Page 1
Adisa et al
Trop J Pharm Res, February 2009; 8 (1): 79
Tropical Journal of Pharmaceutical Research, February 2009; 8 (1): 79-86
© Pharmacotherapy Group,
Faculty of Pharmacy, University of Benin,
Benin City, 300001 Nigeria.
All rights reserved.
Available online at http://www.tjpr.org
Research Article
Simultaneous quantitative determination of
zidovudine and nevirapine in human plasma using
isocratic, reverse phase high performance liquid
chromatography
Vibhuti Kabra1*, Vivek Agrahari1, Chandrabose Karthikeyan2 and
Piyush Trivedi2
1Department of Pharmacy, Shri G. S. Institute of Technology & Science (SGSITS), Indore 452001, 2School of
Pharmaceutical Sciences, Rajiv Gandhi Technical University (RGTU), Bhopal 462036, Madhya Pradesh, India.
Abstract
Purpose: To develop a sensitive and rapid reverse phase high performance liquid chromatography
(HPLC) method for the measurement of the levels of zidovudine (ZVD) and nevirapine (NVP) in human
plasma.
Methods: Standard stock solutions for HPLC analysis were prepared by dissolving ZVD and NVP in
methanol. In the HPLC measurement, sample detection was carried out at 246 nm using an ultraviolet
(UV)-photo diode array (PDA) detector. Plasma sample pretreatment consisted of protein precipitation
extraction with methanol. The compounds were separated using a mobile phase consisting of a pH 3.0
solution (obtained by adjusting the pH of water with orthophosphoric acid): acetonitrile (73:27 v/v) on a
Phenomenex LUNA C18, column (250×4.6 mm i.d., 5µm) at a flow rate of 0.9 mL min-1. The total run
time for the assay was 10.2 min. The method was validated over the range of 300-9600 ng mL-1 and
200-6400 ng mL-1 for ZVD and NVP, respectively.
Results: The lowest limits of quantification (LLOQ) and of detection (LOD) were 300 and 63 ng mL-1 for
ZVD and 200 and 17 ng mL-1 for NVP, respectively. The method was found to be accurate, with
accuracy ranging from -10.92 to +9.57 % and precise, with intra-day, inter-day as well as analyst to
analyst precision of 0.68 to 9.38 %. Extraction recoveries of the drugs from plasma were 91.39, 95.01,
89.51 % for ZVD and 90.93, 93.26, 92.13 % for NVP, for LQC (low quality control), MQC (medium
quality control) and HQC (high quality control) samples, respectively. Stability data revealed that the
drugs were stable in plasma under various test conditions.
Conclusion: This assay can be suitably used for the determination of zidovudine (ZVD) and nevirapine
(NVP) in human plasma and should be useful in HIV clinical trials and clinical therapeutic drug
monitoring (TDM) programs. It would also be potentially useful in the determination of pharmacokinetic
profiles and in bioequivalence studies in HIV research.
Keywords: Assay, Zidovudine, Nevirapine, Human plasma, Reverse phase high-performance liquid
chromatography.
Received: 30 July 2008 Revised accepted: 24 October 2008
*Corresponding author: E-mail: vibhuti.kabra@rediffmail.com; Phone: +91-9827500875.
Page 2
Kabra et al
Trop J Pharm Res February 2009; 8 (1): 80
INTRODUCTION
Major advances have been accomplished in
recent years in the treatment of patients
infected with human immunodeficiency virus-
type 1 (HIV-1). However, many patients
experience treatment failure within one year
after initiation of antiretroviral therapy1.
Combination therapy has now become the
standard line of treatment to manage acquired
immunodeficiency syndrome (AIDS)2. The
need for such a therapy has arisen due to the
development of resistance by human
immunodeficiency virus (HIV), to single anti-
HIV drugs and also in order to minimize the
dose-dependent side effects produced by
these drugs3. Many drugs have been tested
for their activity against HIV-1. Nevirapine
(NVP), a representative of a new class of
antiretroviral drugs - the non-nucleoside
reverse transcriptase inhibitors (NNRTIs) -
i.e., 11-cyclopropyl-5,11-dihydro-4-meth yl-
6H-dipyrido- [3,2-b:2,3 e] [1,4] diazepin-6-one,
and zidovudine (ZVD), i.e., 3-azido- 3-
deoxythymidine, are synthetic nucleoside
analogues used for the treatment of HIV
infections (Fig. 1).
Several multi-drug high-performance liquid
chromatography (HPLC) assays that measure
a number of NNRTIs have been described in
the literature in recent years. Numerous
analytical methods, such as HPLC with UV
detection, mass spectrometry detection and
immunoassay, have been reported for ZVD
and NVP but they either use a gradient
method for separation or a complicated liquid-
liquid extraction method for preparation of
plasma samples and also often involve
complicated chromatography techniques. In
the literature, numerous methods to
individually quantify zidovudine4-7 and
nevirapine8-11 have been described. A few
methods to estimate zidovudine and
nevirapine simultaneously have also been
reported12–17. Some of these methods used
liquid–liquid12-14 and solid phase extraction
procedure for the extraction of the drugs from
plasma15–17. This could increase the time and
cost of the assay. However, a combination of
several characteristics makes this assay a
unique and a useful analytical tool for
quantification of these drugs.
The objective of this work was to devise a
simultaneous technique for the quantitative
evaluation of zidovudine (ZVD) and nevirapine
(NVP) in human plasma which involves rapid
protein precipitation extraction and based on
simple chromatographic conditions with
detection at a single wavelength (246 nm)
over a short run time.
Page 3
Kabra et al
Trop J Pharm Res February 2009; 8 (1): 81
EXPERIMENTAL
Reagents and chemicals
Zidovudine and nevirapine were kindly
provided by Ranbaxy Laboratories Ltd,
Dewas, (M.P.), India. Methanol and
acetonitrile were of HPLC grade and
purchased from Merck Ltd, New Delhi, India.
Water used was of HPLC grade water (Mili Q
Water) was purchased from Ranbaxy
Laboratories Ltd, (New Delhi, India). Human
plasma was obtained from Blood Bank
Department, Bhopal Memorial Hospital &
Research Centre, Bhopal, (M.P.), India. All
other chemicals were of analytical grade and
used without further purification.
Instrumentation
The HPLC system (Shimadzu, Japan)
consisted of a LC-10AT VP pump, a SPD-10A
VP, PDA detector, a Phenomenex, Luna C18
(250mmX4.6mm, 5µm) column, a
Phenomenex, HPLC guard cartridge system
and a Class LC10/M10A software.
Chromatographic conditions
The chromatographic analysis was performed
at ambient temperature on a Phenomenex
LUNA analytical column with a mobile phase
composed of water (pH 3.0, adjusted with
orthophosphoric acid): acetonitrile (73:27, v/v)
and was isocratically eluted at a flow rate of
0.9 mL min-1. A small sample volume of 20 µL
was used for each sample run, being injected
into the HPLC system. The chromatogram
was monitored with UV detection at a
wavelength of 246 nm.
Preparation of the calibration standards,
mixed standard and quality control (QC)
samples
Standard stock solutions were prepared by
dissolving 60 mg of ZVD and 40 mg of NVP in
100 mL of methanol to obtain final
concentrations of 600 and 400 µg mL-1 for
ZDV and NVP, respectively (stock-A). From
this stock, 1 mL was taken and diluted to 10
mL with methanol to give 60 µg mL-1 of ZVD
and 40 µg mL-1 of NVP standard solutions,
respectively (Stock-B). Aliquots of stock-B
were further diluted to obtain different
concentrations: 300, 600, 1800, 3000, 4800,
6000, 7800, 9600 ng mL-1 for ZDV and 200,
400, 1200, 2000, 3200, 4000, 5200, 6400 ng
mL-1 for NVP. To prepare standard spike
plasma stock solutions, 1 mL was taken from
stock-A, and diluted to 10mL with blank
plasma to give 60 µg mL-1 of ZVD and 40 µg
mL-1 of NVP (stock-C). From the working
standard solution stock-C, different
concentrations were prepared by taking 0.1,
0.2, 0.4, 0.6, 0.8, 1.2, 1.6, and 1.8 mL, and
then the volume was made up, in each case,
to 2 mL with blank plasma. For all the
concentrations, 8 mL of precipitating agent
(methanol) was added to obtain the final
concentrations of 300, 600, 1800, 3000, 4800,
6000, 7800, 9600 ng mL-1 for ZVD and 200,
400, 1200 2000, 3200, 4000, 5200, 6400 ng
mL-1 for NVP.
QC samples at three different levels of HQC
(high quality control, 6000/4000 ng mL-1),
MQC (medium quality control, 3000/2000 ng
mL-1), LQC (low quality control, 600/400 ng
mL-1) and LLOQ (lowest limit of quantitation,
300/200 ng mL-1) for ZVD and NVP,
respectively, were selected to perform
different validation parameters.
Sample pretreatment and extraction
procedure
Drugs were extracted from plasma samples
using a protein precipitation technique.
Methanol was selected as the precipitating
agent. Each plasma sample gave satisfactory
values for recovery with a single extraction.
The satisfactory result was obtained when 1:4
ratios of plasma and methanol were mixed
thoroughly, vortexed at room temperature and
centrifuged at 12000 rpm for 10 min at 4°C.
The clear supernatant liquid was decanted,
filtered through a 0.45 µm syringe filter and
injected (20 µL) into HPLC system.
Page 4
Kabra et al
Trop J Pharm Res February 2009; 8 (1): 82
Limit of detection (LOD) and lowest limit of
quantification (LLOQ)
The limit of detection (LOD) was defined as
the concentration that yields a signal-to-noise
ratio of 3. The lowest limit of quantification
(LLOQ) was calculated to be the lowest
analyte concentration that could be measured
with a signal-to-noise ratio of 10. To determine
LLOQ and LOD, plasma samples were spiked
with decreasing concentrations of the analytes
and analyzed. The LLOQ and LOD were
observed 300 and 63 ng mL-1 for ZVD while
for NVP the values were 200 and 17 ng mL-1,
respectively.
Linearity of method
Calibration plots for the analytes in plasma
were prepared by spiking drug-free plasma
with standard stock solutions to yield
concentrations of 300-9600 ng mL-1 for ZVD
and 200-6400 ng mL-1 for NVP. The solutions
were injected in replicates (n = 5) into the
HPLC column while keeping the injection
volume constant (20 µL). Calibration curves
were constructed by using ratios of the
observed analyte peak area versus
concentration of analyte. Intercept, slope and
correlation coefficient (r2) were determined by
linear regression data analysis, which were
then used to calculate the analyte
concentration in each sample.
Precision and accuracy of method
The inter-day, intra-day, analyst to analyst
precision and accuracy of the assay were
determined by assaying three QC samples
and LLOQ in replicates (n = 5) for each drug.
Precision was reported as percent relative
standard deviation (% RSD) and accuracy
was as % nominal concentration and % bias.
Recovery of zidovudine and nevirapine
from plasma
Recovery from plasma was determined for QC
samples of each drug by comparing the peak
area of each analyte after extraction with the
respective non-extracted standard solutions at
the same concentration. The percentage of
the drug recovered from the plasma samples
was determined by comparing the peak height
ratio after extraction with those of unextracted
sample containing same concentrations of the
drugs as in plasma.
Selectivity
Selectivity is the ability of an analytical method
to differentiate and quantify the analyte in the
presence of other components in the sample.
To evaluate the selectivity of the assay, blank
samples of the appropriate biological matrix
(plasma) were prepared from six different
sources.
Stability of method
Stability of drugs in biological fluids is a
function of the sample storage conditions,
chemical properties of the drug, matrix and
the container system18. Blank samples were
spiked with appropriate aliquots of diluted
ZVD and NVP stock solutions to prepare LQC
and HQC samples. The stability of drugs was
evaluated under conditions likely to be
encountered during actual sample handling
and analysis. These samples were kept to
evaluate different stability parameters such as
stock solution stability (SSS, 6 h at room
temperature), bench top stability (BTS, 12 h at
room temperature), post processing stability
(PPS, over the maximum time, i.e., from the
completion of sample work-up to the
completion of data collection), freeze and
thaw stability (FTS, subjected to three freeze–
thaw cycles of -20 0C during 24 h) and long-
term storage stability (LTS, subjected to
freeze storage at -20 0C during the entire
period covered by the bioanalytical study, i.e.,
from the first day of sample preparation to the
last day of sample analysis). For each
concentration and storage conditions three
replicates were analyzed. The concentration
of ZVD and NVP after each storage period
was related to the initial concentration as
determined for the samples that were freshly
prepared and processed immediately.
Page 5
Kabra et al
Trop J Pharm Res February 2009; 8 (1): 83
RESULTS
At the chromatographic conditions selected for
this method, the retention time for blank
plasma (Fig. 2) and extracted peaks of ZVD
and NVP (Fig. 3) were 2.7 ± 0.3 min, 5.2 ± 0.3
min and 9.5 ± 0.3 min, respectively. The
correlation coefficients for the linearity of the
present method were 0.9997 and 0.9995 for
ZVD and NVP respectively. The lowest limit of
quantification and limit of detection were found
to be 300 and 63.0 ng mL-1 and 200 and 17.0
ng mL-1 for ZVD and NVP, respectively. The
recovery of the extraction procedure for ZVD
was 95.39 %, 94.01 % and 89.51 % while for
NVP, it was 94.93 %, 93.26 % and 92.13 %
for HQC, MQC & LQC, respectively (Table 1).
The accuracy (% nominal conc.) for HQC,
MQC, LQC and LLOQ, samples were found to
be in the range 93.42 to 109.79 and 89.04 to
105.15 for ZVD and NVP, respectively. The
accuracy data are shown in the Table 1 and
were well within the acceptance limit. The
precision data for HQC, MQC, LQC & LLOQ
samples were analyzed in replicates (n = 5)
(Table 1). For intra-day, it was 1.47 to 8.59 %
and 2.42 to 7.15 % for ZVD and NVP,
respectively. For inter-day, the range was
1.93 to 9.38 % and 1.35 to 8.98 %, for ZVD
and NVP, respectively, while for analyst to
analyst precision, it was in the range 0.68 to
5.94 % and 1.79 to 6.82 % for ZVD and NVP,
respectively.
Table 1: Accuracy, precision and mean recovery data for the developed method
QC Accuracy Precision Mean
Samples Recovery (%)
% N.C. a % Bias Intra day Inter day A-to-A b
ZVDc LLOQ d 104.38 4.38 8.59 9.38 4.50 -
LQC e 97.70 -2.30 2.34 7.03 5.94 91.39
MQC f 109.79 9.79 5.51 4.37 2.62 95.01
HQC g 93.42 -6.58 1.47 1.93 0.68 89.51
NVPh LLOQ 89.08 -10.92 6.26 5.92 5.50 -
LQC 98.44 -1.56 7.15 8.98 3.72 90.93
MQC 106.15 6.15 2.42 2.67 6.82 93.26
HQC 100.80 0.80 4.13 1.35 1.79 92.13
a % Nominal concentration; b analyst to analyst; c zidovudine; d lowest limit of quantitation; e low quality control; f medium
quality control; g high quality control; h nevirapine.
Table 2: Stability of the developed method at various conditions
Stability Zidovudine Nevirapine
conditions LQC HQC LQC HQC
% N.C.a % C.b % N.C. % C. % N.C. % C. % N.C. % C.
SSS c 92.68 4.94 98.45 -1.13 103.73 2.83 92.69 -5.31
BTS d 95.51 -2.23 88.60 -7.52 97.15 6.25 94.92 -1.63
PPS e 103.53 5.21 99.35 -3.23 91.59 -9.69 99.40 1.11
FTS f 96.58 3.51 91.96 -5.43 99.57 8.95 104.10 5.49
LTS g 107.25 8.28 102.74 6.26 109.50 11.97 105.60 -0.51
a % Nominal concentration; b % change; c stock solution stability; d bench top stability; e post processing stability; f
freeze & thaw stability; g long term stability
End of preview.
