1.0E-04 1.5E-04 2.0E-04 2.5E-04 3.0E-04 10000 20000 30000 [A o ] / A C T ( m o l/ L) 1/[Do] mol-1.L y = 0.6838x + 0.3406 R² = 0.9979 0.0 0.3 0.6 0.9 -0.7 -0.2 0.3 0.8 A b so rb a n ce Volume of standard solution (ml) Cstdx Vstd= Cunkx Vunk -(50x--0.4981)=Cunk x 0.5ml Cunk= 49.81ml a -0.4981 y = 0.6142x + 0.6369 R² = 0.9968 -0.5 0.0 0.5 1.0 1.5 2.0 -1.3 -0.6 0.2 0.9 1.6 A b so rb a n ce Volume of standard solution(ml) Cstd x V std=Cunkx Vunk -(50ppmx-1.0369)=Cunk x1.0ml Cunk =51.845 ppm b -1.0369