Development and Validation of a Simultaneous
HPLC Method for Assay and Dissolution of Bisoprolol fumarate and Amlodipine besylate in Pharmaceutical Dosage.
Dr.
Shalini Pant* and Krishan
Pal
1Principal, S.S.D.P.C.
Girls Post Graduate College, Roorkee-247667, Uttarakhand,
India
2K.L.D.A.V. (P.G.) College, Roorkee Haridwar, Uttrakhand India
-247667
ABSTRACT:
A fast and robust RP-HPLC method was developed for
simultaneous determination of bisoprolol fumarate and amlodipine besylate in pharmaceutical dosage form. A mixture of buffer
prepared by 0.4ml of TEA and 3.12 gm of Sodium dihydrogen
orthophosphate in 1000 ml water adjusted to pH 3.0ą0.05 and acetonitrile
(50:50) was used as mobile phase at 1.0 ml/min flow rate. A widely used stationary phase i.e. C18
column (5μ, 250×4.6 mm ID) in pharmaceutical industry was selected and UV
detection was performed at 230 nm. retention time was obtained as 4.4 minutes
and 6.4 minutes for bisoprolol and amlodipine respectively. Linearity was established in the
range 1.3μg/ml to 10.8μg/ml. Correlation coefficients were 0.9999 and
0.9988 for bisoprolol and amlodipine
respectively. Mean recoveries were obtained as 99.25% and 99.96% for bisoprolol fumarate and amlodipine besylate respectively.
KEYWORDS: Bisoprolol fumarate, amlodipine besylate, RP-HPLC
INTRODUCTION:
Bisoprolol fumarate is a synthetic beta1
-selective cardioselective adrenoceptor
blocking agent. The chemical name for bisoprolol fumarate is (ą)-1-[4-[[2-(1-methylethoxy) ethoxy]
methyl]phenoxyl-3-[(1-methylethyl)amino]-2-propanol(E)-2-butenedioate
(2:1). It is a white crystalline powder, which is readily soluble in water,
methanol, ethanol, and chloroform1.
It is official in USP2.
Amlodipine besylate, a long-acting
calcium channel blocker, is chemically described as
3-ethyl-5-methyl(ą)-2-[(2-aminoethoxy)methyl]-4-(2-chlorophenyl)-1,4-dihydro-6-methyl-3,5-pyridine
dicarboxylate, mono benzenesulphonate.
Amlodipine besylate is a
white crystalline powder. It is slightly soluble in water and sparingly soluble
in ethanol3.
It is official in BP4. Beta blocker plus calcium channel
blocker combinations have utility in certain cardiovascular diseases like
angina pectoris, myocardial infarction and hypertension. A tablet formulation
containing bisoprolol fumarate
and amlodipine besylate has
been recently introduced on the market.
Various methods for determination of bisoprolol by fluorimetry5,6, HPLC79
and densitometry1012
are reported in literature.
Also HPTLC1316, HPLC1723,
spectrophotometry 2430 methods are
reported for determination of amlodipine alone or in
combination with other drugs. Combination of amlodipine
and Bisoprolol Spectrophotometry32.
But literature survey did not reveal method for simultaneous
determination of bisoprolol and amlodipine
at normally available conditions. The aim of this study was to develop a fast,
precise, accurate, rugged and robust RP-HPLC method for simultaneous
determination of bisoprolol and amlodipine
in tablets for assay and dissolution. Criteria employed for assessing the
suitability of proposed method was cost effectiveness as well as fast analysis
of routinely used analysis parameter.
MATERIAL
AND METHOD:
A liquid chromatographic system comprising of Waters 2695
separation module and Waters 2996 PDA detector (Waters Corporation, Milford,
USA) connected to Empower chromatography software for processing the data
generated were used and LC-2010CHT (Shimadzu) connected with LC solution
software. Reference standards of bisoprolol fumarate and amlodipine besylate were used. HPLC grade acetonitrile
was purchased from Qualigens (Part of thermo Fisher Scientfic USA). Chemicals are used same ExcelaR
grade was used and membrane filter paper Milipore.
The combination tablets containing bisoprolol fumarate and amlodipine besylate (Concor AM, Merck, Zabesta-AM TABLETS- ACCENT Pharma)
were procured from the market.
A buffer solution was prepared by adjusting the pH
3.0ą0.05 of 0.4 ml TEA and 3.12 gm of Sodium dihydrogen
orthophosphate with 10% orthophosphate solution. The mobile phase was filtered
and degassed mixture of buffer pH 3.0 and acetonitrile
(50:50, v/v). Luna C18 column (5 μ, 250×4.6 mm), as well as Hibar(R) RP-18e (5
μ, 250×4.6 mm), was used as stationary phase. A constant flow of 1.0
ml/min was maintained throughout the analysis. Detection was carried out using
PDA/UV detector at 230 nm.
The description
of the in vitro dissolution profiles was calculated by using model-independent
method33-34 in this study, as
model-independent approaches, two fit factors were applied to the dissolution
data that compare the dissolution profiles of a pair of drug product. These fit
factors directly compare the difference between the percent drug dissolved per
unit time for a test and reference product. The fit factors are f1 (difference
factor) and f2 (similarity factor).
Experimental work
Preparation of standard
solution: A combined
standard stock solution of bisoprolol fumarate and amlodipine besylate was prepared dissolving in 5 ml of Acetonitrile and finaly make up
with mobile phase to obtain the solution having 250 μg/ml
concentrations. 2ml of standard stock solution was diluted to 100 ml with
mobile phase to obtain a 5.0 μg/ml solution of bisoprolol and amlodipine and
used as working standard for assay analysis. 2ml of standard stock solution was
diluted to 100 ml with mobile phase. Further 5 ml dilute to 10ml with
dissolution medium and used as working standard for dissolution analysis.
Preparation of sample
Solution: Twenty tablets
were weighed and crushed to fine powder. An accurately weighed portion of the
powder equivalent to 10 mg of bisoprolol fumarate and 10 mg of amlodipine
was taken in 100 ml volumetric flask, about 10 ml of acetonitrile
was added to it and flask was kept in an ultrasonic bath for 2 min with
intermittent swirling. These solutions were then diluted to the mark with
mobile phase and filter through whatman no 1 filter
paper. 5ml of the filter solution was diluted to 100 ml with mobile phase and
mixed. This solution was filtered through Millipore membrane filter and used
for assay analysis. Dissolution analysis carried out in 900 ml of 0.01N
Hydrochloric Acid at 75 RPM, 20minutes for Bisoprolol
and 30 minutes for Amlodipine. Dissolution sample
further diluted 5ml to 10 ml with mobile phase.
Procedure: 20μl of working standard and sample
solution were injected into the chromatograph and the peak areas were recorded
for assay analysis. While 50μl of working standard and sample solution
were injected into the chromatograph and the peak areas were recorded for
Dissolution analysis. The amount of each active was computed by external
standard quantification method.
RESULT
AND DISCUSSION:
In order to optimize the LC separation of bisoprolol and amlodipine,
initially, mobile phases of buffer and acetonitrile were
used. The retention behavior of both the drugs was studied with respect to pH
of buffer solution in the range of 2.5 to 5.0, and aqueous composition of
mobile phase. Retention of both the drugs was found slightly dependant on pH of
buffer (slight increase in retention with increase in pH). Bisoprolol
was found relatively less sensitive to aqueous composition as against amlodipine, which was found more sensitive to aqueous
composition. A 5% percent increase in aqueous composition resulted in 1.05 and
1.31 times increase in retention for bisoprolol and amlodipine respectively. The buffer solution of pH 3.0 and
mobile phase composition of buffer: acetonitrile
(50:50) was found most appropriate for separation of bisoprolol
and amlodipine on Luna C18-2 column and Hibar (R) RP18e. Flow rate was optimized based on column
efficiency. Bisoprolol and amlodipine
were well resolved in reasonable time of about 10 minutes run time. The
retention times were 4.43minutes and 6.42minutes respectively. The resolution
between bisoprolol and amlodipine
was 7.46 the dilution of analytes with mobile phase
helped to minimize the interference due to blank peaks. The wavelength of 230
nm was selected for the UV detection because at this wavelength there was
maximum overlap of the spectra of bisoprolol and amlodipine.
To ascertain
effectiveness of system suitability test, six replicate injections of freshly
prepared working standard solution were injected into the chromatograph and
relative standard deviation (RSD) of peak areas was calculated. System
suitability parameters such as tailing factor, resolution and theoretical
plates of a typical chromatogram are tabulated in Table1.
Table No -1 System Suitability (n=6)
|
Parameter |
Bisoprolol
Fumarate |
Amlodipine
Besylate |
|
Retention Time
(min) |
4.4380ą0.0040 |
6.4245ą0.0065 |
|
Theoretical
Plates (N) |
6373.3265ą53.1866 |
6856.0577ą42.7427 |
|
Tailing Factor |
1.3693ą0.0123 |
1.6167ą0.0360 |
|
Resolution |
0.00 |
7.4582ą0.0257 |
|
Area Of Peak |
125413.50 |
237418.17 |
|
RSD of peak Area |
0.5800 |
1.2551 |
Linearity (described
by equation and corresponding correlation coefficient) was determined using
eleven calibration levels for both the compounds (at 25-200% levels). The
concentrations of calibration solutions of both the drugs were from 1.30 to 10 μg/ ml. The method of linear regression was used for
data evaluation. Peak area of standard compounds was plotted against respective
concentrations. The limit of Detection (LOD) and limit of Quantification (LOQ)
of the developed method were determined by injecting blank (n=6) and standard
solutions (n=6) using the developed RP-HPLC method during system suitability
Test and calculated on based of signal to noise ration of blank finally
verified at nearest concentration. The LOD is the smallest concentration of the
analyte that gives a measurable response (signal to
noise ratio of 3). Method Precision were observed intraday as well as inter
days (table-2).
The LOD for Bisoprolol Fumarate and Amlodipine Besylate were found to
be 0.022 and 0.022 μg /ml respectively. The LOQ
is the smallest concentration of the analyte, which
gives response that can be accurately quantified (signal to noise ratio of 10).
The LOQ was 0.066 and 0.066μg/ml Bisoprolol Fumarate and Amlodipine Besylate respectively. To confirm the accuracy of the
proposed method, recovery experiments were carried out by standard addition
technique. Three different levels of standards were added to pre-analyzed
tablet samples in triplicate. The mean percentage recoveries of bisoprolol and amlodipine were
99.25% and 99.96% respectively.
Table No -2 Validation Parameters:
|
Ingredient |
Bisoprolol
Fumarate |
Amlodipine
Besylate |
|
Linearity Range |
1.3878 to 11.1022 |
1.361 To 10.885 |
|
Correlation
Coefficients |
0.9999 |
0.9988 |
|
R˛ |
0.9998 |
0.9976 |
|
Limit of
Detection (mcg) |
0.022 |
0.022 |
|
Limit of quantitation (mcg) |
0.067 |
0.066 |
|
Recovery (n=3) |
99.25% SDą0.9350 |
99.96% SDą0.6688 |
|
Precision
(RSD) |
||
|
Intraday (N=6) |
0.283% |
0.780% |
|
Inter day (n=12) |
0.482% |
0.722% |
The proposed
method was subjected to robustness studies with respect to change in pH of
buffer (ą0.5 units), change in mobile phase composition (ą10%), change in
column temperature (ą5°C) and change in flow rate (ą0.1 ml).The method was
found robust with respect to variability in above conditions (table -3).
Table No -3
(A) Bisoprolol Fumarate
(n=6)
|
Parameter |
Retention
Time |
Tailing
factor |
Theoretical
Plate |
Area |
RSD (n=6) |
|
Initial
conditions |
4.4380 SD
ą0.0040 |
1.3693 SD
ą0.0123 |
6373 SD
ą53 |
125413 |
0.5800 |
|
Flow 0.9
ml |
4.9336 SD
ą0.0040 |
1.4028 SD
ą0.0073 |
6117 SD
ą83 |
143482 |
0.8691 |
|
Flow 1.1 ml |
4.0230 SD
ą0.0178 |
1.2372 SD
ą0.0115 |
6475 SD
ą24 |
118297 |
0.2998 |
|
Temperature
20°C |
4.4485 SD
ą0.0071 |
1.3830 SD
ą0.0109 |
6307 SD
ą70 |
125820 |
0.4915 |
|
Temperature
30°C |
4.4488 SD
ą0.0110 |
1.3163 SD
ą0.0148 |
6503 SD
ą72 |
124525 |
0.2788 |
|
Solvent
-10% |
4.6423 SD
ą0.0116 |
1.3273 SD
ą0.0047 |
6064 SD
ą41 |
133635 |
0.8968 |
|
Solvent
+10% |
4.2098 SD
ą0.0129 |
1.2919 SD
ą0.0036 |
6638 SD
ą114 |
113583 |
1.0771 |
Table No -3
(B) Amlodipine
Besylate (n=6)
|
Ingredient |
Retention
Time |
RRT |
Resolution
|
Tailing
factor |
Theoretical
Plate |
Area |
RSD (n=6) |
|
Initial
conditions |
6.4245 SD
ą0.0065 |
1.4476
SDą0.0003 |
7.4582
SDą0.0257 |
1.6167
SDą0.0360 |
6856 SD
ą42 |
237418 |
1.2551 |
|
Flow 0.9
ml |
7.1351 SD
ą0.0045 |
1.4462
SDą0.0009 |
8.0525
SDą0.0394 |
1.6328
SDą0.0143 |
6393 SD
ą25 |
264270 |
0.2471 |
|
Flow 1.1
ml |
5.8256 SD
ą0.0037 |
1.4481
SDą0.0061 |
7.0267
SDą0.0147 |
1.5524
SDą0.0034 |
6909 SD
ą72 |
206761 |
1.2309 |
|
Temperature
20°C |
6.4382 SD
ą0.0196 |
1.4473
SDą0.0034 |
7.3887
SDą0.0143 |
1.6247
SDą0.0184 |
6741 SD
ą60 |
258765 |
0.9777 |
|
Temperature
30°C |
6.3942 SD
ą0.0554 |
1.4373
SDą0.0035 |
7.5368
SDą0.0194 |
1.5583
SDą0.0081 |
6927 SD
ą41 |
249555 |
0.1736 |
|
Solvent
-10% |
8.3173 SD
ą0.0110 |
1.7916
SDą0.0057 |
8.0830
SDą0.0451 |
1.5392
SDą0.0190 |
6433 SD
ą76 |
295660 |
0.3986 |
|
Solvent
+10% |
5.1215 SD
ą0.0050 |
1.2166
SDą0.0043 |
6.1828
SDą0.0382 |
1.4837
SDą0.0179 |
7224 SD
ą39 |
193409 |
0.3083 |
Table No -3
(C) effect of pH
|
pH |
Retention
Time |
RRT of Amlodipine |
Resolution
Amlodipine |
Tailing
Factor |
Theoritical Plate |
|||
|
Bisoprolol |
Amlodipine |
Bisoprolol |
Amlodipine |
Bisoprolol |
Amlodipine |
|||
|
2.50 |
4.326 |
6.200 |
1.433 |
7.412 |
1.333 |
1.542 |
6632 |
6952 |
|
3.00 |
4.433 |
6.416 |
1.447 |
7.483 |
1.364 |
1.567 |
6445 |
6889 |
|
3.50 |
4.727 |
6.755 |
1.429 |
7.685 |
1.392 |
1.483 |
6385 |
6982 |
Table No -4
Comparison
|
Parameter |
Bisoprolol Fumarate |
Amlodipine Besylate |
||
|
Concor-AM |
Zabesta-AM |
Concor-AM |
Zabesta-AM |
|
|
Assay |
4.96mg |
4.98mg |
5.04mg |
4. 991mg |
|
(99.25%) |
(99.55%) |
(100.77%) |
(99.82%) |
|
|
SDą0.2809 |
SDą0.4824 |
SDą0.7856 |
SDą0.6413 |
|
|
Dissolution |
87.25% |
85.23% |
89.36% |
86.25% |
|
Similarity
Factor |
51.31 |
52.41 |
||
|
Difference
factor |
10.79 |
13.01 |
||
The content of
actives found in the commercial brand of tablets (Concor
AM, Merck, Zabesta-AM TABLETS- ACCENT Pharma) by proposed method. The low values of RSD indicates
that method is precise for assay and dissolution (Table -4). Intermediate
precision was studied using different column, HPLC instrument and performing
the analysis on different day.
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Š A&V Publication all right reserved
Research
Journal of Pharmaceutical Dosage Forms and Technology. 4(1): Jan. - Feb., 2012,
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