A Simple RP-HPLC Method to Simultaneously Assay the
Contents of Lamivudine, Tenofovir, and Nevirapine in Fixed Dose
Combined Oral Antiviral Medicines
Phoebe Esinam Goku ,
Emmanuel Orman ,
Anna Naa Kwarley Quartey,
Joseph Kwasi Adu ,
and Reimmel Kwame Adosraku
Department of Pharmaceutical Sciences, School of Pharmacy, Central University, Accra 23321, Ghana
Department of Pharmaceutical Chemistry, School of Pharmacy, University of Health and Allied Sciences, Ho 23321, Ghana
Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences,
Kwame Nkrumah University of Science and Technology, Kumasi 23321, Ghana
Correspondence should be addressed to Emmanuel Orman; firstname.lastname@example.org
Received 3 July 2020; Accepted 10 August 2020; Published 8 September 2020
Academic Editor: Patricia E. Allegretti
Copyright ©2020 Phoebe Esinam Goku et al. is is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
An accurate and rapid reverse HPLC method has been developed and validated for the simultaneous quantiﬁcation of lamivudine,
nevirapine, and tenofovir disoproxil fumarate. Suitable separation was achieved on Phenomenex Synergi C18 (250 ×4.6 mm, 4 μm)
using mobile phase, methanol (50%): ammonium acetate buﬀer (adjusted to pH 2.80) (40%): acetonitrile (10%) in an isocratic mode.
e drugs were detected at 270 nm with a ﬂow rate of 1.0 ml/min, and the retention times were found to be 3.26, 5.42, and 7.55
minutes for lamivudine, nevirapine, and tenofovir disoproxil fumarate, respectively. e developed method was validated per ICH
guidelines. Good linearity was obtained within the concentration ranges of 10–59 µg/ml, 7–42 µg/ml, and 15–90 µg/ml with a
correlation coeﬃcient of not less than 0.990. e % RSD values for precision (intraday and interday) and accuracy studies were found
to be less than 2%. e results obtained from quantitative analysis conform to USP content requirements for marketed tablet dosage
forms, RICOVIR-LN, and tenofovir disoproxil fumarate/lamivudine tablets. e method is therefore useful for routine quality
control of antiretroviral tablet dosage forms containing tenofovir disoproxil fumarate, lamivudine, and nevirapine.
HIV/AIDS is a major public health issue and as such forms a
signiﬁcant part of the Sustainable Development Goals, with
the aim of ensuring healthy lives and promoting well-being
for all at all ages . e introduction of highly active
antiretroviral therapy (HAART), a treatment regimen
comprising of the combination of three or more anti-
retroviral drugs , has revolutionized the management of
the disease condition, with the resultant dramatic reduction
in mortality rates and the incidences of opportunistic in-
fections . In Ghana and other parts of the world, three of
the widely used antiretrovirals (ARVs) in HAART include
lamivudine (3TC), nevirapine (NVP), and tenofovir
disoproxil fumarate (TDF). It may be argued that a lasting
impact on morbidity and mortality on people living with
HIV/AIDS (PLWHA) could be assured if the quality of
ARVs was adequately monitored. With recent reports of
substandard medicines available in Ghanaian health facili-
ties , it has become particularly important to increase
surveillance on the quality of all medicines procured for
public health facilities, especially for antiretrovirals, used for
a signiﬁcant public health intervention programme. is
therefore calls for the development and validation of reliable
analytical methods to achieve such quality control purposes.
In the past, most of the methods developed had focused
on the detection and quantitation of ARVs in biological
samples [5, 6]; just a handful had concentrated on methods
Journal of Chemistry
Volume 2020, Article ID 4618360, 9 pages
for the oral dosage formulations [7–9]. In as much as these
methods have provided useful tools to assess the qualities of
some ARVs, it is necessary that very eﬃcient alternative
methods with the target scope of ARVs in the HAART
programme be developed to achieve similar quality control
outputs. e aim of the current study therefore was to
develop a simple, aﬀordable, and reliable method for the
quality assessment of ARVs used in the HAARTprogramme
in Ghana and other parts of the world, where applicable.
us, the method when developed would be used for the
analyses of 3TC, NVP, and TDF in monotherapies and ﬁxed-
dose combination products as well.
3TC is chemically known as 4-amino-1-[(2R,5S)-2-
. It is a nucleoside reverse transcriptase inhibitor and is
available for the treatment of HIV-I and HIV-II infections,
as well as hepatitis B virus . TDF is a derivative of
adenosine S′-monophosphate and is a prodrug .
Chemically, it is known as [[(1R)-2(6-amino-9H-purin-9-
yl)-methylethoxy] methyl] phosphonate, bis (iso-
propyloxycarbonyloxymethyl ester), fumarate (1 : 1). It in-
hibits both HIV-I and HIV-II. It is a nucleotide analogue
that is available for use in antiretroviral therapy . NVP,
on the other hand, belongs to the non-nucleoside reverse
transcriptase inhibitor class of ARVs. Its chemical name is
11-cyclopropy l-4-methy l-5, 11-dihydro [3, 2b: 2′, 3′-e] [1,4]
diazepin-6-one hemihydrate. It is approved for the treat-
ment of HIV infections in adults and children in combi-
nation with other antiretroviral agents .
2. Materials and Methods
2.1. Standards and Samples. e working standards used for
the study included conﬁrmed 3TC (assay: 102.0%), NVP
(assay: 100.1%), and TDF (assay: 100.2%) (Table 1), which
were donated by Danadams Pharmaceutical Industry
Limited, Spintex, Ghana. e drug products, samples A1
(containing TDF 300 mg/3TC 300 mg tablets + NVP
200 mg) and A2 (containing TDF 300 mg/ 3TC 300mg) both
claimed to be manufactured by Mylan Laboratories Limited,
India, were used.
2.2. Chemicals and Reagents. e reagents and solvents used
to prepare the samples and carry out the analyses were of
analytical and HPLC grades, respectively. Solvents including
acetonitrile and methanol were procured from Fisher Sci-
entiﬁc, United Kingdom. Ammonium acetate and glacial
acetic acid purchased from VWR International Limited and
Merck House, respectively, were also used for the analysis.
Puriﬁed water was freshly produced in-house, terminally
sterilized with ultraviolet radiation, and ﬁltered through a
0.45 µm membrane ﬁlter before being used to prepare all
solutions and buﬀers.
2.3. Equipment and Instrument. e HPLC system used
comprised of Shimadzu prominence UFLC series system,
consisting of LC-20A quaternary pump (part G1311 A),
in-line vacuum degasser (part no. G1322A), and
SPD-20A ultraviolet detector. Data acquisition was per-
formed by LC solutions software (version A.10.02 Build
1757). e chromatographic separation was carried out
using on a C18 Phenomenex Synergi column (250 ×4.6 mm;
4µm). An electronic analytical balance (Mettler Toledo,
AB204-S/FACT), a digital pH meter (Mettler Toledo Seven
Compact pH/Ion S220), and a sonicator were also used.
2.4. Preparation of Solutions
2.4.1. Buﬀer Preparation. A 500 ml of 0.02 M ammonium
acetate buﬀer was prepared by weighing a determined
quantity of ammonium acetate powder, dissolving with
some amount of distilled water, and transferred into a 500 ml
volumetric ﬂask. e pH was adjusted to 2.8 with acetic acid,
and with continuous stirring, the solution was topped up
with distilled water to the required volume. It was then
ﬁltered with a 0.45 µm membrane ﬁlter.
2.4.2. Mobile Phase/Diluent Preparation. e solvent system
used was a mixture of buﬀer (pH �2.8), methanol, and
acetonitrile in the ratio 40 : 50 : 10 (v/v), respectively. is
solution was also used as the diluent to prepare solutions of
the standards and samples.
2.4.3. Preparation of Standard Solutions. A stock standard
solution containing 75 mg of 3TC, 35 mg of NVP, and 50 mg
of TDF was prepared in a 100 ml volumetric ﬂask. e
content in the ﬂask was sonicated at 37°C for 10 minutes
before topping up to the required volume with the diluent,
after cooling. e resulting solution was ﬁltered with a
0.45 µm membrane ﬁlter. 3 ml of the stock solution was
pipetted to prepare 50 ml of working standard solution
containing 45 µg/ml of 3TC, 21 µg/ml of NVP, and 30 µg/ml
2.4.4. Preparation of Sample Solutions. Solutions of pow-
dered product samples A (containing TDF and 3TC) and B
(containing two formulations, with B
containing TDF and
3TC and B
containing NVP) were prepared for analysis. For
samples A and B
, a weight of each containing an equivalent
of 75 mg of 3TC was weighed and transferred into a 100 ml
volumetric ﬂask. About 50 ml of methanol was added,
sonicated for 10 minutes at 37°C, and made up to volume
with the diluent. e resulting solution was then ﬁltered
through a Whatman No. 1 ﬁlter paper, discarding the ﬁrst
5 ml of the ﬁltrate. 3 ml of the resulting ﬁltrate was pipetted
and transferred into a 50 ml volumetric ﬂask and made up to
volume using the diluent. For sample B
, a weight containing
an equivalent of 35 mg of NVP was transferred into a 100ml
volumetric ﬂask and a similar procedure as described above
was employed to prepare the sample B solution.
2.5. Development and Validation of Method. e method for
detection, separation, and quantitation of the three anti-
retroviral drug substances in the product was developed
empirically (S1), and validated in accordance with the
2Journal of Chemistry
International Council for Harmonisation (ICH) Q2(R)
guidelines . e validation parameters investigated in-
cluded linearity and its range, limits of detection and
quantitation, speciﬁcity, stability of test solution, robustness,
accuracy, and precision.
2.5.1. Speciﬁcity/Selectivity. Speciﬁcity and selectivity were
assessed by comparing the chromatograms from a matrix
without expected analytes (blank sample) with that of
matrices containing the expected analytes (that is, 3TC,
NVP, and TDF) [13, 14]. e selectivity/speciﬁcity was
conﬁrmed by comparing mean ±SD of the retention times
for the analytes using one-way ANOVA followed by Tukey’s
post hoc test .
2.5.2. Accuracy. Accuracy was investigated by determining
recoveries of the analytes at three concentration levels (80%,
100%, and 120%) [16, 17].
2.5.3. Precision. Precision was demonstrated by determin-
ing repeatability (intraday precision) and intermediate
precision of the method. Intraday precision was conﬁrmed
Table 1: Chemical data on drug substances considered in the study.
substance Code Chemical structure Mol
wt. (g/mol) cLog P pKa Solubility
229.3 −1.46 13.90
Soluble in water,
sparingly soluble in
soluble in ethanol
635.5 2.65 3.75
Slightly soluble in
water, soluble in
slightly soluble in
266.3 2.65 2.8
in water, sparingly
soluble or slightly
soluble in methylene
soluble in methanol
Journal of Chemistry 3
from the peak areas of the analytes, from triplicate injections
of three dilute concentrations of the standard solution
within the same day (that is, 15 µg/ml, 45 µg/ml, and 90 µg/
ml for 3TC; 7 µg/ml, 21 µg/ml, and 42 µg/ml for NVP; and
10 µg/ml, 30 µg/ml, and 59 µg/ml for TDF). Intermediate
precision, on the other hand, involved studying the variation
in response at 100% concentration of the working standard
on three diﬀerent days. e results obtained were statistically
analysed by determining the relative standard deviations and
carrying out ANOVA, where applicable .
2.5.4. Linearity and Range. e linearity of the developed
method was investigated by injecting six concentrations
prepared from the stock standard solution, to obtain con-
centrations for 3TC, NVP, and TDF within the ranges of
15 µg/ml–90 µg/ml, 7 µg/ml–42 µg/ml, and 10 µg/ml–59 µg/
ml, respectively . Triplicate determinations were carried
out for each test concentration and the peak areas, reported
as mean ±SD were plotted against test concentrations.
Statistical analysis was performed by the least-squares
method . Linearity was predicted by estimating the
regression coeﬃcient (R
) and the linear regression y-in-
tercept of the response versus concentration plot. e re-
gression model was also tested for ﬁtness by determining the
level of signiﬁcance of the F-value of the model in an
ANOVA at 5% risk level . Additionally, the residual plots
for the sets of test data were generated.
2.5.5. Limit of Detection (LOD) and Limit of Quantitation
(LOQ). e limits of detection and quantiﬁcation were
determined from the intercept on the y-axis slope from the
linear regression model derived from the linearity test .
e formulae below were used for calculating the LOD and
LOQ. e results are shown in Table 2.
LOD �3.3×standard deviation of the response
slope of the calibration curve ,
LOQ �10 ×standard deviation of the response
slope of the calibration curve .
2.5.6. Robustness. e robustness of the developed method
was tested by monitoring the eﬀects of deliberate changes in
the ﬂow rate (±0.1 ml/min) and the wavelength of detection
(±2 nm) on the general performance of the developed
method [13, 18].
2.5.7. Stability of Test Solution. e stability of the working
standard solution was assessed over a 6-hour period at room
temperature by monitoring the peak areas of the drug
substances with time .
2.6. Analysis of Commercial Products. e contents of 3TC,
NVP, and TDF in samples A, B
, and B
as prepared above
were assessed from peak areas from triplicate injections of
the samples and applying the linear regression models ob-
tained to the recorded peak areas.
3. Results and Discussion
3.1. Method Development. e chromatographic method
was developed empirically, guided by the physicochemical
properties like acid dissociation constant (pKa) and partition
coeﬃcient (cLogP) (Table 1). e conditions comprising
suitable column, mobile phase composition, wavelength for
analyte detection, ﬂow rate, column temperature, and in-
jection volume were determined empirically by monitoring
the resolution, peak symmetry, and run time for the analytes
(S1). e investigations resulted in the choice of a mobile
phase system consisting of methanol (50%), ammonium
acetate buﬀer pH of 2.8 (40%) and acetonitrile (10%), and a
ﬂow rate of 1 ml/min, among others (Table 3), which were
then used to develop the chromatographic method
3.2. Method Validation. e results from the validation are
summarized in Table 2. In establishing the speciﬁcity and
selectivity of the developed method, the chromatogram
generated from the blank sample showed only noises, with
no apparent peak observed within 0–20 minutes (S2). Upon
injection of the working standard solutions (containing 3TC,
NVP, and TDF), three resolved peaks were observed. It was
further shown that the retention times for each of the
analytes were diﬀerent from each other (p<0.0001; Table 2).
e outcome showed that the method was capable of in-
dependently detecting the three analytes and distinguishing
one from the other, thus speciﬁc and selective. In order to
evaluate the quantifying power of the method, its accuracy
was also determined. It was shown that the percentage re-
coveries obtained, complied with the acceptance criteria of
98%–102% [13, 14] (with minimum deviations) over the
concentration range, 80%–120% of test concentration for the
three analytes (Tables 2 and S3).
e responses for 3TC, NVP, and TDF were
showed to be linear within the ranges 15 µg/ml–90 µg/ml,
7µg/ml–42 µg/ml, and 10 µg/ml–59 µg/ml, respectively
(Table 2 and Figure 2). e regression coeﬃcient of
) obtained for the abovementioned analytes
was 0.9959, 0.9948, and 0.9971, respectively. F-values
from the linear regression models were also shown to be
signiﬁcant, further demonstrating strong correlations
between the concentration of the analytes and peak area
responses (Table 2). e method was shown to detect at
least 5.5032 µg/ml, 3.1496 µg/ml, and 3.9267 µg/ml of 3TC,
NVP, and TDF, respectively. However, quantitation could
only be carried out with the method when their con-
centrations were at least 16.6762 µg/ml, 9.5443 µg/ml, and
10.0433 µg/ml, respectively (Table 2).
Method precision was demonstrated with repeatability
and intermediate precision (Tables 2 and S4). It was ob-
served in both evaluations that the RSDs for the method
responses (peak areas) were less than 2%, proving their
consistency and precision [12, 13]. In addition, it was
4Journal of Chemistry
Table 2: Results from validation carried out on the developed method.
Validation parameter 3TC NVP TDF Remarks
(mean ±SD) 3.269 ±0.0074 min (N�5) 5.423 ±0.0015 min (N�5) 7.555 ±0.0024 min (N�5)
Retention times are signiﬁcantly
diﬀerent from each other
�1.102e+ 006; p<0.0001).
Method selectively and speciﬁcally
identiﬁes the three analytes.
36 µg/ml �99.30% ±0.34 (N�3) 16.8 µg/ml �102.99% ±0.45∗
(N�3) 24 µg/ml �101.75% ±0.28 (N�3) Developed method accurately estimates
the content of the analytes. Acceptance
45 µg/ml �100.80% ±0.32 (N�3) 21 µg/ml �99.71% ±0.72 (N�3) 30 µg/ml �98.58% ±0.71 (N�3)
54 µg/ml �97.90% ±0.46∗(N�3) 25.2 µg/ml �98.22% ±0.51 (N�3) 36 µg/ml �98.18% ±0.26 (N�3)
equation Y�14487x+ 38521 Y�9363x+ 11417 Y�5283x+ 8162
Acceptance criteria: [R
linearity of responses established for the
speciﬁed analytes concentration ranges.
0.9959 0.9948 0.9971
Sy.x 25214 8543 5062
F-value, pvalue 3900, p<0.0001 3090, p<0.0001 5429, p<0.0001
Range 15 µg/ml–90 µg/ml 7 µg/ml–42 µg/ml 10 µg/ml–59 µg/ml
LOD 5.5032 µg/ml 3.1496 µg/ml 3.9267 µg/ml
LOQ 16.6762 µg/ml 9.5443 µg/ml 10.0433 µg/ml
(3 conc terms) RSD �0.4000% ±0.2291, N�3 RSD �0.7967% ±0.342, N�3 RSD �0.9467% ±0.6704, N�3Acceptance criteria: [RSD <2.0%].
Two-way ANOVA showed that the
diﬀerences in the responses produced
from the diﬀerent concentrations on
diﬀerent days not were signiﬁcant
(p≥0.05). Developed method responses
(3 analysts for
RSD �0.52%–1.70%, N�9;
RSD �0.41%–1.94%, N�9;
RSD �0.29%–1.63%, N�9;
time �3.416 ±0.019 min and
3.131 ±0.131 min at 0.90 ml/min
and 1.1 mil/min, respectively.
(ii) % deviation of peak area <±5%
time �8.375 ±0.033 min and
3.417 ±0.022 min at 0.90 ml/min
and 1.1 mil/min, respectively.
(ii) % deviation of peak area <±5%
time �9.573 ±0.011 min and
6.783 ±0.036 min at 0.90 ml/min
and 1.1 mil/min, respectively.
(ii) % deviation of peak area < ±5%
Deliberate changes in ﬂow rate resulted
in retention times of the analytes
maintaining their distinction from each
other. Hence, peaks maintained
resolutions at 0.90 mil/min
�60322; p<0.0001] and 1.10 mil/
Deliberate changes in the wavelength
detection did not show signiﬁcant
diﬀerences in peak areas (RSD <2%; %
deviation <5%). In addition,
proportionate detection was maintained
(p>0.005). Method is fairly robust.
∗Minimal deviations of results from acceptance criteria.
Journal of Chemistry 5
Table 3: Optimised chromatographic conditions adopted for the validation.
Column Phenomenex Synergi C18 (250 ×4.6 mm, 4 µm)
Mobile phase Acetonitrile (10%): ammonium acetate buﬀer (adjusted to pH 2.8) (40%): methanol (50%)
Diluent Acetonitrile (10%): ammonium acetate buﬀer (adjusted to pH 2.8) (40%): methanol (50%)
Flow rate 1 ml/min
Column temperature 25°C
Injection volume 10 µL
Wavelength 270 nm
0.0 2.5 5.0 7.5
10.0 12.5 15.0
Figure 1: Chromatogram showing optimised separation of lamivudine (3.216min), nevirapine (5.344 min), and tenofovir disoproxil
fumarate standard (7.430 min) (from left to right) using the developed method.
Conc of 3TC (ug/ml)
20 40 60 80 100
1400000 y = 14487x + 38521
R2 = 0.9959
Conc of nevirapine (ug/ml)
10 20 30 40
400000 y = 9363x + 11417
R2 = 0.9948
Conc of tenofovir (ug/ml)
10 20 30 40 50 60
y = 5283x + 8162
R2 = 0.9971
Conc of 3TC (ug/ml)
Figure 2: Proof of linearity of the developed method. (a) A
, (b) B
, (c) C
, (d) A
, (e) B
, and (f) C
6Journal of Chemistry
Figure 3: Stability of analytes in test solution within 6-hour period. (a) 3TC, (b) NVP, and (c) TDF.
Table 4: Percentage content obtained for sampled products.
Analyte Content (%) Acceptance criteria (%) Inference
Sample A TDF 97.50 ±0.75 Passed
3TC 97.72 ±0.09 Passed
TDF 98.55 ±0.17 90–110 Passed
3TC 105.33 ±0.85 Passed
NVP 99.20 ±1.17 Passed
0.0 2.5 5.0 7.5 10.0 12.5 15.0
C:\Users\GSA\Documents\DRUGS\2016–02–03\1 A 1.lcd
Figure 4: Continued.
Journal of Chemistry 7
observed from the intermediate precision determinations
that the peak areas recorded on diﬀerent days for the dif-
ferent concentrations adopted were not signiﬁcantly dif-
ferent from each other (F
(Tables 2 and S4).
In testing for the robustness of the method, it was ob-
served that deliberate changes in the ﬂow rate did not
signiﬁcantly aﬀect the resolution of the peaks, as they
remained well separated from each other (F
p<0.0001 and F
�1949; p<0.0001). Upon changes
eﬀected to the wavelength of detection, the deviations ob-
served were also not signiﬁcant (that is, % deviations < ±5%)
(Tables 2 and S5). us, the method was considered robust.
e analytes in the test solution were also found to be stable
within the period of analysis, which in the current study was
estimated to be 6 hours (Figure 3).
3.3. Assay of Sampled Products. e validated method dem-
onstrated its usability in the analysis of commercial products.
e outcome of such investigation showed that the products
complied with content assay speciﬁcations as indicated in the
United States Pharmacopeia  (Table 4 and Figure 4).
An accurate, precise, and selective reverse HPLC method
for the simultaneous estimation of lamivudine, nevir-
apine, and tenofovir disoproxil fumarate has been de-
veloped and validated. e method was validated per the
ICH guidelines and passed all tests for the various vali-
dation parameters. e developed method is speciﬁc and
selective as well as robust, accurate, and precise. e
HPLC method was successfully applied to quantitatively
estimate the active pharmaceutical ingredients in
commercial products A
(containing TDF 300 mg/ 3TC
300 mg tablets + NVP 200 mg) and A
300 mg/ 3TC 300 mg).
Processed data used to support the ﬁndings of this study are
included within the article. Some of the data related to the
validation of the developed method are also included within
the article while others are included as Supplementary Data
Conflicts of Interest
e authors declare that they have no competing interests.
e authors are grateful to Danadams Pharmaceutical In-
dustry Limited, Spintex, Accra, for the donation of the pure
antiretroviral APIs used in this research. e authors are also
thankful to Mr. Brown of Ghana Standards Authority for
providing HPLC instrument and all reagents used.
S1: investigations carried out to determine suitable chro-
matographic conditions in method development. S2: spec-
iﬁcity/selectivity. S3: results for accuracy studies involving
analytes. S4: precision repeatability—triplicate injections of
three diﬀerent concentrations of the analytes, to investigate
the eﬀect of concentration change on the precision of the
method. S5: robustness examining eﬀects from change in
ﬂow rate. (Supplementary Materials)
0.0 2.5 5.0 7.5 10.0 12.5 15.0
Figure 4: Chromatogram showing the peaks of lamivudine and tenofovir in a sample B
and nevirapine in sample B
. Retention times are
3.27 and 7.55 minutes, and 5.42 minutes, respectively.
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