Introduction
Isradipine is a calcium channel blocker of the dihydropyridine class. It is usually prescribed for
the treatment of high blood pressure in order to reduce the risk of stroke and heart attack [1].On a
milligram-per-milligram basis, Isradipine is the most potent drug of this class. It was approved
by the FDA in December 1990 for the treatment of hypertension [2]. The mechanism of action is
similar to other calcium-channel antagonists, acts by inhibiting the influx of extracellular
calcium across the cell membrane of myocardial and vascular smooth muscle. The serum
calcium levels remain unchanged. It increases myocardial oxygen supply (secondary to coronary
vasodilatation) and also decreases myocardial oxygen demand (secondary to decreased afterload
and lack of an increase in heart rate). These effects seem to best explain the benefit of it and
other dihydropyridine in the treatment of angina. It also causes vasodilatation in coronary,
skeletal, and cerebral vasculature. More recent research in animal models suggests that isradipine
may have potential uses for treating Parkinson's disease [3] The drug is commercially available in
tablet dosage form (2.5 mg) with also controlled release 5 mg and 10 mg in capsule dosage
form.[3]
Isradipine-3-methyl5-propan-2-yl4-(2,1,3-benzoxadiazol-4-yl)-2,6-dimethyl-1,4dihydropyridine3,5-dicarboxylate is a yellow colored crystalline powder and it is insoluble in water, but freely
soluble in methanol& chloroform . Molecular weight of Isradipine is 371.387 g/mol and formula
is C19H21N3O5. It’s structure is given in Figure 1[3, 4,].
MATERIALS AND METHODS
Instrument, Chemicals, Reagents and Samples
Analysis of Isradipine was carried out on UV-Visible double beam Spectrophotometer
(Shimadzu UV-1800). Sonicator (Lifecare.pvt.ltd) was used for solubilization of tablet.
Isradipine reference standard was kindly gifted by Shasun Pharmaceuticals Limited, Chennai,
India. Methanol (AR Grade) was purchased from loba chemie pvt. Ltd, Mumbai, India. was used
as a diluent.
Selection of wavelength
In order to ascertain the wavelength of maximum absorption (λmax) of the drug, solutions of the
drug (10μg/ml) in methanol were scanned using spectrophotometer within the wavelength range
of 200 – 400 nm against methanol as blank. The resulting spectrum is shown in figure 2 and the
absorption curve showed characteristic absorption maxima at 327 nm for Isradipine.
Standard Solution Preparation
10 mg Isradipine was accurately weighed and transferred to 100ml amber colored volumetric
flask & diluted up to the mark with methanol to produce a stock solution of 100 μg/ml
concentration. 0.5ml of aliquot was diluted to 10mlwith methanol to give solution having 5
μg/ml concentrations. Similarly, solutions having concentrations of 10, 15, 20, 25 and 30 μg/ml
were prepared which were used for the construction of calibration curve (Tablet No 1).
Synthetic tablet preparations
Synthetic tablets were prepared by mixing excipients (Avicel PH 101 100mg, hydroxyl methyl
cellulose 35mg, acriflow Q.S) and labeled amount 10mg of isradipine.
Sample solution Preparation
Twenty tablets were weighed accurately and powdered. Tablet powder equivalent to 10 mg of
Isradipine was weighed and transferred to 100 ml amber colored volumetric flask separately.
About 40 ml of methanol was added and sonicated for 20 minutes for complete solubilization of
drug, the volume was made up to the mark with the same solvent resulting solution of 100 μg/ml
concentration. The solution was filtered through whatman filter paper. 2.0mlof filtrate was
diluted up to 10 ml with methanol and the Absorbance of tablet solution (20μg/ml) was recorded
against methanol as the blank at 327 nm.
Method Validation [4, 5, 8].
Validation can be defined as establishing documented evidence, which provides a high degree of
assurance that a specific activity will consistently produce a desired result or product meeting its
predetermined specifications and quality characteristics. The method was validated for several
parameters like linearity, accuracy, precision, Ruggedness, Robustness, Limit of detection
(LOD), and Limit of quantification (LOQ) according to ICH guidelines.
Linearity
For quantitative analysis of Isradipine, the linearity curve was plotted. Linearity range of
Isradipine was established in concentration range of 5μg/ml to 30 μg/ml. The slope and intercept
along with its correlation coefficient is given in Figure-4.
Specificity and selectivity
Specificity is the ability to assess unequivocally the analyte in the presence of components which
may be expected to be present. The spectra obtained from tablet solution and standard solution
containing an equivalent concentration of Isradipine showed that there was no any interference
from excipients. Therefore it could be concluded that developed method is highly selective and
specific. There was no placebo interference found in methanol (Figure-5)
Precision
The precision of an analytical procedure expresses the closeness of agreement (degree of scatter)
between a series of measurements obtained from multiple sampling of the same homogeneous
sample under the prescribed conditions. Precision of the method was determined in terms of
repeatability and intraday and interday precisions. The amounts of Isradipine was found by the
number of replicates of both pharmaceutical preparations (n=3) performed by repeatability
(intraday) and intermediate precision (inter-day) and reported as percent relative standard
deviation (% RSD). For this, 20 μg/ml concentration solutions were measured three times in day
and same was measured in next three days. The percent relative standard deviation was
calculated (Table 2.1 and 2.2).
Repeatability
Repeatability expresses the precision under the same operating conditions over a short interval of
time. Repeatability of the method was determined by analyzing six samples of same
concentration of drug. Absorbance was measured of each solution. The results are shown in
Table 3.
Accuracy / Recovery
The accuracy of an analytical procedure expresses the closeness of agreement between the value
which is accepted either as a conventional true value or an accepted reference value and the
value found. To ensure accuracy of the method, recovery studies were performed by standard
addition method at 80%, 100%, and 120% level to preanalyzed samples and subsequent solutions
were reanalyzed. At each level, three determinations were performed. The absorbance was
measured at wavelength maxima and the amount of drug recovered from the formulation was
calculated. Each level was repeated three times (n = 3). Recovery study was carried out by
adding a known amount of pure drug to the preanalyzed formulation and the proposed method
was followed. From the amount of drug found, percentage recovery was calculated (Table 4).
Recovery study was carried out at three levels 80%, 100% and 120% for the formulation
concentration of 20 μg/ml.
LOD & LOQ
LOD (k = 3.3) and LOQ (k = 10) of the method were established according to ICH definitions.
In this study LOD and LOQ were based on the standard deviation of the response and the slope
of the corresponding curve using the following equationsLOD = 3.3∂/s
LOQ = 10 ∂
Where ∂ is the standard deviation of the intercept and S is the slope of the calibrations curve.
LOD and LOQ of method are reported in Table 5.
Robustness
The robustness of an analytical procedure is a measure of its capacity to remain unaffected by
small, but deliberate variations in method parameters and provides an indication of its reliability
during normal usage. To determine the robustness of the method, the experimental conditions
were deliberately altered and assay was evaluated. The effect of detection wavelength was
studied at ±2 nm. For changes of conditions, the sample was assayed in triplicate. The results are
shown in Table 6.
Stability
Solution was kept at room temperature for one day to evaluate the stability of Isradipine in
standard solution and tablet solution. For standard solution, similarity factor was calculated from
initial to one day and was found within the acceptance criteria of 0.98 to 1.02. A formula for
similarity factor is mentioned below (Table 8).
𝐒𝐢𝐦𝐢𝐥𝐚𝐫𝐢𝐭𝐲 𝐟𝐚𝐜𝐭𝐨𝐫 =
𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝐢𝐧𝐢𝐭𝐢𝐚𝐥 𝐬𝐭𝐚𝐧𝐝𝐚𝐫𝐝 𝐬𝐨𝐥𝐮𝐭𝐢𝐨𝐧
𝐀𝐛𝐬𝐨𝐫𝐛𝐚𝐧𝐜𝐞 𝐨𝐟 𝐛𝐞𝐧𝐜𝐡 𝐭𝐨𝐩 𝐬𝐭𝐚𝐛𝐥𝐞 𝐬𝐭𝐚𝐧𝐝𝐚𝐫𝐝 𝐬𝐨𝐥𝐮𝐭𝐢𝐨𝐧
For tablet solution, % assay of Isradipine tablet solution were estimated against freshly prepared
standard solution. The difference in % assay of Tablet preparations from initial to one day was
found to be within the limits(difference in % assay value from initial should be not more than
3.0%)(Table 9).
RESULT AND DISCUSSION
The UV spectrum of isradipine in Methanol has maximum absorption (λmax) at 327 nm. The
absorbance of excipients in tablet solution did not interfere with isradipine. As a result, 327nm
wavelength was selected for quantitative analysis and validation. The drug obeyed Beer–
Lambert’s law in the concentration range of 5–30 μg/ml with regression 0.9996 at 327nm. The
overall % recovery was found to be 99.82% at 327nm, which reflect that the method is specific
and selective. The developed method was found to be precise as the %RSD values for intraday
and interday precision were found to be less than 2%. The developed method was found to be
precise, specific, linear and accurate during method validation. Bench top stability (up to 1 day)
of standard and tablet preparation were established by keeping the preparation at room
temperature and the preparations were found to be stable. The results are summarized in table 10.
CONCLUSION:
The developed UV spectrophotometric method for the determination of isradipine has the
advantage of being fast and applicable over a wide concentration range with high precision and
accuracy. The developed UV spectrophotometric method is cheaper, simpler and faster than
other UV, LC and GC methods for analysis of isradipine in the pharmaceutical preparations. The
method was validated as per the ICH guidelines. The results of the validation were found to be
satisfactory and therefore this method can be applied successfully to analyze drug formulations.
REFERENCES
1. Hansson L, Zanchetti A, Calcium antagonists in hypertension, Drugs Vol.40 (1990)
Page No 1–71.
2. http://www.accessdata.fda.gov/drugsatfda_docs/nda/pre96/19-546_Isradipine.cfm
3. Rees K, Stowe R et al, Anti-hypertensive drugs as disease-modifying agents for
Parkinson's disease: evidence from observational studies and clinical trials,
Cochrane Database Syst Rev. Vol.11(2011),CD008535
4. British Pharmacopoeia, H. M. Stationary Office, London, 2009, II, 1196, 2768.
5. Gennaro R., Remington ;The Science and Practice of Pharmacy, Mack Publishing
Company, Pennsylvania, 19th edn., 1995,vol-1,437-490.
6. International Conference on Harmonization, Draft Guideline on Validation
Procedure, Definition and Terminology Federal Register, Vol.(60)1995.
7. ICH, Q2A validation of analytical procedure, Methodology International
Conference on Harmonization, Geneva, October 1994.
8. Nijhu RS, Akhte DT, Development and validation of UV spectrophotometric
method for quantitative estimation of nitroglycerin in pharmaceutical dosage form,
International Current Pharmaceutical Journal 2011, Vol.1(1) Page no. 1-5.
9. k.chaitanya et al, Isocratic-Reverse Phase Liquid Chromatographic method for the
quantification of isradipine by UV detection in tablets, European Journal of Applied
Engineering and Scientific Research, Vol.1 (4) (2012) page no:220-227
10. G.aswini et al, development and validation of isradipine in bulk and its
pharmaceutical f formulation by RP-HPLC method, Vol.3 (9) page no: 131-133.
ACKNOWLEDGMENT
We would like to thank Shasun Pharmaceuticals Limited, Chennai, India, for providing pure
drug sample of isradipine for this study and the Ramanbhai Patel College of Pharmacy,
CHARUSAT, Gujarat, India, for providing us the research facility.
Figures:
Figure 1 Structure of Isradipine
Figure 2 UV Spectrum of 10μg/ml Isradipine in Methanol
Absorbance
1
0.8
0.6
y = 0.0311x + 0.0016
R² = 0.9996
0.4
0.2
0
0
10
20
30
Concentration (μg/mL)
Figure 4 Linearity curve of Isradipine (5-30 μg/mL)
Figure 5 Spectra of Placebo (in Methanol)
Tables:
40
Table 1: Linearity of Isradipine (5-30 μg/ ml) (λ max.: 327 nm)
Conc. (μg/mL)
5
10
15
20
25
30
Linearity Equation
Correlation Coefficient
Slope
Intercept
Mean Response ± S.D. *
0.155 ± 0.0023
0.317 ± 0.0031
0.467 ± 0.0044
0.626 ± 0.0059
0.769 ± 0.0066
0.941 ± 0.0051
% R.S.D.
1.4712
0.9774
0.9343
0.9562
0.8565
0.5411
y = 0.031x + 0.001
0.999
0.031
0.001
* n= 6
Table 2: Precision
2.1. Intraday Precision:
Conc.
(μg/mL)
10
20
30
Absorbance
0.321
0.318
0.316
0.629
0.625
0.620
0.948
0.941
0.935
Conc.
(μg/mL)
10.28
10.19
10.12
20.15
20.02
19.86
30.37
30.15
29.95
Mean absorbance Mean
Conc.
± S.D.
(μg/mL) ± S.D.
0.3183
10.1987
±
±
0.0025
0.0806
0.6247
20.013
±
±
0.0045
0.1444
0.9413
30.1583
±
±
0.0065
0.2084
% R.S.D.
0.7905
0.7218
0.6912
2.2. Interday Precision
Conc.
(μg/mL)
10
20
30
Absorbance
0.312
0.317
0.316
0.631
0.620
0.627
0.940
0.944
0.935
Conc.
(μg/mL)
9.99
10.16
10.12
20.22
19.86
20.08
30.12
3024
2996
Mean absorbance Mean
Conc.
± S.D.
(μg/mL) ± S.D.
0.315
10.0919
±
±
0.0026
0.0847
0.626
20.0557
±
±
0.0056
0.1783
0.9397
30.1049
±
±
0.0045
0.1444
% R.S.D.
0.8399
0.8894
0.4799
Table 3: Repeatability
No.
1
2
3
4
5
6
Mean
S.D.
% R.S.D.
Conc. (μg/mL)
Absorbance
0.631
0.620
0.627
0.635
0.625
0.620
0.6263
0.006
0.9562
20
Conc. (μg/mL)
20.22
19.86
20.09
20.34
20.02
19.86
20.0664
0.1919
0.9562
Table 4: Accuracy (Recovery Study)
Level of Sample
Recover Conc.
y
(μg/mL)
10
80
10
10
10
100
10
10
10
120
10
10
Std.
Added
(μg/mL)
8
8
8
10
10
10
12
12
12
Total
amount
(μg/mL)
18
18
18
20
20
20
22
22
22
Absorbanc
e
0.562
0.560
0.564
0.624
0.619
0.625
0.689
0.685
0.686
Amount
Recovered
(μg/mL)
17.99
17.92
18.06
19.99
19.83
20.02
22.07
21.94
21.97
%
Recover
y
99.94
99.55
100.3
99.95
99.15
100.1
100.32
99.73
99.86
Mean %
Recover
y
99.93
99.73
99.97
Table 5: Limit of Detection (LOD) & Limit of Quantification (LOQ)
LOD (μg/mL)
0.1028
Drug
Isradipine
LOQ (μg/mL)
0.3115
Table 6: ROBUSTNESS
Conc.
(µg/mL)
20
Average
S.D.
% R.S.D.
Absorbance
325 nm
0.628
0.631
0.629
329 nm
0.622
0.624
0.621
Conc. µg/mL
325 nm
329 nm
0.636
0.618
0.632
0.616
0.626
0.624
% Assay
325 nm
0.630
0.632
0.626
329 nm
0.62
0.624
0.621
0.6296
0.0015
0.2425
0.6223
0.0015
0.2454
0.6313
0.0050
0.7972
0.6293
0.0030
0.4854
0.621666
0.002
0.334
0.6193
0.0041
0.6722
Table 7: ANALYSIS OF THE MARKETED DRUG FORMULATION
Tablet
Synthetic Tablet
Drug
Isradipine
Label Claim(mg)
10 mg
%Assay± S.D.
99.83 ± 0.5116
%RSD
0.5124
*n=6
Table 8: Standard Solution Bench Top Stability
Conc. (μg/mL)
20
Initial Absorbance(t=0 Absorbance after
Similarity Factor
hour)
one day (t=24 hour)
0.626
0.632
0.991
0.620
0.625
0.992
0.631
0.639
0.987
Table 9: Tablet Solution Stability
Initial
Conc.
Initial
Conc.
(μg/mL) Absorbance
(μg/mL)
0.618
19.81
20
0.625
20.03
0.617
19.77
%
Assay
99.05
100.15
98.85
Abs.
Conc. After
after 24 24
hr.
hr.
(μg/mL)
0.623
19.97
0.629
20.16
0.623
19.97
% Assay
after 24
hr.
99.85
100.8
99.85
Difference
in
%
Assay
0.8
0.65
1.0
Table 10: Summary of Validation Parameters
Parameter
λ max
Linearity
Equation
R2
LOD
LOQ
Repeatability (%RSD, n = 6)
Intraday Precision (%RSD, n = 3)
Interday Precision (%RSD, n = 3)
% Recovery
Standard solution stability (Similarity
Factor)
Tablet solution stability
(Assay value difference from initial)
% Assay ± S.D. (n=5)
Isradipine
327 nm
5- 30 μg/mL
y = 0.031x + 0.001
0.999
0.1028 μg/mL
0.3115 μg/mL
0.9562
0.7345
0.7364
99.88
0.99
0.8167
99.83 ± 0.5116