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).
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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.
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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
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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.
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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.
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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.
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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
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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.
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Where k ` is the pseudo irst order rate constant.
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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
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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
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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).
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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.
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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).
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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).
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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).
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Vol. 5 No. 2
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KInetIc spectrofluorometrIc determInatIon of certaIn calcIum channel BlocKers
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