Determination of Tropicamide and Hydroxyamphetamine in
Opthalmic formulations by RP-HPLC and its Validation
Devadasu Ch*,
Sravya G
Department of Pharmaceutical Analysis, Koringa College
of Pharmacy,
Korangi- 533461 Dist-East Godavari (Andhra Pradesh)
India.
ABSTRACT:
The
aim of this study was to develop a simple and suitable analytical method for
determination of tropicamide and hydroxyamphetamine in ophthalmic preparations.
A fast, sensitive, and reliable RP-HPLC
method involving cyber lab HPLC System with UV detection was developed
and validated for the determination and quantification of Tropicamide, an antimuscarinic agent used as eye drops for
refractive examinations and
Hydroxyamphetamine. Chromatography was performed on the Inertsil -ODS
C18 (250 x 4.6mm, 5μ) column using filtered and mixed Degassed Methanol:
Buffer (75:25) as a mobile phase with a flow rate of 1.0ml/min and an effluent
of 257nm. Retention times for Tropicamide 3.047min, and Hydroxyamphetamine
4.313. The method was found to be linear
(r2> 0.999) in the range of 20-80 µg/ml for the drug candidates selected.
KEYWORDS: Method
Development, Validation, Tropicamide, Hydroxyamphetamine, RP-HPLC.
INTRODUCTION:
Tropicamide1, (R,S)-N-ethyl-3-hydroxy-2-phenyl-N-(pyrid-4-ylmethyl)propionamide. Tropicamide is
indicated for causing mydriasis (pupil dilation) and cycloplegia (paralysis of
the eye's ciliary muscle) in diagnostic procedures, such as assessment of
refractive errors and examination of the eye's fundus. Hydroxyamphetamine hydrobromide is an indirect acting
sympathomimetic agent which causes norepinephrine to be released from the
terminals of the adrenergic nerves, leading to mydriasis (Fig. 1). Several
analytical methods have been reported from the literature, for determination of
tropicamide by HPLC in human aqueous humour2, formulations3,
in API with impurities4 and with hydroxyamphetamine hydrobromide5,6.
Spectroscopic estimation of tropicamide by derivative7 and AUC8
methods have also been reported in the literature.
The
main aim of the project work is to develop a novel RP-HPLC method which is able
to separate and quantify the drug candidates selected for study viz.,
Tropicamide and Hydroxyamphetamine present in its pure form as well as
formulation and validate the method by ICH Q2 (R1)9 guidelines with
demonstrable accuracy, linearity, precision and robustness.
A B
Fig.
1: Chemical structure of
Tropicamide (A) and Hydroxyamphetamine (B)
MATERIAL
AND METHODS:
Chemicals
and reagents:
All
the chemicals and reagents used in this study were of analytical grade.
Tropicamide was obtained from Malladi Drugs and Pharmaceuticals, Chennai,
India. Dipotassiumhydrogen phosphates and Potassiumdihydrogen phosphate were
obtained from Merck specialties private limited, Mumbai. HPLC grade
acetonitrile and water were obtained from Qualigens.
Instrumentation:
A liquid
chromatograph, CYBERLABTM equipped with LC100 pump LC100
UV LC detector was used for separation. The column used in this study is Analytical
packed with BDS, 5µm, size: 250x4.6mm and the column effluents were monitored
and integrated using WS-100 Work station software. ELICO SL 159
double beam UV visible spectrophotometer with 1 cm quartz cells was used for
spectral studies.
Mobile
Phase:
Phosphate
degassed buffer pH 3.4 and Methanol 25:75 V/V.
Preparation
of standards and working solutions:
Preparation
of solution for stocks:
Reference
solution: The solution was prepared by dissolving 20.0mg of precisely weighed
Tropicamide and 25.0mg of hydroxyamphetamine in mobile process, separately in
two volumetric flasks of 100.0 mL and sonicating for 20min. From the above
solutions take 10.0 mL of each solution into a 50.0 mL volumetric flask and
then make up for 10min with mobile phase and sonicated.
Preparing
Standard Working Solution:
In combination with Tropicamide and Hydroxyamphetamine
above, stock solutions equal to 20ppm to 80ppm in respect of both drugs were
prepared, sonicated and filtered through 0.45μ membrane.
Standard
Solution preparation for trials:
Weighing
down 10 mg of Tropicamide and hydroxyamphetamine drugs and dissolving in 10ml
of Mobile phase taken in two 10ml volumetric flasks separately and sonicated
for 20 minutes to get 1000ppms and 1ml of each solution was taken into a 10ml
volumetric flask and diluted to 10ml with mobile phase.
Method Validation
System Suitability:
The system suitability test was performed using 20
μL of standard solution for five replicate injections before analysis of
samples and the chromatogram was recorded.
Retention time (Rt), number of theoretical plates (N), tailing
factor (T), and peak asymmetry (AS), resolution (RS) were
evaluated for five replicate injections of the drug.
Specificity:
435 mg placebo with 250 mL ACN was taken in 500 mL
volumetric flask and the volume was adjusted with water. Prepared blank,
placebo, standard solution with diluent are analyzed to check the interference.
LOD and LOQ
LOD = 3.3σ / S
LOQ = 10 σ / S
Where σ is the standard deviation in the
intercept and S is the slope of the calibration curve
Linearity:
A series of solutions are being formulated
using Tropicamide and hydroxyamphetamine working standards at 20 ppm to 80 ppm
target concentration levels.
Precision:
Repetition (Repeatability):
a.
Device accuracy (System
precision):
Standard
solution prepared and injected five times, as per test form.
b.
Precision method (Method
precision):
Six
sample preparations were prepared individually using single as per test
process, and each solution was injected.
Intermediate
precision (analyst to analyst variability):
The
prepared proposed system suitability solution was injected into the
chromatographic system at finalized condition and variable conditions like flow
variation, pH variation of buffer, column oven temperature variation,
wavelength variation.
Accuracy (% Recovery):
A research was performed on accuracy. Drug Assay was
performed in triplicate as per test method with equal amounts of Tropicamide
and hydroxyamphetamine in each volumetric flask for each spike stage to get the
concentration of tropicamide and hydroxyamphetamine equivalent to 50 percent,
100 percent, and 150 percent of the labeled quantity as per test method. The
average recuperation rate of tropicamide and hydroxyamphetamine was estimated.
Robustness studies:
Effect
of Flow Variation:
(Effect
of variation of flow rate):
A
research was carried out to determine the impact of flux rate variability.
Using flow speeds, 1.0 ml / min and 1.2ml / min, standard solution prepared
according to the test method was injected into HPLC network. The parameters of
suitability for the device were tested and found to be within the 1.0ml / min
and 1.2 ml / min flow limits. Tropicamide and Hydroxyamphetamine were resolved
from all other peaks and the retention times were comparable to those obtained
for the 1.0ml / min mobile process.
RESULTS
AND DISCUSSION:
Method
development and optimization:
The
extent of the organic and aqueous phases were adjusted to attain a specific and
reliable assay method for the determination of Tropicamide and Hydroxyamphetamine
with less run time, short retention time and the sharp peak. The UV absorption of the selected drug candidates shown good response at 257 nm for detection of analytes (Fig. 2). Keeping in view all the trials, a mobile phase
composed of phosphate buffer pH 3.4 and methanol was used in different
proportions until to get the best separation conditions, and a ratio of 25:75
v/v, has been found suitable for the study. The optimized chromatographic
conditions was given in table 1.
Fig.
2: UV absorption spectrum of Tropicamide in Ethanol and water
Method
validation:
The
method was validated with particular parameters like specificity, linearity,
precision, LOD & LOQ, accuracy and robustness.
Table
1: Optimised chromatographic conditions
|
Parameters
|
Method
|
|
Stationary phase (column)
|
Inertsil -ODS C18(250 x 4.6 mm, 5 µ)
|
|
Mobile Phase
|
Methanol : Buffer (75:25)
|
|
Flow rate (ml/min)
|
1.0 ml/min
|
|
Run time (minutes)
|
10 min
|
|
Column temperature (°C)
|
Ambient
|
|
Volume of injection loop (ml)
|
20
|
|
Detection wavelength (nm)
|
257nm
|
|
Drug RT (min)
|
3.047min for Tropicamide and
4.313 for hydroxyamphetamine.
|
System
suitability studies:
It
was observed from the data tabulated above, that system suitability parameters
i.e., tailing factor < 2.0 and theoretical plates are more than 2000. Both
parameters were found to be within the acceptance criteria. Hence it can be
concluded that the preferred system is suitable. The results were presented in
table 2. Chromatograms representing system suitability were presented in Fig.
3.
Table
2: Data of System Suitability
|
Injection
|
RT
|
Peak Area
|
USP Plate count
|
USP Tailing
|
RT
|
Peak Area
|
USP Plate count
|
USP Tailing
|
|
|
Tropicamide
|
Hydroxyamphetamine
|
|
1
|
3.049
|
1788455
|
15023.84
|
1.08
|
4.316
|
3682978
|
8325.87
|
1.024
|
|
2
|
3.047
|
1786594
|
15010.54
|
1.05
|
4.312
|
3687548
|
8384.54
|
1.012
|
|
3
|
3.042
|
1783458
|
15036.87
|
1.04
|
4.309
|
3684584
|
8314.87
|
1.03
|
|
4
|
3.048
|
1784579
|
15027.25
|
1.06
|
4.317
|
3688455
|
8372.78
|
1.04
|
|
5
|
3.047
|
1785982
|
15084.65
|
1.05
|
4.313
|
3686878
|
8392.08
|
1.02
|
|
Mean
|
3.052
|
1786086
|
15036.82
|
1.05
|
4.35
|
3686076
|
8358.87
|
1.03
|
|
SD
|
0.002
|
1839.51
|
-------
|
-------
|
0.0024
|
2014.80
|
-------
|
-------
|
|
% RSD
|
0.075
|
0.102
|
-------
|
-------
|
0.04
|
0.054
|
-------
|
-------
|
Fig.
3: Chromatogram for system suitability
Fig.
4: Chromatogram of placebo for specificity
Fig.
5: Chromatogram showing for specificity in separation of two drugs
Table
3: Results of specificity *N=3
|
Name of
sample
|
Retention time (min)
|
Area
|
Efficiency (N)
|
Tailing factor
|
|
Tropicamide
|
Hydroxyamphetamine
|
Tropicamide
|
Hydroxyamphetamine
|
Tropicamide
|
Hydroxyamphetamine
|
Tropicamide
|
Hydroxyamphetamine
|
|
Diluent
|
Not found
|
Not found
|
Not found
|
Not found
|
Not found
|
Not found
|
Not found
|
Not found
|
|
Standard
|
3.049
|
4.316
|
1786594
|
3684584
|
15036.81
|
8325.87
|
1.032
|
1.035
|
|
Placebo
|
Not found
|
Not found
|
Not found
|
Not found
|
Not found
|
Not found
|
Not found
|
Not found
|
|
% RSD*
|
0.21
|
0.20
|
0.81
|
0.15
|
0.35
|
0.43
|
0.31
|
0.35
|
Table
4: Results of linearity
|
Tropicamide
|
Hydroxyamphetamine
|
|
Concentration, µg/mL
|
Area
|
Concentration, µg/mL
|
Area
|
|
0
|
0
|
0
|
0
|
|
20
|
894432
|
20
|
1842747
|
|
30
|
1341649
|
30
|
2764121
|
|
40
|
1788865
|
40
|
3685494
|
|
50
|
2210846
|
50
|
4550784
|
|
60
|
2683298
|
60
|
5528464
|
|
70
|
3130514
|
70
|
6449615
|
|
80
|
3577730
|
80
|
7370988
|
|
Regression analysis
|
|
Slope
|
44690
|
Slope
|
92068
|
|
y-Intercept
|
-1770
|
y-Intercept
|
-3939
|
|
Correlation Coefficient
|
0.999
|
Correlation Coefficient
|
0.999
|
Specificity:
Specificity
part (A) includes the interference from blank and placebo. No interference was
observed at the retention time of selected drug candidates. Therefore it can be
concluded that no interference due to diluent, placebo and standard for the
quantification of Tropicamide and Hydroxyamphetamine in formulations. Hence,
the method is specific and selective. The results are shown in Fig. 4, 5 and
table 3.
Linearity:
The
proposed method is linear over the concentration range 20-80µg/mL and
20-80µg/ml for Tropicamide and Hydroxyamphetamine respectively. The correlation
coefficient between the concentration of selected drugs and their
chromatographic peak response (area) was highly impressive and found to have R˛
= 0.999. This regression analysis indicates that the method has excellent
linearity over the wide concentration range. The calibration curves were given
in fig. 6 and 7. The results of the linearity for both the drugs were shown in
table 4 and the representative chromatograms were given in Fig. 8-12.
Fig.
6: Calibration curve for Tropicamide Fig.
7: Calibration curve for hydroxyamphetamine
Fig.
8: Chromatogram for linearity solution of 20 ppm
Fig.
9: Chromatogram for linearity solution of 30 ppm
Fig.
10: Chromatogram for linearity solution of 40 ppm
Fig.
11: Chromatogram for linearity solution of 50 ppm
Fig.
12: Chromatogram for linearity solution of 60 ppm
Table
5: Intra-assay precision of Tropicamide and Hydroxyamphetamine
|
Injection
|
Peak
Area
|
%
Assay
|
Peak
Areas
|
%
Assay
|
|
|
Tropicamide
|
Hydroxyamphetamine
|
|
1
|
1788697
|
100.16
|
3688559
|
100.26
|
|
2
|
1784568
|
99.92
|
3684565
|
100.15
|
|
3
|
1782564
|
99.81
|
3687045
|
100.22
|
|
4
|
1786200
|
100.02
|
3686038
|
100.19
|
|
5
|
1787240
|
100.07
|
3685528
|
100.18
|
|
6
|
1785621
|
99.98
|
3686788
|
100.21
|
|
Mean
|
1785815
|
99.99
|
3686420
|
100.20
|
|
SD
|
2128.211
|
0.119
|
1377.282
|
0.037
|
|
%
RSD
|
0.119
|
0.119
|
0.037
|
0.037
|
Fig.
13: Repeatability chromatogram
Table
6: Inter-day precision study
|
Injection
|
Peak
Area
|
%
Assay
|
Peak
Areas
|
%
Assay
|
|
1
|
1786598
|
100.04
|
3687548
|
100.23
|
|
2
|
1786903
|
100.06
|
3683598
|
100.13
|
|
3
|
1786035
|
100.01
|
3684875
|
100.16
|
|
4
|
1787312
|
100.08
|
3686582
|
100.21
|
|
5
|
1787964
|
100.11
|
3685648
|
100.18
|
|
6
|
1788450
|
100.14
|
3684575
|
100.15
|
|
Mean
|
1787210
|
100.07
|
3685471
|
100.18
|
|
SD
|
890.610
|
0.049
|
1431.484
|
0.038
|
|
%
RSD
|
0.049
|
0.049
|
0.038
|
0.038
|
Fig.
14: Chromatogram representing intermediate precision
Limit
of detection and limit of quantification:
The
limit of detection and quantitation was determined from the calibration
curve and it was found satisfactory since the lowest amount that can be
detected by this method was 0.135 µg/mL and the minimum concentration of the
analyte that can be quantified was found as 0.411 µg/mL. Similarly, the LOD and
LOQ was found as 0.072 µg/ml and 0.218 µg/ml for Hydroxyamphetamine. From the
linearity plot the LOD and LOQ are calculated by the following formula:
Precision:
Three different sample concentrations and triplicate
of each concentration in linearity range were taken for intra and inter day
precision studies. The % RSD for intra-day precision of the sample (n=6) was
found as 0.119 and 0.037for Tropicamide and Hydroxyamphetamine respectively.
The interday precision was achieved by performing the method in between the
days and the % RSD was found as 0.049 and 0.038 for Tropicamide and
Hydroxyamphetamine respectively. In all the cases studied, the % RSD has been
found by the proposed method was within 2.0 % that has indicated a consistency
in its precision and can be seen in the results of precision studies in table 5
and table 6 for intra-day and interday precisions respectively. Chromatograms
representing the precision studies were given in fig. 13-14.
Accuracy:
Recovery
studies were performed on three different levels at 50, 100, and 150 in three
replicates in each level in the present study. Standard drug was spiked to the
pre-analyzed sample and injected into an
HPLC system to determine the amount recovered by the proposed method. The %
recovery values were observed to be in the range of 99.98 % - 100.27 % and %
RSD less than 0.1. The recovery results indicated that the method had an
acceptable level of accuracy. Results for the accuracy study were shown in
table 7 and the chromatograms were given in fig. 15-17.
Table
7: Data of Accuracy
|
% of Spiked level
|
Amount added
(ppm)
|
Amount found
(ppm)
|
% Recovery
|
Amount added
(ppm)
|
Amount found
(ppm)
|
% Recovery
|
|
Tropicamide
|
Hydroxyamphetamine
|
|
50% Injection 1
|
20
|
20.05
|
100.27
|
20
|
20.05
|
100.27
|
|
50% Injection 2
|
20
|
20.06
|
100.30
|
20
|
20.10
|
100.51
|
|
50% Injection 3
|
20
|
20.06
|
100.28
|
20
|
20.08
|
100.40
|
|
100 % Injection 1
|
40
|
39.99
|
99.98
|
40
|
40.07
|
100.18
|
|
100 % Injection 2
|
40
|
40.02
|
100.05
|
40
|
40.08
|
100.20
|
|
100% Injection 3
|
40
|
40.04
|
100.11
|
40
|
40.06
|
100.15
|
|
150% Injection 1
|
60
|
60.09
|
100.15
|
60
|
60.09
|
100.15
|
|
150% Injection 2
|
60
|
60.11
|
100.19
|
60
|
60.08
|
100.14
|
|
150% Injection 3
|
60
|
60.17
|
100.29
|
60
|
60.06
|
100.11
|
Fig.
15: Chromatogram for Accuracy at 50 % level
Fig.
16: Chromatogram for Accuracy at 100 % level
Fig.
17: Chromatogram for Accuracy at 150 % level
Robustness:
The
robustness of a method is evaluated by varying method parameters such as flow
rate variation, column temperature, buffer pH and the results are shown in
table 25.A change of ± 0.1 mL flow rate has lead to change in retention time
from 2.877 min to 4.68 min for Tropicamide and Hydroxyamphetamine without
altering
tailing factor and plate count respectively. In this study, a slight change in
column temperature and pH of the buffer solution n does not influence the
method. Table 8 and 9 shows the change in variables during robustness study.
Fig. 18-20 shows the Chromatograms for robustness studies.
Table
8: Data for Effect of variation in flow rate (Tropicamide)
|
Flow
rate
|
0.8
mL/min
|
1.0
mL/min
|
1.2
mL/min
|
|
Injection
|
Std
Area
|
Tailing
factor
|
Std
Area
|
Tailing
factor
|
Std
Area
|
Tailing
factor
|
|
1
|
1706894
|
1.175
|
1786854
|
1.18
|
1848653
|
1.18
|
|
2
|
1704687
|
1.168
|
1785684
|
1.17
|
1846878
|
1.17
|
|
3
|
1708645
|
1.178
|
1788465
|
1.16
|
1847845
|
1.14
|
|
4
|
1706989
|
1.189
|
1787854
|
1.15
|
1843675
|
1.15
|
|
5
|
1707545
|
1.174
|
1786538
|
1.17
|
1847854
|
1.18
|
|
6
|
1708522
|
1.138
|
1787805
|
1.16
|
1843789
|
1.16
|
|
Mean
|
1707213
|
1.170
|
1787200
|
1.17
|
1846449
|
1.17
|
|
SD
|
1442.70
|
0.017
|
1024.71
|
0.010
|
2178.76
|
0.015
|
|
%RSD
|
0.084
|
1.476
|
0.057
|
0.88
|
0.117
|
1.312
|
Table
9: Data for Effect of variation in flow rate (Hydroxyamphetamine)
|
Flow
rate
|
0.8mL/min
|
1.0
mL/min
|
1.2
mL/min
|
|
Injection
|
Area
|
Tailing
factor
|
Area
|
Tailing
factor
|
Area
|
Tailing
factor
|
|
1
|
3654824
|
1.048
|
3684575
|
1.056
|
3714865
|
1.06
|
|
2
|
3658456
|
1.087
|
3685018
|
1.086
|
3716894
|
1.04
|
|
3
|
3659848
|
1.080
|
3683698
|
1.078
|
3716034
|
1.05
|
|
4
|
3654897
|
1.068
|
3687805
|
1.035
|
3714852
|
1.06
|
|
5
|
3655980
|
1.085
|
3682579
|
1.096
|
3715654
|
1.06
|
|
6
|
3654798
|
1.078
|
3684785
|
1.029
|
3713520
|
1.045
|
|
Mean
|
3656467
|
1.074
|
3684743
|
1.063
|
3715303
|
1.053
|
|
SD
|
2171.37
|
0.014
|
1746.79
|
0.027
|
1163.03
|
0.010
|
|
%RSD
|
0.059
|
1.351
|
0.047
|
2.603
|
0.031
|
1.020
|
Fig.
18: The robustness chromatogram with flow rate 0.8 ml / min
Fig.
19: The robustness chromatogram with for flow rate 1.0 ml / min
Fig.
20: The robustness chromatogram with flow rate 1.2 ml / min
CONCLUSION:
The
empirical method was developed by analyzing diverse parameters. First of all,
the average absorbance was observed at 257 nm Tropicamide for and 250 nm for
Hydroxyamphetamine. Typical wavelength would be 257 nm, and peak purity was
superb. Injection volume was selected as 20μl which gave a good peak area.
Inertsil C18, a solid peak type chosen by ODS, was the column used for
analysis. The ambient temperature was identified as being sufficient for the
drug solution's quality. The new recovery over the same range as 98.0-100.03
was found to be linear and reliable. It has been found that accuracy of both
the tool and the procedure is accurate and within range. The detection limit
was found to be 0.135Tropicamide, and 0.072 for Hydroxyamphetamine. The
linearity analysis was, it was found to be correlation coefficient and curve
fitting. The analytical method observed linearity of both the drugs over the
target concentration range of 20-80 ppm. The analytical has passed sturdiness
as well as robustness tests. In both occasions the relative standard deviation
was very satisfying.
ACKNOWLEDGEMENT:
The
authors are grateful to the management, Koringa educational society, Korangi,
for providing us the facilities.
CONFLICT OF INTEREST:
The authors declare no conflict of interest.
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