Spectrophotometric determination of amitriptyline hcl in pure and pharmaceutical forms

Article abstract of DOI:10.4067/S0717-97072014000100005

Five spectrophotometric methods for determination of Amitriptyline HCl have been developed, validated and applied for the assay of the drug in pharmaceuticals. Methods A, B and C are based on ion pair complexation of drug, in acidic buffers, with triphenylmethane dyes viz., Bromothymol blue (BTB), Bromophenol blue (BPB) and Bromocresol purple (BCP). The complexes are extracted into chloroform and absorbance is measured around at 415 nm as function of concentration of the drug. The stoichiometry of the complex is found 1:1 in each case. Method D depends upon charge transfer complexation of neutralized drug with iodine which produces iodide ion whose absorbance at 366 nm is measured as function of concentration of the drug. This complex, too, has 1:1 composition as determined by Job's method. Method E is developed on the basis of oxidation of the drug with alkaline KMnO4 which generates green colored manganate ion with ?max 610 nm. As the intensity of green color increased with increasing time kinetics of the reaction is followed and calibration curves are constructed by using initial rate and fixed time methods. Excellent recovery studies with high accuracy and precision indicate that the methods can be successfully used in industries for the assay of drug in pure form and pharmaceuticals.

Full text of DOI:10.4067/S0717-97072014000100005

J. Chil. Chem. Soc., 59, Nº 1 (2014)
SPECTROPHOTOMETRIC DETERMINATION OF AMITRIPTYLINE HCl IN PURE AND
PHARMACEUTICAL FORMS
1
1
2
*
K. SUSMITHA , M.THIRUMALACHARY ,T.CHARAN SINGH AND G VENKATESHWARLU
1
Department of Chemistry, Jawaharlal Nehru Technology University Kukatpally, Hyderabad-500 085,
2
Department of Chemistry, G.Narayanamma Institute of Science & Technology
*
Department of Chemistry, Nizam College, Hyderabad 500001, A.P. India
(Received: December 18, 2012 - Accepted: January 15, 2013)
ABSTRACT
Five spectrophotometric methods for determination of Amitriptyline HCl have been developed, validated and applied for the assay of the drug in
pharmaceuticals. Methods A, B and C are based on ion pair complexation of drug, in acidic buffers, with triphenylmethane dyes viz., Bromothymol blue (BTB),
Bromophenol blue (BPB) and Bromocresol purple (BCP). The complexes are extracted into chloroform and absorbance is measured around at 415 nm as function
of concentration of the drug. The stoichiometry of the complex is found 1:1 in each case. Method D depends upon charge transfer complexation of neutralized drug
with iodine which produces iodide ion whose absorbance at 366 nm is measured as function of concentration of the drug. This complex, too, has 1:1 composition
as determined by Job’s method. Method E is developed on the basis of oxidation of the drug with alkaline KMnO which generates green colored manganate ion
4
with λmax 610 nm. As the intensity of green color increased with increasing time kinetics of the reaction is followed and calibration curves are constructed by using
initial rate and fixed time methods. Excellent recovery studies with high accuracy and precision indicate that the methods can be successfully used in industries for
the assay of drug in pure form and pharmaceuticals.
Keywords: Spectrophotometry, Amitriptyline HCl , Bromothymol blue, Bromophenol blue, Bromocresol purple, Iodine, Alkaline KMnO4
1
. INTRODUCTION
syndrome, diabetic neuropathy, post-traumatic stress disorder and for migraine
prophylaxis. Various analytical methods developed for determination of
4
– 11
12
Amitriptyline HCl (AMT) chemically [3-(10, 11-dihydro-5H-dibenzol
a, d] cyclohept-5-ylidene) propyldimethylamine] hydrochloride constitute
an important class of neurotherapeutics belonging to first generation of
amitriptyline HCl viz., spectrophotometric
, spectrofluorometric , flow
1
3
14 15,16
[
injection method , atomic absorption spectroscopic ,conductometric
,
1
17
18
19
voltammetric , UPLC-MS/MS MEPS by LC&GC-MS , Electrogenerated
,
20 21
2,
3
antidepressant drug
It may be used to treat depression, irritable bowel
Chemiluminescence and HPLC methods have been enumerated.
Table I. Comparison with other quantification methods for the determination of Amitriptyline hydrochloride
S.No.
Method
Spectrophotometric {AMIYTP(βCD)(PEG)3}
CT Complexation with TCNQ
Extractive spectrophotometry
molybdedum and thiocyanate
MEPS by LC & GC-MS
Electrogenerated Chemiluminescence
Spectrofluorimetry
λ max (nm)
242
Working range
Remarks
-1
1
2
3
0.1-1.0μgmL
costly reagents
-1
842
10 - 300μgmL
-1
1-30 μgmL
low sensitivity
-
1
4
5
6
7
8
0.08 – 0.360 μgmL
-
1
0.09 – 0.24 μgmL
Involves
Expensive
instruments
-
1
0.05 – 1.3 μgmL
-1
HPTLC
2.47-7.5μgmL
-
1
RP-HPTLC
0.01- 0.035 μgmL
7
,12,19,20
From the comparison table it is clear that the methods used so far for quantification of AMT suffer from involving costly equipment
or costly chemicals
4,6
8
or instruments with low sensitivity and not useful for routine laboratory analysis. Therefore it seems necessary to develop a sensitive, simple and rapid
determination of Amitriptyline HCl. In the present communication we report five quantification methods viz., A, B, C, D, and E which have been developed and
validated for quantification of Amitriptyline HCl both in pure and pharmaceutical forms.
o
2
. EXPERIMENTAL
desiccator (mp 113.6 -3 C). Iodine in 1, 2 dichloroethane was freshly prepared
(
-3
daily) by dissolving 254mg solute in 50mL of solvent (4.0×10 M). The drug
2
.1 Instruments
was procured from Aurobindo Pharmaceiticals, Hyderabad, as gift sample.
The UV--Visible spectra of the samples were recorded on SHIMADZU
40 double beam, Thermo Nicolet 1000 and ELICO 159 UV-Visible single
beam spectrophotometers using quartz cells of 10 mm path length. An Elico
1
Neutralisation is carried out by dissolving 100 mg of AMT in 10mL of
distilled water and solution is transferred into 100 mL separating funnel and
rendered alkaline with 5mL of 0.45M NaOH solution.The liberated base is
extracted with three times , each with 25mL of 1,2 Dichloroethane.The solution
is further diluted with same solvent to obtain the working solution.
2.3. Methods A, B and C
model Li-120 pH meter was used for pH measurement.
2
.2 Materials
HPLC grade chloroform, 1, 2 Dichloroethane (DCE), Analytical grade
(
AR) HCl, Sodium acetate, KMnO , Sodium hydroxide and dyes viz., a) BTB
4
b) BPB c) BCP supplied by Sd Fine Chemicals, Mumbai were used in the
study. Iodine (BDH, Poole, UK) was twice sublimed and preserved in vacuum
The methods A, B and C are based on the interaction of the drug with
Bromothymol blue (BTB), Bromophenol blue (BPB) and Bromocresol purple
e-mail: venkateshwarlugoud@yahoo.com
2265

J. Chil. Chem. Soc., 59, Nº 1 (2014)
(
(
BCP) respectively, to form chloroform extractable ion pair complexes
Scheme 1) which absorb around 415 nm. (Fig. 1) The absorbance of this band
increases with increasing the concentration of the drug and formed a basis for
the quantification of the drug. The dyestuffs were used as 0.025% solutions in
doubly distilled water. Sodium acetate-hydrochloric acid buffers of pH 2.8, 2.5
and 2.5 were prepared by mixing 50ml of 1.0M sodium acetate solution with
4
9.50 mL, 50.50mL and 50.50 mL of 1.0 M HCl solution respectively and
diluted to 250 mL with doubly distilled water. The pH of each solution was
adjusted to an appropriate value with the aid of a pH meter.
-
Figure 2. Absorption spectra (2) reaction product of
5 μg mL
1
Amitriptyline hydrochloride with iodine in 1, 2 Dichloroethane (3) iodine
2
.5. Method E Kinetic method
Bromothymol blue: R =isopropyl, R =CH
3
1
2
The method depends on the oxidation of the drug with alkaline KMnO
4
Bromophenol blue: R = Br, R = H Bromocresolpurple: R = .CH , R = H
-2
1
2
1
3
2
(1x10 M) to produce Manganate ion which absorbs at 610 nm (Fig.3) and
formed a basis for quantification of drug. A solution of 0.45M NaOH is used
to produce required alkalinity. Mixing the solutions of permanganate and the
drug slowly developed green color and hence kinetics of the reaction was
followed spectrophotometrically. The initial rate and fixed time methods are
followed for the determination of AMT. The literature survey reveals that
Amitriptyline undergoes oxidation at exocyclic double bond and gives rise to
Scheme 1 Amitriptyline HCl – dye complex
2
2
dibenzosuberone . (Scheme -3)
Figure1. Absorption spectra of Amitriptyline Hydrochloride - dyes com-
plex extracted into 10 mL chloroform:
-
1
(
(
(
(
a) drug = 20 µg mL + 5 mL of 0.025% BTB + 5 mL of pH 2.8 buffer;
-1
b) drug = 20 µg mL + 5 mL of 0.025% BPB + 5 mL of pH 2.5 buffer;
-2
Figure 3. Absorption spectra of (a) Drug (b) KMnO (1 x10 M) (c)
4
-1
c) drug = 22.5 µg mL + 5 mL of 0.025% BCP + 5 mL of pH 2.5 buffer,
-1
Reaction product of 10μg mL Amitriptyline hydrochloride with alkaline
-1
d) drug = 0 µg mL + 5 mL of 0.025% Dye + 5 mL of buffer
KMnO4
2
.4. Method D
The method depends upon the interaction of neutralized drug with Iodine
that generates iodide ion having an absorption band at 366 nm. (Fig. 2) The
absorbance of this band increases with increasing the concentration of the drug
and formed a basis for the quantification of the drug. Mixing the solution of
iodine prepared in DCE with AMT resulted in a change of violet color of io-
dine into light brown to pale yellow and as a consequence, absorption spectra
-
exhibited a new band of 366nm. This is attributed due to I3 ion formed by the
interaction of iodine with drug. (Scheme 2).
D + I2
DI (Outer complex) fast……….…1
2
+ -
DI (Outer complex)
( DI ) I (inner complex ) slow……….2
Scheme – 3 Oxidation of Amitriptyline HCl with alkaline KMnO4
2
+
-
+
-
(DI ) I (inner complex ) + I
( DI ) + I fast…………3
2
3
2
.6. Calibration curves for method A, B and C
Scheme – 2
Different aliquots of drug solution were transferred into 125 mL separating
2
266

J. Chil. Chem. Soc., 59, Nº 1 (2014)
funnel. To this 5 mL of buffer, 5 mL of dye were added and total volume was
made up to 20 mL with water. 10 mL of chloroform was added and the contents
were shaken for 5 min. The two layers were allowed to separate for 5 min
and the organic layer was separated and absorbance of yellow colored solution
which is stable at least for 3 hr is measured at 415 nm against blank similarly
prepared. The same procedure of analysis is followed either for assay of pure
drug or for dosage form. The calibration graphs are linear over a range of drug
2.8. Calibration curve for method E
The calibration curves are constructed by using initial rate method and
fixed time method.
2.8.1. Initial rate method
Aliquots of 1 to 8 mL of Amitriptyline HCl solution containing 2.5 mg
-1
mL of drug were pipetted into a series of 10 mL standard flasks. To each
-2
flask, 1.0 mL of 0.45 M NaOH and 1 mL of 1x 10 M potassium permanganate
were added successively and then diluted with distilled water at 25 ± 1 C. The
-1
-1
-1
0
1
.25 - 25μgmL (BTB), 1.5 – 25μgmL (BPB), 2.0 – 25μgmL (BCP).
.7. Calibration curve for method D
Into separate 10mL of volumetric flasks different aliquots of AMT solution
2
contents of each flask were mixed well, and the increase in absorbance as a
function of time was measured at 610 nm. The initial rate of the reaction at
different concentrations was evaluated by deferring the slope of the tangent
to the absorbance-time plot. The calibration graphs in the range 6.25 -
was transferred followed by the addition of 1mL of iodine solution. The volume
was completed using the 1, 2 dichloroethane solvent and the absorbance was
measured against reagent blank at 366nm and calibration curve is linear over a
1
50μgmL were obtained by plotting the initial rate of reaction versus the molar
-1
range of drug 2.5 - 25μgmL
concentration of the Amitriptyline HCl (Fig . 4)
2
.8.2. Fixed time method
A fixed time of 25 min was selected for the fixed time method. At this time
the absorbance of reaction mixture was measured at 610 nm against a reagent
blank prepared similarly. The calibration curve was obtained by plotting the
absorbance against concentration of Amitriptyline HCl.
2
2
.9. Assay of drug in pharmaceuticals
.9.1. Procedure for the assay of pure drug
Five different solutions of pure drug in the range of calibration curve
were selected and the recovery experiments were performed. The recoveries
and their relative standard deviation are tabulated in Table III.
2
.9.2. Procedure for the assay of dosage forms
Five tablets of Elavil 10 mg with composition Amitriptyline HCl -10
mg, microcrystalline cellulose, croscarmellose sodium, magnesium stearate,
colloidal silicon dioxide, hydroxyl propyl methyl cellulose, polyethylene
glycol, carnauba wax are powdered and dissolved in doubly distilled water
and stirred thoroughly, filtered through a Whatman No. 42 filter paper. This
solution was transferred into 100 mL standard volumetric flask and diluted
with doubly distilled water as required. Different solutions of drug in the
range of calibration curve were chosen and its quantify was estimated using
the calibration curve and the results of the recovery studies are tabulated in
Table IV.
Figure 4. Calibration curves
Table II Performance data for the proposed methods.
Parameters
A
B
C
D
366
E
610
λmax (nm)
415
410
415
-
1
Beer’s law limit (μg mL )
1.25-25
0.0169
0.059
0.1164
0.9997
0.0150
0.80
1.5 -25
0.0131
0.0766
0.1140
0.9988
0.0320
1.30
2.0 - 25
0.0147
0.0680
-0.0710
0.9944
0.0200
0.97
2.5 – 25
0.014
0.071
0.0107
0.9987
0.021
0.9
6.25 - 50
0.0823
0.0121
0.0192
0.9967
0.02
-
2
Sandell sensitivity (μg cm )
Slope, a
Intercept b
Correlation coefficient (r)
Standard deviation of intercepts (% n=6)
-
1)
Limit of detection, (μgmL
Limit of quantification, (μgmL
5.4
-1)
2.40
3.90
2.91
2.9
16.2
Y=
Y=
Y=
Y=
Y=
Regression equation Y = a*C +b
0
.059*C+ 0.1164
0.0766*C+ 0.114
0.068*C-0.071 0.071*C+0.010
0.0121*C+0.019
-1
Y= a*C + b, Where C is the concentration in μgmL
2
267

J. Chil. Chem. Soc., 59, Nº 1 (2014)
Table III. Application of proposed methods for the determination of AMT in pure form.
A
B
C
D
E
-1
Amount taken (μg mL )
10.00
10.00
15.00
20.00
25.00
30.00
10.00
15.00
20.00
25.00
30.00
10.00
15.00
20.00
25.00
30.00
10.00
15.00
20.00
25.00
30.00
1
2
2
3
5.00
0.00
5.00
0.00
Amount found
9
.98
10.22
10.15
9.98
9.99
-1
(μg mL )
1
2
2
2
5.04
0.06
5.28
9.99
14.96
19.99
15.07
20.09
14.96
19.97
15.01
19.99
25.05
25.10
24.19
25.05
30.05
30.05
29.87
30.08
Recovery (%)
99.8
102.2
101.5
99.32
99.9
1
1
00.27
99.73
100.47
100.45
100.4
100.74
100.50
100.20
99.56
100
1
00.3
99.95
99.95
01.12
100.2
100.2
9
9.97
100.17
100.45 ± 0.99
100.33 ±
0.44
100.17
100.60 ± 0.51
100.33 ±
0.44
100.2
Mean ± SD
100.29 ± 0.50
100.33 ±
100.06 ± 0.608
100.33 ±
0.44
100.05 ± 0.141
100.33 ±
0.44
4
Reference method
Mean ± SD
0
.44
t-test*(2.306)
F-test*(5.05)
0.134
1.29
0.247
0.896
0.804
1.35
5
1.3
1.9
0.9
2
3
Each result is the average of five separate determinations. *Values in parenthesis are the tabulated t and F values at p=0.05
Table IV. Application of proposed methods for the determination of AMT in Pharmaceutical form.
A
B
10.00
C
10.00
20.00
30.00
40.00
50.00
9.95
D
E
10.00
20.00
30.00
40.00
50.00
9.97
-1
Amount taken (μg mL ) (Elavil – 10mg)
10.00
10.00
20.00
30.00
40.00
50.00
9.98
2
3
4
5
0.00
0.00
0.00
0.00
20.00
30.00
40.00
50.00
-1
Amount found
(μg mL )
9.98
10.02
2
0.02
9.96
9.00
9.60
19.95
20.15
30.15
39.77
50.22
99.5
20.03
29.95
39.55
49.4
19.98
29.88
39.88
50.8
2
3
4
30.10
40.05
50.11
Recovery (%)
99.8
100.2
99.80
99.7
1
00.1
9.87
99.75
100.75
100.5
99.43
100.44
100.12 ± 0.61
100.15
99.83
99.9
9
100.33
100.13
100.22
100.13 ± 0.22
99.6
9
7.5
9.2
98.88
99.7
9
98.80
101.6
100.1 ± 0.845
Mean ± SD
99.29 ± 1.06
99.49 ± 0.613
4
Reference method Mean ± SD
100.33 ±
100.33 ±
0.44
100.33 ±
0.44
100.33 ±
0.44
100.33 ±
0.44
0
.44
t-test*(2.306)
F-test*(5.05)
1.617
5.8
0.045
0.2
0.01
2.28
0.539
3.6
1.9
1.9
2
3
*
Values in parenthesis are the tabulated t and F values at p=0.05
2
268

J. Chil. Chem. Soc., 59, Nº 1 (2014)
3
. RESULTS AND DISCUSSION
3.5. Optimization of the factors affecting the absorbance.
The factors effecting the absorbance of ion pair complexes like pH and
volume of the dye, in methods A, B and C have been optimized. 1.8 mL of
BTB and buffer of pH 2.8, 1.6 mL of BPB and buffer of pH 2.5 and 1.7 mL
of BCP and buffer of pH 2.5 are found to be optimal for methods A, B and C
respectively. However 5 mL of each dye is used, at optimal pH, in the study
to ensure complete extraction of the drug. Similarly the 1 mL of iodine for
3
.1. Methods A, B and C
AMT forms ion-pair complexes in acidic buffer with dyestuffs viz., BTB,
BPB and BCP which are quantitatively extracted into chloroform. Ion-pair
complexes of drug with dyes absorbed maximally at 415 nm (Fig. 1). AMT
contains a tertiary nitrogen atom and in strongly acidic medium it exists as
a cation and sulphonic acid group present in the dyes that is the only group
undergoing dissociation in the pH range 1-5. Finally the protonated AMT forms
ion-pairs with the dyestuff which is quantitatively extracted into chloroform.
method D and 1 mL of KMnO , 1 mL of 0.45 NaOH for method E are found to
4
be optimal and hence are used in the study.
3.6. Validation of the proposed methods
3
.2. Stoichiometry
The proposed method have been validated in terms of guidelines proposed
2
9
In order to establish molar ratio between AMT and dyestuffs used, the
by ICH viz., selectivity, specificity, accuracy, precision, limits of calibration
curve, LOD, LOQ, robustness, ruggedness and regression equation. The
student t-test and variance F-test have been performed in comparison with a
reference method. To test the reproducibility of the proposed methods, five
replicate determinations of 15.0µg mL of AMT were made. The coefficient of
variation was found to be less than 1.2% for all the procedures.
2
4
Job’s method of continuous variation has been applied . In this method,
-5
solutions of drug and dyestuff with identical molar concentrations [8 x 10 M]
were mixed in varying volume ratios in such a way that the total volume of
each mixture is the same. The absorbance of each solution was measured and
plotted against the mole fraction of the drug, [drug]/[drug]+[dyestuff] . This
measurement showed that 1:1 complex was formed.
The proposed methods have been successfully applied to the determination
of AMT in pharmaceutical preparations. The results obtained and shown in
The Job’s method of stoichiometry is also applied for Iodine with AMT
which indicated the charge transfer complex formed is of 1:1 composition (Fig
4
Table II were compared to those obtained by a reference method by means
5
).
of t-test at 95% confidence level. In all cases, the average results obtained by
proposed methods and reference method were statistically identical, as the
difference between the average values had no significance at 95% confidence
level.
The proposed methods are simple, sensitive and reproducible and can be
used for routine analysis of AMT in pure form and in formulation
4
. CONCLUSIONS
The proposed spectrophotometric methods present selective and simple,
specific and inexpensive analytical procedures for determination of AMT, in
pure or in tablet dosage forms without interference from common excipients.
Moreover, the developed methods are time saving and do not require elaborate
treatments associated with chromatographic methods. These attributes, make
them suitable for routine analysis in quality control laboratories.
ACKNOWLEDGEMENTS
Authors are thankful to the Head, Department of Chemistry and
Principal Nizam College, Head Department of Chemistry, Jawaharlal Nehru
Technological University Hyderabad, for providing laboratory facilities for
this work.
Figure 5. Jobs continuous variation plot-
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.
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4
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.3. Formation constants
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.
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-
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1
1
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28
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A ]/d = I/K [D ] ε + 1/ε
0
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1
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0
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2
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