Development and validation of LC‐MS/MS method for

simultaneous determination of sofosbuvir and daclatasvir in

human Plasma: Application to pharmacokinetic study
Ola M. Abdallah1,2 | Ahmed M. Abdel‐Megied3 | Amira S. Gouda4

1Analytical Chemistry Department, Faculty of

Pharmacy, Al‐Azhar University (Girls), Cairo,
Abstract
Egypt
A simple and highly sensitive liquid chromatography–tandem mass spectrometry (LC‐MS/MS)
2Pharmaceutical Chemistry Department,
bioanalytical method was developed and fully validated for the first time for the simultaneous
Faculty of Pharmacy, Badr University in Cairo
(BUC), Badr City, Cairo, Egypt determination of newly discovered antiviral drugs, namely sofosbuvir (SOF) and daclatasvir (DAC) in
3Pharmaceutical Analytical Chemistry human plasma. Tadalafil (TAD) was used as internal standard (IS). SOF, DAC and TAD (IS) were extracted
Department, Faculty of Pharmacy and from plasma using liquid–liquid extraction technique with methyl tert‐butyl ether. The chromatographic
Pharmaceutical Manufacturing, Kafrelsheikh
separation was carried out using ZorbaxSB‐C18 column (4.6 × 50 mm,5 μm) and 5 mM ammonium
University, Kafrelsheikh City, Egypt
4Zi‐diligence Research Center, Cairo, Egypt
formate buffer (pH 3.5)–acetonitrile (50:50, v/v) as mobile phase in an isocratic elution mode pumped

Correspondence
Ahmed M. Abdel‐Megied, Pharmaceutical
Analytical Chemistry Department, Faculty of
Pharmacy and Pharmaceutical Manufacturing,
Kafrelsheikh University, Kafrelsheikh City, Egypt.
Email: dr_ahmed80@hotmail.com,
dr_ahmed80@pharm.kfs.edu.eg
1 | INTRODUCTION
at a flow rate 0.7 mL min−1. The quantitation was performed on API4500 triple quadrupole tandem mass
spectrometer with positive electrospray ionization interface in multiple reaction monitoring mode.
Validation was applied according to US Food and Drug Administration guidelines for bio‐analytical
methodswith respect to linearity, precision, accuracy, selectivity, carry‐over, stability and dilution
integrity. Linearity was obtained over concentration ranges of 0.3–3000 and 3–3000 ng mL−1 for SOF and
DAC, respectively, by applying a weighted least‐squares linear regression method (1/x2). The proposed
method could be applied successfully in bioequivalence and/or clinical studies for therapeutic drug

Hepatitis C virus (HCV) infection and its monitoring of patients undergoing dual combination therapy as the latter combination proved more

complications affect more than 150 million efficacious and powerful tool for the complete treatment of hepatitis C genotype 3 within 16 weeks.

people worldwide, especially in Egypt, which The suggested method has been applied successfully to pharmacokinetic studies with excellent assay

has the highest prevalence of HCV in the ruggedness and reproducibility.

world, estimated nationally about 14.7%

KEYWORDS
(Kandeel et al., 2017; Souri, Jalalizadeh, &
Shafiee, 2007). Thus prevention of HCV must daclatasvir, hepatitis C, human plasma, LC‐MS/MS, pharmacokinetic study, sofosbuvir

become a national priority as it is considered the main cause of liver anti‐HCV activity in vitro (Product leaflet, 2013). Currently, SOF is the most

cirrhosis and hepatocellular carcinoma (Thein, Yi, Dore, & Krahn, 2008). Up used antiviral agent owing to its high pangenotypic effectiveness, alone or

until 2011, interferon (PEG‐α‐INF) with ribavirin was the only combination in combination with others such as daclatasvir (DAC), which inhibits the

available in global market. This protocol of treatment was effective against HCV nonstructural protein 5A (NS5A) by interfering with the replication and

genotype 1 and associated with severe side effects (Palumbo, 2011). A new impairing phosphotidylinositol‐4‐kinase III‐α activation. However, NS5A

antiviral class, sofosbuvir (SOF), is an oral nucleotide analog that inhibits has no known enzymatic functions and so it is difficult to understand its

the RNA polymerase of the HCV virus by being a defective substrate (Sofia mode of action (Belema et al., 2014; Smith, Regal, &

et al., 2010). The major metabolite of SOF, GS‐331007, was proven to lack
Mohammad, 2016; Figure 1). There are eight metabolites of DAC, one of which (BMS‐805215) has some activity; however, it is 100‐fold less potent than the
parent drug (Product monograph, 2016). Exposure of

Abbreviations: DAC, daclatasvir; HCV, hepatitis C virus; MRM, multiple reaction

monitoring; NS5A, nonstructural protein 5A; SOF, sofosbuvir; TAD, tadalafil.
Biomedical Chromatography. 2018;e4186. wileyonlinelibrary.com/journal/bmc
Copyright © 2018 John Wiley & Sons, Ltd. 1 of 9 https://doi.org/10.1002/bmc.4186
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FIGURE 1 Chemical structures for (a) sofosbuvir (SOF) and (b) daclatasvir (DAC)
the SOF metabolite (GS‐331007) was similar in the presence and absence
of DAC, while SOF exposures were ~35% higher in the presence of DAC
(http://www.hcvdruginfo.ca/downloads/
Hepatitis%20Cint_new%20NS5A%20inhibitors.pdf). The published
methods for determination of SOF and/or DAC in human plasma are HPLC
(Baker, El‐Kafrawy, Mahrous, & Belal, 2017; Hassib, Taha, Elkady, &
Barakat, 2017; Nannetti et al., 2017; Srinivasu, Kumar, & Thirupathi, 2016),
LC‐MS/MS (Abdallah, Abdel‐Megied, & Gouda, 2017; Ariaudo et al., 2016;
Jiang et al., 2015; Nebsen & Elzanfaly, 2016; Qu, Wang, Zeng, Lub, & Yin,
2015; Rezk et al., 2015, 2016, b; Shi et al., 2015) and electrochemical
detection (Azab & Fekry, 2017). To author's knowledge, this bio‐analytical
method is the only LC‐MS/MS method for determination of SOF and DAC in
human plasma with application to a pharmacokinetic study for both. Owing
to extensive use of these drugs in combination therapy, the monitoring of
SOF and DAC concentrations in patients undergoing HCV therapy is crucial,
especially to determine drug interaction potential. Treatment optimization
through therapeutic drug monitoring has already been reported to increase
the efficacy of the therapy, and could lead to a cost saving as well.
The present work aimed to develop a simple and highly sensitive LC‐
MS/MS bio‐analytical method with high accuracy and precision for
simultaneous determination of SOF and DAC in human plasma. The method
was thoroughly validated according to new World Health Organization bio‐
analytical guidelines (Bristol‐Myers‐Squibb, 2014; WHO Prequalification
Team – Medicines, 2015).
2 | EXPERIMENTAL
2.1 | Chemicals and reagents
SOF was purchased from Optemus Ltd (Telangana, India). DAC was
purchased from Virupaksha Organics Ltd (Mumbai, India). Tadalafil (IS) was
purchased from SMS Ltd (Hyderabad, India). Methanol, acetonitrile and
ammonium formate (HPLC grade) were obtained from Sigma‐Aldrich
(Milwaukee, USA). Methyl tert‐butyl ether (HPLC grade) was purchased
from Scharlab S.L. Chemicals (Barcelona, Spain).
ABDALLAH ET AL .
Ultra‐pure water (resistivity > 18 MΩ cm−1 at 25°C and TOC < 5 ppb) was
obtained from a MilliQ UF‐Plus system (Millipore, Bedford, MA, USA). Blank
plasma samples were obtained from (a) Vacsera Holding Co. (Giza, Egypt),
(b) Dr Mohamed El Shabrawishy Hospital (Giza, Egypt), (c) Mataria Institute
for Kidney and Urology (Cairo, Egypt), (d) El Demerdash Hospital (Cairo,
Egypt) and (e) El‐Zeraien Hospital (Giza, Egypt) and El‐Helal Hospital (Cairo,
Egypt).
2.2 | Pharmaceutical formulations
Solvaldi® 400 mg tablets were manufactured by Gilead Sciences (Co. Cork,
Ireland) for Gilead Sciences international (Cambridge, UK). Daklinza® 60 mg
film‐coated tablets (Bristol‐Myers Squibb Pharma EEIG, UK) were labeled
to contain 65.92 mg daclatasvir dihydrochloride, equivalent to 60 mg DAC
per tablet.
2.3 | Instruments
Quantitative analysis was performed using an Exion LC™ chromatographic
system (ABSciex, USA). The detection was performed using a triple
quadrupole mass spectrometry API4500 (ABSciex, Canada) operated in
positive electrospray ionization using multiple reaction monitoring (MRM)
mode. The curtain, ion source gas 1 and ion source gas 2 pressures were
set at 30, 30 and 20 psi, respectively. The source temperature was 600°C
and the ion‐spray capillary voltage was 4000 V. The collision gas was set at
8 psi. The optimization of the analyte‐dependent MS/MS parameters was
performed via direct infusion of standards into the MS at a flow rate of 10
μL/min. Data acquisitions were performed with Analyst 1.6.3 software
Hotfixes® (ABSciex) to control all parameters of HPLC and MS.
2.4 | Liquid chromatographic and mass
spectrometric conditions
Chromatographic separations were achieved using a Zorbax SB‐C18 column
(4.6 × 50 mm, 5 μm; Agilent Technologies, CA, USA) and the mobile phase
was pumped with an isocratic elution program consisting of 5 m M
ABDALLAH ET AL . 3 of 1
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ammonium formate buffer pH 3.5–acetonitrile (50:50, v/v) was delivered
at flow rate 0.7 mL min−1. Each component of the mobile phase was
degassed before use in an ultrasonic bath for 10 min. The column
temperature was maintained at 25°C with an injection volume of 3 μL. The
MRM mode with positive electrospray ionization interface was used by
detection of the ions at transition pairs of m/z 530.0 → 243.3 for SOF, m/z
739.5 → 565.3 for DAC and m/z 390.2 → 268.2 for tadalafil (TAD) (IS) which
are represented in Table 1.
2.5 | Standard solutions and calibration curves
The primary stock solutions (300 μg mL−1 of SOF and DAC, and 100 μg mL−1
of TAD, IS) were completed with methanol and stored at −20°C for 2 weeks.
Appropriate dilutions were then made in methanol to produce working
solutions of 30,000 ng mL−1of SOF and DAC and 10 μg mL−1 of TAD (IS). For
each analyte, quality control (QC) samples were prepared at three levels
high (QCH), medium (QCM) and low (QCL) at concentrations 2400, 1200,
0.9 ng mL−1 for SOF and 2400, 1200, 9 ng mL−1 for DAC.
2.6 | Sample preparation
Before analysis, the plasma samples were thawed at room temperature.
−1
Anliquot of 0.5 mL plasma was spiked with 50 μL of 10 μg mL IS then
vortex mixed for 30 s. Liquid–liquid extraction was carried out by adding
3.5 mL methyl tert‐butyl ether, and mixed for 4 min. Finally, samples were
centrifuged at 4500 rpm at ambient temperature for another 5 min. The
clear upper layer was separated carefully and evaporated at 45°C under a
vacuum stream, then it was reconstituted with 300 μL of acetonitrile–water
in the proportion 40:60, v/v, and finally, 3 μL was injected into the LC‐
MS/MS system.
chromatographic interference from endogenous plasma components
between the analytes and IS.
2.7.2 | Linearity and range
Calibration curves were constructed by plotting the ratio of peak area of
SOF and DAC with TAD (IS) against the nominal concentration of calibration
standards. Concentrations used for SOF were 0.3, 3, 15, 30, 150, 300, 900,
1500 and 3000 ng mL−1 and 3, 15, 30, 150, 300, 900, 1500 and 3000 ng mL−1
for DAC. Construction of calibration plots was performed using weighted
least‐squares linear regression method (1/x2) by measuring the peak area
ratio of the analytes to the IS. Linearity was achieved with correlation
coefficients 0.9996 and 0.9984 for SOF and DAC, respectively. The QC
samples concentrations and analytes under investigation were calculated
according to the calibration plots. The set of acceptance criteria deviated
±15% from the nominal value for back‐calculated standard concentrations
except for LLOQ, which was set as ±20%.
2.7.3 | Carry‐over
Blank plasma samples were injected after calibration standard at the ULOQ
to make sure that it did not affect the accuracy and the precision of the
proposed method. For all three analytes, the carry‐over should be <20% of
the peak response of the LLOQ.
2.7.4 | Precision and accuracy
Assay run precision was determined as intra‐ and inter‐day precision using
six replicates (n = 6) for intra‐day and 18 replicates (n = 18) for inter‐day,
expressed as relative standard deviation (RSD). The accuracy of the method
was expressed as relative error (RE) and was within ±10.06% for the
analytes. It was expressed with an acceptance criterion of ±15% except for
the LLOQ, which was set as ±20%.

2.7 | Method validation

2.7.5 | Extraction recovery and matrix effect
Validation of the proposed method was performed with respect to The relative recoveries (RE) of SOF, DAC and TAD (IS) from real human
selectivity, precision, accuracy, matrix effect, stability, dilution integrity, plasma were determined by comparing the peak area before extraction and

TABLE 1 LC‐MS/MS parameters selected for the quantification of SOF, DAC and TAD (IS)
Analyte Q1a (m/z) Q3b (m/z) Declustering potential (V) Entrance potential (V) Collision energy (V) Cell exit potential (V)

SOF 530.0 243.3 80.0 10.0 26.2 9.0

DAC 739.5 565.3 80.0 10.0 63.7 13.5

TAD (IS) 390.2 268.2 59.0 10.0 16.0 9.0

DAC, daclatasvir; SOF, sofosbuvir; TAD, tadalafil.
a
Q1, precursor ion.
b Q3, product ion.
carry‐over and application to incurred plasma samples as per the guidelines the response of unextracted standards at equivalent concentrations. The IS
of the US Food and Drug Administration (2001; Zimmer, 2014). recovery was similarly assessed. The matrix effect (ME) was demonstrated
by applying a comparison between the peak areas obtained from samples
2.7.1 | Selectivity with those obtained from the pure standard solution at equivalent

The selectivity of the proposed method was evaluated by analyzing six concentrations. The process efficiency was estimated as PE = [ME × RE/100]

different sources of human plasma to demonstrate the lack of (Matuszewski, Constanzer, & Chavez‐Eng, 2003). All parameters were
evaluated at three QC levels in six replicates (n = 6). The estimation of IS‐
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normalized matrix factor was obtained by dividing the matrix factor of each
analyte by the matrix factor of the IS.
2.7.6 | Stability
Analyte stability in human plasma was evaluated by analyzing three
replicates (n = 3) at the low and high concentrations. The samples were
exposed to different conditions of temperature with different time
intervals and the results were compared with those obtained from freshly
prepared samples. Stability of analytes in plasma was evaluated at room
temperature after 24 h. The extracted samples were kept in the
autosampler at 4°C and evaluated after 72 h. The QC samples were frozen
and stored at −80°C for 100 days. The samples were evaluated after three
freeze–thaw cycles, stored at −80°C for 12 h and thawed unassisted at
room temperature. The results should be within 15% of nominal
concentrations.
2.7.7 | Dilution integrity
Dilution integrity could be demonstrated through spiking the matrix with
an analyte concentration above the LLOQ and diluting this sample with the
same blank matrix (at least five determinations per dilution factor). SOF and
DAC were spiked separately into real human plasma samples. Furthermore,
these solutions were diluted in six replicates (n = 6) by 2‐ and 4‐fold with
human plasma. The acceptance criteria must have accuracy of 100 ± 15%
and precision ≤15%.
2.7.8 | Incurred plasma samples reanalysis
The use of calibration standards and QC samples during validation may not
mimic the actual study samples. Therefore reanalysis of study samples was
done to evaluate the accuracy of incurred samples. The percentage
difference between the initial concentration and the concentration
measured during the repeat analysis should not be more than 20% of their
mean.
2.8 | Pharmacokinetic study
The aim of this study is to investigate the pharmacokinetic parameters of
SOF and DAC in three healthy male subjects. The study was conducted
according to the Declaration of Helsinki for biomedical research involving
human subjects (https://www.wma.net/ policies‐post/wma‐declaration‐
of‐helsinki‐ethical‐principles‐for‐medical‐research‐involving‐human‐
subjects/, last accessed May 2017), which indicates that all volunteers
should be informed about the protocol, purpose and risk of the study then
give written consent to participate. In addition, Zi Diligence Research
Center (Cairo, Egypt) reviewed and approved all experimental procedures.
Volunteers were prohibited from eating for 12 h before the study but water
was freely available. Blood samples (3.0 mL) were collected from a forearm
vein into vacutainers containing K3EDTA at (pre‐dose) 0.00, 0.25, 0.50, 0.75,
1.00, 1.25, 1.50, 1.75, 2.00, 2.50, 3, 4, 5, 6,8, 10, 12, 24, 36, 48 and finally
72 h after simultaneous co‐administration of a single dose Sovaldi® 400 mg
ABDALLAH ET AL .
and Daklinza® 60 mg tablets. The samples were immediately centrifuged at
3500 rpm for 10 min. The plasma obtained was stored at −80°C until
analysis. All pharmacokinetic parameters were estimated using the
validated Kinetica® 5.1 SP1software.
3 | RESULTS AND DISCUSSION
3.1 | Development of sample preparation
Development of a procedure for plasma extraction is considered the
cornerstone of any bio‐analytical study in order to reach the maximum
extraction recovery with minimum matrix effects, thus enhancing the
sensitivity and accuracy of LC‐MS/MS analysis. The procedure used for the
extraction of any drug from biological matrices usually requires the removal
of proteins and any interfering species prior to the HPLC analysis.
Therefore, many trials were performed to achieve the best recovery with
the lowest matrix effect. For simplicity and time‐saving, a protein
precipitation technique using methanol, acetonitrile was tried but the
results were unsatisfactory owing to high matrix effects and low recovery
at low concentrations. Therefore, liquid–liquid extraction using methyl tert‐
butyl ether, dichloromethane and diethyl ether was also applied. It was
found that methyl tert‐butyl ether is the most reproducible solvent with
the best recovery and minimal matrix effect.
3.2 | Optimization of the chromatographic
conditions
Optimization of the proposed method was required several trials using
different mobile phases and stationary phases. Columns such as Kinetex®
C8 column (100 × 4.6 mm, 5 μm), Kinetex® C8 column (50 × 4.6 mm, 5 μm),
Zorbax Eclipse XDB‐C18 column (4.6 × 100 mm, 3.5 μm) and Zorbax SB‐C18
column (4.6 × 50 mm, 5 μm) were tried. The C8 columns resulted in low
resolution between SOF and matrix peaks or long retention time for DAC.
Using Zorbax Eclipse XDB‐C18 column (4.6 × 100 mm, 3.5 μm) resulted in
long run time while the Zorbax SB‐C18 column (4.6 × 50 mm, 5 μm) was the
most suitable one since it produced symmetrical peaks with high resolution
and high sensitivity within a reasonable time of analysis of <2.5 min.
Preliminary trials with mobile phase compositions containing different
ratios of organic and aqueous phases were tested in an isocratic mode.
Different trials were carried out at different values of pH, 2–7, of the mobile
phase, but all attempts were futile. A 0.1% aqueous solution of formic acid,
ammonium acetate pH 2–6 with different ratios of acetonitrile and
methanol was tested; the results were unsatisfactory owing to high matrix
interference that leads to relative low responses, especially for SOF. It was
a challenge to find a suitable LC‐MS/MS method for both analytes owing to
different physiochemical properties where they behave in different ways in
plasma extraction and chromatographic separation. The best conditions
were achieved with isocratic elution using a ZorbaxSB‐C18 column (4.6 × 50
mm, 5 μm). A mixture of ammonium formate buffer (pH 3.5; 5 m M) and
acetonitrile (50:50, v/v) was used as the mobile phase pumped at a flow
rate of 0.7 mL min−1. The peaks achieved were well defined and resolved
ABDALLAH ET AL . 5 of 1
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free from tailing with retention times 1.23, 1.09 and 1.72 min for SOF, DAC 3.4.3 | Carry‐over
and TAD (IS), respectively, as shown in (Figure 2). Blank plasma samples were injected after calibration standards at ULOQ
(3000 ng mL−1) for SOF and DAC. The carry‐over in the blank samples was
found to be <20% of LLOQ.
3.3 | Optimization of mass parameters
Both analytes and internal standard produced strong signals in the positive‐ 3.4.4 | Precision and accuracy

FIGURE 2 Mass chromatograms of (a) blank plasma, (b) blank plasma spiked with LLOQ and (c) plasma samples of subject at peak concentration
ion mode rather than the negative‐ion mode. Therefore, ESI+ (Cmax) after Assay run precision was determined as intra‐ and inter‐day precision, which
oral co‐administration of SOF/DAC, 400/60 tablets was performed using six replicates (n = 6) for intra‐day and 18 replicates (n
= 18) for inter‐day, expressed as RSD. The intra‐day and inter‐day precisions
ranged from 1.54 to 15.97% for the analytes.

mode was appropriate for the detection both analytes and IS. The The accuracy of the method expressed as RE was within ±10.06% for the

predominant protonated ions [M + H]+ of SOF, DAC and TAD (IS) in the Q1 analytes. The results are summarized in Table 3.

full‐scan MS spectra were at m/z 530.0, 739.5 and 390.2, respectively, as

shown in Figure 3. The collision energy and other MRM parameters were 3.4.5 | Extraction recovery and matrix effect
optimized. The following product ions were selected: 243.3, 565.3 and Matrix effect was evaluated using blank plasma samples obtained from six
268.2 for SOF, DAC and TAD (IS), respectively. different batches. The matrix factor for each lot was calculated by
comparing the analytes and (IS) peak areas in extracted plasma samples

3.4 | Method validation with those in the neat equivalent standard concentration. The IS‐
normalized matrix factor, CV %, was found not to exceed10.38% for both
3.4.1 | Selectivity
analytes, indicating that any enhancement from the human plasma should
Representative chromatograms are shown in Figure 3 and indicate that
be negligible. The relative recovery is the real recovery, which could be
there were no interference peaks observed from human plasma at the
calculated by comparing the area ratio response of extracted plasma
retention times of the analytes and IS. The retention times of SOF, DAC and
samples (spiked before extraction) with the post‐extraction (spiked after
TAD (IS) were 1.23, 1.09 and 1.72 min, respectively. Six plasma (n = 6)
extraction) samples. The recoveries across QCs ranged from 83.13 to
samples were extracted to demonstrate the sensitivity of the proposed
91.28% for both analytes and IS as presented inTable 4.
method.

3.4.6 | Stability
3.4.2 | Calibration curve and quantitation range
Stability of analytes in human plasma was evaluated under different
The calibration plots were found to be linear over the ranges of 0.3–3000
storage conditions at low and high concentrations with three
and 3.0–3000 ng mL−1 for SOF and DAC, respectively, as shown in Table 2.
determinations for each. Samples were exposed to different temperature
Blank and zero samples were assayed in order to verify the absence of
conditions for different time intervals and the results were compared with
interference. The accuracy values for LLOQ were acceptable, ranging from
those obtained from freshly prepared samples. Spiked plasma samples
97.50 to 100.20% for both analytes while precision was acceptable with
were stable after storage at room temperature for 24 h. The extracted
RSD < 0.96%.
samples were analyzed and found to be stable after keeping in the
autosampler at 4°C for 72 h. QC samples were frozen at −80°C and
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remained stable for 100 days. Samples were stable after three freeze–thaw
cycles after storing at −80°C for 12 h and thawing unassisted at room
temperature. During repeated freezing and thawing, it was observed that
substance loss was negligible, as summarized in Table 5.
3.4.7 | Dilution integrity
Plasma samples were spiked at concentrations 4800 and 9600 ng mL−1 for
both SOF and DAC. Blank plasma samples were diluted 2‐ and 4‐fold at six
determinations per dilution. Precision RSD was between

FIGURE 3 Representative spectra for (a) SOF, (b)

DAC and (c) IS (tadalafil)

TABLE 2
Linearity results for SOF and DAC in human
plasma 2.35 and 4.51%, while accuracy results
ranged from 93.49 to 103.90%
Linear range for the analytes.
Analyte Run (ng mL−1) Linear equation R2
SOF 1 0.3–3000 y = 0.00140x + 0.00277 0.9984 3.4.8 | Incurred plasma samples
2 y = 0.00148x + 0.00317 0.9964 reanalysis
Reanalysis of study samples was done to evaluate
3 y = 0.00153x + 0.00128 0.9998
the accuracy of incurred samples. The percentage
DAC 1 3–3000 y = 0.00170x + 0.00104 0.9984 difference from initial concentration to the final
2 y = 0.00136x + 0.00214 0.9998 concentrations was measured during the repeat
analysis, and ranged from 4.56 to 14.07% for the
3 y = 0.00145x + 0.00113 0.9968
analytes, as shown inTable 6.

TABLE 3 Intra‐ and Inter‐day accuracy and precision results for SOF and (ng mL−1) RE (%) CV (%) RE (%) CV%
DAC
Concentration Intra‐day Inter‐day SOF LLOQ 0.3 −4.93 2.42 0.91 5.42
Analyte QCL 0.9 −2.57 3.57 1.52 3.40
ABDALLAH ET AL . 7 of 1
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QCM 1200 10.06 1.54 4.20 4.46 4 | APPLICATION TO PHARMACOKINETIC

QCH 2400 7.86 3.85 2.52 8.19
STUDY

DAC LLOQ 3 0.34 3.98 −5.74 15.97 The validated LC‐MS/MS bioanalytical method was successfully applied to
QCL 9 7.25 4.67 6.52 9.02 determine SOF and DAC in human plasma for pharmacokinetic study in

QCM 1200 −6.85 2.16 0.36 4.78 three healthy male volunteers, following oral co‐administration of tablets
containing SOF (400 mg) and DAC (60 mg) in a fasting condition. The mean
QCH 2400 −6.23 4.34 −1.43 8.20
plasma concentration–time profiles are illustrated in (Figure 4). The
n 6 18 maximum plasma concentrations (Cmax) for SOF and DAC were 1837.65 ±
541.27 and 1308.19 ± 6.22 ng mL−1 and were achieved at 0.42 ± 0.12 and
1.63 ± 1.24 h, respectively. The area under the concentration–time curve
(AUC0–t) for SOF and DAC
TABLE 4 Matrix effect, relative recovery and process efficiency for SOF and DAC
Concentration Matrix effect Relative recovery Process efficiency
(ng mL−1)

ME (%) CV (%) RE (%) CV (%) PE (%) CV (%)

Analyte

SOF QCL 0.9 100.98(100.15)a 6.84 (3.96) 83.13 (89.61)b 12.45 (4.56) 84.15 (89.69)c 2.25 (2.65)

QCM 1200 99.64 (100.53)a 3.34 (2.60) 86.99 (83.91)b 4.40 (5.40) 86.66 (84.36)c 2.51 (6.46)

QCH 2400 98.11 (98.54)a 2.99 (2.29) 90.75 (88.32)b 5.52 (8.54) 89.01 (87.03)c 4.53 (9.24)

DAC QCL 9 98.21 (100.43)a 3.98 (8.19) 91.28 (85.89)b 6.49 (7.90) 89.48 (86.23)c 4.32 (5.39)

QCM 1200 97.52 (100.53)a 1.34 (2.60) 90.98 (83.91)b 6.90 (5.40) 88.71 (84.36)c 5.52 (6.46)

QCH 2400 98.34 (98.54)a 2.52 (2.29) 88.80 (88.32)b 1.35 (8.54) 87.32 (87.03)c 2.13 (9.24)

n 6 6 6

a
Values of matrix effect for internal standard. b
Values of relative recovery for internal standard.
c
Values of process efficiency for internal standard.

TABLE 5 Stability results for SOF and DAC in plasma at different conditions
Short‐term stability at room Freeze–thaw stability at Long term stability at Processed sample stability at
temperature (24 h) −80 °C (three cycles) −80 °C(100 days) 4 °C(72 h)

Concentration Accuracy CV Stability Accuracy CV Stability Accuracy CV Stability Accuracy CV Stability

Analyte (ng mL−1) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%) (%)
SOF QCL 0.9 100.90 7.81 102.38 101.36 4.82 102.84 101.92 4.55 103.41 96.66 4.72 90.99
QCH 2400 102.77 9.05 96.62 104.98 2.65 98.70 104.58 0.24 98.32 100.59 3.80 93.18

DAC QCL 9 103.05 4.13 95.70 103.57 4.31 96.19 105.02 7.21 97.54 108.85 4.05 104.58
QCH 2400 93.12 2.40 95.63 96.28 5.66 98.88 95.10 3.31 97.66 100.19 2.97 105.03

n 3 3 3 3

TABLE 6 Incurred sample reanalysis

SOF DAC
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Incurred Initial concentration Repeat concentration Difference

sample (ng mL−1) (ng mL−1) (%)
reanalysis Initial concentration Repeat concentration Difference
(ng mL−1) (ng mL−1) (%)
833.96 914.50 9.21 1098.02 1040.09 5.42
383.09 441.08 14.07 1312.58 1390.89 5.79
78.02 86.77 10.62 1200.02 1273.66 5.95
5.52 6.01 8.55 251.58 227.08 10.24
0.357 0.339 5.28 79.97 76.40 4.56
9.94 11.28 12.67 73.93 65.13 12.66

FIGURE 4 Mean plasma concentration (± SD) following co‐administration of single oral dose of SOF/DAC tablets contain 400/60 mg SOF/DAC:
(a) SOF; (B) DAC values from different reported studies ranged from 0.5 to 2 h, and it was
observed that there was no disparity between the obtained values in our
study compared with the other reported ones (https://www.accessdata.
TABLE 7 Pharmacokinetic parameters (results ± SD)
fda.gov/drugsatfda_docs/nda/2014/205834Orig1s000ClinPharmR. pdf;
Parameters SOF DAC
German, Mathias, Brainard, & Kearney, 2016; Kirby, Symonds, Kearney, &
Cmax (ng mL−1) 1837.65 ± 541.27 1308.19 ± 6.22
Mathias, 2015; Rezk, Bendas, et al., 2016; Smolders et al., 2016). For DAC,
Tmax (h) 0.42 ± 0.12 1.63 ± 1.24 Cmax, AUC0–∞, T1/2 and Tmax results from different reported studies showed

T1/2 (h) 1.56 ± 1.21 11.16 ± 1.71 values in the ranges 928–1396 ng mL−1, 9486–14,572 ng h mL−1, 11.9–15 h
and 0.75–2 h, respectively. The results were in close agreement with those
AUC0–t (ng h mL−1) 1322.14 ± 437.61 15,668.80 ± 1308.29
reported in our study
AUC0–∞(ng h mL−1) 1322.69 ± 436.90 15,825.55 ± 1394.77
(https://www.accessdata.fda.gov/drugsatfda_docs/nda/
Ke (h−1) 0.64 ± 0.50 0.06 ± 0.01 2015/206843Orig1s000ClinPharmR.pdf; Bifano et al., 2011; Kawakami et
CL [mg/(ng/mL h)] 5.33 ± 1.76 0.063 ± 0.006 al., 2016; Smolders et al., 2016). It was noted in the present study that there
Vz [mg h/(ng/mL h)] 807.95 ± 803.77 58.68 ± 0.09 is no apparent effect of SOF on DAC exposure, while DAC modestly
increases SOF exposure, with the results are in close agreement with the
Vss [mg h/(ng/mL h)] 246.84 ± 78.06 53.61 ± 3.82
earlier reported studies (Eley et al., 2013) as DAC and SOF are required with
Cmax, Maximum plasma concentration; Tmax, time to peak concentration; T1/2,
elimination half‐life; AUC, area under the concentration–time curve; Ke, co‐administration.
elimination constant; CL, clearance; Vz, Apparent volume of distribution during
terminal phase; Vss, Apparent volume of distribution at steady state.
5 | CONCLUSION
was 1322.14 ± 437.61 and 15,668.80 ± 1308.29 ng h mL−1, respectively. The
AUC0–∞ for SOF and DAC was 1322.69 ± 436.90 and 15,825.55 ± 1394.77 ng The present LC‐MS/MS method was fully validated according to US Food
h mL−1, respectively. The elimination half‐life (T1/2) for SOF and DAC was and Drug Administration guidelines and successfully applied to support
1.56 ± 1.21 and 11.16 ± 1.71, respectively, as presented in Table 7. pharmacokinetic and clinical studies. The new bio‐analytical method for
The presented values were compared with earlier reported studies for simultaneous determination of SOF and DAC is considered a reliable, robust
SOF and DAC separately. For SOF, the pharmacokinetic results reported and patient‐friendly and is expected to be used widely. The application of
from different studies with respect to Cmax, AUC0–∞ and T1/ 2 were in the incurred sample reanalysis in the validation protocol helped to ensure the
ranges 622–928.7 ng mL−1, 629–1202.47 ng h mL−1 and 0.4–1.24 h, validity of results and supports the use of the proposed method as a routine
respectively. Regarding Cmax values, it has been observed that there was a assay for bioequivalence studies, which will be very useful in therapeutic
significant increase in Cmax compared with that found in the literature when drug monitoring.
administered alone. AUC0–∞ increased slightly upon co‐administration with
DAC and the half‐life was slightly longer. Time to peak concentration (Tmax) ORCID
ABDALLAH ET AL .
Ahmed M. Abdel‐Megied http://orcid.org/0000-0003-0180-0918
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How to cite this article: Abdallah OM, Abdel‐Megied AM, Gouda AS.
Development and validation of LC‐MS/MS method for
simultaneous determination of sofosbuvir and daclatasvir in human
Plasma: Application to pharmacokinetic study. Biomedical
Chromatography. 2018;e4186. https://doi.org/10.1002/

bmc.4186