Download The present developed Stability Indicating RP

Stability-Indicating UPLC Method for Simultaneous Quantification of Emtricitabine,
Efavirenz and Tenofovir Disoproxil Fumarate in pharmaceutical dosage forms
A. KIRAN KUMAR* and G. NAGESWARA RAO
Department of Inorganic and Analytical Chemistry, Andhra University, Visakhapatnam530003, India
Corresponding mail id: [email protected]
ABSTRACT
Developing a single analytical method for estimation of individual drugs from a
multidrug composition is a very challenging task. This paper describes a strategy for the
systematic development of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC)
methods with the objective of reducing analysis time and maintaining good efficiency. A
simple, rapid, precise, and reliable simultaneous stability indicating UPLC method was
developed for the separation and estimation of three drugs, Emtricitabine, Efavirenz and
Tenofovir Disoproxil Fumarate in bulk drug mix and pharmaceutical dosage forms.
Chromatography was carried on an Acquity UPLC BEH Phenyl (2.1 x 100mm, 1.7 µm)
column using gradient composition of 0.1% Orthophosphoric acid (OPA) as Mobile Phase A
and 100% gradient grade Acetonitrile as Mobile Phase B. The flow rate was 0.5 mL/min and
effluents were monitored at 265 nm. The retention times of the Emtricitabine, Tenofovir
disoproxil fumarate and Efavirenz were about 0.68, 1.34 and 2.10 min, respectively. The
detector response was linear over the ranges of 10-150% level of standard concentration for
Efavirenz, Tenofovir and Emtricitabine. The method was validated for linearity, precision,
accuracy, solution stability, ruggedness, and post degradation studies were performed. The
results of specificity studies indicated no interference from excipients, impurities, and
degradation products under various stress conditions and assured that the peak response was
due to a single component only. Hence, the present method is cost-effective, faster, and can
be used for the routine analysis of these drugs in pure and formulations.
Keywords: Emtricitabine, Tenofovir disoproxil fumarate, Efavirenz, UPLC.
Introduction
Developing a single analytical method for estimation of individual drugs from a
multidrug composition is a very challenging task. This paper describes a strategy for the
systematic development1-5 of ultrahigh-pressure liquid chromatographic (UHPLC or UPLC)
methods with the objective of reducing analysis time and maintaining good efficiency.
Emtricitabine6 is chemically known as 4-amino-5-fluoro-1-[(2R, 5S)-2-(hydroxymethyl)-1, 3oxathiolan-5-yl] pyrimidin-2-one. Efavirenz7 is chemically known as (4S)-6-chloro-4(cyclopropylethynyl)-1, 4-dihydro-4-(trifluoromethyl) 2-H-3, 1-benzoxazin 2-one. Tenofovir
disoproxil fumarate8 is chemically know as 9-[(R)-2-[[bis [[isopropoxycarbonyl] oxy]
methoxy] phosphonyl] methoxy] propyl] adenine fumarate. Emtricitabine, Tenofovir
disoproxil fumarate, and Efavirenz are a novel formulation combining fixed doses of the
nucleoside reverse transcriptase inhibitors. Emtricitabine (200 mg) and Tenofovir disoproxil
fumarate (300 mg) with the non-nucleoside reverse transcriptase inhibitor Efavirenz (600 mg)
represents the first once-daily, one-tablet antiretroviral regimen. It is official in MartindaleThe Extra pharmacopoeia.
Literature
The literature search indicated that many spectrophotometric11-12 and HPLC13-16 methods
were available for individual and a combination of three drugs with other drugs. Based on the
literature search Gangrade et al.5 proposed a validated UPLC method to determine
Emtricitabine, Tenofovir and Efavirenz simultaneously in synthetic mixture form.
Chromatographic separation was achieved on a BEH Phenyl column using a mixture of
buffer pH 6.5, Methanol and Acetonitrile in the ratio of 45:27.5:27.5 (v/v) at a wavelength of
260 nm. In the present study the authors made efforts to develop a stability indicating
method, which had less retention time, specific and higher selectivity using 0.1% OPA and
Acetonitrile, for Emtricitabine, Tenofovir and Efavirenz, simultaneously.
Development and Optimization of Stability-Indicating HPLC Method
Reagents and chemicals
Analytical grade reagents such as Acetonitrile, orthophosphoric acid, Hydrochloric acid,
sodium hydroxide, hydrogen peroxide and HPLC grade water were procured from Merck
India.
Preparation of mobile phase
Mobile Phase A (0.1% Orthophosphoric acid): 1 ml of Orthophosphoric acid was added to
1000 ml of HPLC water and filtered through 0.22 µ membrane filter.
Mobile Phase B: 100% gradient grade Acetonitrile.
Diluent preparation: Mobile phase A and Mobile Phase B were mixed in the ratio
20:80%v/v.
Preparation of Standard solution
40 mg of Emtricitabine, 60 mg of Tenofovir and 120 mg of Efavirenz were weighed
accurately and transferred to a 200 mL volumetric flask, about 140 mL of diluents were
added and sonicated for 10 min with intermediate shaking. The solution was cooled to
room temperature, diluted up to volume with diluent and mixed well.
Equipment and Chromatographic conditions
The UPLC system used for method development, degradation studies, and validation was
Waters 2695 separation module consisting of binary pump plus autosampler, autoinjector;
SM4 E 07 SM 4094 A (Singapore), online degasser, column oven, and 2996 photodiode array
(PDA) detector. The output signal was monitored and processed using Empower software,
Waters Corporation, Milford, USA (Database Version 6.10.01.00). An Acquity UPLC BEH
Phenyl (2.1 x 100mm, 1.7 µm) column was used for LC studies and to develop the SIAM
(Stability Indicating Assay Method). The flow rate of mobile phase was 0.5 mL/min. The
elution mode was gradient (Table 1) and the column temperature was maintained at 40°C,
and the detection was monitored at a wavelength 265 nm. The injection volume was 10 µL.
Typical chromatograms of blank and standard are given in Figures 4, 5.
Method Validation Parameters: The optimized method was validated in accordance with
the ICH Q2 (R1) guidelines and reported.
Specificity: The results of forced degradation studies of each drug in the presence of their
degradation products indicated a high degree of specificity of this method for Emtricitabine
Efavirenz, and Tenofovir. No interference was observed with blank, placebo and known
impurities with three main peaks.
Linearity and Range: The linearity was established over the ranges of 10-150% level of
standard concentration for Emtricitabine Efavirenz, and Tenofovir. Correlation coefficients
(𝑅2) were found to be 0.999 for all the analytes. Typically, the means of the regression
equations were y = 2567.6x + 4480.4, y = 458.45x + 5181.5 and y = 2367.8x + 5231 for
Emtricitabine Efavirenz, and Tenofovir. Linearity plots are given in Figures 6-8.
Accuracy
Accuracy of the method was determined by performing the recovery experiment for spiked
three analytes to placebo at 50% level, 100% level and 150% level of sample concentration
and the recovery was found good. The recoveries of Emtricitabine Efavirenz, and Tenofovir
were found to be within acceptable ranges of 100 ± 2%. The results are presented in Table 5.
PRECISION
Prepared six samples and calculated the assay for three analytes. The %RSD values for the
intraday and inter day precision were ≤2% confirming that the method was sufficiently
precise. The results are presented in Table 6.
Robustness Test: Robustness of the method was checked by small deliberate changes made
in the method parameters such as wavelength (±2 nm), mobile phase ratio (±2%), flow rate
(±0.1 mL) and temperature (40°C ±2°C) but these changes did not affect the method results.
The results are presented in Table 7.
The Assay
The assay value for the marketed formulation was found to be within the limits as
listed in Table: 8. The low %RSD value indicated the suitability of the method for routine
analysis of Emtricitabine Efavirenz, and Tenofovir in pharmaceutical dosage forms.
Concentration of Emtricitabine is 200 ppm, Tenofovir is 300 ppm and Efavirenz is 600 ppm)
Weighed and finely powder 20 tablets, transfer accurately weighed portion of a powder,
equivalent to one tablet into a 100mL volumetric flask, added about 80mL of diluent,
sonicated for 10 min with intermediate shaking. Diluted up to volume with diluent and mixed
well. Centrifuged the above solution at 4000RPM for 10minutes and then filtered the
supernatant solution through 0.45µm syringe filter. Further diluted 5mL of the above filtered
solution to 50mL with diluent and shake well. Chromatogram of Placebo and sample were
given as Figures 9 and 10.
Stability Studies
The International Conference on Harmonization (ICH) guideline 9-10 entitled stability testing
of new drug substances and products requires that stress testing be carried out to elucidate the
inherent stability characteristics of the active substance. In this study, the drugs were exposed
to different chemical and physical degradation conditions such as 0.5N HCl (acid hydrolysis),
0.05N NaOH (base hydrolysis), 0.3% H2O2 (oxidation), heat (thermal decomposition) and
UV-light (radiation decomposition) for specified time, and then diluted as similar as standard
dilution, and then chromatograms were obtained under the similar chromatographic
conditions, the percent of degradation was calculated from the peak area of the
chromatograms. In the study of acid or base hydrolysis, an amount of fine powdered sample
equivalent to one tablet was transferred into 100 mL volumetric flask and added 10 mL of
freshly prepared 0.5 N HCl/0.0.5 N NaOH, shaken well and allowed for 10minutes at bench
top and neutralized with 0.5N HCl/0.05N NaOH and added about 80 mL of diluent, sonicated
for 10 min with intermediate shaking. Cool to room temperature, dilute up to volume with
diluent and mixed well. Centrifuged the above solution at 4000 RPM for 10 min and then
filtered the supernatant solution through 0.45 µm syringe filter. Further dilute 5ml of the
above filtered solution to 50 ml with diluent and shake well.
In case of peroxide degradation same amount of sample was transferred into 100 mL
volumetric flask, added 10 mL of freshly prepared 0.3% H2O2 and kept on bench top for 5
min added about 80 mL of diluent, sonicated for 10 min with intermediate shaking. Cool to
room temperature, dilute up to volume with diluent and mixed well. Centrifuged the above
solution at 4000 RPM for 10 min and then filtered the supernatant solution through 0.45 µm
syringe filter. Further dilute 5 mL of the above filtered solution to 50 mL with diluent and
shake well. In the study of thermal or Humidity or UV-light degradation, exactly same
amount of fine powdered sample was accurately transferred into a clean and dry watch glass,
placed in an oven at 105˚C for 48 h or UV cabinet-254 nm or Humidity (90%RH at 25°C) for
168 h. (Then removed from the oven or UV chamber and allowed to stand for some time at
room temperature.) The weighed and transferred into 100 mL volumetric flask and added
about 80 mL of diluent, sonicated for 10 min with intermediate shaking. Cool to room
temperature, dilute up to volume with diluent and mixed well. Centrifuged the above solution
at 4000 RPM for 10 min and then filtered the supernatant solution through 0.45 µm syringe
filter. Further dilute 5 mL of the above filtered solution to 50 mL with diluent and shake well.
Injected into UPLC and chromatograms were obtained under optimized conditions. A study
of forced degradation was carried out to evaluate the stability of the drugs in formulations
and the results of degradation and stability of drugs were presented in Tables 9-11.
Conclusions
The present developed Stability Indicating RP-UPLC method was found to be simple, rapid,
accurate and specific for the determination of Emtricitabine, Efavirenz and Tenofovir
disoproxil fumarate in tablet dosages. Finally the simplicity of sample preparation and the
shorter chromatographic runtime gives the method capability for high sample throughput.
From the results of all the validation parameters we can conclude that the present method can
be useful for quality control in any quality control and testing laboratory with desired
precision and accuracy.
References
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Academic Researcher: Validation View Point. Analytical Letters. 1999; 32, 10671080.
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Young and Raymond. J. Weigand, www.alltech web.com
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IFPMA, Geneva.
4. FDA, Guidance for Industry: Impurities in Drug Product, Draft Guidance, Center for
Drug Evaluation and Research (CDER), 1998.
5. Manish G, Avinash K, Ravi Y, Pravish T, Development and validation of uplc
method for emtricitabine, tenofovir and efavirenz in pharmaceutical preparation,
Analytical Chemistry, 2010,Vol. 9, Issue 2,247-251.
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8. http://www.chemspider.com/Chemical-Structure.54859.html
9. International Conference on Harmonization. 1996. Photo stability testing of new drug
substance and products Q1B. International Conference on Harmonization, IFPMA,
Geneva.
10. International Conference on Harmonization. 2003. Stability testing of new drug
substances and products Q1A (R2). International Conference on Harmonization,
IFPMA, Geneva.
11. S. Venkatesan and N. Kannappan, Simultaneous Spectrophotometric Method for
Determination of Emtricitabine and Tenofovir Disoproxil Fumarate in ThreeComponent Tablet Formulation Containing Rilpivirine Hydrochloride, International
Scholarly Research Notices, 541727, 2014, 1-8.
12. K. Anandakumar, K. Kannana, T. Vetrichelvan, Development and Validation of
Emtricitabine and Tenofovir Disoproxil Fumarate in pure and in Fixed Dose
Combination by UV Spectrophotometry, Vol. 6, No 3, July-September 2011, 10851090.
13. Dittakavi R, Bommakanti VP, Tummala VBR, Yadlapalli SR, Golkonda R, Stability
Indicating RP-UPLC Method for Assay of Emtricitabine and Tenofovir Disoproxil
Fumarate in Bulk and Dosage Forms, American Journal of Analytical Chemistry,
2015,vol-6,807-821.
14. N. Srinivasa Rao, Dr. Venkataramana, P. Srinivas, R. Meghana, P. Anitha, Method
Development and Validation for Simultaneous Estimation of Lamivudine, Tenofovir
Disoproxil Fumarate and Efavirenz in Combined Tablet Dosage form by RP-HPLC,
World Journal of Pharmacy and Pharmaceutical Sciences, Volume 3, Issue 10, 16581671,Oct 2014.
15. Shweta H and Sunil RD, Development and validation of a stability-indicating LC
method for the determination of tenofovir disoproxil fumarate in pharmaceutical
formulation, Songklanakarin J. Sci. Technol. 2012 Dec; 34 (6), 615-622.
16. Prashant S. Devrukhakar, RB, Nalini S, and K. V. Surendranath, A Validated
Stability-Indicating RP-HPLC Method for the Simultaneous Determination of
Tenofovir, Emtricitabine, and a Efavirenz and Statistical Approach to Determine the
Effect of Variables, Hindawi Publishing Corporation ISRN Chromatography 878295,
8, 2013.
Figure: 2 Efavirenz
Figure:1 Emtricitabine
Figure: 3 Tenofovir
Chemical Structures of Emtricitabine, Efavirenz and Tenofovir
Figure: 4 Typical Chromatogram of Blank
Figure: 5 Typical Chromatogram of Standard
1000000
y = 2567.6x + 4480.4
R² = 0.9999
500000
0
0
50
100
150
200
250
Concentration (µg/mL)
300
350
Figure: 6 Linearity of Emtricitabine
Mean Peak Area
Mean Peak area
Linearity of Emtricitabine
Linearity of Efavirenz
500000
y = 458.45x + 5181.5
R² = 0.9996
400000
300000
200000
100000
0
0
200
400
600
Concentration (µg/mL)
Figure: 7 Linearity of Efaverinz
800
1000
Mean Peak Area
Linearity of Tenofovir
1500000
y = 2367.8x + 5231
R² = 0.9999
1000000
500000
0
0
100
200
300
400
Concentration ( µg/mL)
Figure: 8 Linearity of Tenofovir
Figure: 9 A Typical Chromatogram of Placebo
Figure: 10 A Typical Chromatogram of Sample
500
Table 1: Gradient Programme
S.No
1
2
3
TIME (minutes)
Initial
2.0
2.2
%A
88
15
88
%B
12
85
12
CURVE
Initial
6
6
4
3.0
88
12
6
Table 2: Linearity for Emtricitabine
% Linearity level
10
50
80
100
120
150
Concentration (ppm)
20
40
100
160
200
240
Response
53360
104826
264275
418770
520670
619734
Table 3: Linearity for Efaverinz
% Linearity level
10
20
50
80
100
120
150
Concentration (ppm)
60.00
120.00
300.00
480.00
600.00
720.00
900.00
Response
30076
58155
145416
229022
282947
333979
414547
Table 4: Linearity for Tenofovir
% Linearity level
10
20
50
80
100
120
150
Concentration (ppm)
30
60
150
240
300
360
450
Response
73108
144217
364062
578304
719454
855781
1066446
Table 5: Accuracy
S.No
Level
1
2
3
50 %
100 %
150 %
Emtricitabine
% Recovery
100.7
100.1
99.0
Efavirenz
% Recovery
99.8
99.1
98.2
Tenofovir
% Recovery
99.4
99.0
98.5
Table 6: Precision
Intraday Precision
Name of
Component
Conc.
(µg/ml)
Mean Amt
Found
(µg/ml)
±SD
Emtricitabine
Tenofovir
Efavirenz
200
300
600
99.733
99.800
99.911
0.115
0.100
0.038
Inter-day Precision
%RSD
Amt.
Found
(µg/ml)
±SD
%RSD
0.116
0.100
0.039
99.600
99.733
99.889
0.200
0.058
0.038
0.201
0.058
0.039
Table 7: Robustness
Chromatographic
conditions
Flow rate
(0.5mL/mi
n)
Tenofovir
263
99.59
Theoret
ical
Plates
11999
267
99.80
11235
1.2
99.82
108742
0.9
98.93
38547
1.1
0.09%
98.91
11458
1.1
98.90
112147
1.0
99.28
39874
1.1
0.11%
99.48
11248
1.0
100.25
109874
1.2
100.02
38745
1.2
0.490
100.50
11148
1.1
99.87
107485
1.4
99.94
39854
1.1
0.51
99.89
11247
1.1
99.58
108954
1.3
98.95
35247
1.0
38°C
99.25
11147
1.2
98.79
110214
1.0
100.21
39852
1.2
42°C
99.51
11874
1.1
99.85
101247
1.0
100.30
38417
1.1
%
assay
Wave
length
(265nm±2
nm)
Mobile
Phase
Variation
(0.1%
OPA)
Efavirenz
Emtricitabine
Asym
metry
%
assay
Theoret
ical
Plates
Asym
metry
%
assay
Theoretica
l Plates
Asy
mm
etry
1.1
99.16
109727
1.0
99.74
39448
1.0
Temperatu
re (40°C)
Table 8: Assay of Synthetic Mixture of Components
Drug
Emtricitabine(200mg),
Labeled
Amount
(µg/ml)
200
Amount
taken(µg/ml)
Mean(± S.D)
199.224±0.01050
600
300
Efavirenz (600mg)
% Label
Claim
%RSD
99.612
0.4217
599.196±0.01527
99.866
0.3059
299.466±0.0057
99.822
0.0771
and Tenofovir
(300mg)
Table 9: Degradation data of Emtricitabine
Degradation conditions
Emtricitabine
%Assay
% Degradation
Purity angle
Purity
Threshold
Unstressed Sample
0.5N HCl at Bench top for 5min
99.7%
N/A
0.140
0.282
94.80%
4.90%
0.144
0.285
0.05N at Bench top for 5 min
94.20%
5.50%
0.152
0.286
Thermal 105°C for 48 h
92.80%
6.90%
0.171
0.287
UV Light at 254nm for 168 h
93.30%
6.40%
0.149
0.281
90.50%
9.20%
0.138
0.28
96.10%
3.60%
0.235
0.27
Humidity(90%RH at 25°C) for
168 h
0.3% H2O2 at Bench top for 5
min
Table 10: Degradation data of Efavirenz
Degradation conditions
Efavirenz
%Assay
% Degradation
Purity angle
Purity
Threshold
Unstressed Sample
0.5N HCl at Bench top for 5min
99.9%
N/A
2.265
8.033
94.90%
5.00%
1.985
7.849
0.05N at Bench top for 5min
94.40%
5.50%
1.9
7.698
Thermal 105°C for 48 h
96.10%
3.80%
2.452
7.698
UV Light at 254nm for 168 h
95.10%
4.80%
2.078
8.182
93.50%
6.40%
2.141
7.891
95.80%
4.10%
2.196
8.084
Humidity(90%RH at 25°C) for
168 h
0.3% H2O2 at Bench top for 5
min
Table 11: Degradation data of Tenofovir
Degradation conditions
Tenofovir
%Assay
% Degradation
Purity angle
Purity
Threshold
99.0%
N/A
0.127
0.369
89.90%
9.10%
0.298
0.3382
0.05N at Bench top for 5 min
89.30%
9.70%
0.298
0.388
Thermal 105°C for 48 h
88.50%
10.50%
0.16
0.382
UV Light at 254nm for 168 h
94.20%
4.80%
0.143
0.375
95.50%
3.50%
0.137
0.342
91.80%
7.20%
0.237
0.28
Unstressed Sample
0.5N HCl at Bench top for 5
min
Humidity(90%RH at 25°C) for
168 h
0.3% H2O2 at Bench top for 5
min