Simultaneous UV
Spectrophotometric Method for the Determination of Tenofovir,
Efavirenz and Lamivudine in Bulk and Combined Dosage
Form
S. Vidyadhara, RLC Sasidhar*,
B. Venkateswara Rao, P. Ratna Kumari
Chebrolu Hanumaiah Institute of
Pharmaceutical Sciences, Chowdavaram, Guntur – 19, Andhra Pradesh, India.
*Corresponding Author E-mail: rlcsasidhar@gmail.com
ABSTRACT:
The aim of the present
work is to develop a simple, accurate, precise and reproducible UV
spectrophotometric method for the simultaneous determination of Tenofovir, Efavirenz and Lamivudine
in bulk and pharmaceutical dosage form. The stock solutions of the standard
drugs were prepared in methanol followed by the further required dilutions with
distilled water. This method involves the formation and solving of simultaneous
equations at 260nm, 347nm and 272 nm, as absorbance maxima of Tenofovir, Efavirenz and Lamivudine
respectively. Beer’s law obeyed the concentration range of 10 – 40mcg/mL, 5 – 20 mcg/mL and 5 – 20 mcg/mL for Tenofovir, Efavirenz and Lamivudine respectively. The results of analysis were
validated statistically and by recovery studies. The % RSD for the recovery
study was less than 2. The proposed method can be effectively used for the
simultaneous estimation of these three drugs in bulk and combined tablet dosage
form.
KEYWORDS:
Tenofovir, Efavirenz and Lamivudine,
Simultaneous equation method, Method validation.
INTRODUCTION:
Tenofovir(TNF) is Bis{[(isopropoxycarbonyl)oxy]methyl} ({[(2R)-1-(6-amino-9H-purin-9-yl)-2-propanyl]oxy}methyl)
phosphonate. Tenofovir is
an antiretroviral medication used to prevent and treat HIV/AIDS and to treat
chronic hepatitis B The active substance is tenofovir,
while tenofovir disoproxil
is a prodrug that is used because of its better
absorption in the gut. Many analytical methods for TNF alone or in combination
with other drugs including spectroscopic and chromatographic methods are
reported in literature(1-3).
Figure 1: Chemical structure of Tenofovir.
Efavirenz (EFV), (s)-6-chloro-(cyclopropyl
ethynyl)-1, 4-dihydro-4-(trifluoro
methyl)-2H-3, 1-benzoxazin-2-one. Efavirenz is a non-nucleoside reverse
transcriptase inhibitor (NNRTI). It is used as part of highly active
antiretroviral therapy (HAART) for the treatment of a human immunodeficiency
virus (HIV) type 1. Various analytical methods have been reported for the assay
of AMB alone or in combination with other anti- hypertensive agents in
pharmaceutical formulations. They include UV spectroscopy, high performance
liquid chromatography, high performance thin layer chromatography, LC – MS
and LC - MS/ MS(4-8).
Figure 2: Chemical structures of Efavirenz.
Lamivudine (LMV) is 4-amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one.Lamivudine
is an antiretroviral medication used to
prevent and treat HIV/AIDS . It is also used to treat chronic hepatitis B when
other options are not possible. It is effective against both HIV-1 and HIV-2.
It is typically used in combination with other antiretrovirals.
Various analytical methods have been reported for the assay of LMV alone or in
combination with other agents. They include UV spectroscopy, high performance
liquid chromatography. All three drugs are official in IP. Literature survey
revealed that there are several methods were reported for the estimation of
TNF, EFV and LMV individually as well as in combination with some other drugs.
The aim of the present study was to develop accurate, precise and sensitive
method for simultaneous UV spectrophotometric estimation of TNF, EFV and LMV in
bulk and in combined tablet dosage form (9-13).
Figure 3: Chemical structure of Lamivudine.
MATERIALS AND METHODS:
Instrumentation:
The analytical estimations were carried out
on LAB INDIA double beam UV - Visible spectrophotometer with pair of 10 mm
matched quartz cells. Glassware used were of ‘A’ grade and were soaked
overnight in a mixture of chromic acid and sulphuric
acid, rinsed thoroughly with double distilled water and dried in hot air oven.
Reagent and chemicals:
Analytical grade standard samples of TNF,
EFV and LMV were obtained as a gift samples from Mylan
laboratories. All solvents used for the study were of analytical grade and
commercially obtained from S.D. Fine chem. Limited, Mumbai. A combination of
TNF (300mg), EFV (600mg) and LMV (300mg) in tablet formulation was procured
from local pharmacy manufactured as Vonaday by Emcure Pharmaceutical Pvt. Ltd, India.
Experimental Condition:
Based upon the solubility studies for the
three drugs methanol was found to be the common solvent for the drugs. Hence
the stock solution was prepared in methanol and further dilutions were made up
with distilled water.
Preparation of standard stock solution:
25 mg of TNF, EFV, and LMV were accurately weighed and transferred in to 25 mL volumetric flasks separately. Dissolved in methanol and
made up to the volume to 25 mL with the same. These
solutions were observed to contain 1000 mcg/mL of
TNF, EFV and LMV respectively.
Study of
Spectral and Linearity Characteristics:
The standard stock solutions of TNF, EFV
and LMV were further diluted with distilled water to get the concentration of
10 mcg/ mL of each and the solutions were scanned
between the range 200 - 400 nm in 1cm cell against distilled water as blank and
the overlain spectra was recorded.
In quantitative estimation of three
components by simultaneous equation method, three wavelengths i.e. 260nm,
λ max of TNF; 247nm, λ max of EFV and 272nm, λmax
of LMV were analysed from their maximum λ max. A set of three simultaneous equations were
framed using absorptivity coefficients at selected
wavelengths. The concentrations of three drugs in the mixture were calculated
using the following equations.
Cx= 
CY= 
CZ= 
Where,
A1, A2 and A3
are absorbance of sample solution at 260 nm, 247 nm and 272 nm, respectively.
ax1, ax2 and ax3
are absorptivity coefficients of TNF at 260 nm, 247
nm and 272 nm, respectively.
ay1, ay2 and ay3
are absorptivity coefficients of EFV at 260 nm, 247
nm and 272 nm, respectively.
az1, az2 and az3
are absorptivity coefficients of LMV at 260 nm,
247 nm and 272 nm, respectively.
Cx, Cy and Cz
are concentrations of TNF, EFV and LMV respectively in mixture.
The aliquot portions of standard stock
solution of HCT, AMB and TEL were transferred into 100 mL
volumetric flasks individually and made up to the volume with distilled water.
The calibration curves for TNF, EFV and LMV were prepared in the concentration
range of 10-40µg/ml, 5-20µg/ml and 5-20µg/ml, respectively at all selected
wavelengths. The simultaneous equations were constructed as follows by using absorptivity coefficient values.
At 260nm,
A1=
200.2 Cx + 51.3 CY +130.2 CZ
At 247nm,
A2=
70.6 Cx +
400.5CY + 21CZ
At 272nm,
A3=
75 Cx + 26.1 CY + 400.2 CZ
Analysis of Tablet Formulation:
Twenty tablets were weighed and average
weight was found. The tablets were triturated to a fine powder. An accurately
weighed quantity of powder equivalent of
LMV was transferred in to 100 mL volumetric flask and
added a minimum quantity of methanol to dissolve the substance and made up to
the volume with the same. The solution was sonicated
for 15 minutes, centrifuged for another 15 minutes at 100 rpm and filtered through
Whatmann filter paper No. 41. From the clear
solution, further dilutions were made by diluting 1.0 mL
into 100 mL with distilled water to obtain 10 mcg/ mL solution of which
is also contains 30 mcg/ mL of TNF and 60 mcg/ mL of EFV theoretically. The absorbance of sample solutions
were measured at all selected wavelengths. The content of HCT, AMB and TEL in
sample solutions of tablet were calculated. This procedure was repeated for six
times.
Method validation (14-15):
The methods were validated with respects to
linearity, LOD (Limit of detection), LOQ (Limit of quantitation),
precision, accuracy and ruggedness.
Linearity:
Linearity was checked by preparing standard
solutions at six different concentration ranges from 10-40 mcg/ mL of TNF, 5-20 mcg/ mL of EFV
and 5-20 mcg/ mL of LMV. Calibration curves (n = 6)
were plotted between concentration and absorbance of drugs. Optical parameters
were calculated.
Sensitivity:
The limit of detection (LOD) and limit of quantitation (LOQ) parameters were calculated using the
following equations; LOD = 3.3σ/ s and LOQ = 10σ/ s, where σ is
standard deviation of y intercept of calibration curve (n = 6) and s is slope
of regression equation.
Precision:
The precision of the method was confirmed
by repeatability and intermediate precision. The repeatability was performed by
the analysis of formulation was repeated for six times with the same
concentration. The amount of each drug present in the analytical sample was
calculated. The % RSD was calculated. The intermediate precision of the method
was confirmed by intraday and inter day analysis i.e. the analysis of
formulation was repeated three times in the same day and on three successive
days. The amount of drugs was determined and % RSD also calculated.
Accuracy:
To check the accuracy of the developed
method and to study the interference of formulation excipients,
analytical recovery experiments were carried out by using standard addition
method in three different concentrations. From the total amount of drug found,
the percentage recovery was calculated. This procedure was repeated for three
times for each concentration. The % RSD was calculated.
Ruggedness:
The ruggedness test of analytical assay
method is defined as the degree of reproducibility of test results obtained by
the analysis of the same samples under a variety of normal test conditions such
as different labs, different analysis, different lots of reagents etc.
Ruggedness is a measure of reproducibility of test results under normal
expected operational conditions from laboratory to laboratory and from analyst
to analyst. In present study, determination of TNF, EFV and LMV were carried
out by using different instruments and different analysts.
RESULTS AND DISCUSSION:
The proposed method is based on spectrophotometric
simultaneous estimation of TNF, EFV and LMV in UV region using methanol and
distilled water as solvent.
Figure.4: Overlain UV spectra of TNF, EFV and LMV
The isobestic
point for the three drugs is taken as 260nm. The overlain spectra showed the
maximum absorbance at 260 nm for TNF, 247 nm for EFV and 272nm for LMV (Figure
4). This was involved in the construction and solving of simultaneous
equations using absorptivity coefficient values. The
stability was performed by measuring the absorbance of same solution at
different time intervals. It was observed that TNF, EFV and LMV were stable for
up to 4 hours at all selected wavelengths.
Beer’s law obeyed in the concentration
range of 10-40 mcg/ mL, 5-20 mcg/ mL
and 5-20 mcg/ mL for TNF, EFV and LMV, respectively.
The correlation coefficient values were found above 0.999, which shows that
absorbance of all the drugs was linear with concentration. The optical
characteristics such as Beer’s law limits, correlation coefficient, slope, intercept,
Sandell’s sensitivity and molar absorptivity
were calculated and are summarized in (Table 1).
The LOD and LOQ were found to be 0.48 and
1.45,0.42 and 1.29,0.44 and 1.35 for
TNF, EFV and LMV, respectively. The low values indicate the sensitivity of the
simultaneous equation method.
The percentage assay values present in
tablet formulation was found to be 99, 99.02,100 for TNF, EFV and LMV,
respectively (Table 2). Precision of
the method was confirmed by the repeated analysis of formulation for six times.
The % RSD values were found to be 0.44, 0.79 and 0.46 for TNF, EFV and LMV,
respectively (Table 2). The low %
RSD values indicated that all the three drugs showed good agreement with the
label claim ensures the precision of the method (Table 3).
Further, the precision of the method was
confirmed by Intraday and Inter day analysis. The % RSD values for intraday and
inter day analysis was found to be 0.14 and 0.20 for TNF ,0.8
and 0.7 for EFV and 0.10 and
0.11 for LMV, respectively. Hence the precision
of the method was further confirmed.
Table 1: Spectral and linearity characteristics data
|
Parameters
|
TNF*
|
EFV*
|
LMV*
|
|
λmax (nm)
|
260 nm
|
247 nm
|
272 nm
|
|
Linearity
range (mcg/ mL)
|
10-40
|
5-20
|
5-20
|
|
Correlation
coefficient (r2)
|
0.999
|
0.999
|
0.999
|
|
Molar absorptivity (L mol-1 cm-1)
|
200.2
|
400.5
|
400.2
|
|
Sandell’s sensitivity (μg/ cm2/
0.001A.U)
|
0.04
|
0.02
|
0.02
|
|
Slope (m)
|
0.022
|
0.044
|
0.045
|
|
Intercept
(c)
|
0.003
|
0.007w
|
0.003
|
|
Regression
equation (y = mx
+ c)
|
y = 0.022x
+ 0.003
|
y = 0.044x + 0.007
|
y= 0.045x
+ 0.003
|
|
LOD (mcg/ mL)
|
0.48
|
0.42
|
0.44
|
|
LOQ (mcg/ mL)
|
1.45
|
1.29
|
1.35
|
|
Standard
Error
|
0.0004
|
0.0020
|
0.0016
|
* Mean of six observations.
Table
2: Results of analysis of tablet formulation.
|
Parameters
|
TNF
|
EFV
|
LMV
|
|
Labeled Claim (mg)
|
300
|
600
|
100
|
|
% Assay*
|
99
|
99.02
|
100
|
|
SD
|
0.0017
|
0.0023
|
0.0021
|
|
%RSD
|
0.4489
|
0.7914
|
0.4659
|
* Mean of
six determinations.
Table 3: Intermediate Precision and Ruggedness of the
method.
|
Parameters
|
% RSD
|
|
TNF
|
EFV
|
LMV
|
|
Intraday Precision (n=6)
|
0.14-0.19
|
0.8-0.9
|
0.10-0.16
|
|
Interday precision (n=6)
|
0.20-0.35
|
0.7-0.8
|
0.11-0.17
|
|
Different
instruments (n=6)
|
|
|
|
|
Instrument 1
|
0.17-0.19
|
0.7-0.9
|
0.74-0.96
|
|
Instrument 2
|
0.14-0.19
|
0.8-0.9
|
0.77-0.92
|
|
Different
analyst (n=6)
|
|
|
|
|
Analyst 1
|
0.14-0.29
|
0.5-0.7
|
0.32-0.45
|
|
Analyst 2
|
0.18-0.25
|
0.5-0.8
|
0.30-0.39
|
Table 4: Recovery studies
|
Recovery
level
%
|
Amount
of Standard drug added (µg/ml)
|
Amount
of test added(µg/ml)
|
Total
Amount Recovered (µg/ml)
|
%
Recovery w/w
|
|
TNF
|
EFV
|
LMV
|
TNF
|
EFV
|
LMV
|
TNF
|
EFV
|
LMV
|
TNF
|
EFV
|
LMV
|
|
80
|
4
|
6
|
6
|
20
|
10
|
10
|
23.90
|
15.97
|
15.95
|
99.58
|
99.81
|
99.6
|
|
100
|
10
|
10
|
10
|
20
|
10
|
10
|
30.04
|
20.05
|
20.06
|
100.1
|
100.25
|
100.3
|
|
120
|
16
|
14
|
14
|
20
|
10
|
10
|
35.98
|
23.96
|
24.02
|
99.9
|
99.84
|
100.0
|
*Mean of six observations
The developed method was validated for
Ruggedness. The analysis of formulation was done by using different instruments
and different analysts. The % RSD values were found to be less than 2
indicating that the method was more rugged. The results of analysis of
intermediate precision and ruggedness are shown in (Table 3).
In order to check the accuracy of the
developed method, known quantities of standard drugs of TNF, EFV and LMV in
three different concentrations were added to its pre-analysed
sample and analysed by the developed method. The
percentage recovery was found to be in the range of 99.58-99.9% for TNF,
99.81-100.25% for EFV and 99.6-100.0% for LMV. The results of recovery studies
are shown in Table
4. The % RSD values for TNF, EFV and LMV were found to be 0.49, 0.39 and
0.38, respectively. The low % RSD values confirm that there is no interference
due to the excipients used in formulation. This
ensures the accuracy of the method.
From validation, the developed method was
found to be simple, rapid, economical, precise, accurate and rugged. Hence the
proposed method could be effectively applied for the routine analysis of TNF,
EFV and LMV in bulk and in combined tablet dosage form.
ACKNOWLEDGEMENTS
The
authors are thankful to the Mylan Labs limited,
Hyderabad for providing gift sample of Telmisartan, amlodipine besylate and hydrochlorothiazide and
also to the management of Chebrolu Hanumaiah Institute of Pharmaceutical Sciences, Chowdavaram, Guntur for providing facilities and great
support to carry out the research work.
REFERENCES:
1.
Saminathan J and Vetrichelvan T.
Development and Validation of HPTLC Method for Simultaneous Estimation of Emtricitabine, Rilpivirine and Tenofovir
Disoproxil Fumarate in
Combined Dosage form. Bangladesh Pharmaceutical Journal. 19(1); 2016: 114-121.
2.
Syeda K, Vidya Sagar G. Determination of Tenofovir
and Emtricitabine by Simultaneous Equation
Spectrophotometric Method. IOSR
Journal of Pharmacy and Biological Sciences. 11(3); 2016:111-115.
3.
Abdelhay MH, Gazy AA, Shaalan RA and Ashour HK.
Selective RP-HPLC DAD Method for Determination of Tenofovir
Fumarate and Emtricitabine
in Bulk Powder and in Tablets. Acta Chromatographica. 2015: 41–54.
4.
Amit Patel, Ami
Patel and Ashlesha
M. Development and of Validation an
Accurate and Precise LC-MS/MS Method for Determination of Tenofovir
and Emtricitabine in Human Plasma. International
Journal of Pharmaceutical, Chemical and Biological Sciences, 5(1); 2015:
372-380.
5.
Sunitha PG, Kaliappan I. Validated First-order Derivative
Spectrophotometry for Simultaneous Determination of Emtricitabine
and Tenofovir Disoproxil Fumarate in Pharmaceutical Dosage Form. Journal of Drug
Delivery and Therapeutics. 4(2); 2014: 9-11.
6.
Sampreeth Kumar G, Pritosh P, Arabinda P and Subrahmanyam KV. A New Method Development and Validation of
Efavirenz by RP-HPLC. International Journal of Innovative Pharmaceutical
Sciences and Research. 2(12); 2014:
2962-2970.
7.
Srinath A, Sneha B, Akhila
A, Rayees Ahmed and Kulkarni
RG. Method Development and Validation for Simultaneous Estimation of Lamivudine, Tenofovir and
Efavirenz in combined tablet dosage form by RP-HPLC and UV-Spectroscopic
Method. International Journal of
Pharmaceutical Sciences and Research. 5(12); 2014:5491-5497.
8.
Prathap B, Akalanka D and Srinivasa Rao GH. Analytical Method Development and Validation of Invitro Dissolution Studies of Efavirenz, Lamivudine and Tenofovir Disoproxil Fumarate in
Pharmaceutical Dosage form by RP-HPLC. Journal of Global Trends in Pharmaceutical Sciences. 5(2) ; 2014: 515–520.
9.
Vanaja P, Anusha N and Giri Prasad VS.
Development and Validation of a RP-HPLC Method for Simultaneous Estimation of Lamivudine, Tenofovir Disoproxil Fumarate and Efavirenz
in a combined tablet dosage form. International Journal of Pharmacy and Pharmaceutical Sciences. 5(3); 2013: 1-5.
10.
Anandakumar K, Abirami G, Murugan S and Ashok B. RP-HPLC method for
simultaneous estimation of lamivudine, tenofovir disoproxil fumarate and efavirenz in tablet formulation. Journal of
Analytical Chemistry. 68(9);
2013: 815-821.
11.
Soumya B, Manish Kumar T,
Raghunandhan N. Simultaneous Determination of Tenofovir disoproxil fumarate and Lamivudine by UV
Spectrophotometric Method. International
Journal of Pharmacy and Pharmaceutical Science Research. 2(1); 2012: 9-15.
12.
Indian Pharmcopoeia, Vol. II, Govt. of India, Ministry of Health
and Family Welfare, Published by the Controller of Publication, New Delhi.
2007, 714, 318.
13.
.British
Pharmacopoeia, Vol. I, Her Majesty’s Stationary Office, London, 2009, 137, 565.
14.
ICH
Q2A; Guidelines on validation of analytical procedure; Definitions and
terminology, Federal Register, 1995, 60, 11260.
15.
ICH
Q2B; Guidelines on validation of analytical procedure; Methodology, Federal
Register, 1996, 60, 27464.
