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A Simple RP-HPLC Method to Simultaneously Assay the Contents of Lamivudine, Tenofovir, and Nevirapine in Fixed Dose Combined Oral Antiviral Medicines


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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. The 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. The 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. The % RSD values for precision (intraday and interday) and accuracy studies were found to be less than 2%. The results obtained from quantitative analysis conform to USP content requirements for marketed tablet dosage forms, RICOVIR-LN, and tenofovir disoproxil fumarate/lamivudine tablets. The method is therefore useful for routine quality control of antiretroviral tablet dosage forms containing tenofovir disoproxil fumarate, lamivudine, and nevirapine.
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Research Article
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;
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
properly cited.
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 1059 µg/ml, 742 µg/ml, and 1590 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.
1. Introduction
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 [1]. e introduction of highly active
antiretroviral therapy (HAART), a treatment regimen
comprising of the combination of three or more anti-
retroviral drugs [2], has revolutionized the management of
the disease condition, with the resultant dramatic reduction
in mortality rates and the incidences of opportunistic in-
fections [3]. 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 [4], 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-
[10]. 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 [11]. TDF is a derivative of
adenosine S-monophosphate and is a prodrug [11].
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 [11]. 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 [11].
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
of TDF.
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 [12]. 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 [15].
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
Lamivudine 3TC
229.3 1.46 13.90
Soluble in water,
sparingly soluble in
methanol, slightly
soluble in ethanol
635.5 2.65 3.75
Slightly soluble in
water, soluble in
methanol, very
slightly soluble in
Nevirapine NVP
266.3 2.65 2.8
Practically insoluble
in water, sparingly
soluble or slightly
soluble in methylene
chloride, slightly
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 [12].
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 [12]. 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 [13]. 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 [13]. 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 [12].
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 [13].
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
(Figure 1).
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/ml90 µg/ml,
7µg/ml42 µg/ml, and 10 µg/ml59 µg/ml, respectively
(Table 2 and Figure 2). e regression coefficient of
correlation (R
) 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
Retention time
(mean ±SD) 3.269 ±0.0074 min (N5) 5.423 ±0.0015 min (N5) 7.555 ±0.0024 min (N5)
Retention times are significantly
different from each other
1.102e+ 006; p<0.0001).
Method selectively and specifically
identifies the three analytes.
% recovery
(mean ±SD)
36 µg/ml 99.30% ±0.34 (N3) 16.8 µg/ml 102.99% ±0.45
(N3) 24 µg/ml 101.75% ±0.28 (N3) Developed method accurately estimates
the content of the analytes. Acceptance
criteria: [98.00%–102.00%].
45 µg/ml 100.80% ±0.32 (N3) 21 µg/ml 99.71% ±0.72 (N3) 30 µg/ml 98.58% ±0.71 (N3)
54 µg/ml 97.90% ±0.46(N3) 25.2 µg/ml 98.22% ±0.51 (N3) 36 µg/ml 98.18% ±0.26 (N3)
Linearity and
equation Y14487x+ 38521 Y9363x+ 11417 Y5283x+ 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, N3 RSD 0.7967% ±0.342, N3 RSD 0.9467% ±0.6704, N3Acceptance 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
(p0.05). Developed method responses
were precise.
(3 analysts for
3 days)
RSD 0.52%–1.70%, N9;
1.786, p0.1756
RSD 0.41%–1.94%, N9;
2.890, p0.0520.
RSD 0.29%–1.63%, N9;
0.08900, p0.9847
(i) Retention
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%
(i) Retention
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%
(i) Retention
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/
min [F
1949; p<0.0001].
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.
Condition Description
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
Det.A Ch1
C:\Users\GSA\Documents\DRUGS\20160203\Carryover 1.lcd
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
Peak area
Conc of tenofovir (ug/ml)
Peak area
10 20 30 40 50 60
y = 5283x + 8162
R2 = 0.9971
Conc of 3TC (ug/ml)
0 20406080100
Conc (ug/ml)
0 1020304050
Conc (ug/ml)
0 20406080
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
Peak area
Upper limit
Lower limit
Peak area
Upper limit
Lower limit
Peak area
Upper limit
Lower limit
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
Sample B
TDF 98.55 ±0.17 90–110 Passed
3TC 105.33 ±0.85 Passed
Sample B
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
Det.A Ch1
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
0.08900, p0.9847)
(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 [19] (Table 4 and Figure 4).
4. Conclusion
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
(containing TDF
300 mg/ 3TC 300 mg).
Data Availability
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.
Supplementary Materials
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)
C:\Users\GSA\Documents\DRUGS\2016–02–03\2B 1.lcd
Det.A Ch1
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.
8Journal of Chemistry
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Journal of Chemistry 9
... Method validation. The developed method was validated in accordance with the ICH Guideline Q2(R1) [21] ; following recommendations from literature [22][23][24] . ...
... Specificity and selectivity. Specificity and selectivity were assessed by comparing the chromatogram from a matrix without expected analytes with that of a matrix containing the expected analytes (that is, 1.2 mg/mL of PSD and 0.08 mg/mL of CPM) [24] . The selectivity was confirmed by comparing the Mean ± SD of the retention times for the analytes using Student t -test analysis [22] . ...
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Common cold medicines usually present as multi-component products and constitute one of the largest groups of ‘over-the-counter’ medicines marketed in Ghana. Due to their wide patronage and easy accessibility, their quality control is of utmost importance to medicine consumers. In this study, the Design of Experiment (DOE) concept is adopted to develop a chromatographic method for the simultaneous assay of pseudoephedrine hydrochloride and chlorphenamine maleate in commercially available products. The separation of the drugs was achieved on an Agilent Eclipse Plus C18 analytical column (4.6 × 150 mm; 5 µm) and detected at 252 nm, while maintaining column temperature at 30 oC. The method parameters, including acetonitrile concentration in the mobile phase, mobile phase pH and flow rate were optimized using a Central Composite Design study, while monitoring the method attributes, asymmetric factors, selectivity, retention times, and resolution. The outcome showed a good correlation between experimental and predictive values throughout the modeled Design Space, with a Desirability of 1.000. The conditions optimized included a mobile phase containing acetonitrile, 50 mM aqueous acetate buffer solution (pH = 3.19) and triethylamine (15.34:84.65:0.01 v/v/v), and a flow rate of 1.437 mL/min. The method was then validated following the International Council for Harmonization Q2(R) guidelines to establish linearity and range, accuracy, precision, robustness, and stability of the test solution. The developed method was successfully applied to estimate pseudoephedrine hydrochloride and chlorphenamine maleate contents in single and fixed-dose formulations commercially available (N = 38).
... (1) Specificity/Selectivity. is was demonstrated by comparing the chromatograms from the diluent (mobile phase system) with that from the working standards at a concentration level of 80 μg/mL for quinidine, cinchonine, cinchonidine, dihydroquinine, dihydroquinidine, and cryptolepine (100% working) and 400 μg/mL for quinine sulfate [22]. ...
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Quinine- and cryptolepine-based antimalarials serve as valuable alternatives to artemisinin-based combination therapies (ACTs) in Ghana. Their use, however, is associated with adulteration and substandard quality challenges. An HPLC method targeting quinoline and indoloquinoline antimalarial alkaloids was developed, validated, and applied to evaluate herbal and pharmaceutical antimalarial formulations (HPAFs) and starting materials (APIs). The separation/quantitation of the alkaloids (including quinine, quinidine, cinchonine, cinchonidine, dihydroquinine, dihydroquinidine, and cryptolepine) was achieved on a Zorbax SB-CN column (250 mm × 4.6 mm, 5 μm), with an isocratic elution system of methanol: trifluoroacetic acid (0.1%, v/v) (15 : 85, v/v) at 1.5 mL/min and 223 nm. Method validation was according to ICH Q2(R1) guidelines. It was then used to assess the quality of APIs (n = 3) and HPAFs (n = 44) including quinine-based pharmaceutical antimalarial formulations (QBPAFs) (n = 23) and herbal antimalarial products (HAMPs). The method was found to be specific, selective, accurate, precise, and robust toward the alkaloids with linearity achieved within specified concentration ranges (r2 > 0.995 for all analytes). Analyte stability ranged between 6 and 12 hours. All the APIs contained quinine
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Cholesterol plays a key role in the synthesis of bile acids and steroid hormones in the human body. However, excessively high levels are usually implicated in cardiovascular diseases. For this reason, it is essential to monitor exposure to high levels of it in products meant for human consumption, and this calls for the need to develop analytical methods to detect them. e use of Liebermann-Burchard reaction in this study has been explored to develop a simple, reliable, and robust quantitative colorimetric method to assay cholesterol, and hence provide a good alternative to chromatographic methods. e developed method was validated and used to determine the contents of cholesterol in selected dairy products on the Kumasi Metropolis market. e method demonstrated a good linearity (2 = 0.996) over concentration range of 0.01-0.08 mg/ml. It was also shown to be and robust. e limit of detection (LOD) and limit of quantiication (LOQ) were determined to be 0.00430 mg/ml and 0.01304 mg/ml, respectively. Ten selected brands of canned milk (B1-B5) and fresh yoghurt products (A1-A5) were then assayed using the developed method. e results showed that three products from each category had cholesterol contents above the allowable content of 5 mg/100 g in dairy products. e study thus has proposed a simple colorimetric method that can be adopted by dairy products manufacturing facilities to rapidly determine cholesterol contents during manufacturing in order to monitor the safe consumption of their products, and eliminate or minimize possible future health hazards.
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A multiple reaction monitoring liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) method for the simultaneous determination of five antiretroviral drugs i.e. emtricitabine, tenofovir, efavirenz, lopinavir and ritonavir using commercially sterilized human blood plasma as the matrix and a THF/water solvent system was developed for therapeutic drug monitoring purposes and partially validated using the United States Food and Drug Administration (US FDA) guidelines. The analytical performance characteristics that were examined were limits of detection (LODs), lower limits of quantification (LLOQs), upper limits of quantification (ULOQs), selectivity, percent mean recoveries, precision measured as percent relative standard deviations and accuracy. Optimized LC-MS/MS parameters were employed for this purpose. The percent mean recoveries were between 77.3 and 90.1% using solid phase extraction (SPE) for sample preparation. These recoveries were obtained at three spike levels i.e. the LOD, the LLOQ and the ULOQ. The precision of the SPE technique was within an acceptable range of <15% i.e. between 2.7 and 9.2% at the LLOQ. Accuracy was calculated as the percent deviation of the mean value from the true value and the values ranged between 9.9 and 22.7%. The SPE technique satisfied all the requirements of the US FDA guidelines except for efavirenz whose accuracy was slightly higher than recommended at the LLOQ i.e. 22.7% as opposed to 20%. The limits of detection using SPE also fell below the clinically relevant therapeutic range (3–8 mg L−1) for most of the drugs and therefore the method could be useful for therapeutic drug monitoring in HIV patients.
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Aim: The objective of this study was to develop a reliable, accurate and precise Titrimetric - UV spectrophotometric method for the assay of fixed dose combination formulations involving Paracetamol, Caffeine and Ibuprofen. Study Design: Experimental. Place and Duration of Study: Quality Control Department of SALOM Pharmacy Limited between June, 2015 and January, 2016. Methodology: The method employed an extraction of Ibuprofen from a fixed dose combination product using petroleum ether (40 – 60°C) and its evaporation followed by titration. The remaining solution was basified with 1 M NaOH and Caffeine extracted with chloroform. The extract was then evaporated and the Caffeine assayed at 273 nm. Finally, the resulting solution was diluted with distilled water and Paracetamol assayed at 257 nm. The developed method was validated as per International Conference on Harmonisation specifications [Q2 (R1)]. Results: Linearity was observed in the concentration range of 3.75 - 9 μg/ml and 4.5 - 10.8 μg/ml for Paracetamol and Caffeine respectively and a titre value range of 4.80 - 11.70 ml for Ibuprofen. Conclusion: The results demonstrated that the method is accurate, precise, specific, and robust, hence can be suitably applied for simultaneous quantification of Paracetamol, Caffeine and Ibuprofen in laboratory prepared mixtures and in commercial preparation (capsules, caplets and tablets).
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A new method using high-performance liquid chromatography coupled with ultra violet detection (HPLC-UV) was developed and validated for the simultaneous quantification of atazanavir, dolutegravir, darunavir, efavirenz, etravirine lopinavir, raltegravir, rilpivirine and tipranavir in human plasma. For the first time we reported here the development and validation of an HPLC-UV assay to quantify the frequently administered nine antiretroviral compounds including dolutegravir and rilpivirine. A simple solid phase extraction procedure was applied to 500 μL aliquots of plasma. The chromatographic separation of the drugs and internal standard (quinoxaline) was achieved with a gradient of acetonitrile and sodium acetate buffer on a C-18 reverse-phase analytical column with a 25 min analytical run time. Calibration curves were optimised according to the therapeutic range of drug concentrations in patients, and the coefficient of determination (r2 ) was higher than 0.99 for all analytes. Mean intraday and interday precisions (% RSD) for all compounds were less than 15.0 %, and the mean accuracy (% deviation from nominal concentration) was also found to be less than 15.0 %. Extraction recovery range was between 80% and 120% for all drugs analyzed. The solid phase extraction and HPLC-UV method enable a specific, sensitive, and reliable simultaneous determination of nine antiretroviral agents in plasma. Good extraction efficiency and low limit of HPLC-UV quantification make this method suitable for use in clinical trials and therapeutic drug monitoring.
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Chlorpheniramine maleate-paracetamol-caffeine tablet formulation is one of the common over-the-counter drugs used for the treatment of cold and cough. A reversed-phase high-performance liquid-chromatography method has been successfully developed for the simultaneous determination of chlorpheniramine maleate, paracetamol and caffeine in a drug formulation. The RP-HPLC method employed a Phenomenex C18 reversed phase column (Luna 5µ, 250 × 4.6 mm) with an isocratic mixture of methanol and 0.05 M dibasic phosphate buffer pH 4.0 in the ratio of (30:70; v/v) as the mobile phase. The column temperature was kept at 30 °C. The flow rate was 1.0 mL/min and detection was by means of a UV detector at wavelength of 215 nm. All the active components were successfully eluted with mean retention times of 2.4, 4.2, 7.2 min for chlorpheniramine maleate, paracetamol and caffeine respectively. The method was found to be linear (R(2) > 0.99), precise (RSD < 2.0 %), accurate (recoveries 97.9-102.8 %), specific, simple, sensitive, rapid and robust. The validated method can be used in routine quality control analysis of fixed dose combination tablets containing chlorpheniramine maleate, paracetamol and caffeine without any interference by excipients.
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Analytical validation is an essential component in allowing a laboratory to ensure routine acceptable performance of analytical methods. Despite the considerable amount of important published work on this subject, diversity still prevails in the employed methodologies because validation of an analytical method depends on the specific purpose of that method. This can lead to difficulties in validation approaches and the interpretation of results. Aiming to assist in the planning of validation methods, we discuss relevant approaches of various parameters in quantitative high-performance liquid chromatographic methods and validation fields in pharmaceutical analysis. Moreover, this article provides several up-to-date examples that should be useful as an introduction to analytical validation for practical applications in academic research or the industrial sector.
Objective To assess the quality of antibiotics sampled from authorized traders (ATs) (i.e, hospitals/health centres, pharmacies, and licensed chemical shops) and unauthorized traders (UATs) (mainly street vendors) in Ghana and to explore the health seeking behaviour of medicine consumers. Methods The contents of 14 active pharmaceutical ingredients (APIs) in 348 sampled products were determined using a validated liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) method. Data on health seeking practices were collected through entry and exit interviews, and field observations from ATs and UATs. Results It was observed that 66.38% of all sampled antibiotic products were substandard; they either contained less (<90%) or more API (>110%) than the label claimed. Medicines from UATs recorded substantially less API than those from ATs (F(2,419) = 43.01, p < 0.0001). For example, 90.54% of street vendor samples contained <90% of the API. 75.93% of consumers often sought self‐treatment with drugs without a prescription from UATs, as they perceived UATs as easily accessible, trustworthy and knowledgeable, and their medicines as inexpensive. These consumers also thought of the formal health care providers as alternative sources. Conclusions Consumers who purchase from UATs are at high risk of receiving substandard medicines. The quality of medicines in the national healthcare system, in the supply chain and in the distribution system needs to be monitored regularly to reduce the incidence of substandard medicines and their impact on antimicrobial resistance. The fight against substandard medicines needs to incorporate a full understanding of socioeconomic factors that drive consumer decisions regarding their health and choice of healthcare providers.