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Vijaya Bhaskar Reddy.T et al. / Journal of Pharmacy Research 2012,5(7),3896-3899
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A stability indicating RP-HPLC method for the determination
of amitriptyline hydrochloride in pure and dosage forms
1
Vijaya Bhaskar Reddy.T1, Ramu.G1, Ravindra Babu.N1 and Rambabu.C1*
*Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar, Guntur, Andhra Pradesh, India
Received on:07-04-2012; Revised on: 12-05-2012; Accepted on:16-06-2012
ABSTRACT
A simple isocratic RP-HPLC method was developed for the determination of amitriptyline HCl in pure and dosage forms. A Waters HPLC equipped with
Alliance 2695 separation module and 2487 dual wavelength UV-Visible detector and Symmetry C18 (4.6x150mm, 3.5 µm, Make: XBridge) HPLC column
thermostated at 30oC was used in the present investigation. A mixture of potassium dihydrogen orthophosphate buffer of pH 3.0±0.05 and acetonitrile in
the ratio 35:65 was used as mobile phase with a flow rate and injection volume were 0.8ml/min. and 20µl respectively. The data were acquired at 239nm. The
proposed method was linear in the range of concentration 20-60µg/ml and the correlation coefficient is found to be 0.9990. The mean recovery of the
substance was found to be 100.2%. The values of LOD and LOQ for amitriptyline HCl were found to be 0.015 and 0.052µg/ml respectively. The method
has higher sensitivity towards the determination of the amitriptyline HCl. The developed method is found to be accurate and precise as indicated by recovery
studies and % RSD not more than 2.0. The proposed method can be used as an alternative method for routine analysis in quality control.
Keywords: RP-HPLC, Amitriptyline HCl, Linearity, LOD, LOD, Correlation coefficient
INTRODUCTION
Amitriptyline HCl (ATL) is used to treat depression, mainly melancholic,
endogenous, or when anxiety or insomnia coexists. It is the most widely used
tricyclic antidepressant(TCA) and has at least equal efficacy
against depression as the newer class of SSRIs according to a study from
early 2001.1 Amitriptyline is used for a number of medical conditions
including: depressive disorders, anxiety disorders, attention deficit hyperactivity disorder, migraine prophylaxis, eating disorders, bipolar disorder, postherpetic neuralgia, and insomnia.2It is also used as a preventive for patients
with recurring biliary dyskinesia .3 and in the treatment of nocturnal enuresis in
children. ATL may be prescribed for other conditions such as cyclic vomiting
syndrome post-traumatic stress disorder (PTSD), chronic pain, tinnitus,
chronic cough,carpal tunnel syndrome (CTS), fibromyalgia, vulvodynia,
interstitial cystitis, male chronic pelvic pain syndrome, irritable bowel
syndrome (IBS), diabetic peripheral neuropathy, neurological pain, laryngeal
sensory neuropathy, chronic fatigue syndrome and painful paresthesias related
to multiple sclerosis. Typically lower dosages are required for pain modification of 10 to 50 mg daily. A randomized controlled trial published in June
2005 found that amitriptyline was effective in functional dyspepsia that did
not respond to a first-line treatment (famotidineor mosapride).4
The chemical structure of the drug is 1-Propanamine,3-(10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5-ylidene)-N,N-dimethyl-,hydrochloride with
molecular formula and molecular weight C20H23N·HCl and 313.86g/mole
respectively. It is available in different brand names such as Amitrol, Elavil,
Endep, Levate, Tryptizol, Vanatrip. Amitriptyline Hydrochloride contains
not less than 99.0percent and not more than 100.5percent of C20H23N·HCl,
calculated on the dried basis. The chemical structure of the drug is given in
Fig.1. Several techniques have been adopted for the determination of amitriptyline, including spectrophotometry 5,6 , high-performance liquid
*Corresponding author.
Rambabu.C
Department of Chemistry,
Acharya Nagarjuna University,
Nagarjuna Nagar, Guntur,
Andhra Pradesh, India
chromatography7,8gas chromatography9, capillary electrophoresis10 and
voltammetry11 and potentiometric method using ionselective electrodes12,13
fluorescence polarization immunoassay method 14, UV spectrophotometric
method 15, combination with other drugs 16,17 and UV method 18
MATERIALS AND METHODS
Experimental
A Waters HPLC equipped with Alliance 2695
separation module and 2487 dual wavelength
UV-Visible detector was used for the separation and quantification. An analytical column;
Symmetry C18 (4.6 mm ID x 150mm, 3.5
µm, Make: XBridge) was used in the analysis. Chromatographic software Empower-2 Fig.1 The chemical strucwas used for data collection and processing. ture of Amitriptyline HCl
Elico-SL159 model, 2nm high resolution,
double beam, 1cm length quartz coated optics and wavelength range1901100nm is used for measuring absorption spectrum.
Chemicals and Reagents
Acetonitrile and methanol of HPLC grade procured from Merck (India) were
used. Potassium dihydrogen orthophosphate and orthophosphoric acid were
all of AR grade (Merck, India) HPLC grade water obtained from Millipore
system was used throughout the analysis. The investigated sample, amitriptyline HCl was obtained as a gift sample from Dr. Reddy’s Laboratory,
Hyderabad, India.
About 7.0 grams of potassium di hydrogen phosphate is transferred into a
beaker dissolved in 1000ml with HPLC water, sonicated, filtered through
0.45µm filter and the pH of the solution was adjusted to 3.0±0.05 by adding
a few drops of ortho phosphoric acid. The mobile phase was prepared by
mixing about 350ml buffer and 650 ml of HPLC grade acetonitrile. The mixer
was degassed in ultrasonic water bath for 5 minutes, filtered through 0.45µm
filter.
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Vijaya Bhaskar Reddy.T et al. / Journal of Pharmacy Research 2012,5(7),3896-3899
Table 1: System suitability parameters
S.No.
System suitability parameter
Value of the parameter
1
2
3
Number of theoretical plates
Tailing factor
Retention time
2578.10
1.30
2.533
Table 2: Precision of the developed method
Injection
Concentration
Peak Area
Injection-1
Injection-2
Injection-3
Injection-4
Injection-5
40µg/ml
40µg/ml
40µg/ml
40µg/ml
40µg/ml
Average
Standard Deviation
% RSD
2953021.000
2956623.000
2979601.000
2975186.000
2974030.000
2967692.000
11999.120
0.404
Table 3: Linearity of peak area against concentration of the drug
S.No.
Concentration
Peak Area
1
2
3
4
5
6
Slope
Intercept
Correlation Coefficient
LOD
LOD
0.0
20.0
30.0
40.0
50.0
60.0
0
1503401
2288209
2975614
3745564
4372123
73363
35380
0.9990
0.015µg/ml
0.052 µg/ml
Fig.2 Absorption spectrum of amitriptyline HCl in methanol
Table 4: Accuracy of the proposed method
%Concentration
(at specification
Level)
Peak
Area
Amount
Added
(mg)
Amount
Found
(mg)
%
Recovery
Mean
Recovery
50%
100%
150%
1514396
2999235
4344466
5.0
10.0
14.5
5.0
9.99
14.4
100.9%
99.9%
99.8%
100.2%
Preparation of standard and sample
About 10.0mg of 99.80 percent pure amitriptyline HCl was accurately
weighed transferred into a 10ml volumetric flask, 7.0ml of diluent was added,
sonicated to dissolve and filtered through 0.45µm filter. Further 0.4ml of the
above solution was pipette out into 10ml volumetric flask and diluted to the
mark with diluent, sonicated and filtered. The average weight of five amitriptyline HCl tablets was calculated, made them as homogeneous powder, an
amount of the powder equivalent to 10 mg of amitriptyline HCl was weighed,
transferred into a 10ml volumetric flask, dissolved in 7ml of diluent, sonicated to dissolve, made up to the mark, mixed well and filtered through
through 0.45µm filter. Further 0.4ml of the sample solution was pipette out
into 10ml volumetric flask and diluted to the mark with diluent, sonicated
and filtered.
Operating conditions
The analysis was carried out under the isocratic conditions. The data were
acquired at 239nm for 50min. and processed by use of Empower software
data handling system. A mixture of buffer and acetonitrile in the ratio 35:65
(v/v) was used as diluents in the preparation of analytical solutions. Amitriptyline HCl working standard (40µg/ml) solution was used as system suitability solution. 20µl of system suitability solution, blank, five replicate injections of diluted standard solution and sample solution were separately
chromatogramed.
Fig.3Atypical chromatogram of standard drug
Linearity
The linearity plot of peak areas versus concentration was drawn for amitriptyline HCl and is presented in Fig.5. The linear regression data for the drug
tested is given in Table 2. The data shown in Table 2 is confirmed that the
detector response at 239 nm was linear over the range studied for the drug.
The limit of detection (LOD) and limit of quantitation (LOQ)
The limit of detection (LOD) and limit of quantitation (LOQ) were determined for amitriptyline HCl from the standard deviation of the peak area and
slope of the linearity data. The values of LOD and LOQ for amitriptyline
HCl were found to be 0.015 and 0.052µg/ml respectively. The results were
depicted in Table 2.
Precision and Accuracy
System precision was verified using diluted solution standard solution, which
was analysed for five times and R.S.D. of amitriptyline HCl peak areas was
evaluated and found to be 0.404%. Precision of the method was studied for
repeatability and intermediate precision. Repeatability was determined by
analyzing five separate Amitriptyline HCl sample solutions the %R.S.D.was
found to be 0.404% and given in Table 2. The intermediate precision of the
method was determined on five separate sample solutions prepared from
same lot by spiking by different days. The %R.S.D was evaluated and found
to be 0.410% which was within the acceptance criterion of NMT 10%
R.S.D. The results are presented in Table-5. Accuracy of the method was
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proposed conditions. Only one parameter was changed while the other was
kept unaltered. The mean and R.S.D. were evaluated. The difference between
the mean values from the repeatability mean results was found to be 10%.The
studies indicated no effect on the determination of the drug. Therefore the
test method is robust for the quantification of the drug.
Table 5: Intermediate precision
Injection
Concentration
Injection-1
40µg/ml
Injection-2
40µg/ml
Injection-3
40µg/ml
Injection-4
40µg/ml
Injection-5
40µg/ml
Average
Standard Deviation
%RSD
Area
3002113
2996759
3003927
3002104
3028438
3006668
12462.1
0.410
Table 6: Study of degradation of the drug in the presence of different
degradation conditions
Degradation
Parameter
Peak Area
Peak Area
of Standard
% of
Recovery
% of drug
Degraded
0.1M HCl
0.1 M NaOH
Thermal
Peroxide
2780828
2721026
2601420
2541617
2990138
2990138
2990138
2990138
92.99
91.00
86.99
84.99
7.00
9.00
13.00
15.00
Fig.4 A typical chromatogram of sample
Study of degradation
The percent of drug degraded in the presence of acid, base, thermal and
peroxide conditions were studied. The drug standard was prepared in 0.1NHCl
solution, 0.1N NaOH and 1% peroxide solutions and kept at room temperature for 48 hours. The drug standard in mobile phase is kept at 45oC for 36
hours. The amount of drug recovered of degraded is calculated by comparing
the area of the standard with that of the area of the degraded sample. The
results are presented in Table 6.
RESULTS AND DISCUSSION
The system suitable parameters such as tailing factor (1.3) and number of
theoretical plates (2578) are found to be within the limits. A typical chromatogram for the standard and sample were presented in Fig.3 and Fig.4
respectively. The retention time of the component was found to be 2.256min.
The intra-day precision or inter-day precision of a method was expressed in
terms of statistical parameters such as standard deviation and %RSD. The
%RSD was calculated for five replicate measurements and found to be less
than 2.0. Inter-day precision of the method was determined by carrying out
the experiment on different days using same instrument and same column
under similar chromatographic conditions. The results are presented in Table2. The proposed method was linear in the range of concentration 20-60µg/ml
and the correlation coefficient is found to be 0.9990. A calibration curve was
constructed by plotting concentration against peak area (Fig.5). The correlation coefficient, slope and intercept were presented in Table-3.The accuracy
of the method was determined from recovery experiments. The recovery
studies were carried out at three different concentration levels (50%, 100%
and 150% of target concentration). The percentage recovery of the drug at
three different concentration levels and the mean percent of recovery are
presented in Table-4. Robustness of the proposed method is checked by
making slight deliberate change in the flow rate and mobile phase composition is made to evaluate the impact on the method.
CONCLUSIONS
A simple isocratic RP-HPLC method was developed for the determination of
Amitriptyline HCl in pharmaceutical formulations as per the ICH guidelines.
The method has higher sensitivity towards the determination of the Amitriptyline HCl. The developed method is found to be accurate and precise as
indicated by recovery studies and % RSD not more than 2.0. Recovery
studies are performed at 50%, 100% and 150% concentration levels are
found to be within the limits mentioned as per ICH Guidelines. The proposed method was found to be simple, precise, accurate and robust. Therefore the method can be used for routine analysis in quality control.
Fig. 5 Linearity plot of peak area against concentration of the drug.
determined by analyzing Amitriptyline HCl sample spiked at three different
concentration levels 50, 100 and 150% of each in triplicate at the specified
limit. The mean recovery of the substance was found to be 100.2% and the
data was given in Table 4.
Robustness
To evaluate the robustness of the method, the influence of small and premeditated alteration of analytical parameters on the quantification of the
drug substance and selectivity was studied. The parameters selected were
flow rate and mobile phase composition at 10% variation to that of the
ACKNOWLEDGEMENTS
The authors are grateful to Pharma Train, an analytical testing laboratory,
Hyderabad for providing laboratory facilities, Dr. Reddy’s Laboratory for
gifted samples and university authorities for providing provision for research work.
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Source of support: Nil, Conflict of interest: None Declared
Journal of Pharmacy Research Vol.5 Issue 7.July 2012
3896-3899