DEVELOPMENT AND VALIDATION OF A
SPECTROPHOTOMETRIC AREA UNDER CURVE METHOD FOR
ESTIMATION OF AMITRIPTYLINE HYDROCHLORIDE IN BULK
AND TABLET DOSAGE FORM
A. A. ANSARI1, H. K. JAIN2*, K. N. GUJAR2, S. D. MULE2
1
Department of Pharmaceutical Chemistry, STES’s Sinhgad College of Pharmacy,
Vadgaon (Bk), Off. Sinhgad Road, Pune -411041, Maharashtra, India.
Department of Quality Assurance Techniques, STES’s Sinhgad College of Pharmacy,
Vadgaon (Bk), Off. Sinhgad Road, Pune -411041, Maharashtra, India.
2
*Name and postal address of corresponding author:
Dr Hemant Kumar Jain, Department of Quality Assurance Techniques, STES’s Sinhgad
College of Pharmacy, Vadgaon (Bk.), Off. Sinhgad Road, Pune-411041, Maharashtra, India
E-mail: hemantkjain2001@yahoo.co.in
1
Abstract
Amitriptyline hydrochloride is a tricyclic antidepressant drug. The present work describes
an accurate and precise UV spectrophotometric area under curve method for quantitation of
Amitriptyline. This method was based on area under curve of UV spectrum between 234 to
244 nm. The analytical method was validated as per International Conference on
Harmonization (ICH) guidelines. The method has followed linearity in the range of 10-20
µg/ml. The value of correlation coefficient (R) of regression equation was 0.999. Intra-and
Inter-assay precisions as well as relative standard deviation were satisfactory. Recovery
studies indicated accuracy of the method. Statistical analysis of results suggests that the
developed method is suitable for routine estimation of Amitriptyline Hydrochloride in
pharmaceutical formulations.
Keywords
Amitriptyline Hydrochloride, Tricyclic antidepressant, Estimation, UV spectrophotometric,
Area under curve, Validation.
Introduction
Amitriptyline hydrochloride (AMI) is a tricyclic antidepressant drug. This drug is mainly
used for treatment of depression [2] and other indications are anxiety disorders, bulimia and
chronic pain. Chemically, AMI is 3-(10, 11-dihydro-5h-dibenzo [a, d] cycloheptane-5ylidine)-N, N dimethyl 1-propanamine [1] (Fig.1). AMI is official in Indian Pharmacopoeia
(IP), British Pharmacopeia (BP) and United States Pharmacopeia (USP). Official methods
have been used non-aqueous titrations [3&4] and acid-base titration [5] for assay of AMI.
Various other techniques have been reported for analysis of AMI, such as estimation by
UV[6], voltametry[7] and HPLC[8,9], dissolution studies in presence of other drugs[10],
simultaneous estimation with chlordiazepoxide[11], as well as multivariate calibration
method[12]. There are only few papers available for estimation of AMI using UV
spectrophotometery, but most of these techniques require a sophisticated data processing
facility. In this context, we wish to further explore UV spectrophotometry using area under
curve calculation. The present work describes development and validation of a simple UV
spectrophotometric area under curve method for assay of AMI in bulk and tablet dosage
form.
Materials and Methods
Instrumentation
Shimadzu UV 1800 (Japan) double beam spectrophotometer with matched quartz cells and
UV Prob Software interface was employed for this work. Shimadzu AX200 (Japan) digital
balance and Spectrolab UCB 40 (Germany) ultrasonicator, were also used.
2
Chemicals and Reagents
A gift sample of Amitriptyline hydrochloride active pharmaceutical ingredient was
procured from Astron Research Ltd. (Ahmadabad Gujarat). Commercially available tablets
(Triptomer containing 10 mg of AMI) were purchased from local market. Analytical grade
of methanol (solvent) was used. It was purchased from Merck India Ltd.
Spectrophotometric method
Selection of solvent was done on the basis of solubility and stability of AMI at room
temperature. AMI was accurately weighed (10 mg) and transferred to 100 mL volumetric
flask. Then this drug was dissolved in 20 ml of methanol (solvent) and volume was made
up to mark using solvent for the preparation of standard stock solution. This solution was
further diluted with same solvent to obtain working standard solutions i.e. 4, 8, 12, 16, and
20 μg/mL. These solutions were scanned within 400-200 nm in spectrum mode. Here
wavelength range, concentration range was determined on the basis of linear relationship
between area and corresponding concentration (Figure 2).
Area under curve (Area calculation)
This method is based on calculation of integrated value of absorbance with respect to
wavelength in indicated range. Area calculation processing item calculates the area
bounded by the curve and horizontal axis13. Here horizontal axis represents baseline.
𝜆1
Area calculation (α+β) =∫𝜆2 𝐴𝑑𝜆
Whereas, α is area of portion bounded by curve data and a straight line connecting the start
and end point, β is area of portion bounded by a straight line connecting the start and end
point on curve data and horizontal axis, λ1 and λ2 are wavelenghts representing start and end
point of curve region. In this study area was integrated between wavelength ranges from
234 to 244 nm.
Preparation of calibration curve
Working solutions solutions were scanned from 400 to 200 nm and area under curve (AUC)
was integrated14 in the range of 234-244 nm. The calibration curve was plotted between
area under curve against concentration (Figure 3).
Assay of Tablet Dosage Form
Twenty tablets were accurately weighed and average weight was calculated. The tablets
were crushed into a fine powder. Powder equivalent to 10 mg of amitriptyline was weighed
accurately and transferred into a 100 ml volumetric flask. It was dissolved with about 40 ml
methanol. The contents of volumetric flask were sonicated for about 30 minutes and
volume was made up to mark with methanol. The solution was filtered using Whatmann
3
filter paper (No.41). The first 10 ml of filtrate was discarded and suitable aliquot was
diluted to obtain solution of 15µg/ml concentration. The area of this solution was measured
at 234-244 nm (Table 1).
Method Validation
Validation of an analytical procedure is the process by which it is established by laboratory
studies that the performance characteristics of the procedure meet the requirements for the
intended analytical application. The proposed method was validated for various parameters
such as linearity, precision, accuracy, Limit of detection (LOD), Limit of Quantitation
(LOQ) according to ICH Q2 (R1) guidelines [15].
Linearity and Range
The working standard solutions were prepared by diluting stock standard solution with
methanol to give a concentration range of 4 to 20 µg/ml. The spectrums of these solutions
were recorded and area under curve was integrated in wavelength range 234-244 nm. The
relationship between area under curve (as a dependant variable) and concentration of
standard working solution (as an independent variable) were established by simple linear
regression method. The regression equation was obtained and this relationship is presented
in the calibration curve (Figure 3). The range of solution has been decided according to
correlation coefficient of regression equation.
Precision
Intraday precision was studied by measuring the area of a standard solution of 15 µg/ml
concentration at six independent series in the same day. Inter-day precision studies were
performed by measuring the area of standard solution of 15 µg/ml concentration on three
subsequent days. The percentage relative standard deviation (%RSD) was calculated (Table
2).
Accuracy
The method was applied to drug sample and recovery studies were performed where AMI
corresponding to 80, 100, and 120% of label claim was present. Three determinations at
each level were performed and results were expressed as percent recovery (Table 3).
Limit of Detection (LOD) and Limit of Quantitation (LOQ)
Six sets of known concentrations (10-20 µg/ml) were prepared. Calibration curves were
plotted for each set. LOD and LOQ were calculated using the formulae as
SD
LOD  3.3
S
SD
LOQ  10
S
Where, S is value of slopes of calibration plot and SD is calculated using values of y
intercepts of regression equations.
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Results and Discussion
244
The generated regression equation was ∫𝜆234 Ad = 0.0131C + 0.0117 with R2 value as
244
0.9988. Where, ∫𝜆234 Ad is area under curve between 234 to 244nm, C is concentration
and R is correlation coefficient. Values of correlation coefficient suggest that the method is
linear in the range 4-20 µg/ml of drug. Result obtained by Assay of amitriptyline
hydrochloride that applicability of developed method to tablets, as an average amount
found was 100.28% with %RSD (0.161s) (Table 1). The percent relative standard deviation
(% RSD) for inter and intraday precision were found to be 0.2657 and 0.2967, respectively,
which is under acceptable range. This showed that developed method is precise (Table 2).
The limit of detection and limit of quantitation values were found to be 0.1588µg/ml and
0.4813µg/ml, respectively. This suggests that lowest amount of drug that can be detected
using this analytical procedure is 0.1588µg/ml and lowest amount of drug in a sample that
can be quantitatively determined with suitable precision and accuracy is 0.4813µg/ml. The
Percent (%) recovery was calculated as amount of found drug/added drug X 100. %
Recovery was calculated in the range of 99.46 to 100.66 % (with mean 100.05%). This
indicated good accuracy of developed method (Table 3). There was no interference from
the excipients of tablet formulation. The validation parameters are summarized in Table 4.
Conclusion
UV Spectrophotometric area under curve method for estimation of Amitriptyline
Hydrochloride was developed. Validation parameters suggest that the proposed method
showed acceptable accuracy, precision, selectivity and linear concentration range.
Statistical analysis for the results proved that the method can be successfully applied for the
routine estimation of amitriptyline hydrochloride in tablets.
ACKNOWLEDGEMENTS:
The authors are thankful to Astron Research Ltd. (Ahmadabad, India) for supplying API of
Amitriptyline Hydrochloride as a gift sample, President of this society and Principal of
Sinhgad College of Pharmacy, Pune (India), for providing required facilities to carry out
this work.
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References:
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methods for the determination of imipramine, amitriptyline and perphenazine in content
uniformity and drug dissolution studies. J Pharm Biomed Anal 2005; 37(2): 249-58.
11. Patel S, Patel NJ. Spectrophotometric and chromatographic simultaneous estimation of
amitriptyline hydrochloride and chlordiazepoxide in tablet dosage forms. Indian J Pharm
Sci 2009; 71: (4):472-76.
12. Sarrafi AHM, Khodakarami Z, Karkeabadi MH. Simultaneous Spectrophotometric
Determination of Amitriptyline Hydrochloride and Chlordiazepoxide in Pharmaceutical
Tablets by Multivariate Calibration Method, E-Journal of Chemistry 2009; 6 (S1): S111-16.
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6
Figures:
Fig. 1
Fig. 1: Chemical structure of Amitriptyline Hydrochloride.
7
Fig. 2
Fig. 2: Representative chromatogram of 20 µg/ml of standard solution of
Amitriptyline hydrochloride.
8
Fig. 3
Fig. 3: Calibration curve of Amitriptyline hydrochloride.
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TABLES:
Table 1: Assay of tablet dosage form.
Tablet solution containing (µg/ml) % Found Mean % Found* % RSD*
100.10
15
15
100.33
15
100.41
100.28
0.1615
*n=3
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Table 2: Precision data of Amitriptyline hydrochloride.
Parameters
Intra-day
precision
Inter-day
precision
Sample solution concentration
(µg/ml)
15
15
Area (Mean ± S.D)*
0.2111±0.0005
0.2124±0.0006
%RSD
0.2658
0.2968
*n=6
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Table 3: Accuracy data of Amitriptyline hydrochloride.
Accuracy Level
I (80%)
II (100%)
III (120%)
*n=3
Amount added
(µg/ml)
% Recovery*
Mean %
Recovery*
% RSD*
12
15
18
100.66±0.0634
99.46±0.0794
100.02±0.0865
100.05%
0.0764
12
Table 4: Summary of Validation Parameters.
Parameters
Amitriptyline Hydrochloride
Area range (nm)
234-244
Linearity range
Regression equation
(y = mx+c)
Correlation coefficient (r2)
Slope (m) ± SD*
Intercept (c) ± SD*
LOD (µg/ml )
LOQ (µg/ml )
4-20 µg/ml
Precision % RSD*
Intraday
Interday
% Recovery
AUC = 0.0131C + 0.0117
0.9988
0.0131
0.0117
0.1588
0.4813
0.2657
0.2967
100.05%
*n=6
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Titles of Figures:
Fig. 1: Chemical structure of Amitriptyline Hydrochloride.
Fig. 2: Representative chromatogram of 20µg/ml of standard solution of Amitriptyline HCl.
Fig. 3: Calibration curve of Amitriptyline hydrochloride.
Titles of tables:
TABLE 1: Assay of Tablet Dosage Form.
TABLE 2: Precision data of Amitriptyline hydrochloride.
TABLE 3: Accuracy data of Amitriptyline hydrochloride.
TABLE 4: Summary of Validation Parameters.
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