InternatIonal journal of BIomedIcal scIence ORIGINAL ARTICLE Kinetic Spectroluorometric Determination of Certain Calcium Channel Blockers via Oxidation with Cerium (IV) in Pharmaceutical Preparations M. I. Walash, F. Belal, N. El-Enany, A. A. Abdelal Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura, Egypt AbstrAct A simple and sensitive kinetic spectroluorometric method was developed for the determination of some calcium channel blockers namely, verapamil hydrochloride, diltiazem hydrochloride, nicardipine hydrochloride and lunarizine. The method is based upon oxidation of the studied drugs with cerium (IV) ammonium sulphate in acidic medium. The luorescence of the produced Ce (III) was measured at 365 nm after excitation at 255 nm. The different experimental parameters affecting the development and stability of the reaction product were carefully studied and optimized. The luorescence-concentration plots were rectilinear for all the studied compounds over the concentration range of 0.01 to 0.12 µg mL -1. The limits of detections for the studied compounds ranged from 2.93 × 10 -3 to 0.012 µg mL -1 and limits of quantiication from 9.76 × 10 -3 to 0.04 µg mL -1 were obtained. The method was successfully applied to the analysis of commercial tablets. The results obtained were in good agreement with those obtained with reference methods. (Int J Biomed Sci 2009; 5(2):146-157) Keywords: verapamil hydrochloride; diltiazem hydrochloride; nicardipine hydrochloride; Cerium (IV) InTroduCTIon Verapamil hydrochloride (VP), diltiazem hydrochloride (DLT), nicardipine hydrochloride (NC) and lunarizine (FZ) (Fig. 1) are widely used calcium channel blockers. VP, DLT and NC are currently used for the .management of angina pectoris, and also used in the treatment of hypertension. As for FZ, it is used for migraine prophylaxis, for vertigo and vestibular disorders and for peripheral and cerebral vascular disorders (1). Several analytical methods have been reported for the determination of VP either per se or in pharmaceutical prepara- Corresponding author: F. Belal, Department of Analytical Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura, Egypt. Fax: +20502247496; E-mail: ffbelal@yahoo.com. Received November 7, 2008; Accepted March 6, 2009 146 June 2009 Vol. 5 No. 2 tions including: spectrophotometry (2-4), luorometry (5, 6), voltammetry (7), HPLC (8-13) and capillary electrophoresis (14). VP is the subject of a monograph both of the British Pharmacopoeia, BP (15); and the United State Pharmacopoeia, USP (16). Both the BP and USP recommend non-aqueous titration for the raw material and spectrophotometric measurement at 278 nm for the tablets. The USP (16), on the other hand, recommend HPLC method for its formulations. Regarding DLT, various methods have been applied for its determination in its formulations such as spectrophotometry (17, 18), voltammetry (19), HPLC (20- 23). The BP (15) recommends non-aqueous titration for the raw material, The USP (16), on the other hand, recommend HPLC method for its formulations. Several methods have been utilized for the quantitative estimation of NC in its pharmaceutical preparations including: spectrophotometry (24, 25), voltammetry (26, 27), HPLC (28-31) and capillary electrophoresis (32). Int J Biomed Sci w w w.ijbs.org KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers H3C CN N H N O OMe F H3C O CH3 N N CH3 CH3 H N COOCH2CH2N H3COOC H OMe MeO Flunarizine NO2 O CH3 H 3C Nicardipine Hcl Diltiazem Hcl Verapamil Hcl CH2 S H3C OMe N CH3 F H MeO Figure 1. Structural formulae of the studied drugs. Relatively few methods have been described for the determination of FZ in its formulations viz spectrophotometry (33, 34), luorometry (35), voltammetry (36) and HPLC (37-39). The proposed method depends simply on oxidation of all the studied drugs with Ce (IV) in acidic medium and measuring the intensity of the formed Ce (III) at 365 nm after excitation at 255 nm. Compared with the reported spectroluorimetric methods (5, 6) for VP, the proposed method is more sensitive, since the working concentration range of the reported methods ranged from 1-10 µg mL-1 (5). In the other method, the determination was conducted on 1 µg mL-1 with detection limit in the range of 0.040.1 µg mL-1 (6). However, the proposed method is highly sensitive, it could measure as low as 0.01-0.12 µg mL-1. Similarly, for FZ, the working concentration range of the reported methods ranged from 0.94-7.1 µg mL-1 (35), and poor sensitivity compared with the proposed method. To the best of our knowledge, no spectroluorometric method has been reported for the analysis of DLT and NC up till now. This initiated the present study. ExpErImEnTAL Apparatus The luorescence spectra and measurements were recorded using a Perkin-Elmer UK model LS 45 luminescence Spectrometer, equipped with a 150 Watt Xenon arc lamp, gratting excitation and emission monochromators for all measurements and a Perkin-Elmer recorder. Slit widths for both monochromators were set at 10 nm. A 1 cm quartz cell was used. w w w.ijbs.org materials and reagents All reagents and solvents were of Analytical Reagent Grade. • Verapamil hydrochloride, diltiazem hydrochloride, nicardipine hydrochloride and lunarizine pure samples were purchased from Sigma (St. Louis, Mo, USA) and used as received. • Cerium (IV) ammonium sulphate, (BDH, Pool, UK), 5 × 10 -4 M aqueous solution was freshly prepared in 1.0, 1.25 and 1.5 M sulphuric acid. • Sulphuric acid, (Prolabo, France), 1.0, 1.25 and 1.5 M aqueous solutions. Standard Solutions Stock solutions of VP, DLT and NC were prepared by dissolving 10.0 mg of each of the studied compounds in 100.0 mL of distilled water, while stock solution of FZ was prepared by dissolving 10.0 mg of FZ in 100.0 mL of 2 M H2SO4 solution, and was further diluted with the same solvent as appropriate. The standard solutions were stable for 10 days when kept in the refrigerator. General procedures Aliquot volumes of VP, DLT, NC and FZ standard solutions covering the working concentration range cited in table 1 were transferred into a series of 10 mL volumetric lasks; followed by speciied volumes of 5 × 10 -4 M Ce (IV) solution as shown in table 1. The lasks were heated in a thermostatically controlled water-bath at 100 °C for speciied time (table 1). The solutions were cooled and diluted to the mark with distilled water. A blank experiment was performed simultaneously. The relative luorescence Int J Biomed Sci Vol. 5 No. 2 June 2009 147 KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers intensity (FI) of the solutions was measured at 365 nm after excitation at 255 nm. The corrected FI was plotted vs inal concentration of the drug (µg mL-1) to get the calibration graphs. Alternatively, the corresponding regression equations were derived. AppLICATIonS procedure for dosage forms An accurately weighed quantity of the mixed contents of 10 capsules or powdered tablets equivalent to 10.0 mg of VP, DLT and NC was transferred into small conical lask and extracted with 3 × 30 mL of distilled water while FZ capsules were extracted with 3 × 30 mL of 2 M H 2SO4 solution. The extract was iltered into 100 mL volumetric lask. The conical lask was washed with few mLs of distilled water. The washings were passed into the same volumetric lask and completed to the mark with the same solvent. Aliquots covering the working concentration range cited in table 1 were transferred into 10 mL volumetric lasks. The “General Procedures” were then applied and the nominal content of capsules or tablets was determined either from a previously plot- ted calibration graph or from the corresponding regression equation. rESuLTS And dISCuSSIon Recently, ceric (IV) has been frequently utilized as a useful reagent for the determination of pharmaceutical compounds, such as antivirals (41), some psychoactive drugs (42), aztreonam (43) and isoxsuprine hydrochloride (44). As the luorescence intensity of the formed Ce (III) increases with the time, this fact was used as a basis for a useful kinetic method for the quantitative determination of certain calcium channel blockers in pharmaceuticals. In the present study, oxidation of the studied drugs with Ce (IV) in an acid medium yields an equivalent amount of luorescent Ce (III) which exhibits maximum luorescence at 365 nm after excitation at 255 nm. Figure 2 illustrates the resulting luorescence spectra of the produced Ce (III) in an acidic medium. The oxidation products were found to be non luorescent product. This conirmed the luorescence induced in the oxidation of the investigated drugs with Ce (IV) was not attributed to their oxidation products; however, it was mainly due to the formation of Ce (III). Table 1. Performance data of the proposed method Verapamil HCl diltiazem HCl nicardipine HCl Flunarizine 0.02-0.12 0.01-0.06 0.02-0.12 0.04-0.12 Volume of 5 × 10 M Ce (IV), mL 0.5 1 1.5 0.8 Concentration of H2SO4 (M) 1.25 1 Time of heating (min) 25 20 6.33 × 10 -3 3.1 × 10 -3 Parameter -1 Concentration range (µg mL ) -4 Limit of detection (LOD) (µg mL-1) 1.5 1.5 15 25 2.93 × 10 -3 0.012 Limit of quantiication (LOQ) (µg mL ) 0.02 0.01 Correlation coeficient (r) 0.9998 0.9998 0.9999 0.9998 Slope 4260.00 7380.233 2992.857 3340.00 Intercept 7.133 9.512 3.670 4.60 Sy/x 3.110 3.210 0.344 2.530 -1 9.76 × 10 -3 0.04 Sa 26.750 32.100 2.924 26.820 Sb 37.020 32.380 4.095 40.000 RSD (%) 1.252 1.220 0.260 1.020 % Error 0.511 0.550 0.106 0.460 Student’s t test 1.19 0.28 1.47 0.11 F test 3.24 1.14 2.96 1.48 Sy/x, Standard deviation of the residuals; Sa, Standard deviation of the intercept; Sb, Standard deviation of the slope; % Error, %RSD/√n; RSD% = Relative standard deviation. 148 June 2009 Vol. 5 No. 2 Int J Biomed Sci w w w.ijbs.org KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers opTImIzATIon oF ExpErImEnTAL CondITIonS The spectroluorometric properties of Ce (III) as well as the different experimental parameters affecting its formation and its stability were carefully studied and optimized. Such factors were changed individually while the others were kept constant. These factors included the Ce (IV) concentration, type of acid and its concentration, heating time, temperature and diluting solvents. Effect of Ce (IV) concentration The inluence of Ce (IV) volume on the luorescence intensity of the formed Ce (III) was studied using increasing volumes of 5 × 10 -4 M Ce (IV) solution. It was found that maximum and constant luorescence intensity was attained upon using 0.5, 1, 1.5 and 0.8 mL for VP, DLT, NC and FZ, respectively as shown in (Fig. 3) and Table 1. Fluorescence intensity 500.0 450 (A) (A`) 400 350 300 Effect of acid type and its concentration The oxidation reaction of Ce (IV) have to be performed in acid medium to prevent precipitation of Ce (OH)3. Different acids such as, sulphuric acid, hydrochloric acid, nitric acid and perchloric acid were tested to determine the most suitable one for the reaction. Nitric acid is not preferred to be used owing to the inhibitory effect of nitrate ions on the luorescence of Ce (III) (44). In the presence of hydrochloric acid, perchloric acid and sulphuric acid, the reaction rate and the luorescence of Ce (III) were found to be high. However, hydrochloric acid and perchloric acid gave high blank readings, so sulphuric acid was selected for this study. The effect of sulphuric acid concentration on the luorescence intensity was studied using concentrations ranging from 0.25 to 3 M (Fig 4). The results are abridged in Table 1. Effect of temperature and heating time Oxidation of the studied drugs with Ce (IV) was carried out at different temperature setting, using a thermostatically controlled water bath, ranged from ambient temperature, 40, 60, 80ºC and boiling water bath for period of times ranging from 5 to 60 min. the results are shown in (Fig. 5) and Table 1. 250 200 150 100 50 0.0 200.0 220 240 260 280 300 320 340 360 380 400 420 440 460 480.0 Wavelength, nm Figure 2. Excitation and emission spectra of induced Ce(III) by oxidation of 0.06 µg mL-1 DLT with Ce (IV) where: (A), Excitation spectrum; (A`), Emission spectrum. Effect of diluting solvents Dilution with different solvents such as water, methanol, acetonitrile, dimethyl sulfoxide and dimethyl formamide was attempted. It was found that, water was the best solvent for dilution as it gave the highest luorescence intensities and the lowest blank reading, moreover its choice adds to the advantages of the proposed method. Distinct and sharp decrease in the luorescence intensities was attained upon using acetonitrile and methanol, while 500 500 400 400 300 ∆F ∆F 300 200 200 100 100 0 0.00 0.25 0.50 0.75 1.00 1.25 -4 Volume of 5 × 10 di l ti azem ni cardi pi ne 1.50 1.75 2.00 M Ce(IV), ml fl unari zi ne 2.25 2.50 0 0.00 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 concentrati on of H 2sO4 , M verapami l Figure 3. Effect of volume of 5 × 10 -4 M Ce (IV) on the relative luorescence intensity. ●, 0.06 µg mL-1 DLT; □, 0.1 µg mL-1 VP; ∆, 0.1 µg mL-1 FZ; ■, 0.1 µg mL-1 NC. w w w.ijbs.org Int J Biomed Sci Ni cardi pi ne Hcl Verapami l Hcl Fl unari zi ne Di l ti azem HcL Figure 4. Effect of molar concentration of H2SO4 on the relative luorescence intensity. ●, 0.06 µg mL-1 DLT; □, 0.1 µg mL-1 VP; ∆, 0.1 µg mL-1 FZ; ■, 0.1 µg mL-1 NC. Vol. 5 No. 2 June 2009 149 KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers dimethyl sulfoxide and dimethyl formamide quenched the luorescence completely. The rate of the reaction was found to be dependent on the concentration of the studied drugs. The rates were followed with various concentrations in the range of 0.020.12 µg mL-1 for VP and NC, 0.01-0.06 µg mL-1 for DLT and 0.04-0.12 µg mL-1 for FZ keeping Cerium (IV) and H2SO4 acid concentrations constant at the recommended levels mentioned before. The rate of the reaction was found to obey the following equation: Rate of the reaction = ∆ F/ ∆ t = k`[drug]n (a) where K` is the pseudo-order rate constant and n is the order of the reaction. The rate of the reaction may be estimated by the variable time method measurement (45), where F is the luorescence intensity and t is the time in seconds. Taking logarithms of rates and drug concentrations (Table 2), the previous equation is transformed into: 500 400 ∆F 300 200 100 0 0 10 20 30 40 50 60 70 Time of heating, minute nicardipine diltiazem flunarizine STudy oF THE KInETIC pArAmETErS verapamil Figure 5. Effect of the heating time on the relative luorescence intensity. ∆, 0.06 µg mL-1 DLT; ●, 0.1 µg mL-1 VP; □, 0.1 µg mL-1 FZ; ■, 0.1 µg mL-1 NC. Table 2. Logarithm of the rate for different concentrations of the studied drugs by the proposed method Compound Verapamil HCl Diltiazem HCl Nicardipine HCl Flunarizine 150 Log ∆F/∆t Log [drug] -1.22 -7.39 -0.92 -7.09 -0.76 -6.91 -0.63 -6.79 -0.54 -6.69 -0.46 -6.61 -1.16 -7.65 -0.89 -7.35 -0.59 -7.05 -0.50 -6.96 -0.43 -6.88 -1.15 -7.42 -0.86 -7.11 -0.69 -6.93 -0.57 -6.81 -0.47 -6.71 -0.39 -6.63 -1.04 -7.00 -0.86 -6.83 -0.74 -6.70 -0.65 -6.61 -0.57 -6.53 regression equation Correlation coeficient rate constant (S -1) order of reaction (n) Log rate=5.950+0.970 log C 0.9999 891251 0.970 Log rate=6.104+0.950 log C 0.9998 1270574 0.950 Log rate=6.037+0.970log C 0.9999 1088930 0.970 Log rate=5.834+0.981 log C 0.9998 682339 0.981 June 2009 Vol. 5 No. 2 Int J Biomed Sci w w w.ijbs.org KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers Log (rate) = Log ∆ F/∆ t = Log k` + n Log [drug] (b) A plot of log reaction rate versus log concentration of the drug is shown in Figure 6 at 365 nm after excitation at 255 nm, where the slope (n) is the order of the reaction and the intercept ( Log K') is the logarithm of the rate constant (Fig. 6). A Plot of log reaction rate versus log drug concentration resulted in a pseudo-order rate constant and irst order of the reaction which are abridged in Table 2. These results indicated that the reaction is pseudo irst order reaction, depending on the drugs concentration. SELECTIon oF THE bEST KInETIC mETHod Several kinetic techniques were adopted for the selection of the best method. Rate constant, ixed luorescence and ixed time methods (46, 47) were tried and the most suitable analytical method was selected taking into account the applicability, the sensitivity, i.e. the slope of the calibration graph and the correlation coeficient (r). 10 -7 M, 2.22 × 10 -8 – 1.33 × 10 -7 M, 3.88 × 10 -8 – 2.33 × 10 -7 M and 9.89 × 10 -8 – 2.97 × 10 -7 M, respectively were plotted and all appeared to be rectilinear. Pseudo-irst order rate constants (k`) corresponding to different drug concentrations (C) were calculated from the slopes multiplied by – 2.303 and are presented in Table 3. Regression of (C) versus K` gave equations: k` = -4.195 + 847496 C (r=0.960) for VP k` = -1.305 + 828048 C (r=0.9528) for DLT k` = -1.287 + 735044 C (r=0.9949) for NC k` = -6.498 + 1407295 C (r=0.9963) for FZ where C is the molar concentration of the drugs. Fixed luorescence method Reaction times required to reach speciic luorescence of redox reaction for different concentrations of VP, DLT, Table 3. Application of the rate constant method in the quantiication of the studied drugs by the proposed method Compound rate constant method Graphs of log absorbance versus time for VP, DLT, NC and FZ concentration in the range of 4.07 × 10 -8 – 2.44 × A Verapamil HCl -0.25 -1.00 Log ∆F/∆t Log ∆F/∆t -0.50 -0.75 Diltiazem HCl -0.50 C Nicardipine HCl D -0.5 -0.25 Log ∆F/∆t Log ∆F/∆t -4.080 × 10 -4 1.22 × 10 -7 -4.053 × 10 -4 1.63 × 10 -7 -4.023 × 10 -4 2.04 × 10 -7 -4.000 × 10 -4 2.44 × 10 -7 -1.283 × 10 -3 0.22 × 10 -8 -1.262 × 10 -3 0.44 × 10 -8 -1.235 × 10 -3 0.89 × 10 -8 -3 1.11× 10 -7 -1.184 × 10 -3 1.33 × 10 -7 -1.263 × 10 -3 0.39 × 10 -8 -1.225 × 10 -3 0.78 × 10 -8 -1.200 × 10 -3 1.16 × 10 -7 -1.176 × 10 -3 1.55 × 10 -7 -0.7 -1.139× 10 -3 1.94 × 10 -7 -0.8 -1.124 × 10 -3 2.33 × 10 -7 -0.9 -4 0.99× 10 -8 -6.296 × 10 -4 1.48 × 10 -7 -6.218 × 10 -4 1.98 × 10 -7 -6.162 × 10 -4 2.47 × 10 -7 -6.070 × 10 -4 2.97 × 10 -7 Flunarizine -1.00 -6.351 × 10 -1.0 -1.25 -7.75 -7.50 -7.25 -7.00 -6.75 -6.50 -6.25 Log concentration 0.81 × 10 -8 -0.6 -0.50 -0.75 0.41 × 10 -8 -4 -1.215 × 10 -1.25 -8.00 -7.75 -7.50 -7.25 -7.00 -6.75 -6.50 Log concentration -1.50 -7.75 -7.50 -7.25 -7.00 -6.75 -6.50 -6.25 Log concentration -4.190 × 10 -4 -0.75 -1.00 -1.25 [drug] -4.100 × 10 B -0.25 k ` (S -1) -7.25 -7.00 -6.75 -6.50 Log concentration -6.25 Figure 6. Plot of Log reaction rate (Log ∆F/∆t) versus Log concentration of: A, VP; B, DLT; C, NC; D, FZ. w w w.ijbs.org Where k ` is the pseudo irst order rate constant. Int J Biomed Sci Vol. 5 No. 2 June 2009 151 KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers NC and FZ in the range of 1.50 × 10 -7 – 2.04 × 10 -7 M, 4.43 × 10 -8 – 1.36 × 10 -7 M, 8.91 × 10 -8 – 1.77 × 10 -7 M and 1.48 × 10 -7 – 2.97 × 10 -7 M, respectively were recorded. A preselected values of the luorescence 301, 155, 140 and 181 for VP, DLT, NC and FZ were ixed, respectively and the time was measured in seconds. The reciprocal of time (1/t) versus the initial concentration of drug was plotted. Table 4 and the following equations of the calibration graphs were obtained: 1/t = -1.553 × 10 -3 + 15762.88 C (r=0.9977) for VP 1/t = -4.571 × 10 -4 + 2760 5.75 C (r=0.9972) for DLT 1/t = -7.922 × 10 -4 + 21257.45 C (r=0.9999) for NC -3 1/t = -1.573 × 10 + 16597.49 C (r=0.9984) for FZ where C is the molar concentration of the drugs and t = time in second. Fixed time method At a preselected ixed time, which was accurately determined, the luorescence was measured. Calibration graphs of luorescence versus initial concentrations of VP, DLT, NC and FZ at ixed times for VP and FZ at 5, 10, 15, 20, 25 minutes, for NC at 5, 10, 15 minutes and for DLT at 5, 10, 15, 20 were established with the regression equations and correlation coeficients assembled in Table 5. It is clear that the slope increases with the time and the most acceptable values of the correlation coeficient (r) was chosen as the most suitable time interval for measurement. As a conclusion, the ixed time method was chosen for quantiication because it gives the best correlation coeficient in a reasonable time. determined by evaluating the lowest concentration of the analyte that can be readily detected and was found to be 6.33 × 10 -3, 3.10 × 10 -3, 2.93 × 10 -3 and 0.012 µg mL-1 for Table 4. Application of the ixed luorescence method in the quantiication of the studied drugs by the proposed method Compound Verapamil HCl Diltiazem HCl Nicardipine HCl Flunarizine 152 June 2009 Vol. 5 No. 2 1/t (sec.-1) [drug] 1200 8.33 × 10 -4 1.50 × 10 -7 900 1.11 × 10 -3 1.71 × 10 -7 600 1.67 × 10 -3 2.04 × 10 -7 1200 8.33 × 10 -4 4.43 × 10 -8 600 1.67 × 10 -3 8.09 × 10 -8 300 3.33 × 10 -3 1.36 × 10 -7 900 1.11 × 10 -4 8.91 × 10 -8 600 3.30 × 10 -3 1.16 × 10 -7 300 6.80 × 10 -3 1.94 × 10 -7 1200 8.33 × 10 -3 1.48 × 10 -7 600 1.67 × 10 -3 1.90 × 10 -7 300 3.33 × 10 -3 2.97 × 10 -7 Table 5. Application of the ixed time method in the quantiication of the studied drugs by the proposed method Compound Verapamil HCl AnALyTICAL pErFormAnCE The luorescence-concentration plots for the studied drugs were linear over the range cited in Table 1. Linear regression analysis of the data gave the following equations: F = 7.133 + 4260.00 C (r=0.9998) for VP F = 9.512 + 7380.23 C (r=0.9998) for DLT F = 3.67 + 2992.86 C (r=0.9999) for NC F = 4.60 + 3340.00 C (r=0.9998) for F where F is luorescence intensity, C is the concentration of the drug (µg mL-1) and r is correlation coeficient. The limit of quantiication (LOQ) was determined by establishing the lowest concentration that can be measured according to ICH Q2B recommendations (49), below which the calibration graph is non linear and was found to be 0.02, 0.01, 9.76 × 10 -3, 0.04 µg mL-1 for VP, DLT, NC and FZ, respectively. The limit of detection (LOD) was Time (sec.) Diltiazem HCl Nicardipine HCl Flunarizine Int J Biomed Sci time (min.) regression Equations correlation Coeficient 5 F = 5.667 + 2621.429 C r=0.9991 10 F = 3.267 + 3048.571 C r=0.9991 15 F = 5.467 + 3574.286 C r=0.9992 20 F = 14.200 + 3811.429C r=0.9993 25 F = 7.133 + 4260.000 C r=0.9998 5 F = -2.814 + 2533.721 C r=0.9991 10 F = 12.116 + 3952.326 C r=0.9995 15 F = 4.535 + 5290.698 C r=0.9996 20 F = 9.512 + 7380.233 C r=0.9998 5 F = -2.80 + 1554.286 C r=0.9997 10 F = 7.80 + 2188.571 C r=0.9998 15 F = 3.67 + 2992.857 C r=0.9999 5 F = -1.00 + 1510.00 C r=0.9993 10 F = -1.20 + 2395.00 C r=0.9993 15 F = -4.40 + 2750.00 C r=0.9994 20 F = 6.40 + 2865.00 C r=0.9996 25 F = 4.60 + 3340.00 C r=0.9998 w w w.ijbs.org KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers VP, DLT, NC and FZ, respectively. LOQ and LOD were calculated according to the following equations (49): LOQ = 10 ó /S LOD = 3.3 ó /S where ó is the standard deviation of the intercept of regression line and S is the Slope of the calibration curve. The proposed method was evaluated by studying the accuracy as percent relative error and precision as percent relative standard deviation. The results are abridged in Table 1. VALIdATIon oF THE mETHod Linearity The proposed method was tested for linearity, speciicity, precision, and reproducibility. Linear regression equations were obtained. The regression plots showed a linear dependence of FI value on drug concentrations over the range cited in Table 1. The table also clariies the lower detection limits as well as the slopes and intercepts. Validation of the method was evaluated by statistical analysis of the regression line regarding standard deviation of the residuals (Sy/x), the intercept (Sa), and the slope (Sb). The small values of the igures point out to the low scattering of the points around the calibration curve. Statistical analysis (50) of the results, obtained by the proposed and the comparison methods (15, 35, 51) using Student’s t-test and variance ratio F-test, shows no signiicant difference between the performance of the two methods regarding the accuracy and precision, respectively. Accuracy and precision The intra-day precision was evaluated through replicate analysis of pure samples of 0.04, 0.06 and 0.08 µg mL-1 VP as a model example. Each concentration was analyzed three times. The mean percentage recoveries are shown in Table 6. The repeatability and reproducibility of the proposed method are fairly good as indicated by small values of standard deviation (SD). The inter-day precision was evaluated through replicate analysis of pure 0.08 µg mL-1 VP on three successive days. The percentage recoveries based on the average of three separate determinations are abridged in Table 6. The accuracy of the proposed method was evaluated by analyzing standard solutions of the studied drugs. The results obtained by the proposed method were compared with those given by the comparison methods (15, 16, 35, 51). w w w.ijbs.org robustness of the method The robustness of the method adopted in the proposed method is demonstrated by the constancy of the luorescence intensity with the minor changes in the experimental parameters such as 5 × 10-4 M Ce (IV) volume, 0.5 ± 0.1 mL, 1 ± 0.2 mL, 1.5 ± 0.25 mL and 0.8 ± 0.2 mL for VP, DLT, NC and FZ, respectively and change in the concentration of sulphuric acid, 1.25 ± 0.25 M, 1 ± 0.1 M, 1.5 ± 0.2 M and 1.5 ± 0.25 M for VP, DLT, NC and FZ, respectively. These minor changes that may take place during the experimental operation didn’t affect the luorescence intensity. pHArmACEuTICAL AppLICATIonS The proposed method was applied to the determination of the studied drugs in their dosage forms. The speciicity of the method was investigated by observing any interference encountered from the common excipients, such as talc (20 Table 6. Precision and accuracy of the proposed method for spectroluorometric determination of pure VP regimen Parameters concentration concentration recovery Added (µg/mL) Found (µg/mL) % Intra-day 0.040 0.0392 98.00 0.0395 98.75 0.0398 99.50 ( x- ) 98.75 ± S.D. 0.75 0.060 0.0598 99.67 0.0603 100.50 0.0607 101.17 ( x- ) 100.45 ± S.D. 0.75 0.080 Inter-day 0.0799 99.88 0.0789 98.63 0.0805 100.63 ( x- ) 99.71 ± S.D. 1.01 st 1 day nd 0.080 0.0788 98.50 2 day 0.0815 101.88 3rd day 0.0808 101.00 ( -x ) 100.46 ± S.D. 1.75 Each result is the average of three separate determinations. Int J Biomed Sci Vol. 5 No. 2 June 2009 153 KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers mg), lactose (15 mg), starch (15 mg), avisil (15 mg), gelatine (0.7 mg) and magnesium stearate (10 mg). These excipients did not interfere with the proposed method (Table 7). The results of the proposed method were compared with those obtained using the comparison methods (15, 16, 35, 51). Statistical analysis (50) of the results obtained using Student’s t-test and variance ratio F-test revealed no signiicant difference between the performance of the two methods regarding the accuracy and precision, respectively (Table 7). mECHAnISm oF THE rEACTIon The Stoichiometry of the reaction between the studied drugs and cerium (IV) was studied adopting the limiting logarithmic method (53) (Fig. 7). The luorescence intensities of the reaction product were alternatively measured in the presence of excess Ce (IV) and the studied drugs. Plots of log [drugs] versus log ∆F and log [Ce (IV)] versus log ∆ F gave straight lines, the values of the slopes were 0.97: 0.1.60 for VP, 0.99: 1.52 for DLT, 0.97: 1.54 for NC and 0.982: 1.51 for FZ (drug: Ce (IV)). Hence, it is concluded that, the molar reactivity of the reaction is 1: 2 i.e. the reaction proceeds in ratio of 1: 2. Based on the above fact and by analogy to previous reports (41), proposals for the reactions between the studied drugs and Ce (IV) shown in the following igures (Figs. 8, 9, 10, 11). ConCLuSIon The present work describes a validated spectroluorometric method for the determination of the studied drugs without interference from common excipients. Hence, it could be applied for the routine quality control of the studied drugs either in bulk or in their corresponding dosage forms. The methodology appears to be straightforward and results are relevant. Another advantage is that, comparing to the existing spectroluorometric methods, the proposed method is several times more sensitive. From economic point of view, the proposed method is simple, rapid and inexpensive besides the use of water as diluting solvent. So, it is a good alternative to the reported methods and to high cost HPLC methods. Table 7. Application of the proposed method to the determination of the studied drugs in dosage forms Comparison methods proposed method (15, 16, 35, 51) Compound concentration taken Concentration found recovery recovery % % (µg mL -1) (µg mL -1) Isoptin 80 mg tabletsa 0.06 0.0606 101.00 98.58 (VP HCl 80 mg/tablet) 0.08 0.0809 101.125 99.29 0.10 0.0996 99.60 99.82 Mean ± S.D. Student’s t-test F-test Isoptin retard 240 mg tabletsa (VP HCl 240 mg/tablet) 100.58 ± 0.85 99.23 ± 0.62 1.734 (2.776) 1.90 (19.00) 0.06 0.0604 100.67 99.29 0.08 0.0798 99.75 98.23 0.10 0.0992 99.23 98.19 Mean ± S.D. 99.88 ± 0.73 98.57 ± 0.63 Student’s t-test 2.30 (2.776) F-test 1.38 (19.00) Verapamil 40 mg tabletsb 0.06 0.0593 98.83 99.29 (VP HCl 40 mg/tablet) 0.08 0.0795 99.38 98.94 0.10 0.1007 100.70 98.55 Mean ± S.D. 99.64 ± 0.96 98.93 ± 0.37 Student’s t-test 1.20 (2.776) F-test 6.43 (19.00) Each result is the average of three separate determinations. Figures between parenthesis are the tabulated t and F values, respectively at p=0.05 (50). aProducts of the Arab Drug Company, Cairo, Egypt); bProduct of El-Nasr Pharmaceutical Chemical Company (Cairo, Egypt); cProduct of Lusofarmaco, Cairo, Egypt). 154 June 2009 Vol. 5 No. 2 Int J Biomed Sci w w w.ijbs.org KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers Table 7. Application of the proposed method to the determination of the studied drugs in dosage forms (Continued) Comparison methods (15, 16, 35, 51) proposed method Compound concentration taken (µg mL -1) Altiazem Retard 60 mg tabletsc (DLT HCl 60 mg/tablet) Concentration found (µg mL -1) recovery % 0.02 0.0199 99.50 98.72 0.04 0.0406 101.50 100.25 0.05 0.0507 101.40 100.62 100.80 ± 1.13 99.86 ± 1.01 Mean ± S.D. Student’s t-test F-test Delay-tiazem SR 90 mg capsules d (DLT HCl 90 mg/capsule) 1.25 (19.00) 0.0197 98.50 97.72 0.0403 100.75 101.15 0.05 0.0508 101.60 98.48 100.28 ± 1.60 99.12 ± 1.80 F-test (DLT HCl 120 mg/ capsule) (2.776) 0.02 Student’s t-test Delay-tiazem SR 120 mg capsules 1.070 0.04 Mean ± S.D. d recovery % 0.832 (2.776) 1.27 (19.00) 0.02 0.0201 100.50 99.74 0.04 0.0405 101.25 100.90 0.05 0.0504 Mean ± S.D. 100.80 98.90 100.85 ± 0.38 99.85 ± 1.00 Student’s t-test 1.614 F-test (2.776) 6.94 (19.00) Delay-tiazem SR 180 mg capsulesd 0.02 0.0204 102.00 100.94 (DLT HCl 180 mg/capsule) 0.04 0.0404 101.00 100.00 0.05 0.0505 Mean ± S.D. 101.00 101.13 101.33 ± 0.58 100.69 ± 0.61 Student’s t-test F-test Pelcard 50 capsulese (NC HCl 50 mg/capsule) 1.312 (2.776) 1.11 (19.00) 0.06 0.0603 100.50 100.00 0.08 0.0810 101.25 101.39 0.10 0.1013 101.30 101.45 101.02 ± 0.45 100.95 ± 0.82 Mean ± S.D. Student’s t-test 0.130 (2.776) F-test 3.32 (19.00) 99.00 100.82 f Sibelium capsules (FZ HCl 5 mg/capsule) 0.06 0.0594 0.08 0.0806 100.75 101.20 0.10 0.1012 101.20 98.77 100.32 ± 1.16 100.26 ± 1.31 Mean ± S.D. Student’s t-test 0.059 (2.776) F-test 1.27 (19.00) Each result is the average of three separate determinations. Figures between parenthesis are the tabulated t and F values, respectively at p=0.05 (50). dProducts of GlaxoWellcome Company, El-Salam City, Cairo, Egypt; eProduct of Global Napi& Makram Mehany, Cairo, Egypt); f Product of Janssen Cilag Company, Cairo, Egypt, Under License of Janssen Pharmaceutica-Belgium). w w w.ijbs.org Int J Biomed Sci Vol. 5 No. 2 June 2009 155 KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers B A 2.7 2.6 +4 2.75 2.5 Log ∆ F of Ce Log ∆ F of verapamil 3.00 2.50 2.25 2.4 2.3 2.00 2.2 1.75 -8.00 -7.75 -7.50 -7.25 -7.00 -6.75 -6.50 2.1 -5.1 -6.25 -5.0 -4.9 -4.8 -4.7 -4.6 -4.5 Log concentration Log concentration Figure 7. Stoichiometry of the reaction between VP and Ce (IV) adopting limiting logarithmic method. A, Log (VP] vs log ∆ F; B, Log (Ce (IV)] vs log ∆ F. CH3 H 3C CH3 CH3 CN H 3C OMe N CH3 CN + 2Ce + H2O +4 + + 2Ce 3 + 2H + O OMe MeO OMe N OMe MeO OMe OMe Figure 8. Proposal for the reaction between VP and Ce (IV). H3C H3 C O O H O H H 3C H3C O N N CH3 CH3 O N H O +2Ce+4+ H2O +2 Ce+3 + 2H+ N H S S MeO O MeO Figure 9. Proposal for the reaction between DLT and Ce (IV). NO2 NO2 CH3 CH3 H COOCH2CH2N H3COOC CH2 H3C N +2Ce+4+ H2O COOCH2CH2N H3COOC CH2 CH3 H3C N +2 Ce+3 + 2H+ CH3 H Figure 10. Proposal for the reaction between NC and Ce (IV). O F N F N N +2Ce+4+ H2O N + 2 Ce+3 + 2H+ F F Figure 11. Proposal for the reaction between FZ and Ce (IV). 156 June 2009 Vol. 5 No. 2 Int J Biomed Sci w w w.ijbs.org KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers rEFErEnCES 1. Paritt K., editor In Martindale, “The Complete Drug Reference”, 32th ed., The Pharmaceutical Press, Massachusetts, 1999. 2. Rahman N., Hejaz A., Syed N. Farmaco, 2004;59: 529-536. 3. Rahman N., Hoda M. Anal. and Bioanal. Chem., 2002; 374: 484-489. 4. Khalil S., Kelzieh A. J. Pharm. Biomed. Anal. , 2002; 27:123-131. 5. Esteves da Silva J. C. G., Leitao J. M. M., Costa F. S., Ribeiro J. L.A. Anal. Chim. Acta , 2002; 453: 105-115. 6. McAllister R. G., Howell S. M. J. Pharm. Sci., 1976; 65: 431-432. 7. Aboul Kasim E., Ghandour M. A., El-Haty M. T., Ahmed M. M. J. Pharm. Biomed. Anal., 2002; 30: 921-929. 8. Jhee O. H., Hong J. W., Om A. S., Lee M. H., Lee W. S., Shaw L. M., Lee J. W., Kang J. S. J. Pharm. Biomed. Anal., 2005; 37: 405-410. 9. Rao R. N., Nagaraju V. J. Pharm. Biomed. Anal. , 2003; 33: 335-337. 10. Misllanova C., Hutta M. J. Chromatogr. B., 2003; 797: 91-109. 11. Pelander A., Ojanpera I., Laks S., Rasanen I., Vuori E. Anal. Chem. 2003; 75:5710-5718. 12. Mullett W. M., Walles M., Levsen K., Borlak J., Pawliszyn J. J. Chromatogr. B., 2004; 801: 297-306. 13. Alçyn O. Y, Niyazi Y., Sibel O. A., Nci B. Farmaco. 2000; 55: 376382. 14. Li S. L., Vigh G. Electrophoresis. 2003; 24: 2487-2498. 15. British Pharmacopeia Vols. I and II, Her Majesty’s Stationery Ofice, London, UK, 2000 (Through electronic version). 16. The United States Pharmacopoeia XXVII., National Formulry XXII Rockville, USP Convention Inc., MD, 2004. 17. Rahman N., Hejaz-Azmi S. N. J. Pharm. Biomed. Anal. 2000; 24: 3341. 18. Rahman N., Azmi S. N. H. Microchem. J. 2000; 65: 39-43. 19. Ivanovskaya E. A., Bobleva Y. V. and Karpov R. S. J. Anal. Chem. 2000; 55: 1077-1079. 20. Gil-Agusti M., Carda-Broch S., Garcia-Alvarez-Coque C., EsteveRomero j. J. Chromatogr. Sci. 2000; 38: 521. 21. Quinones I., Cavazzini A., Guiochon G. J. Chromatogr. A. 2000; 877: 1-11. 22. Cahill J. D., Furlong E. T., Burkhardt M. R., Kolpin D., Anderson L. G. J. Chromatogr. A. 2004; 1041: 171-180. 23. Quaglia M. G., Donati E., Fanali S., Bossu E., Montinaro A., Buiarelli F. J. Pharm. Biomed. Anal. 2005; 37: 695-701. 24. Escrig-Tena I., Rodriguez L. A., Esteve-Romero J., Garcia-AlvarezCoque M. C. Talanta. 1998; 47:43-52. 25. Ragno G., Vetuschi C. Pharmazie. 1998; 53: 628-631. 26. Ozkan S. A., Uslu B., Aboul-Enein H. Y. Crit. Rev. Anal. Chem. 2003; 33: 155-181. 27. Atkosar Z., Altiokka G., Tuncel M. Pharmazie. 1997; 52: 959-960. 28. Gong L., Wang S. C., He L. C. Yaowu. Fenxi. Zazhi. 2003; 23: 399401. w w w.ijbs.org View publication stats Int J Biomed Sci 29. Fernandez C. M., Veiga F. J. B. Biomed. Chromatogr. 2003; 17: 33-38. 30. Uno T., Ohkubo, T., Sugawara K. J. Chromatogr. B: Biomed. Appl. 1997; 698: 181-186. 31. Meiling Q., Peng W., Xin J. J. Chromatogr. B. 2006; 830: 81-85. 32. Pomponio R., Gotti R., Fiori J., Cavrini V., Mura P., Cirri M., Maestrelli F. J. Pharm. Biomed. Anal. 2004; 35: 267-275. 33. Chen Z. S., Yang Y. G., Wang W. S., Yu B. H., Wang D. X. Yaowu. Fenxi. Zazhi. 1995; 15: 57-58. 34. El-Walily A. F. M., El-Gindy A., Wahbi A. A. M. J. Pharm. Biomed. Anal. 1995; 13: 53-58. 35. Kelani K., Bebawy L. I., Abdel-Fattah L. J. Pharm. Biomed. Anal. 1999; 18: 985-992. 36. Uslu B., Yilmaz N., Erk N., Ozkan S. A., Senturk Z., Biryol I. J. Pharm. Biomed. Anal. 1999; 21: 215-220. 37. Martinez-Algaba C., Bermudez-Saldana J. M., Villanueva-Camanas R. M., Sagrado S., Medina-Hernandez M. J. J. Pharm. Biomed. Anal. 2006; 40: 312-321. 38. Tang L. F., Wu J. H., Li L. C., Chen H. Y., Tan B. Y., Li Z. W. Fenxi. Ceshi. Xuebao. 2000; 19: 75-77. 39. John L. Z., David M., Anna A., Linyee S. J. Pharm. Biomed. Anal. 2005; 37: 757-762. 40. Sun Y., Nakashima M. N., Takahashi M., Kuroda N., Nakashima K. Biomed. Chromatogr. 2002; 16: 319-326. 41. Darwish I. A., Khedra A. S., Askala H. F., Mahmoud R. M. Farmaco. 2005; 60: 555-562. 42. Mohamed F. A., Mohamed H. A., Hussein S. A., Ahmed S. A. J. Pharm. Biomed. Anal. 2005; 39: 139-146. 43. Mahgoub H. J. Pharm. Biomed. Anal. 2003; 31: 767-774. 44. Alarfaj, N. A. A. J. Pharm. Biomed. Anal. 2002; 28: 331-335. 45. Kashaba R. Y. J. Pharm. Biomed. Anal. 2002; 27: 923-932. 46. Yatsimirskii K. B. editor In “Kinetic Methods of Analysis”, Pergamon Press, London. 1966, p.43. 47. Persez-Bendito D., Silva M. editors In “Kinetic Methods in Analytical Chemistry”, John Wiley and Sons, NY, 1988, p. 44. 48. Ingle J. D., Crauch S. R. Anal. Chem. 1971; 43: 697. 49. Guidance for Industry Bioanalytical method Validation, US Department of Health and Human Serves, Food and Drug Administration. (2001) Center for Drug Evaluation and Research, Rockville, MD, May. http://www.fda.gov/eder/guidance/4252fnl.pdf (accessed September 1, 2004). 50. Miller J.C., Miller J. N. editors In “ Statistics for analytical Chemistry”, Wiley, New York. 1984. 51. Mustafa A. A., Al-Rashood K. A., Hagga M. E. M., Gad-Kariem E. A., Al-Awady M. E. Pak. J. Sci. Ind. Res. 1994; 37: 1. 52. Huang H., Li H. Yaowu. Fenxi. Zazhi. 1990; 10: 359-360. 53. Rose J. editor In “Advanced Physicochemical Experiments”, Pitman, London. 1964, p.67. Vol. 5 No. 2 June 2009 157