Analytical Method Development and Validation for Simultaneous Estimation of Lercandipine and Atenolol Tablet Dosage Form by RP-HPLC

147
* Corresponding author: Mahaboob subhani syed.
E-mail address: subhanimahaboob13@gmail.com
IJPAR |Volume 3 | Issue 1 | Jan-Mar -2014 ISSN: 2320-2831
Available Online at: www.ijpar.com
[Research article]
Analytical Method Development and Validation for Simultaneous
Estimation of Lercandipine and Atenolol Tablet
Dosage Form by RP-HPLC
*Mahaboob Subhani Syed, Ch.Bala Sekhar Reddy, M.Sarbudeen.
Vagdevi college of Pharmacy, Guruzala, Guntur, A.P., India.
ABSTRACT
A rapid high performance liquid chromatographic method has been development and validation for the
estimation of Lercanidipine and Atenolol stimultaneously in combined dosage form. A kromosil C-8 column
having dimensions of 4.6µmx250mm and particle size of 5µm in isocratic mode , with mobile phase containing
a mixture of Di- Potassium Hydrogen Phosphate and Acetonitrile in ratio of (70:30v/v) (pH adjusted to pH
6.5±0.5 using phosphoric acid was used . Detection was done at 215 nm using PDA detector. The mobile phase
was pumped at a flow rate of 1.0µl/min and injection volume is10µl. The selected chromatographic conditions
were founds to effectively separate Lercandipine (Rt 3.8min)(2)
and Atenolol (Rt: 6.1 min) having resolution of
7.8. The method was validated in terms of linearity, accuracy, precision, and specificity, limit of detection and
limit of quantitation.
Key Words: Lercanidipine, Atenolol, RP HPLC, Method development, Validation
INTRODUCTION
Atenolol is a (RS)-4-(2hydroxy-3-isopropylamino
pro-poxy) phenyl acetamide is a cardio selective
β1-blocker. It is a selective β-adrenergic receptor
blocking agent without membrane stabilizing of
intrinsic sympathomimetic (partial agonist)
activities. This preference effect is not absolute
however, and at higher doses. Atenolol inhibits β2-
adreno receptors, chiefly located in the bronchial
and vascular musculature (1)
.
Lercandipine is chemically 2-{(3,3-diphenylpropyl
methyl amine}-1,1-dimethylethyl , methyl-1,4-
dihydro-2,6-dimethyl-4- (3-nitro phenyl )-3,5-
pyridine decarboxylic ester.(5)
It is a new third
generation calcium channel antagonist used as anti-
hypertensive agent. Atenolol alone and
combination with other drugs is reported to be
estimated by HPLC(5)
in pharmaceutical dosage
form. The Uv-spectrophotometry(9)
, gas-liquid
chromatography, HPTLC. capillary zone
electrophoresis, some analytical methods for
quantitative estimation of lercandipine in
pharmaceutical formulation and human plasma (3)
.
A rapid and sensitivity RP-HPLC method for the
stimultaneous determination of lercandipine and
atenolol in tablet dosage form. The method was
validated as per ICH guideline which is mandatory
(1,4)
.

148
Mahaboob Subhani Syed et al / Int. J. of Pharmacy and Analytical Research Vol-3(1) 2014 [147-156]
www.ijpar.com
MATERIAL AND METHODS
Instrument
Liquid chromatography consists of the following
components: Waters Allinance 2695, and rheodyn
injector. Chromatographic analysis was performed
on a KromosilC8 (250mm x4.6µm) i.d and 5µm
particle size the mobile containing mixture of Di-
Potassium hydrogen phosphate and Acetonitrile in
the ratio of 30:70. pH was adjusted with phosphoric
acid to 6.5±0.5. it was filtered with Whatman filter
no1 and degassed for10mins. The flow rate of the
mobile phase was maintained at 1.0µl/min.
Detection was carried out at 215nm in PDA
detector.
Chemicals and Regents.
HPLC water (HPLC grade) – Merck Speciality
Pvt., Mumbai.Acetonitrile (HPLC grade) - Merck
Speciality Pvt., Mumbai.Di-potassium hydrogen
phosphate (Hipur Fine Chemical Industry)
Phosphoric acid (HPLC grade) – Merck Speciality
Pvt., Mumbai.% purity of Lerandipine 99.5%.
%purity of atenolol is 99.63%. Sample-
commercial tablet of lotensyl AT (SunPharma)
Preparation of standard stock solution
A 10 mg of lercanidipine working standard solution
was taken in a 10ml volumetric flask and dissolved
it properly and diluted up to volume with the
diluent, so as to give a concentration of 1000
mcg/ml of lercanidipine and 100 mcg/ml of
atenolol. 10 l of this solution was injected and
chromatogram was recorded. The retention time of
atenolol was found to be 6.112 and lercanidipine
was found to be 3.887 mins.
Preparation of Sample solution
Accurately weighed quantity of powder equivalent
to 10mg of lercanidipine and 50 mg of atenolol
from 20 tablets, was dissolved in10ml volumetric
flask using diluent by sonication and made up to
volume with diluent. The solution was filtered
through the nylon milli-pore filter. Take 10
l of
solution was injected in HPLC according to the
chromatographic conditions and chromatograms
were recorded. The amount of lercanidipine and
atenolol present in each tablet formulation were
calculated by comparing the peak areas of the
standards and reports shown in table.
Amount of drugs in each tablet were found to be:
AreadardtanS
AreaSample
x
dilutiondardtanS
weightdardtanS
x
weightSample
dilutionSample
x
weightAveragex
100
Purity
Each value is the mean of six readings. Acceptance
criteria: 90-110 % w/v.
RESULTS AND DISCUSSION
METHOD VALIDATION(4,5)
The method was validated with reference to ICH
guidelines i.e. Linearity, Accuracy, precision,
specificity, ruggedness respectively.
Specificity
The specificity of the method was evaluated by
analyzing the sample solution spiked with the blank
solution at appropriate levels. The assay result was
unaffected by the presence of extraneous materials.
It was determined by 10
l of blank solution was
injected and t chromatogram were recorded. Take
10mg of atenolol working standard was taken into
10 ml volumetric flask and added 1ml of
lercanidipine standard solution using diluent by
sonication and made up to volume with diluent.
Sample Label-claim
(mg/tab)
Peak Area Amount present % label claim w/v
lercanidipine 10mg 84042 10.04 mg 100.81 %w/v
atenolol 50mg 3438447 50.37mg 97.28 %w/v

149
www.ijpar.com
S.No. Sample Area
obtained
Area
%
1. Blank 0 0
2. lercanidipine 3675994 97.390
3. Atenolol 98524 2.610
Total 3774518 100%
LINEARITY AND RANGE
The linearity of an analytical method is its ability to
Elicit test results that is directly proportional to the
concentration of analyte in sample with in a given
range. The linearity of an analytical method is
determined by mathematical treatment of test result
obtained by analysis of samples with analyte
concentration across claimed range of peak area Vs
concentration was plotted and percentage curve
was calculated. Appropriate aliquots of standards
stock solution of Lercandipine and Atenolol. The
linear range of Lercandipine and Atenolol was
found to be (500mcg-3000mcg), r2
-0.9981 and
(5mcg -30mcg), r2
-0.9996.
Acceptance criteria: Coefficient of co-relation (r2
)
not be less than 99
PRECISION
Precision of the method was validated by
performing replicated assays of the homogeneous
sample. Results were calculated in terms of %RSD
of the content of Lercandipine and Atenolol
A. SYSTEM PRECISION
Preparation of standard solution
A 10 mg of lercanidipine working standard was
accurately weighed and transferred in to 10 ml
volumetric flask and 1ml atenolol working standard
solution was added, dissolve using diluent by
sonication and made up to volume with diluent.
The solution was filtered through the nylon milli-
LINEARITY OF ATENOLOL
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
5mcg 10mcg 15mcg 20mcg 25mcg
CONCETRATION
AREA
area
LINEARITY OF LERCANDIPINE
R2
= 0.9981
0
2000000
4000000
6000000
8000000
10000000
12000000
14000000
500m
cg1000m
cg1500m
cg2000m
cg2500m
cg3000m
cg
CONCENTRATION
AREA
area
Linear (area)

150
www.ijpar.com
pore filter. To obtained final concentration of
1000 mcg/ml of lercanidipin and 100 mcg/ml of
atenolol. About 10
l of the solutions was injected
and chromatograms were recorded.
The standard deviation and relative standard
deviation were calculated from statistical formula
S.D (  ) = 1n
)xx(
2

 
Where x = Sample
x = Mean value of samples
n = No. of Samples
R.S.D (%) =


x x 100
B: Method Precision
The method precision was validated for
intermediate precision by comparing the
performance of method. Six replicate assay of
homogeneous sample were performed using the
same procedure and chromatographic conditions.
Six successive injections of 10 µl of working
sample solution were injected and chromatograms
were recorded.
SYSTEM PRECISION OF ATENOLOL
AND LERCANIDIPINE
S.No PEAK AREAS OF
ATENOLOL LERCANIDIPINE
1 99666 3692555
2 98636 3672587
3 98854 3685378
4 99794 3660673
5 98545 3704044
Mean 99099 3683047
S.D 588.6 16931.6
% RSD 0.59 0.46

151
www.ijpar.com
METHOD PRECISION OF LERCANDIPINE AND ATENOLOL.
S.
No
Weight in
mg
Area obtained Assay value in mg % label claim W/V.
ATEN LERCANI ATEN
LERC
AN
ATEN
LERCA
N
1 694.09 84043 3438449 509.07 58.31 101.83 97.29
2 693.08 83749 3408346 508.04 57.29 100.75 96.59
3 695.16 85149 3537446 510.13 59.64 102.65 99.13
4 694.34 84534 3486501 509.53 58.71 101.96 97.64
5 695.06 83649 3416335 508.65 57.49 100.89 96.92
Mean 101.6 97.5`
S.D 4.650 4.025
RSD 0.67 0.58
ACCURACY
Accuracy was performed by the method of standard
addition at three different levels, multiple level
recovery studies .Accuracy of an analytical method
was the closeness of test result obtained by
developed method to the true value. Pre analyzed
sample was spiked with Lercandipine and Atenolol
in the same proportions as present in tablet dosage
form. Spiked samples were prepared in triplicate at
three intervals a range of 80- 120 percentage of the
target concentration and injected in the HPLC
system.
Acceptance criteria: Percentage recovery should be
within 90-110% w/w.
Preparation of working standard stock
solution
About10 mg of lercanidipine working standard was
weighed and transferred in 10ml volumetric flask
and added 1ml of atenolol standard solution,
dissolved and diluted to volume with the diluent.
Preparation of working mixture solution Aliquot
volume of 8ml, 10ml, 12ml, standard stock solution
was transferred in to 3 different 25 volumetric
flasks. And made up to volume with diluent. That
Relative Standard
Deviation
ATENOLOL LERCANIDIPINE Acceptance Criteria
0.59 0.46 2.0%
RSD ATENOLOL LERCANIDIPINE Acceptance criteria
0.67 0.58 NMT : 2.0%

152
www.ijpar.com
gives 80%, 100%, 120% of working mixture
solution. 10 l of each solution was injected and
chromatograms were recorded. The accuracy data
of atenolol and lercanidipine was shown in tables.
RECOVER DATA OF ATENOLOL
S.No Recovery Area
obtained
Avg.
area
Amt required
in mg
Percentage (%)
recovery w/v
1 80%
72593
73487
75674
73918 40.21 100.05
2 100%
88563
87593
86493
87549 45.57 99.9
3
120%
96542
97685
95492
96573 50.72 99.7
RECOVRY DATA OF LERCANDIPINE
RUGGEDNESS
The ruggedness of an analytical method is degree
of reproducibility of test result obtained by the
analyst under a variety of normal test condition.
Such as different laboratories different analysts
different instruments, lots of reagents different
elapsed assay times, different temperature, different
days etc. The ruggedness of analytical method is
determined by aliquots from homogenous lots of
different analyst using operational and
environmental conditions that may differ but are
S.No Recovery Area
obtained
Avg.
area
Amt required in
mg
Percentage (%) recovery
w/v
1 80%
2865993
2759952
2675317
2675920 7.8 99.5
2 100%
3447786
3375867
3358692
3394115 8.7 99.6
3 120%
4269873
4196385
4289769
4252009 9.8 99.7

153
www.ijpar.com
also within the specified parameters of the assay.
The degree of reproducibility of test results is then
determined as function of assay variables. This
reproducibility may be compared with the precision
of the assay under normal condition to obtain a
measure of the ruggedness of the analytical
method. The assay of lercanidipine and atenolol
was performed in different ways by different
analysts. It was determined by Working standard
solution and working sample solution of
lercanidipine and atenolol were prepared by
different analyst on different days and 10  l of
working standard solution and working sample
solution was injected and chromatograms were
recorded ruggedness of method and report was
shown in tables.
RUGGEDNESS OF METHOD
(DIFFERENT ANALYSTS)
RUGGEDNESS OF METHOD
(DIFFERENT INSTRUMENT)
S.N
o
Analyst Instrument
code
Date of
analysis
Recovery %
Atenolol Lercanidipine
1 Analyst - 1 Water 2695 11/06/13 99.7 99.8
2 Analyst – 1 Prominence 11/06/13 98.9 98.5
ROBUSTNESS
Robustness of an analytical method is measure of
its capacity to remain unaffected by small but
deliberate variation in method parameters and
provides an indication of its reliability during
normal usage. The robustness of an analytical
method is determined by analysis of aliquots from
homogenous lots by differing physical parameters
that may differ but are still within the specified
parameters of the assay. For eg. Change in
physical parameters like flow rate and wavelength.
It is estimated by10  l of various mixed working
standard solution was injected and chromatogram
was recorded and shown in the tables.
REPORT ON CHANGE IN FLOWRATE 0.8 ml/min.
Drugs Avg area in
0.8 ml/min
Avg. area in
1ml/min
Std deviation % RSD
lercanidipine 4156783 3476529 19278 0.49
Atenolol 91672 84973 586.4 0.63
S.No Instrument code Analyst Date of analysis Recovery %
atenolol lercanidipine
1 Waters 2695 Analyst – 1 11/06/13 99.8 99.7
2 Waters 2695 Analyst – 2 11/06/13 98.8 99.3
3 Waters 2695 Analyst – 1 12/06/13 99.9 99.6
4 Waters 2695 Analyst – 2 12/06/13 99.5 98.6

154
www.ijpar.com
REPORT ON SAMPLE CHANGE IN FLOWRATE 1.2 ml/min
Drugs Avg area in 0.8
ml/min
Avg. area in
1ml/min
Std deviation % RSD
lercanidipine 3171793 3476529 23721 0.15
Atenolol 81265 84973 764.5 1.7
REPORT ON CHANGE IN WAVE LENGTH OF 210 nm.
Drugs Avg area in 0.8 ml/min Avg. area in 1ml/min Std deviation % RSD
lercanidipine 3457928 3446798 10874 0.5
atenolol 83376 84568 493.7 0.3
REPORT ON CHANGE IN WAVE LENGTH 220 nm.
Drugs Avg area in 0.8 ml/min Avg. area in 1ml/min Std deviation % RSD
lercanidipine 2647689 3446798 1657 0.54
Atenolol 76657 84568 276.2 0.34
LIMIT OF DETECTION (LOD)
It is the lowest amount of analyte in a sample that
can be detected but not necessarily quantities as an
exact value under the stated experimental
condition. The detection limit is usually expressed
as the concentration of analyte (e.g. parts per
million). It is determined by based on the standard
deviation of response and the slope. The detection
limit was being expressed as LOD =
S
3.3 
, Where
 =the standard deviation of the response.
S= the slope of the calibration curve of the analyte.
LOD
9.42 mcg/ml 1.28 mcg/ml
LIMIT OF QUANTIFICATION
The quantification limit of an analytical procedure
is the lowest amount of analyte in a sample, which
can be quantiated with suitable precision and
accuracy.
Based on the deviation of the response and the
slope.

155
www.ijpar.com
LOQ =
S
10
Where  =the standard deviation
of the response. S= the slope of the calibration
curve of the analyte.
SYSTEM SUITABILITY PARAMETER
For system suitability , five replicate of standard
solutions of Lercandipine and Atenolol working
standard was injected studied the suitability
parameters like Plate Number (N), Resolution (R),
Relative retention time (α) and Peak symmetry of
sample (As) were studied with the help of standard
chromatograms
System
suitability
factors
Atenolol Lercanidipine
Area 109359 3806509
Retention time 6.1 3.8
USP Tailing
factor
1.639 2.157
USP plate count 5772 3471
Resolution (R) 7.8
Capacity factor
( K)
0.59
SUMMARY AND CONCLUSION
A RP-HPLC method was developed for the
estimation of Lercanidipine and Atenolol in tablet
dosage form using HPLC-Waters Alliance 2695
separations module, with Detector – Waters 2996
separations module and column Kromosil C8, 25cm
x 4.6mm, 5 m.Injection volumn was 10µl and the
mobile phase was Buffer solution (Di- Potassium
hydrogen phosphate) and Acetonitrile in ratio
(30:70) having a pH range 6.5±0.05. where as
mobile phase was pumped at a flow rate of
1.0ml/mim maintaning column temparature at
60ºc±1ºc and detected at 215nm using PDA
Dectector.
The peak Retention time of the Lercandipine is
3.8min and Atenolol is 6.1min. The excipientes in
the formulation dose not interfer in the estimation
of active drug. The determination of the
Lercanidipine and Atenolol by RP-HPLCmethod
analysis yielded well resloved peaks with in the
short analysis time of<10min. The valve of
standard deviation were satisfactorily low and
recovery was close to 100%.the corelation co-
efficient of linearity studies were found to be 0.998
for Lercandipine and 0.999 for Atenolol.
The developed method was validated for system
suitability, linearity, precision, accuracy in
accordance with international conference on
harmonization guidelines(ICH).This method was
suitable for the routine analysis of combination
drugs(Lercandipine and Atenolol) in
pharmaceutical formulations.
LOQ
28.55 mcg/ml 3.38 mcg/ml

156
www.ijpar.com
REFERENCES
[1] Sumeet Dwivedi, nirvedUpadhaya, AwadudayY. And pekamwar Sanjay . IJDDHR.ISSN:2231-6078,
Settember(2011) pg:134-137.
[2] G.Mubean, Mamata Pal and M.N. Vimala. International Journal of Pharma and Bio Sciences Vol 1(1)
2010.
[3] Jaya Raj Kumar , Sokalingam Arumugam Dhanaraj Am .J. Pharmatech. ISSN: 2249-3387 , 2012 VOL
2(5).
[4] Panthaganda Sunitha, Kancherla Satyavathi, IRJP, vol 3(11) ISSN :2230-8407.
[5] Deepak Kumar Jain , Prathiba Petal , Abu Salam Khan. IJCHR , ISSN:0974-4290, VOL 3, April- June
2011.
[6] Abdul sallem , P.Permulal D.Boothapathy IJPTRIF ; ISSN:0974-4304, Vol 2(1), pp: 471-474, Jan-
Mar2010.
[7] M.Radha Krishna and Ramesh Jagadesan, IJRPBS; vol.4(3) ISSN:2229-3701, Jul-Sep2013.
[8] A.K. Shah ; H.O Kaila, IJPS; May-June2010.
[9] T.Manikya Sastry and K.Rama Krishna ; IJCT , Vol 16, sep2009, pp:431-436.
[10]Juddy Joseph; M. Sundarapandian; IJPSR 2011 Vol.2(8), pp 2156-2161;ISSN:0975-8232. July2011.
[11]Amit Khandar; Maiteyi Zaveri; JPRHC; VOL 2(3) pp:248-252.
[12]R.S.Murthy, Mariam Ravi Kumar; IJSID ISSN: 2249-5347, Vol2(1) Jan-Feb2012.
[13]Vidya K. Bhusari; Sunil R. Dhaneshwar. URPJ 2011 vol1(3) ;pp:70-76.
[14]M.V. Kumudhavali ; K. Anand Babu , B.Jayakar, DerPharma Chemica 2011, vol3(4) , ISSN:0975-
413X, pp: 63-68.
[15]Venkateswarlu.Ponneri, Thulasamma Paresu. EURJChem 2012,vol3(2) , pp:138-142.
[16]S. AppalaRaju , B. Karadi Inf. J.Chem Sci 2008 ,vol6(1), pp:441-446.
[17]S.Pujol,J.A, A.Aivarez- Lueje, Bio Electrical Laboratory , University of Chile
[18]A.Agrawal , K.Mitial, JPRHC 2010 VOL.2(3) ,PP:263-265.
[19]Nilesh Jain, Ruchi Jain, E.S.Analytical. Chem 2011, vol6(2) ,pp:84-90.
[20]Sherke.O.S, Sable.K.S, IRJP2012,vol3(4), ISSN:2230-8407.
[21]Vishnu. choudhari, Rashmi.H.Mahabal, IJPSR&R,ISSN:0976-044X, vol.3(1) , July-Aug 2010.
[22]Vascular Health and Risk Management 2005 vol1 (3), pp: 173-182.
*******************************

Analytical Method Development and Validation for Simultaneous Estimation of Lercandipine and Atenolol Tablet Dosage Form by RP-HPLC

More Related Content

What's hot (20)

Similar to Analytical Method Development and Validation for Simultaneous Estimation of Lercandipine and Atenolol Tablet Dosage Form by RP-HPLC (20)

More from pharmaindexing (20)

Recently uploaded (20)

Analytical Method Development and Validation for Simultaneous Estimation of Lercandipine and Atenolol Tablet Dosage Form by RP-HPLC