(2019) 13:118
Moustapha et al. BMC Chemistry
https://doi.org/10.1186/s13065-019-0635-2
BMC Chemistry
Open Access
RESEARCH ARTICLE
Two novel UPLC methods utilizing two
different analytical columns and different
detection approaches for the simultaneous
analysis of velpatasvir and sofosbuvir:
application to their co-formulated tablet
Moustapha Eid Moustapha1, Rania Mohamed El‑Gamal2,3*
and Fathalla Fathalla Belal2
Abstract
In the present study two different RSLC columns, Acclaim RSLC 120 C18, 5.0 µm, 4.6 × 150 mm (column A) and
Acclaim RSLC 120 C18, 2.2 µm, 2.1 × 100 mm (Column B) were utilized for the analysis of velpatasvir (VPS) in presence
of sofosbuvir (SFV), where due to the encountered fluorescent properties of VPS fluorescent detection at 405 nm
after excitation at 340 nm (Method 1) was used for its detection where the non‑fluorescent SFV did not interfere. The
same columns were further utilized for the simultaneous determination of SFV and VPS either in bulk form or in their
combined tablet, where UV‑ spectrophotometric detection at 260 nm was selected for the simultaneous analysis of
both drugs (Method 2). A mobile phase consisting of NaH2PO4, pH 2.5 (with phosphoric acid) and acetonitrile in a
ratio of 60:40 v/v was used for both methods. The mobile phase was pumped at a flow rate of 1.0 mL/min when using
column, A and 0.5 mL/min when using column B. The methods showed good linearity over the concentration ranges
of 1.0–5.0 and 2.5–10.0 ng/mL for VPS when utilizing Method 1 A and B respectively. Where the linearity concentration
range was from 30.0–150.0 to 120–600.0 ng/mL for VPS and SFV respectively when applying Method 2. Both methods
1 and 2 were performed by utilizing the two analytical columns. The different chromatographic parameters as reten‑
tion time, resolution, number of theoretical plates (N), capacity factor, tailing factor and selectivity were carefully opti‑
mized. The results show that comparing the performance of the two utilized columns revealed that shorter column
(2.1 mm × 100 mm) with small particle packing was superior to the longer column (4.6 × 150 mm) for the analysis of
the studied drugs allowing a reduction of the analysis time by 70% without any detrimental effect on performance.
This prompts the decrease of the investigation costs by saving money on organic solvents and expanding the overall
number of analyses per day.
Keywords: Velpatasvir, Sofosbuvir, UPLC, Fluorescent detection, UV‑spectrophotometric detection
Introduction
Hepatitis C is an infectious liver disease caused by infection with Hepatitis C Virus (HCV) that is considered a
very dangerous disease, influencing about from three to
five million people in the United States (US) and about
*Correspondence: r_m_elgamal@yahoo.com
2
Department of Pharmaceutical Analytical Chemistry, Faculty
of Pharmacy, Mansoura University, P.O. Box 35516, Mansoura, Egypt
Full list of author information is available at the end of the article
one hundred and seventy million people worldwide. This
disease is asymptomatic in its early stages however if it
becomes chronic it might prompt risky perilous inconveniences, including liver failure, hepatocellular carcinoma
and mortality [1]. Velpatasvir (VPS) is methyl {(2S)-1[(2S,5S)-2-(9-{2-[(2S,4S)-1-{(2R)-[(methoxycarbonyl)
amino]-2-phenylacetyl}-4(methoxymethyl)pyrrolidin-2-yl]1H-imidazol-4-yl}-1,11 dihydro [2] benzopyrano[4′,3′:6,7]
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Moustapha et al. BMC Chemistry
(2019) 13:118
naphtho[1,2-d]imidazol-2-yl)-5-methylpyrrolidin-1-yl]3-methyl-1-oxobutan-2-yl}carbamate, Fig. 1a.
VPS is a Direct-Acting Antiviral (DAA) medication
that plays a significant role in the combination therapy
of chronic Hepatitis C. HCV is a solitary stranded RNA
virus with nine particular genotypes, where, genotype 1 is the most widely recognized type in the United
States, and influencing more than 70% of patients suffering from chronic HCV. Since 2011, the presentation
of Direct Acting Antivirals (DAAs, for example, VPS)
Page 2 of 15
have fundamentally improved chronic hepatitis C treatment. One of the major advantage of VPS is that it has
a noteworthy raised boundary to resistance than its previous generation of NS5A inhibitors, as daclatasvir and
ledipasvir, this accounts for its high potency and efficacy as a treatment for chronic Hepatitis C [2]. Sofosbuvir (SFV) (isopropyl (2S)-2-[(2R,3R,4R,5R)-5-(2,
dioxopyrimidin-1-yl)-4-fluoro-3-hydroxy-4-methyl-tetra
hydrofuran-2-yl] methoxy-phenoxy-phosphoryl] amino]
pro-panoate) is a nucleotide analog NS5B polymerase
Fig. 1 The structural formulae of the studied drugs. a Velpatasvir (VPS), b sofosbuvir (SFV)
Moustapha et al. BMC Chemistry
(2019) 13:118
inhibitor. SFV is a prodrug that is mainly used for the
treatment of HCV, either alone or in combination with
other drugs like, VPS, ribavirin, and ledipasvir [3]
(Fig. 1b).
In June 2016, the American Association for the Study
of Liver Diseases (AASLD) and the Infectious Diseases
Society of America (IDSA) approved VPS and SFV combination (Epclusa) as 1st line therapy for the different six
genotypes of Hepatitis C [4].
Since the drugs are recently approved, their literature
revealed few analytical methods reported up to date,
where, SFV alone was determined by applying chromatographic and spectrophotometric techniques [5, 6]. The
forced degradation behavior of SFV was investigated by
mean of liquid chromatography-tandem mass spectrometry (LC–MS/MS) [7]. Few UPLC-MS/MS techniques
were utilized for the simultaneous analysis of SFV and
other antiviral drugs like ribavirin, ledipasvir or in presence of its metabolite [8–10].
Different RP-HPLC methods were reported for the
simultaneous determination of SFV and VPS either in
bulk, combined tablets or biological fluids [11–14], in
addition to two spectrofluorometric methods that were
recently reported for the assay of VPS in pharmaceutical
tablets and body fluids [15, 16].
The main objective of this work was to develop novel
UPLC methods for the simultaneous analysis of VPS and
SFV utilizing different analytical columns and different
detection approaches.
Experimental
Apparatus
Chromatographic analyses were performed using Thermo
Scientific DIONEX UltiMate 3000 UHPLC Rapid Separation System (Thermo Fisher Scientific Inc., MA, USA),
connected to a quaternary rapid separation pump (LPG3000RS), Ultimate 3000RS autosampler (WPS-3000),
rapid separation diode array detector (DAD-3000RS)
and rapid separation fluorescence detector (DIONEX
Ultimate 3000 RS Flourescence). Data acquisition, peak
integration and calibrations were carried out using
UHPLC, CHROMELEON7 software, Dionex, Thermo
Fisher Scientific, USA. Mobile phases were filtered using
Whatman® Nylon membrane filters 0.2 µm, ø47 mm. The
mobile phase was degassed with a sonicator of type GT
SONIC QTD-series units with digital timer and heater
features, GuangDong GT Ultrasonic Co., Ltd, China. Separation was carried on an Acclaim RSLC 120 C18 2.2um
120A (2.1 × 100 mm) and Acclaim RSLC 120 C1 5.0um
120A (4.6 × 150 mm) (Dionex, USA). Ultrapure water was
obtained from an Evoqua Ultra Clear TP TWF EDI UV
UF TM system, Evoqua Water Technologies, USA.
Page 3 of 15
Materials and reagents
All solvents used in this work were of HPLC grade.
Ultrapure water was used for all preparations. VPS
(≥ 98%) was purchased from BioVision, Milpitas Boulevard, Milpitas, CA 95035 USA). SFV (99.98 ± 0.741) was
obtained from Cayman chemical company, Ann Arbor,
USA) [8]. Acetonitrile and methanol (HPLC grade)
were obtained from Merck (Germany). Phosphoric acid,
analytical grade Merck (Germany). Sodium dihydrogen phosphate (NaH2PO4) was obtained from central
drug house (CDH), New Delhi, India. Phosphoric acid
(0.2 mol/L) solution was used to adjust pH to 2.5.
Dosage form
Epclusa® (sofosbuvir 400 mg/velpatasvir 100 mg) tablets was manufactured by Gilead Sciences International,
Cambridge, UK.
Standard solutions
Stock solutions of concentration 100.0 μg/mL of VPS
and SFV were prepared by dissolving 10 mg of pure drug
in 100 mL methanol using an ultrasonic bath. Working
standard solutions were prepared by suitable dilution of
the stock solutions with mobile phase. All solutions were
stored in the refrigerator to keep their stability.
Chromatographic conditions
Acclaim RSLC columns 120 C18 (120A 4.6 × 150 mm,
5.0um) and Acclaim RSLC 120 C18 (120A 2.1 × 100 mm,
2.2um) were used for methods A and B respectively. A
mobile phase consisting of NaH2PO4, pH 2.5 (with phosphoric acid, 0.2 M) and acetonitrile in a ratio of 60:40
v/v was used for both methods. The mobile phase was
vacuum-membrane filtered through a 0.45 μm Millipore membrane filter and degassed for approximately
10 min before use. The flow rate was 1.0 mL/min when
using column, A and 0.5 mL/min when using column
B. Columns temperature was maintained at 25 °C. For
fluorescence detection of VPS, the detector was set at
340/405 nm (Method 1 A and 1B). While for UV detection of both VPS and SFV the detector was set at 260 nm
(Method 2A and 2B). The injection volume was 10 uL.
Laboratory prepared mixture analysis
Stock solution of (SFV andVPS) was prepared at the ratio
of (4:1), where, 40 and 10 mg of both SFV and VPS were
quantitatively transferred to 100 mL volumetric flask and
the volume was adjusted with methanol. Working standard solutions were prepared by suitable dilution of the
stock solution with mobile phase.
Analysis of the working standard solution was accomplished via adapting procedures cited under “Calibration
Moustapha et al. BMC Chemistry
(2019) 13:118
Page 4 of 15
Fig. 2 Typical chromatograms of VPS 2.5 ng/mL under the described chromatographic conditions (Method 1 A)
Fig. 3 Typical chromatograms of VPS 1.0 ng/mL under the described chromatographic conditions (Method 1 B)
graph construction” section, where, corresponding drug
concentrations were calculated from the derived regression equations.
Calibration graph construction
A calibration curve was created by accurately measuring
volumes of the appropriate drugs working standard solutions delivered into a series of 10 mL volumetric flasks in
order to prepare a set of standard solutions in the range
specified by the method. The standard solutions were
completed to volume with the mobile phase and mixed
thoroughly. Aliquots of 10 μL were injected (triplicate)
into the columns and eluted with the mobile phase under
the optimum chromatographic conditions. The peak area
was plotted against the concentration of the drug in ng/
mL. Consequently, the corresponding regression equations were derived.
Procedures for tablets
A precise weight of the blended content of 10 powdered
tablets equal to 10.0 mg of VPS and 40.0 mg of SFV was
quantitatively conveyed into a 100 mL volumetric flask
and around 30 mL methanol was added. The flask contents were sonicated for 30 min, and made to 100 mL
with the same solvent. The solution was filtered through
cellulose acetate syringe filter. Working standard solutions were prepared by suitable dilution of the filtered
solution with mobile phase.
Analysis of the working standard solution was accomplished via adapting procedures cited under “Calibration
graph construction” section, where, the nominal contents
of the tablet were calculated from the derived regression
equations or the calibration curve.
Results and discussion
VPS was found to exhibit an intense fluorescence at
405 nm, after excitation at 340 nm. As a consequence, we
aimed to utilize this emission band using UPLC coupled
with fluorescence detection, to develop a new method for
its analysis in presence of SFV, the method was applied
for the analysis of the VPS (pure form) in presence of
SFV (Method 1) (Figs. 2 and 3). Moreover, an UPLC with
UV detection was utilized for the simultaneous analysis of VPS and SFV in their pure form as well as in their
combined tablet (Method 2) (Fig. 4a, b). Both methods 1
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Page 5 of 15
Fig. 4 Typical chromatogram of a laboratory prepared mixture of VPS (10 ng/mL) and SFV (40 ng/mL) under the described chromatographic
conditions (Method 2 A and B). a Studied drugs in the mobile phase utilizing column A. b Studied drugs in the mobile phase utilizing column B
and 2 were performed utilizing two different analytical
columns.
Optimization of experimental conditions
Choice of appropriate wavelength
VPS was reported to exhibit a very strong fluorescence
permitting very sensitive detection. The optimum excitation and emission wavelengths were determined via preliminary scanning of its fluorescence in the mobile phase,
VPS was found to exhibit maximum fluorescence intensity at 405 nm after excitation at 340 nm (Fig. 2).
For simultaneous analysis of VPS and SFV, their λmax
were determined through spectrophotometric scan
where 260 nm was chosen as optimum wavelength for
their simultaneous determination.
Mobile phase composition
Several modifications in the mobile phase composition
were carried out in a trial to optimize the selectivity,
efficiency, and resolution of the chromatographic system. These modifications involved, the pH of the mobile
phase, the type and ratio of the organic modifier, column
temperature and the flow rate. The results achieved are
summarized in Tables 1 and 2.
pH of the mobile phase The influence of the pH change
on the different chromatographic parameters studied
was investigated via changing the pH of the mobile phase
and monitoring the consequence change in parameter.
For both methods pH of 2.5 was the optimum pH
resulting in a well-defined peak, optimum resolution of
both drugs by Method 2 and shortest analysis time.
Moustapha et al. BMC Chemistry
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Page 6 of 15
Table 1 Optimization of the chromatographic conditions for determination of VPS by Method 1
Parameter
No. of theoretical plates (N)
Capacity factor (k′)
Tailing factor (Tf)
Column (A)
Column (B)
Column (A)
Column (B)
Column (A)
Column (B)
Room temperature
2900.3
406.2
1.839
2.675
0.532
0.831
30 °C
2941.6
–
1.987
–
0.660
–
40 °C
4547.4
1009.7
2.183
2.038
0.536
0.900
50 °C
5030.4
644.4
2.348
3.179
0.517
0.877
60 °C
–
799.3
–
3.454
–
0.908
2.5
2900.3
406.2
1.839
2.675
0.532
0.831
3.3
3921.4
408.2
2.744
4.150
0.528
0.947
4.0
3305.7
410.6
3.976
6.425
0.491
0.924
0.867
Column temperature °C
pH of mobile phase
Type of organic modifier of Conc 40% (v/v)
Acetonitrile
2720.4
378.8
1.831
2.583
0.556
Methanol
1140.1
365.4
1.843
2.621
0.514
0.907
Ethanol
2304.3
352.9
1.870
2.592
0.413
0.871
Ratio organic modifier: mobile phase (acetonitrile) (v/v)
40:60
3145.5
372.7
1.824
2.554
0.525
0.825
60:40
2720.4
378.8
1.831
2.583
0.556
0.867
80:20
2386.9
314.5
1.844
2.642
0.552
0.906
90:10
2210.2
316.7
1.848
2.654
0.575
0.866
–
452.8
–
4.613
–
Effect of flow rate (mL/min)
0.3
0.5
205.0
1.0
3551.6
1.2
2900.3
1.433
2.376
–
tR
Number of theoretical plates (N) = 5.54 Wh/2
W0.5 ′
Tailing factor (T) = 2f k = (tR− t0) t0,
1.839
0.768
0.750
0.571
–
0.532
–
2
Type of organic modifier of Conc 40% (v/v) Different
organic modifiers of concentration 40% (v/v) were utilized in this study. These include acetonitrile, methanol and ethanol. It was found that acetonitrile was the
organic modifier of choice for both methods resulting in
highest number of theoretical plates, maximum resolution and least tailing factor.
Concentration of organic modifier To study the influence of the concentration of acetonitrile on the proposed analysis methods, its concentration was varied
over the range of (40–90%, v/v). As the percentage of
acetonitirle increases in the mobile phase a marked peak
broadening was noticed, with a concomitant decrease in
the number of theoretical plates. Hence, a concentration of 40% acetonitrile was selected as the optimal concentration where it provides an optimum combination
of peak symmetry, resolution factor and analysis time
(Tables 1, 2).
Flow rate The influence of flow rate on the retention
time and peak shape was investigated for both methods
with utilization of the two comparative columns.
A flow rate of 1.0 mL/min was optimal for both methods when using column, A, where a flow rate of 0.5 mL/
min was optimal for good separation within a reasonable
elution time when using column B. This is mainly due to
the increased back pressure observed when pumping a
mobile phase through columns with small particle size
packing.
The effect of column temperature The column temperature was altered through the study to attain the suitable
temperature for maximum resolution and optimal peak
symmetry. Column temperature was varied over the
range (30–60 °C), it was found that room temperature was
optimal resulting in highest number of theoretical plates,
minimal tailing and best resolution (Tables 1, 2).
Moustapha et al. BMC Chemistry
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Page 7 of 15
Table 2 Optimization of the chromatographic conditions for the determination of SFV by Method 2
Parameter
No. of theoretical plates (N)
Capacity factor (k′)
Column (A)
Column (A)
Column (B)
Column (A)
Column (B)
Column (B)
Tailing factor (Tf)
Column temperature °C
25 °C
4354.9
1.147
4.638
0.475
0.629
40 °C
2950.3
756.4
1014.2
1.057
4.404
0.506
0.772
50 °C
2620.8
677.9
0.989
4.208
0.471
0.73
65 °C
1895.1
468.9
0.871
3.792
0.468
0.679
2.5
4354.9
1014.2
1.147
4.638
0.475
0.629
3.5
3981.2
1089
1.145
4.667
0.526
0.588
5
4617.2
1066.9
1.145
4.667
0.455
0.572
pH of mobile phase
Type of organic modifier of Conc 40% (v/v)
Acetonitrile
4390.4
1004.5
1.236
4.667
0.446
0.679
Methanol
5073.4
1214.5
1.179
4.738
0.474
0.664
Ethanol
3342
925.4
1.175
4.708
0.481
0.65
Ratio organic modifier: mobile phase (Acetonitrile) (v/v)
40:60
4354.9
1100.3
1.147
4.696
0.475
0.677
60:40
1925.3
1004.5
1.173
4.667
0.453
0.679
80:20
7501.7
1648.5
1.187
4.75
0.468
0.647
90:10
8304.6
2064.8
1.19
4.792
0.458
0.653
Effect of flow rate (mL/min)
0.3
1041
8.196
0.5
1090.7
4.613
1
5093.6
1.2
1117.9
tR
Number of theoretical plates (N) = 5.54 Wh/2
W0.5 ′
Tailing factor (T) = 2f k = (tR− t0) t0,
0.816
0.613
1.53
0.287
1.175
0.465
2
Method validation
Table 3 Analytical performance data for the determination
of VPS by Method 1
Parameter
Value
Column (A)
Column (B)
The validity of the proposed UPLC methods was examined in terms of linearity, ranges, limits of detection,
limits of quantification, accuracy, precision, robustness,
specificity, stability of standard solutions and mobile
phase.
Linearity and range (ng/mL)
1.0–5.0
2.5–10.0
Correlation coefficient (r)
1.0
0.9999
Linearity and range
Slope
20,723.82
49,677.50
Intercept
− 745.284
− 21,790.770
Under the above-demonstrated experimental conditions,
a linear relationship was obtained by plotting the peak
areas against the drugs concentrations. The graphs were
found to be rectilinear over the concentration ranges
referred to in Tables 3, 4.
Statistical analysis [17] of the data showed high values
of the correlation coefficient (r) of the regression equation, minute values of the standard deviation of residuals
(Sy/x), of intercept (Sa) and of slope (Sb), and small value
of the percentage relative standard deviation and the percentage relative error (Tables 3, 4). These values demonstrated the linearity of the alignment diagrams.
Sy/x, SD of the residuals
346.597
1808.52
Sa, SD of the intercept
363.51
1871.66
Sb, SD of the slope
109.60
296.12
SD
0.89
0.97
%RSDa
0.888
0.97
%Errorb
0.398
0.435
LODc
0.06
0.12
LOQd
0.18
0.38
a
Percentage relative standard deviation
b
Percentage relative error
c
Limit of detection
d
Limit of quantitation
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Table 4 Analytical performance data for the determination of SFV by Method 2
Parameter
SFV
Column (A)
VPS
Column (B)
Column (A)
Column (B)
Linearity and range (ng/mL)
120–600
120–600
30–150
30–150
Correlation coefficient (r)
1.0000
1.0000
0.9998
0.9999
Slope
0.029
0.029
0.013
0.038
Intercept
− 0.009
− 0.016
− 0.003
− 0.063
Sy/x
SD of the residuals
0.036
0.023
0.014
0.011
Sa
SD of the intercept
0.034
0.022
0.014
0.010
Sb
SD of the slope
0.0001
0.0001
0.0002
0.0001
0.25
SD
0.48
0.28
2.16
%RSDa
0.483
0.283
2.156
0.252
%Errorb
0.216
0.127
0.965
0.113
LODc
3.94
2.47
3.41
0.90
LOQd
11.94
7.47
10.33
2.74
a
Percentage relative standard deviation
b
Percentage relative error
c
Limit of detection
d
Limit of quantitation
Limits of quantitation and limits of detection
Limits of quantitation (LOQ) and limits of detection
(LOD) were evaluated according to ICH Q2R1 recommendations using the following equation [18]:
LOQ = 10Sa/b and LOD = 3.3Sa/b
where Sa = standard deviation of the intercept of the
calibration curves and b = slope of the calibration curves.
The values of LOD and LOQ are summarized in Tables 3
and 4.
Accuracy
To demonstrate the accuracy of the proposed techniques,
the results of the assay of the studied drugs were contrasted with those of the comparison HPLC method [11].
Statistical analysis of the results using Student’s t test and
variance ratio F-test [17] uncovered no huge distinction
between the performance of the methods in regard to
accuracy and precision, individually (Tables 5, 6).
Precision
The intraday precision was assessed through repeat
investigation of various concentrations of the studied
drugs in pure form within the explicit working concentration ranges.
Each sample was investigated three consecutive
times. Likewise, the interday precision was assessed
through triplicate examination of the three specified
concentrations on three progressive days. The results
for both intraday and interday are summarized in
Tables 5 and 6. The relative standard deviations were
found to be very deliberate showing sensible repeatability and intermediate precision of the proposed techniques (Tables 7, 8, 9).
Robustness
For the assessment of the techniques robustness, one
chromatographic parameter was varied while maintaining all others unaltered. The contemplated variables
included; concentration of organic modifier (40% ± 0.1)
and pH of the mobile phase (2.5 ± 0.1). These minor
changes did not affect the chromatographic separation or
the resolution of the studied drugs from each other.
Specificity
Specificity is the capability to estimate unequivocally
the analytes in presence of other components that
might be present [18]. Methods specificity was assessed
by investigating diverse laboratory prepared mixtures
of VPS and SFV at their specified pharmaceutical ratio
(Tables 10, 11). It was additionally demonstrated by
its capacity to determine VPS and SFV in their pharmaceutical tablets without interference from regular
excipients.
Stability of standard solutions and mobile phase
Stock solution stability was studied and evaluated by
quantitation of the drugs in comparison to freshly
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Page 9 of 15
Table 5 Application of Method 1 for the analysis of VPS in its pure forms
Studied drug
VPS
Proposed method
Comparison
method [11]
Amount taken (ng/mL)
Amount found (ng/mL)
% Found
Column (A)
Column (A)
Column (A)
Column (B)
Column (B)
% Found
Column (B)
1.0
2.5
1.016
2.469
101.64
98.79
101.12
2.0
3.5
1.989
3.551
99.47
101.47
102.45
3.0
5.0
2.987
4.980
99.57
99.60
99.78
4.0
7.5
3.992
7.497
99.80
99.96
101.77
5.0
10.0
5.015
10.002
100.30
100.02
99.14
Mean
100.16
99.97
100.85
± SD
0.89
0.97
1.37
t‑test
0.95
1.17
(2.31)*
F‑test
2.39
1.99
(6.39)*
Each result is the average of three separate determinations
* Figures between parentheses are the tabulated t and F values at P = 0.05 [17]
Table 6 Application of Method 2 for the determination of the studied drugs in their pure form
Studied drug
Proposed method
Comparison
method [11]
% Found
Amount taken (ng/mL)
Amount found (ng/mL)
% Found
Column (A)
Column (B)
Column (A)
Column (B)
Column (A)
Column (B)
120.0
120.0
121.208
120.765
101.01
100.64
100.61
200.0
200.0
200.205
199.979
100.10
99.99
99.71
280.0
280.0
279.372
279.766
99.78
99.92
99.82
400.0
400.0
399.597
400.431
99.90
100.11
99.89
600.0
600.0
601.597
600.989
100.27
100.16
100.06
Mean
100.21
100.16
100.02
SFV
± SD
0.48
0.28
0.354
t‑test
0.72
0.72
(2.31)*
F‑test
1.86
1.57
VPS
(6.39)*
30.0
30.0
29.134
30.045
97.11
100.15
96.41
50.0
50.0
51.597
49.907
103.19
99.81
98.24
70.0
70.0
69.821
70.056
99.74
100.08
98.10
100.0
100.0
100.119
99.523
100.12
99.52
99.58
150.0
150.0
150.321
149.981
100.21
99.99
99.47
Mean
100.07
99.91
98.36
± SD
2.16
0.25
1.29
t‑test
2.06
0.95
(2.78)*
F‑test
2.82 (6.39)*
0.0009 (0.156)*
Each result is the average of three separate determinations
* Figures between parentheses are the tabulated t and F values at P = 0.05 [17]
prepared standard solutions. No remarkable variation
was noticed in the response to standard solutions, compared to freshly prepared standards. Furthermore, the
stability of the mobile phase was examined in a similar
method. In both methods the results demonstrated that
sample solutions and mobile phase applied during the
analysis were stable up to 3 days when preserved in the
refrigerator at 4 °C.
Moustapha et al. BMC Chemistry
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Table 7 Precision data for the determination of VPS by Method 1
Parameters
Column (A)
Column (B)
Concentration (ng/mL)
Concentration (ng/mL)
1.0
3.0
5.0
2.5
5.0
10.0
Intraday
% Found
96.83
98.30
99.75
94.23
93.28
99.21
97.78
98.77
99.71
92.00
91.55
98.72
94.82
97.66
99.52
93.23
96.26
98.60
96.48
98.24
99.66
93.15
93.70
98.84
± SD
1.51
0.56
0.12
1.12
2.38
0.32
%RSD
1.5.7
0.57
0.12
1.2
2.54
0.33
%Error
0.9
0.33
0.07
0.69
1.47
0.19
(x)
Inter‑day
% Found
98.24
98.30
99.72
92.97
96.61
99.30
99.44
96.31
100.42
100.22
99.13
100.66
99.44
96.31
100.42
93.72
99.51
99.79
99.04
96.97
100.19
95.46
98.42
99.92
± SD
0.69
1.15
0.40
3.99
1.58
0.69
%RSD
0.70
1.19
0.40
4.17
1.60
0.69
%Error
0.40
0.68
0.23
2.41
0.92
0.40
(x)
Each result is the average of three separate determinations
Table 8 Precision data for the determination of VPS by Method 2
Parameters
Column (A)
Column (B)
Concentration (ng/mL)
Concentration (ng/mL)
30
70
150
30
70
150
Intraday
% Found
95.70
99.06
99.83
101.05
99.51
99.77
93.62
97.15
99.76
92.25
96.37
97.52
100.94
100.58
99.79
95.06
97.59
99.50
96.75
98.93
99.79
96.12
97.82
98.93
± SD
3.77
1.72
0.04
4.50
1.58
1.23
%RSD
3.90
1.74
0.04
4.68
1.62
1.24
%Error
2.25
1.00
0.02
2.70
0.93
0.72
100.21
(x)
Inter‑day
% Found
(x)
98.69
97.19
99.92
100.19
98.41
101.55
98.21
100.13
96.79
103.68
97.25
97.43
98.37
99.87
97.74
98.49
99.77
99.80
99.22
97.92
99.97
98.24
100.19
± SD
2.11
0.64
0.14
1.75
3.02
1.60
%RSD
2.13
0.65
0.14
1.79
3.01
1.61
%Error
1.23
0.38
0.08
1.03
1.74
0.93
Each result is the average of three separate determinations
Moustapha et al. BMC Chemistry
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Page 11 of 15
Table 9 Precision data for the determination of SFV by Method 2
Parameters
Column (A)
Column (B)
Concentration (ng/mL)
Concentration (ng/mL)
30
70
150
30
70
150
Intraday
% Found
98.69
99.41
99.82
99.47
99.77
99.93
103.37
101.32
100.28
100.54
99.53
100.10
100.70
99.55
100.05
100.84
99.95
100.08
100.92
100.09
100.05
100.28
99.75
100.04
± SD
2.35
1.07
0.23
0.72
0.21
0.09
%RSD
2.33
1.06
0.23
0.72
0.21
0.09
%Error
1.34
0.61
0.13
0.42
0.12
0.05
100.34
99.79
99.86
103.21
97.25
100.68
100.35
99.96
100.01
97.48
95.95
99.91
100.38
99.77
99.98
98.07
98.73
99.75
100.36
99.84
99.95
99.59
97.31
100.11
± SD
0.02
0.10
0.08
3.15
1.39
0.50
%RSD
0.02
0.10
0.08
3.17
1.43
0.50
%Error
0.01
0.06
0.05
1.83
0.83
0.29
(x)
Inter‑day
% Found
(x)
Each result is the average of three separate determinations
Table 10 Assay results for the determination of VPS in laboratory prepared mixture with SFV at their pharmaceutical
ratio by Method 1
Combination
SFV/VPS mixture 4:1 (w/w)
Proposed method
Comparison
method [11]
Amount taken (ng/mL)
Amount found (ng/mL)
% Found
Column (A)
Column (A)
Column (B)
Column (A)
Column (B)
VPS
Column (B)
% Found
1.0
2.5
0.989
2.495
98.93
99.78
101.45
2.0
3.5
1.899
3.412
94.99
97.50
100.47
3.0
5.0
2.947
4.913
98.22
98.26
99.12
4.0
7.5
3.933
7.356
98.34
98.09
5.0
10.0
5.003
9.996
100.07
99.96
Mean
98.11
98.72
± SD
1.89
1.09
1.17
t‑test
− 1.82
1.99
(2.45)*
F‑test
2.62 (19.24)*
1.15 (6.94)*
100.35
Each result is the average of three separate determinations
* The figures between parentheses are the tabulated t and F values at P = 0.05 [17]
Applications
Analysis of VPS and SFV in a laboratory prepared mixture
of their pharmaceutical ratio
The reported procedures were effective and applicable for the analysis of VPS in a laboratory prepared
mixture with SFV in addition to their simultaneous
determination at their pharmaceutical ratio (1:4), as
well. The experimental results obtained are expressed
in Tables 12 and 13. The concentrations of each compound in the synthetic mixture were evaluated according to the linear regression equations. The results were
in good agreement with those reported by the reference
method [11].
Moustapha et al. BMC Chemistry
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Page 12 of 15
Table 11 Assay results for the determination of VPS and SFV in their laboratory prepared mixture at their
pharmaceutical ratio by Method 2
Studied drug
Proposed method
Comparison
method [11]
% Found
Amount taken (ng/mL)
Amount found (ng/mL)
% Found
Column (A)
Column (B)
Column (A)
Column (B)
Column (A)
120
120
120.468
119.472
100.39
99.56
200
200
200.140
195.560
100.07
97.78
98.93
280
280
279.720
273.000
99.90
97.50
100.83
400
400
399.520
393.600
99.88
98.40
600
600
588.120
600.420
98.02
100.07
Mean
99.65
98.66
±SD
0.93
1.12
0.95
t‑test
0.38
1.61
(2.45)*
SFV
F‑test
Column (B)
99.98
100.85
1.04
1.38
30
30
30.036
29.817
100.12
99.39
101.45
50
50
49.420
50.305
98.84
100.61
100.47
70
70
68.768
70.455
98.24
100.65
99.12
100
100
99.140
100.130
99.14
100.13
150
150
149.910
149.100
VPS
(6.94)*
99.94
99.40
Mean
99.26
100.04
±SD
0.78
0.62
1.17
t‑test
1.61
0.50
(2.45)*
F‑test
2.25
3.56
(6.94)*
100.35
Each result is the average of three separate determinations
* The figures between parentheses are the tabulated t and F values at P = 0.05 [17]
Table 12 Assay results for the determination of VPS in its co-formulated tablet with SFV by Method 1
Studied drug
®
Epclusa tablet (SFV
400 mg/VPS100 mg)
Proposed method
Comparison
method [11]
Amount taken (ng/mL)
Amount found (ng/mL)
% Found
Column (A)
Column (A)
Column (A)
Column (B)
Column (B)
% Found
Column (B)
1.0
2.5
0.982
2.449
98.20
97.99
102.45
3.0
5.0
2.995
4.827
99.84
96.53
101.78
5.0
10.0
5.049
9.782
100.97
97.82
98.91
Mean
99.67
97.45
101.05
±SD
1.39
0.80
1.88
t‑test
2.74
3.05
(2.78)*
F‑test
1.82
5.55
(19.0)*
Each result is the average of three separate determinations
* The figures between parentheses are the tabulated t and F values at P = 0.05 [17]
Application of the proposed method for quality control
of the studied drugs in commercial dosage forms
The proposed methods were successfully applied for
the determination of VPS and SFV in their commercially available co-formulated tablets (Figs. 5, 6). The
results depicted in Tables 12 and 13 are consistent with
those obtained using the comparison HPLC method
[11]. Statistical analysis using Student’s t-test and variance ratio F-test [17] revealed no meaningful variation
between the performance of the methods concerning the
accuracy and precision, respectively. The favorable percentage recoveries with low standard deviation values
emphasized that the proposed methods were convenient
Moustapha et al. BMC Chemistry
(2019) 13:118
Page 13 of 15
Table 13 Assay results for the determination of VPS and SFV in their co-formulated tablet by Method 2
Studied drug
SFV Epclusa® tablet
(SFV 400 mg/VPS
100 mg)
Proposed method
Comparison
method [11]
Amount taken (ng/mL)
Amount found (ng/mL)
% Found
Column (A)
Column (A)
Column (A)
Column (B)
Column (B)
% Found
Column (B)
200.0
200.0
196.900
199.520
98.45
99.76
99.98
400.0
400.0
389.760
394.920
97.44
98.73
98.93
600.0
600.0
596.760
589.080
Mean
99.46
98.18
100.83
98.45
98.89
100.85
±SD
1.01
0.80
0.95
t‑test
1.83
1.42
(2.78)*
F‑test
VPS
Epclusa® tablet
(SFV 400 mg/VPS
100 mg)
50.0
50.0
48.810
49.955
1.12
1.41
97.62
99.91
(19)*
102.45
100.0
100.0
98.000
98.040
98.00
98.04
101.78
150.0
150.0
148.665
148.185
99.11
98.79
98.91
Mean
98.24
98.91
±SD
0.77
0.94
101.05
1.88
t‑test
2.39
1.76
(2.78)*
F‑test
5.89
3.99
(19.0)*
Each result is the average of three separate determinations
* The figures between parentheses are the tabulated t and F values at P = 0.05 [17]
Fig. 5 Typical chromatogram of VPS in its co‑formulated tablet with SFV under the described chromatographic conditions (Method 1 A and B). a
VPS 2.5 ng/mL utilizing column A. b VPS 1.0 ng/mL utilizing column B
Moustapha et al. BMC Chemistry
(2019) 13:118
Page 14 of 15
Fig. 6 Typical chromatograms of co‑formulated tablet of the studied drugs VPS (10 ng/mL) and SFV (40 ng/mL) under the described
chromatographic conditions (Method 2A and B). a Method 2 utilizing column A. b Method 2 utilizing column B
for the routine determination of the studied compounds
in their commercial dosage form.
Comparison of the two proposed methods
The present work describes two UPLC methods (1 and
2) with the utilization of two analytical columns (A and
B) for the analysis of two antiviral drugs namely VPS and
SFV.
Method 1 can detect only VPS without any interference
from SFV, the method is highly sensitive when compared
to Method 2 and far more selective. In addition, Method
1 A is more sensitive that Method 1 B, however Method 1
B provides shorter analysis time.
Method 2, has the advantage of being able to determine
both drugs at the same time, the method is more sensitive
when compared to previously reported ones, and provide
short analysis time, where Method 2 B can resolve both
drugs in less than 1.5 min. Both methods can be applied
for quality control analysis of both drugs.
Conclusion
The present work represented two convenient UPLC
methods for the determination of VPS and SFV. The
proposed UPLC approaches have been fully validated
and demonstrated accurate assay methods for the determination of VPS and SFV with enhanced sensitivity
and specificity. The good validation criteria of the proposed methods allow their application in quality control
laboratories.
Abbreviations
VPS: velpatasvir; SFV: sofosbuvir; HCV: Hepatitis C Virus; US: United States;
DAAs: Direct Acting Antivirals; AASLD: American Association for the Study of
Liver Diseases; IDSA: Infectious Diseases Society of America; LC–MS/MS: liquid
chromatography–tandem mass spectrometry; UPLC‐MS/MS: Ultra perfor‑
mance liquid chromatography‑tandem mass spectrometry; RP‑HPLC: reversed
phase‑high performance liquid chromatography; RSLC: rapid separation
liquid chromatography; μL: micro liter; μg/mL: microgram per milliliter; v/v:
volume per volume; mL/min: milliliter per minute; LOQ: limits of quantitation;
LOD: limits of detection; ICH: The International Council for Harmonisation of
Technical Requirements for Pharmaceuticals for Human Use; N: no. of theoreti‑
cal plates; k’: capacity factor; Tf: tailing factor; Sa: standard deviation of the
Moustapha et al. BMC Chemistry
(2019) 13:118
Page 15 of 15
intercept; Sb: standard deviation of the slope; SD: standard deviation; %RSD:
percentage relative standard deviation; %Error: percentage relative error.
7.
Acknowledgements
Not applicable.
8.
Authors’ contributions
FB provided the authentic drugs and dosage forms, proposed the subject,
participated in revision of the manuscript. MM designed the assay, con‑
ducted its validation, analysis of the samples, participated in the results. RE,
participated in the study design, assay design, literature review, discussion and
participated in manuscript preparation, results and discussion. All authors read
and approved the final manuscript.
9.
Funding
The research was not funded by any funding body.
11.
Availability of data and materials
All data and materials are available on request (Moustapha Eid Moustapha and
Rania Mohamed El‑Gamal).
Competing interests
The authors declare that they have no competing interests.
Author details
Department of Chemistry, College of Science and Humanities, Prince Sattam
Bin‑Abdul Aziz University, Al‑Kharj 11942, Kingdom of Saudi Arabia. 2 Depart‑
ment of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura
University, P.O. Box 35516, Mansoura, Egypt. 3 Present Address: College of Phar‑
macy, Prince Sattam Bin‑Abdul Aziz University, King Abdullah Road, Al‑Kharj,
Kingdom of Saudi Arabia.
1
10.
12.
13.
14.
15.
Received: 22 May 2019 Accepted: 11 September 2019
16.
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