Spectrophotometric Determination of Imatinib ... - Research Publisher

Chemical Rapid Communications

Vol.2 No.3, December 2014

55

ISSN: 2325-9892 (Print) 2325-9906 (Online) www.researchpub.org/journal/crc/crc.html

Spectrophotometric Determination of Imatinib Mesylate using Charge Transfer Complexs in Pure Form and Pharmaceutical Formulation Sher Shereen. E. Abdel Karima, Raafat. A. Farghalya, Rasha. M. El-Nashar a,b* , Ashraf. H. Abadia* 

recent medications used for the treatment of chronic myeloid leukemia and gastro-intestinal stromal tumor. The introduction of Imatinib mesylate, which targets the kinases presenting with these molecular alterations, has dramatically changed the management of these rare tumors, which were resistant to conventional cytotoxic chemotherapy, both in advanced and localized phases.5 It has opened a new area in cancer therapy and is given orally and chronically, that is why it was chosen as a model drug.6 It is also known as Signal Transduction Inhibitor 571 and as antineoplastic agent. Its target protein is produced by DNA translocation (Philadelphia chromosome), which leads to a fusion protein of Abl with Bcr termed Bcr-Abl.7 Many methods were reported in literature for the determination of imatinib mesylate in bulk, dosage forms and biological fluids, although it is not official in any pharmacopeia some of these methods are HPLC/UV detection 8-10, HPLC/Mass detection11-13, gas chromatography14,15, 16 supercritical fluid chromatography , capillary electrophoresis 17, 18 , voltammetric methods19, 20 and spectrophotometric methods21, 22. However, no Spectrophotometric Colorimetric method using DDQ or CAA have been developed for the determination of imatinib mesylate as a bulk or pharmaceutical formulations. In this work, charge-transfer (CT) reactions are studied spectrophotometrically based on chromogen produced as a result of charge transfer complex formation of the drug with π-acceptors as chloranilic acid and DDQ developing certain colored products that are measured quantitatively. The aim of this work is to introduce a simple, precise and rapid procedure for the simultaneous quantitation of the cited drug in pure and pharmaceutical formulation. The proposed method is suitable for determination of the drug providing economic, less time consuming and more sensitive procedure compared with the reported spectrophotometric method (reference method)21, based on the formation of ion pair complex of imatinib with bromocresol green in acidic buffer followed by their extraction in chloroform.

Abstract— Two new, simple and effective colorimetric methods were developed for determination of Imatinib (mesylate) in pure and pharmaceutical formulations. Charge transfer (CT) interactions between Imatinib as electron donor and 2,3-dichloro-5,6,-dicyano-p-benzo quinone (DDQ) and chloranilic acid (CAA) as π-electron acceptors. The obtained charge-transfer complexes were measured at λmax 462 nm and 537 nm for DDQ and CAA in methylene chloride, respectively. Different variables affecting the reaction were studied and optimized. Under the optimum conditions, Beer’s law was obeyed in the concentration range of the drug 0.25-3.50 mg/ 10 ml and 0.50-5.50 mg/ 10 ml upon using DDQ (0.4% w/v) and CAA (0.3% w/v), respectively, with correlation coefficients 0.996 and 0.998, respectively. The proposed methods showed good linearity, precision, reproducibility and the validity was assessed by applying the standard additions technique with mean percentage recovery 99.90 ± 1.36 in case of DDQ and 100.20 ± 0.91 in case of CAA. Keywords — Charge Transfer Complex, CAA , DDQ, Gleevec, Imatinib Mesylate, Spectrophotometry

I. INTRODUCTION

C

ANCER is the most common cause of mortality and

morbidity in Egypt as well as in other countries as U.K. Despite recent advances in the treatments of cancer, the clinical outcome is yet far away from expectation.1 Chronic Myelogenous Leukemia is a myeloproliferative disorder2, 3, that results from a malignant transformation of progenitor cells leading to clonal proliferation and accumulation of myeloid cells. CML is responsible for 15% of adult leukemias.2 Imatinib mesylate methanesulphonic acid4-[(4-methylpip-erazin-1-yl) methyl]-N-[4-methyl-3[(4-pyridin-3ylpyrim-idin-2-yl)amino]phenyl] benzamide, is a tyrosine kinase inhibitor4, that was found to be one of the most

II. METHODS Submitted March 2014 Department of Pharmaceutical Chemistry , Faculty of Pharmacy and biotechnology, German University in Cairo, Egypt. Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt* *Correspondence to (e-mail:[email protected]) (Email: [email protected])

2.1. Apparatus All absorption spectral measurements were made in the range of 400-800 nm using JASCO V-530 UV/VIS Spectrophotometer, made in Japan. 2.2. Materials and reagents 55



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All chemicals and reagents used were of analytical grade. Doubly distilled water was used. Solvents were of HPLC or spectroscopic grade. Imatinib mesylate was provided by Novartis (Basel, Switzerland) and Hangzhou Hetd Industry Co, Ltd (China). Gleevec® capsules (each containing 100 mg imatinib mesylate) were provided by Novartis. Both 2,3-dichloro-5,6,-dicyano-1,4-benzoquinone (DDQ) and Chloranilic acid (CAA) were provided by Sigma Aldrich (Analytical grade). Methanol and Methylene chloride were provided by El Goumhouria Company for Trading, Medicines, Chemicals and Medical Appliances, Egypt.

III. RESULTS AND DISCUSSION 3.1. Optimization of the complex formation reaction conditions 2,3-dichloro-5,6-dicyano,1,4-benzoquinone and chloranilic acid have been used for the determination of drugs with electron rich nitrogen. The color developed was as a result of the interaction of the drug base as donor and the reagent as πacceptor with the formation of a major chromogen (as a result of formation of charge transfer complex)23, which is reddish orange in case of DDQ and violet in case of CAA. This could be performed at room temperature without the need of heating 24. Different parameters affecting the color development were studied to determine the optimum conditions for the assay procedure.

2.3. Standard solutions a) Imatinib standard solution was prepared at concentration (1mg/ mL) in methylene chloride by weighing 0.058 gm of imatinib mesylate, dissolving in 10 ml distilled water then adding 5 mL NaHCO3 (10%) and finally extraction using methylene chloride. b) 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) 0.4% (w/v) was prepared in methylene chloride. c) Chloranilic acid (CAA) 0.3% (w/v) was dissolved in least amount of methanol (20% of the total volume) and completed to required volume using methylene chloride.

2.4.1. Method A (DDQ method) To a series of 10 mL volumetric flasks, 1 mL 0.4% DDQ was added, followed by 2 mL methanol, then aliquots of Imatinib standard (1mg/ mL) containing 0.25-3.50 mg drug were added. The volume was completed to the mark using methylene chloride. The immediately developed reddish orange color was measured at λmax 462 nm against a blank prepared in a similar manner excluding the drug.

3.1.1. Effect of solvent on the studied complexes Reactions of DDQ with basic drugs were reported in methanol, acetonitrile and chloroform25. While non-polar solvents as benzene and carbon tetrachloride were not suitable for color formation due to the fact that complete electron transfer from the donor to the acceptor takes place in polar solvents26. In this work, maximum color was obtained with methylene chloride in which the DDQ and Imatinib (free base) were freely soluble (main solvent). On using DDQ, 2 mL of methanol were used in complex preparation, to avoid turbidity which was developed upon mixing standard drug solution (in methylene chloride) with DDQ solution directly. Also it was included in the reagent blank, otherwise a shift in absorbance occurs as shown in Figure 1. As for CAA, the reagent was prepared using methanol for dissolution (20% of the total required volume), then completed with methylene chloride (main solvent).

2.4.2. Method B (CAA method) To each 10 mL flask, 2 mL 0.3% CAA was added then followed by 1 mL methanol and the reaction mixture was mixed. Aliquots of Imatinib standard (1mg/ mL) containing 0.50-5.50 mg drug were transferred into the series. The volume was completed to the mark using methylene chloride. The developed violet colour was measured at λ max 537 nm against a blank reagent prepared in a similar manner excluding the drug.

3.1.2. Determination of wavelength at which maximum absorbance occurs (Absorbance spectra) The absorbance spectrum of the complex was recorded against reagent blank in the range of 400-800 nm. The obtained spectrum was compared with that of the drug indicating λ max 462 nm (at which maximum absorbance occurs) for Imb-DDQ. In case of CAA, maximum absorbance occurs for Imb-CAA at 537 nm as shown in Figures 2.

2.5. Analysis of Imatinib mesylate in pharmaceutical dosage forms The content of 10 hard gelatin coated capsules-Gleevec® capsules (100 mg/ capsule)-were mixed together. The average mass of one capsule was found to be 229.60 mg Imatinib (free base), which is equivalent to 119.50 mg Imatinib mesylate. By calculation, 111.40 mg of capsule content were used to prepare a drug solution with concentration (1mg/ mL). The calculated weight is dissolved in the least amount (2-3 mL) of water, followed by 5 mL NaHCO3 (10%) to convert Imatinib mesylate to the free base Imatinib and finally extracted using methylene chloride.

3.1.3. Effect of the amount (concentration) of the reagents on absorbance Using a series of 10 mL solution, 3 mL of imatinib standard solution (1 mg/1 mL) were added, followed by different aliquots of (DDQ solution (0.4% w/v) and 2 mL methanol) or (CAA solution (0.3% w/v) and 1 mL methanol). The volume was completed to 10 mL with methylene chloride. The absorbance was measured at 462 nm or 537 nm against the reagent blank for DDQ and CAA, respectively indicating that maximum absorption occurs with 2.712 mg DDQ (molar ratio 1:2 drug: DDQ ) related to highest drug concentration and with 3.129 mg of CAA (molar ratio 1:3 drug: CAA), related to highest drug concentration. It was clear that the absorbance

2.4. General procedure

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57


increases gradually, then started to be constant on using 509 µL DDQ (0.4% w/v) and 626 µL CAA (0.3% w/v).

The accuracy indicates how close is the measured quantity to its actual (true) value, while precision, is the reproducibility or repeatability. The precision and accuracy of the method were investigated via inter and intraday repeatability of imatinib mesylate. The precision and accuracy of the method are expressed as RSD and recovery %, also reproducibility (day to day) was investigated. Relative standard deviation less than 1.40 % for Imatinib-DDQ complex and Imatinib-CAA complex was obtained. The difference between the obtained and expected value is expressed in terms of recovery %. The student t-test and the F-test can be employed to decide whether the difference between the results obtained by the proposed method and a reference method 21 is accounted for by random errors and for the comparison of the standard deviation of the two methods.27, 28 The one tail F-test method is used in comparison. Comparing the obtained F-and t-values with the tabulated ones as shown in Table 4, the obtained values were lower than the theoretical tabulated values, so there was not any significant differences in the applied methods in comparison to those of the referenced method21, indicating the accuracy and precision of the present work. The ruggedness of the spectrophotometric method was evaluated by carrying out the analysis using two different instruments on different days. The RSD of less than 1.40 % was observed for repetitive measurements in three different day time periods using two different instruments. The results obtained indicated reproducibility. The robustness of the method was explained by the evaluation of the influence of small variation of variables including concentration of the reagent and measuring time. The results showed that the method is robust, the results showed that absorbance started to be constant on using 509 µL DDQ (0.4% w/v) and 626 µL CAA (0.3% w/v) and Imb-DDQ and Imb-CAA complexes remains stable for 40 minutes at room temperature in case of DDQ and 45 minutes in case of CAA.

a) 3.1.4. Effect of time on absorbance Absorbance was measured as a result of the developed colour at time intervals 5-60 minutes for Imb-DDQ and Imb-CAA complexes, respectively showing gradual increase with time reaching maximum (maximum absorbance) within 10 minutes and remains stable for another 40 minutes (50 minutes from the preparation time) at room temperature in case of DDQ, while in case of CAA the absorbance reaches maximum within 5 minutes and remains stable for another 45 minutes (50 minutes from the preparation time at room temperature as shown in Figure 3. 2) 3.2. Linearity The calibration curve was constructed using different aliquots of Imb standard solution 0.25-3.50 mg on using DDQ (0.4% w/v) and 0.50-5.50 mg on using CAA (0.3% w/v). Beer’s law is obeyed over the concentration range of 0.25-3.50 mg/ 10 mL on using DDQ and over the concentration range of 0.50-5.50 mg/ 10 mL in case of CAA, as represented in Figure 4. 3.3. Reproducibility It is a validation for the calibration curve, where different aliquots of drug standard solution 0.75-3.25 mg were used in case of DDQ and 0.75-5.25 mg of drug in case of CAA as in Table 1. 3.4. Application on pharmaceutical dosage forms (Gleevec capsules (100 mg/capsule) Results in Table 2, show that the mean percentage recoveries of labeled Imatinib in Gleevec® capsules were 97.20 ± 1.80 on using DDQ and 95.80 ±0.40 on using CAA. 3.5. Reference method for determination of imatinib mesylate (a previously published method21 The results obtained for the assay on using DDQ and CAA were compared to those of a reference spectrophotometric method based on the formation of ion pair complex of imatinib with bromocresol green in acidic buffer followed by their extraction in chloroform. The absorbance of the chromogen was at 417 nm against the corresponding blank reagent and results are given in Table 3. The method was found to be in need of drying the collected layer over anhydrous Na 2SO4 so the complex was adsorbed and loss occur. Also the complex remained stable for a short period of time, after which the color started to fade (10-15 min.). While in the present work, the drug was extracted first with methylene chloride several times to ensure its complete extraction, then the complex was prepared which remained stable for longer period of time (50 min.).

REFERENCES [1] Sagar, J., Chaib, B., Sales, K., Winslet, M., and Seifalian, A., Role of stem cells in cancer therapy and cancer stem cells: a review Cancer Cell Int, 2007, vol. 7, p. 1-11. [2] Imatinib Mesylate (Gleevec) VHA Pharmacy Benefits Management Strategic Healthcare Group and the Medical Advisory Panel. Nat. PBM Drug Mono. 2002, p. 1-6. [3] Goldberg, S.L. and Masood, A., Identifying and treating imitinib failure in chronic myelogenous leukemia: a practical review of treatment guidelines and available agents. Community Onc., 2009, vol. 6, p. 113-125. [4] Menon-Andersen, D., Mondick, J.T., Jayaraman, B., Thompson, P.A., Blaney, S.M., Bernstein, M., Bond, M., Champagne, M., Fossler, M.J., and Barrett, J.S. Polpulation pharmacokinetics of imitnib mesylate , Cancer Chemother Pharmacol, 2009, vol. 63, p. 229-38. [5] Hunter, T., Treatment for chronic myelogenous leukemia: the long road to Imatinib, J. Clin. Invest., 2007, vol. 117, p. 2036-43.

3.6. Statistical Analysis and Validation of the Proposed Methods In order to be able to ensure that the proposed methods are valid and applicable, the obtained value for the amount of the analyte should be very close to the actual value. 57



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[6] Rochat, B., Fayet, A., Widmer, N., Lahrichi, S.L., Pesse, B., Decosterd, L.A., and Biollaz, J., Imatinib metabolite profiling in parallel to imatinib quantification in plasma of treated patients using liquid chromatography-mass spectrometry, J. Mass Spectrom, 2008, vol. 43, p. 736-52. [7] Ivanovic, D., Medenica, M., Jancic, B., and Malenovic, A., Reversed-phase liquid chromatography analysis of imatinib mesylate and impurity product in gleevec capsules. J. Chromatogr B Analyt. Technol. Biomed. Life Sci., 2004, vol. 800, p. 253-8. [8] Miura, M., Takahashi, N., and Sawada, K., Quantitative determination of imatinib in human plasma with high-performance liquid chromatography and ultraviolet detection. J. Chromatogr. Sci., 2011, vol. 49, p. 412-5. [9] Bouchet, S., Chauzit, E., Ducint, D., Castaing, N., Canal-Raffin, M., Moore, N., Titier, K., and Molimard, M., Simultaneous determination of nine tyrosine kinase inhibitors by 96-well solid-phase extraction and ultra performance LC/MS-MS. Clin. Chim. Acta., 2011, vol. 412, p. 1060-7. [10] Davies, A., Hayes, A.K., Knight, K., Watmough, S.J., Pirmohamed, M., and Clark, R.E., Simultaneous determination of nilotinib, imatinib and its main metabolite (CGP-74588) in human plasma by ultra-violet high performance liquid chromatography. Leuk. Res., 2010, vol. 34, p. 702-7. [11] Teoh, M., Narayanan, P., Moo, K.S., Radhakrisman, S., Pillappan, R., Bukhari, N.I., and Segarra, I., HPLC determination ofimatinib in plasma and tissues after multiple oral dose administration to mice. Pak J. Pharm. Sci., 2010, vol. 23, p. 35-41 (cited in google scholar). [12] Mlejnek, P., Novak, O., and Dolezel, P., A non-radioactive assay for precise determination of intracellular levels of imatinib and its main metabolite in Bcr-Abl positive cells. Talanta, 2011, vol. 83, p. 1466-71. [13] Micova, K., Friedecky, D., Faber, E., Polynkova, A., and Adam, T., Flow injection analysis vs. ultra high performance liquid chromatography coupled with tandem mass spectrometry for determination of imatinib in human plasma. Clin. Chim. Acta., 2010, vol. 411, p. 1957-62. [14] De Francia, S., D'Avolio, A., De Martino, F., Pirro, E., Baietto, L., Siccardi, M., Simiele, M., Racca, S., Saglio, G., Di Carlo, F., and Di Perri, G., New HPLC-MS method for the simultaneous quantification of the antileukemia drugs imatinib, dasatinib, and nilotinib in human plasma. J Chromatogr. B Analyt. Technol, Biomed. Life Sci., 2009, vol. 877, p. 1721-6. [15] Groman, A. and Golebiewski, P., Validation of analytical procedure control of residual ethanol, 2-propanol and ethyl acetate in pharmaceutical substance imatinib. Acta. Pol. Pharm., 2006, vol. 63, p. 414-6. [16] Bui, H., Masquelin, T., Perun, T., Castle, T., Dage, J., and Kuo, M.S., Investigation of retention behavior of drug molecules in supercritical fluid chromatography using linear solvation energy relationships. J. Chromatogr. A, 2008, vol. 1206, p. 186-95.

[17] Rodriguez Flores, J., Berzas Nevado, J.J., Contento Salcedo, A.M., and Cabello Diaz, M.P., Nonaqueous capillary electrophoresis method for the analysis of gleevec and its main metabolite in human urine. J. Chromatogr. A, 2005, vol. 1068, p. 175-82. [18] Rodriguez Flores, J., Berzas, J.J., Castaneda, G., and Rodriguez, N., Direct and fast capillary zone electrophoretic method for the determination of gleevec and its main metabolite in human urine. J. Chromatogr B Analyt. Technol. Biomed, Life Sci., 2003, vol 794, p. 381-8. [19] Diculescu, V.C., Chiorcea-Paquim, A.M., Tugulea, L., Vivan, M., and Oliveira-Brett, A.M., Interaction of imatinib with liposomes: voltammetric and AFM characterization. Bioelectrochem., 2009, vol. 74, p. 278-88. [20] Rodriguez, J., Berzas, J.J., Castaneda, G., and Rodriguez, N., Voltammetric determination of imatinib (gleevec) and its main metabolite using square-wave and adsorptive stripping square-wave techniques in urine samples. Talanta, 2005, vol. 66, p. 202-9. [21] Balaram, V.M., Rao, J.V., Khan, M.M.A., Sharma, J.V.C., and Anupama, K., Visible spectrophotometric determination of imatinib mesylate in bulk drug and pharmaceutical formulations. Asian J. of Chem., 2009, vol. 21, p. 5241-5244. [22] Bende, G., Kollipara, S., Sekar, V., and Saha, R., UV-spectrophotometric determination of imatinib mesylate and its application in solubility studies. Pharmazie, 2008, vol. 63, p. 641-5. [23] Frenzel, W., Enhanced performance of ion-selective electrodes in flow injection analysis: non-Nernstian response, indirect determination, differential detection and modified reverse flow injection analysis. Analyst, 1988, vol. 113, p. 1039–1046. [24] Gunasingham, H. and Fleet, B., Wall-jet electrode in continuous monitoring voltammetry. Anal. Chim., 1983, vol. 55, p. 1409-1414. [25] Abu-Shady, H.A., Hassib, S.T., Hassanein, H.H., and Abadi, A.H., Analysis of some cardiovascular drugs. J. Pharm. Sci., 1995, vol. 36, p. 393-405. [26] Siddiqui, M.R., Tariq, A., Ahmad, A., Chaudhary, M., Shrivastava, S.M., and Singh,R.K., Application of DDQ and p-chloranilic acid for the spectrophotometric estimation of milrinone in pharmaceutical formulations. Asian J. Sci. Res., 2009, vol. 2, p. 135-145. [27] El-Nashar, R.M., Flow injection potentiometric assay of hexoprenaline in its pure state, pharmaceutical preparations and biological samples, J. Auto. Meth. Manag. Chem., 2008. [28] Miller, J.C. and Miller, J.N., Statistics and chemometrics for analytical chemistry. 4th edition ed.: Ellis Horwood, Chichester, U.K. 2001. [29] Karthikeyan R. et al. Research Journal of Pharmacy and Technology, 2009, vol.2, p. 578.

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List of Figures: (A)

(a)

max = 462 nm nm

Figure 1

(A)

Figure 1: Showing the effect of methanol. Blue: 1 mg Imb +4 mg DDQ (CH2Cl2) against blank reagent (DDQ and CH2Cl2). Brown: 1 mg Imb + 4 mg DDQ (CH2Cl2 + methanol) against blank reagent (DDQ, CH2Cl2 and methanol).

(B)

(b)

max = 537 nm BNMNMNM 462462 nm (B)

Figure 2 Figure 2: (a) Absorbance spectra of imatinib (A) and imatinib/DDQ complex (B) and (b) absorbance spectra of imatinib (A) and imatinib/CAA complex (B).

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(a) (a)

(b) Figure 3

(b) Figure 3: Effect of time on absorbance of the charge transfer complex on using DDQ (a) and CAA (b).

Figure 4 Figure 4: Calibration curve of Imatinib on using DDQ (a) and CAA (b).

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Table 1: Reproducibility of results on using DDQ and CAA.

Recovery % Amount taken (mg/10mL)

Average Abs.*

Amount found (mg/10mL)

Recovery %

Amount taken (mg/10mL)

Average Abs.*


0.14

0.74

99.20

On using CAA On using DDQ 0.75

0.22

0.74

98.70

0.75

0.38

1.24

99.00

1.25

0.23

1.26

101.50

1.75

0.55

1.76

100.60

1.75

0.31

1.75

100.30

2.25

0.69

2.20

98.20

2.25

0.39

2.24

99.70

2.75

0.88

2.78

101.10

2.75

0.47

2.75

100.00

3.25

1.05

3.30

101.00

3.50

0.59

3.45

98.80

4.50

0.76

4.54

100.90

5.25

0.89

5.29

100.80

Mean

100.20

*S.D.

0.91

1.25

Mean

99.90

*S.D.

1.36

S.E.

0.55

S.E.

0.32

RSD%

1.36

RSD%

0.90

*Results were the average of triplicate determinations. 61



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Table 2: Application of standard additions technique for determination of Gleevec ® applying spectrophotometric method on using DDQ and CAA. Dosage form



Recovery %



Recovery%

®

On using DDQ

On using CAA

Capsule 1.00

0.98

98.00

1.00

0.96

95.70

1.25

1.20

96.00

1.50

1.45

96.30

1.50

1.44

96.00

2.00

1.91

95.70

1.75

1.75

100.00

2.50

2.38

95.40

2.00

1.91

95.80

3.00

2.87

95.70

Average

97.20

Average

95.80

*S.D.

1.80

*S.D.

0.40

S.E.

0.80

S.E.

0.18

*Results were the average of triplicate determinations.

Table 3: Reference method for determination of imatinib mesylate.

Amount taken (µg/10mL)

Amount found (µg/10mL)

Recovery %

15.00 25.00 30.00

15.16 24.76 30.20

101.00 99.04 100.60

40.00

39.54 Mean *S.D. S.E.

98.90 99.87 0.93 0.46

*Results were the average of 2 determinations.

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Table 4: Test of significance for the proposed methods.

Statistical term

λmax

DDQ method

462

CAA method

537

Refer ence Method [21] 417

Reference Method [22]

Reference [29]

285

251

Pure solution Mean

99.90

100.20

99.87

100.01

-----

Standard Deviation

1.36

0.91

0.93

5.76x10-3

-----

1.36

0.90

0.93

0.10

-----

0.39

-----

Mean

97.20

95.80

99.87

99.65

-----

Standard Deviation

1.80

0.40

0.93

5.76x10-3

-----

1.85

0.41

0.93

0.0005

-----

0.39

-----

Student t test*

(RSD%)

F test* (4,3), (3,4) Student t test* Probability

* Figures between parenthesis are the corresponding tabulated F or t-values

Detection limit 1.389 gm/L and 1.831 gm/L, respectively for DDQ and CAA.

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