Journal of Chromatography B 1117 (2019) 148–157

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

Simultaneous determination of tenofovir alafenamide and tenofovir in T

human plasma by LC-MS/MS and its application to pharmacokinetics study
in clinic
Lizhi Zhao, Zhou Li , Zhen Zhou, Xiuyuan Kang, Baihuan Fang, Huan Ma, Qinghua Ge
⁎

Shanghai Institute of Pharmaceutical Industry, National Pharmaceutical Engineering and Research Center, 1111 Halei Road, Zhangjiang Hi-Tech Park, Shanghai, China

ARTICLE INFO ABSTRACT

Keywords: An ultra performance liquid chromatography-tandem mass spectrometric (UHPLC-MS/MS) method has been
Tenofovir alafenaminde developed for the simultaneous determination of tenofovir alafenamide (TAF) and it's metabolite tenofovir (TFV)
Tenofovir in human plasma. The analytes and inter standards, TAF-d5 and TFV-d6 were extracted from human plasma via
LC-MS/MS protein precipitation (PPT) and only 200 μl plasma was needed. Chromatography separation was achieved on a
Stability
Waters Acquity UHPLC HSS T3 column (100 * 2.1 mm, 1.8 μm) with a total run time of 10 min. A tandem mass
Simultaneous determination
Pharmacokinetic
spectrometric detection was conducted using multiple reaction monitoring (MRM) mode under positive ioni-
zation mode with an electrospray ionization (ESI) interface. The method was developed and validated over the
concentration range of 4.00–400 ng/ml for TAF and 0.400–40.0 ng/ml for TFV, respectively. Each analyte in
acidified plasma was found stable during sample storage, preparation and analytical procedures. The method has
successfully overcame the lack of stability of analytes in plasma samples and been applied to the pharmacoki-
netics study of treatment of 25 mg TAF in 8 healthy volunteers under fasting condition.

1. Introduction
There are 257 million people infected with the hepatitis B virus (HBV)
and over 350 million affected by chronic hepatitis B (CHB) worldwide [1].
Nucleotide analogue (NA), as antiviral treatment drug, has shown to in-
hibit viral replication, halt the progression of HBV, even reverse cirrhosis
and improve survival [2]. Tenofovir alafenamide (TAF, formerly GS-
7340), a prodrug of tenofovir (TFV), is a potent nucleotide analogue in-
hibitor of HBV polymerase/reverse transcriptase, currently recommended
as first line treatment for CHB, including patients harbouring virus.
TAF is a phosphoramidate prodrug of TFV and is efficiently hy-
drolyzed to TFV by intracellular enzymes concluding carboxylesterase 1
(CES 1). TFV is then phosphorylated by cellular kinases to produce
tenofovir diphosphate in hepatocytes [2]. Tenofovir diphosphate is
final active metabolite of TAF, as it is for tenovovir disoproxil fumarate
(TDF) commonly used in treatment for CHB as well. However in clinical
phase 3 trials, TAF was equally potent as an antiretrovirus agent at a
30-fold lower dose (10 mg as compared to 300 mg) than TDF and has
been shown to significantly reduce kidney disturbances and bone mi-
neral density changes [3–7]. Currently FDA had approved a few TAF-
containing products including Venlidy®, Descovy®, Biktarvy®, Gen-
voya® in treatment of HIV and HBV infection [6,8].
Previously a few assays have published for measurement TAF and
TFV in human plasma [9,10], but none of them have focused on the
stability of TAF and TFV in plasma. Due to TAF is an ester drug, it could
be hydrolyzed to TFV easily by enzymes including carboxylesterase in
plasma, which bring a great challenge for the simultaneous determi-
nation of these two drugs. In this article, we described a new method for
the simultaneous determination of TAF and TFV in human plasma and
its application to pharmacokinetics study.
2. Experimental
2.1. Chemicals and reagents
Tenofovir alafenamide (TAF, purtiy:99.3%) and Tenofovir (TFV,
purity:99.0%) were all obtained from Shanghai Desano Biological
Pharmaceutical Co.,Ltd. (Shang Hai, China), d5-tenofovir alafenamide
(TAF-d5, isotopic purity:98.8%) and d6-tenofovir (TFV-d6, isotopic
purity:99.2%) were purchased from Toronto Research Chemicals
(Ontario, Canada). HPLC-grade methanol was product of MERCK
(Darmstadt, Germany). Analytical grade acetic acid and ammonium
acetate were obtained from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai China). HPLC-grade formic acid was purchased from Aladdin

⁎
Corresponding author.
E-mail address: lizhou@nperc.com (Z. Li).

https://doi.org/10.1016/j.jchromb.2019.04.011
Received 8 March 2019; Received in revised form 3 April 2019; Accepted 4 April 2019
Available online 05 April 2019
1570-0232/ © 2019 Elsevier B.V. All rights reserved.
L. Zhao, et al.
Table 1
Optimized mass parameters for analyte and IS.
Analyte MRM(m/z) Dwell time Q1 Pre CV (V) Q3 Pre
(ms) Bias (V) Bias (V)
TFV 288.00 → 176.10 100 −14 −25
TFV-d6 (IS) 294.15 → 182.05 100 −10 −27
TAF 477.20 → 176.15 100 −10 −10
TAF-d5 (IS) 482.20 → 176.10 100 −10 −30
Industrial Corporation (Shanghai China). Purified water used
throughout the study was commercially available (Wahaha®, Hangzhou
Wahaha Co., Ltd., China).
2.2. UHPLC-MS/MS conditions
A UHPLC system (Shimadzu Co., Kyoto, Japan), Consisting of two
LC-30AD pumps, a SIL-30ACMP autosampler, a CTO-20A column oven,
DGU-20A5R Degassing Unit, CBM-20A controler and a Waters Acquity
UHPLC HSS T3 column (100*2.1 mm, 1.8 μm, Waters Co., Milford, MA,
USA) equipped with a guard column (Vanguard™ BEH C18 1.7 μm,
Waters Co., Milford, MA, USA) was used for the chromatographic se-
paration.
Mobile phase ‘A’ and ‘B’ consisted of water and methanol, respec-
tively, both containing 20 mmol/L ammonium acetate (pH:6.89) and
0.1% formic acid. A gradient elution program was started at 5% ‘B’ and
last for 0.5 min followed by a linear gradients to 25% ‘B’ from 0.5 to 2.4
minand to 75% ‘B' form 2.5 to 4.6 min and kept at 100% ‘B’ during 4.7
to 7.0 min, then set to the initial conditions from 7.1 to 10 min. The
total flow rate was 0.4 ml/min from 0 to 4.6 min and 8.1 to 10 min and
0.45 mL/min from 4.7 to 8 min. An on-line motorized six-port divert
valve was used to introduce the LC eluent flow into the mass spectro-
meter over the period of 1.6–5.0 min, while the other eluent flows were
diverted to waste. The injection volume is 10 μl.
The temperatures of the analytical column and auto sampler were
maintained at 40 °C and 4 °C, respectively. Under the current LC–MS/
MS conditions, the two analytes and two IS analytes were well sepa-
rated from interferences in the matrix. The retention times were ap-
proximately 2.54, 2.54, 4.14, 4.14 min for TFV, TFV-d6, TAF, TAF-d5,
respectively.
A LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Co.,
Kyoto, Japan) equipped with an electrospray ionization (ESI) interface
operated in positive ionization mode was used for the mass spectro-
metric detection. A multiple reaction monitoring (MRM) mode was
operated to detect specific precursor and product ions of TAF, TFV,
TAF-d5 and TFV-d6. The operation conditions were optimized by in-
jecting diluted stock solutions of each analyte as follows: nebulizing gas
flow was 3 l/min, heating gas flow was 10 l/min, interface temperature
was 300 °C, DL temperature was 250 °C, heat block temperature was
400 °C, drying gas flow was 10 l/min. The specific parameters for each
analyte are shown in Table 1.
2.3. Preparations of stock solutions, internal standard solutions,calibration
standards, and quality control samples
Primary stock solutions for TAF and TFV were prepared in methanol
and methanol-water (20:80, v/v) at 200 μg/ml, respectively. The in-
dependent stock solutions of all compounds were diluted further with
methanol-water (20:80, v/v) to working standard solutions and quality
control (QC) working solutions. The concentrations of internal standard
(IS) working solutions were 200 ng/ml for TAF-d5 (IS) and 40 ng/ml for
TFV-d6 (IS), respectively. All solutions described above were stored at
4 °C.
The calibration standards were freshly prepared by adding 10 μl
working standard solutions into 204 μl acidified blank plasma (con-
tained 200 μl blank plasma and 4 μl acetic acid). The final calibration
Journal of Chromatography B 1117 (2019) 148–157
concentrations are 4.00, 10.0, 25.0, 50.0, 100, 200, 400 ng/ml for TAF
and 0.400, 1.00, 2.50, 5.00, 10.0, 20.0, 40.0 ng/ml for TFV, respec-
tively.
QC samples were prepared at lower limit quantification, low,
medium and high level (LLOQ, QCL, QCM, QCH) by spiking 204 μl
−19
acidified blank plasma (contained 200 μl blank plasma and 4 μl acetic
−19
−19
acid) with 10 μl QC working solutions. The final calibration con-
−19 centrations are 4.00, 8.00, 40.0, 320 ng/ml for TAF and 0.400, 0.800,
4.00, 32.0 ng/ml for TFV, respectively.
2.4. Sample preparation
Frozen samples of acidified human plasma were thawed in ice-water
bath and a volume of 204 μl plasma sample was transferred to a dis-
posable tube. Then 10 μl IS working solution and 10 μl working solu-
tions was spiked into each sample, followed by precipitation with
800 μl methanol. After vortexed-mixing for 5 min, centrifugation was
applied at 15870 ×g for 10 min. 900 μl upper supernatant was carefully
transferred to a glass tube, and evaporated to dryness under a gentle
stream of nitrogen in water bath at 40 °C. The residue was reconstituted
with 100 μl of methanol-water (20:80, v/v), which containing 2% acetic
acid, and an aliquot of 10 μl was injected into the LC-MS/MS system for
analysis.
2.5. Method validation
The current method was validated for specificity, linearity, intra-
day and inter-day precision, accuracy, matrix effect, recovery, dilution
integrity and stability.
2.5.1. Specificity
The specificity of method was evaluated by analyting six different
sources of blank plasma and one hemolyzed plasma sample to check for
any possible interference with the retention time of analytes and IS.
2.5.2. Carryover
Carryover was assessed by injecting blank sample and LLOQ sample
successively after injecting ULOQ sample (upper limitation of quanti-
fication of method).
2.5.3. Linearity and lower limit quantification (LLOQ)
To evaluate the linearity, calibration standards of seven different
concentrations of TAF and TFV in human plasma were measured. The
concentrations ranged from 4.00 to 400 ng/ml for TAF and
0.400–40.0 ng/ml for TFV. The lower limit quantification of TAF and
TFV were 4.00 ng/ml and 0.400 ng/ml, respectively.
2.5.4. Accuracy and precision
Inter- and intra- assay accuracy and precision were assessed by
analyzing six replicates at four QC concentration levels (LLOQ, QCL,
QCM and QCH) in three separate days. The accuracy was expressed by
percentage of measured concentration to nominal concentration and
precision was expressed by relative standard deviation (RSD).
2.5.5. Matrix effect and recovery
The absolute matrix effect was estimated by the post-extraction
addition method. Unextracted samples were prepared by adding QC
working solution and IS working solution to six different batches of
extracted blank plasma [11].
The matrix effect (ME%) was calculated by comparing the peak area
of the Unextracted sample (B) with the peak area of the aqueous
standard (A), and expressed as (B / A × 100%). After ME% 100% in-
dicates no absolute matrix effect, if ME% > 100%, the signal is en-
hanced, and accordingly, if ME% < 100%, the signal is suppressed.
Relative matrix effects was used to evaluate the variations of dif-
ferent lots of plasma suffered from the matrix effects, and was
149
L. Zhao, et al. Journal of Chromatography B 1117 (2019) 148–157

Fig. 1. MS/MS product ion mass spectra of the protonated molecules [M + H]+ of TAF, TFV and respective internal standards. TAF and TFV were monitored at m/z
477.20 → 176.15 and m/z 288.00 → 176.10, respectively. The transition monitored were m/z 482.20 → 176.10 and m/z 294.15 → 182.05 for TAF-d5 and TFV-d6,
respectively.

150
L. Zhao, et al. Journal of Chromatography B 1117 (2019) 148–157

Fig. 2. Representative chromatograms of blank plasma,TFV,TAF and respective internal standards (A) blank plasma (B) plasma-spiked with TFV at 0.400 ng/ml and
TAF at 4.00 ng/ml (C) volunteer plasma sample obtained 0.5 h after administration of 25 mg TAF, concentrations of TAF and TFV is 292 ng/ml and 1.97 ng/ml.

151
L. Zhao, et al. Journal of Chromatography B 1117 (2019) 148–157

Fig. 2. (continued)

152
L. Zhao, et al. Journal of Chromatography B 1117 (2019) 148–157

Fig. 2. (continued)

153
L. Zhao, et al. Journal of Chromatography B 1117 (2019) 148–157

Table 2 volume of 800 μl plasma was transferred to a cryogenic vials which had
Matrix effects and extraction recovery for TAF and TFV in six different lots of contained 16 μl acetic acid. The vials were capped and vortexed for 3 s.
human plasma (n = 6). Then all samples were stored at −80 °C until assay.
Analyte Analyte Matrix effects (%) Extraction recovery(%)
concentration 3. Results
(ng/mL) Mean ± SD CV(%) Mean% and SD CV(%)

TAF 8.00 95.9 ± 2.06 2.15 78.6 ± 3.13 3.98

3.1. Specificity and selectivity
40.0 96.8 ± 1.28 1.32 80.5 ± 2.77 3.44
320 96.7 ± 0.753 0.78 84.0 ± 1.65 1.96 Abundant protonated molecules of TAF and TFV that formed the
TAF-d5 2.00 102 ± 9.85 9.70 83.9 ± 2.91 3.47 base peak of each mass spectrum were observed from Q1 scans during
TFV 0.800 104 ± 1.89 1.83 68.4 ± 4.60 6.76
the infusion of the neat solution in positive mode. Two [M + H] +
4.00 100 ± 1.95 1.95 68.2 ± 2.31 3.39
32.0 101 ± 0.982 0.97 68.4 ± 2.09 3.06 precursor ions, m/z 288.00 for tenofovir, m/z 477.20 for tenofovir
TFV-d6 10.0 96.4 ± 1.89 1.97 70.2 ± 2.74 3.90 alafenamide, were subjected to collision induced dissociation (CID).
The product ion tandem mass spectra of the protonated molecules of
Matrix effects (%): (peak areas of analytes added post-extraction in six different TAF and TFV are shown in Fig. 1. Mass transition patterns, m/z
lots of plasma)/(mean peak areas of standard) × 100. 477.20 → 176.15 and m/z 288.00 → 176.10 were selected to monitor
Extraction recovery (%): (peak areas of analytes added pre-extraction in
TAF and TFV, respectively. A MS/MS channel of m/z 482.20 → 176.10
plasma)/(peak areas of analytes added post-extraction in plasma) × 100.
and m/z 294.15 → 182.05 were chosen to monitor the internal stan-
CV, (%): coefficient of variation of matrix effects.
dard, TAF-d5 and TFV-d6 respectively.
Under the current LC–MS/MS conditions, the two analytes were
calculated by the coefficients of variation [CV, %] of peak areas of well separated from interferences in the matrix. Chromatograms of
analytes added post-extraction from five different lots of blank plasma. different lots of blank plasma were found to contain no endogenous
Recovery presents the extraction efficiency of a method. It was peak co-eluted with any of the analytes or the internal standard.
evaluated at each QC level by comparing peak areas of QC samples (C) Representative chromatograms of blank samples with or without the
with B and expressed as (C/B × 100%). presence of analytes and internal standard are shown in Fig. 2. In ad-
dition, the “cross-talk” between channels used for monitoring the
2.5.6. Dilution integrity analytes and IS was evaluated by analysis of their individual solution at
Dilution effect was investigated to ensure that plasma samples could high concentration. The responses in the MRM mass transition channels
be diluted with blank matrix without affecting the final concentration. used for quantification were monitored. No “cross-talk” or interference
One concentration plasma sample (1600 ng/mL for TAF and 160 ng/mL between the analytes and IS was observed.
for TFV) were diluted with blank human plasma at dilution factors of 5
in 6 replicates and analyzed. 3.2. Matrix effects and recovery

2.5.7. Stability
The stability of TAF and TFV in spiked samples was investigated.
The stability experiments aimed at testing the effects of possible con-
ditions that the analytes might experience during collection, storage
and analysis, including three cycles of freeze and thaw, stored frozen at
−80 °C, stored in ice-water bath, and storage of extracted samples in an
autosampler (4 °C) and room temperature.
2.6. Application of the analytical method in pharmacokinetic study
The study protocol and informed consent forms were reviewed and
approved by the Medicine Ethics Committee of Shanghai Public Health
Center (Shanghai, China). All subjects provided written informed con-
sent prior to participation in the study.
These volunteers were administered a tablet of tenofovir alafena-
mide (25 mg) in the fasting state, an indwelling intravenous catheter
was inserted following aseptic techniques and blood samples were
drawn for the determination of pre-dose concentrations.
Pharmacokinetics sampling was performed at 0 h, 0.17 h, 0.33 h, 0.5 h,
0.75 h, 1.0 h, 1.33 h, 1.67 h, 2.0 h, 2.5 h, 3.0 h, 4.0 h, 6.0 h, 8.0 h,
12.0 h, 24.0 h, 48.0 h and 72.0 h. Blood (4.0 ml) was collected in
K2EDTA tubes each time and then centrifuged at 1300 ×g for 10 min. A
The results of the matrix effects and recovery are shown in Table 2.
Absolute matrix effects demonstrated that there was no evident matrix
effects on TAF and TFV, which indicated that there was little variance
between different lots of plasma and accurate results coule be obtained.
And the result of recovery indicated that the extraction efficiency for all
the analytes as well as IS was consistent and reproducible.
3.3. Linearity and sensitivity
Seven-point calibration curves were prepared ranging from 4.00 to
400 ng/ml for TAF and from 0.400 to 40.0 ng/ml for TFV, respectively.
The curves were obtained by plotting the peak area ratio of the analytes
to IS against the corresponding concentration of the analytes in the
freshly prepared plasma calibrators. The regression parameters of slope,
intercept and correlation coefficient were calculated by 1/x-weighted
linear regression for TFV and 1/x-weighted quadratic regression for
TAF in Labsolution 5.82 SP1 software used in Shimadzu LCMS 8060.
Excellent linearity was achieved with correlation coefficients >
0.9999 for all validation batches. The results were shown in Table 3.
The concentrations of calibration standards were back calculated to
obtain the accuracy of each calibration point. Concentrations for QC
samples were calculated from the resulting peak area ratios and the

Table 3
Linearity for assay of TAF,and TFV in human plasma.
Analyte Run Linear range (ng/mL) Calibration curve r

2
TAF 1 4.00–400 y = −2.24e-006 × + 0.0115× + 0.00461 0.9999
2 y = −3.54e-006 × 2 + 0.0109× + 0.00118 0.9999
3 y = −3.58e-006 × 2 + 0.0111× + −8.12e-006 0.9999
TFV 1 0.400–40.0 y = 0.116× + 0.00258 0.9999
2 y = 0.118× + 0.000499 0.9999
3 y = 0.119× + 0.00175 0.9999

154
L. Zhao, et al. Journal of Chromatography B 1117 (2019) 148–157

Table 4
Intra-day and inter-day precision and accuracy for assay of TAF and TFV in human plasma.
Analyte ACa (ng/mL) Intra-day (n = 6) Inter-day (n = 18)

b c
MC (ng/mL) RSD (%) A (%) MCb (ng/mL) RSD (%) Ac (%)

TAF 4.00 4.06 ± 0.0611 1.50 101 3.85 ± 0.212 5.49 95.8
8.00 8.03 ± 0.0887 1.12 99.8 7.64 ± 0.318 4.16 95.1
40.0 38.8 ± 1.16 3.01 96.6 38.6 ± 0.993 2.57 96.0
320 319 ± 7.29 2.27 99.3 312 ± 0.91 2.92 97.0
TFV 0.400 0.428 ± 0.0251 5.88 106 0.414 ± 0.0256 6.18 103
0.800 0.850 ± 0.0595 7.00 106 0.812 ± 0.0500 6.15 101
4.00 3.99 ± 0.107 2.67 99.4 4.02 ± 0.114 2.84 99.9
32.0 31.7 ± 1.00 3.17 98.5 31.9 ± 0.926 2.91 99.1

a
AC: Analyte concentration.
b
MC: Measured concentration.
c
A: Accuracy.

regression equation of the calibration curves. The LLOQ of the method
is 4.00 ng/ml for TAF and 0.400 ng/ml for TFV, respectively, which is
sensitive enough for the pharmacokinetics study.
3.4. Precision and accuracy
The intra-day precision and accuracy were determined by the re-
plicate analyses of QC samples (n = 6) at four level concentrations
(LLOQ, QCL, QCM and QCH) in three separate days. All replicate of the
QC samples at each concentration level from three separate validation
batches were used to evaluate the inter-day precision. The assay pre-
cision and accuracy results were shown in Table 4. The intra-day pre-
cision was within 7.00% and the inter-day precision was within 6.18%.
The assay accuracy was 95.1–106.0% of the nominal values.
3.5. Stability
The stability experiments were aimed at testing the effects of pos-
sible conditions that the samples might experience between collection
and analysis. The stability of TAF and TFV in plasma was investigated
and the results indicated that the analytes were found to be stable after
three cycles of freeze and thaw and for 6 h in ice water bath and for at
least 75 days at −80 °C. The stability of processed samples indicated
that TAF and TFV were stable when kept in the room temperature for
24 h and in the auto sampler (4 °C) for 66 h.
3.6. Dilution effect
The results indicates that concentrations above the curve can be
determined accurately and precisely when diluted 1:5 in appropriate
blank plasma.
study, the transformation from TAF to TFV can be prevented by adding
acetic acid to volume of 2% into plasma samples and we proved as
follows. Two kinds of plasma were prepared, one was normal plasma
and the other was acidified with acetic acid. Then plasma samples
containing only TAF were prepared and the concentrations were
8.00 ng/ml and 320 ng/ml, respectively. After storage at −80 °C for
75 days, the plasma samples were determined and the results are shown
in Table 6. In the normal plasma samples the concentration of TFV
increased significantly while that of TAF tend to decline. However in
the acidified plasma samples no TFV was detected and there was no
significant change in the concentration of TAF. It indicates that acid-
ifying plasma with 2% volume of acetic acid can inhibit the conversion
from TAF to TFV during storage.
Two recent reports have shown that the concentration of TAF was
20–30 times higher than that of TFV in plasma samples following a
single dose of TAF 25 mg [4,7]. In this case, even if a small amount of
TAF is converted to TFV, the concentration of TFV that determined may
increase significantly and accurate PK parameters cannot be obtained.
Andrew J.Ocque et al. and Pamela Hummert et al. [9,10] have de-
scribed good stability of TAF and TFV in normal plasma. This may be
dependent on the linear range designed in these two studies. In these
two studies, either the linear range of TAF is the same as that of TFV, or
the linear range of TAF is much smaller than that of TFV. The linear
range designed in our study is more reasonable for the determination of
clinical samples. Meanwhile the problem of stability of plasma samples
was found and solved.
Thus, in order to obtain accurate and reliable results and pharma-
cokinetic parameters in clinical studies, it is necessary to acidify all the
plasma samples with 2% acetic acid.
4.2. Optimization of chromatography conditions

3.7. The results of pharmacokinetics study
8 volunteers (18–45 years old) including six males and two non-
pregnant, non-lactating females were enrolled our study. The pharma-
cokinetics parameters such as Cmax, AUC0→∞ were calculated for TAF
and TFV. Average plasma concentration-time curves of TAF and TFV
was shown in Fig. 3. The pharmacokenitics parameters are presented in
Table 5.
4. Discussion
4.1. Study of stability
At the beginning of method development, we found that the con-
centration of TFV in the normal plasma samples increased during the
storage period. That's because TAF is a kind of phosphoranidate com-
pound and it transforms to TFV in weakly alkaline plasma. Through our
TFV is strong polar compound and its' retention behavior on reverse
chromatography column is weak. In the initial stage of gradient elution,
low ratio of methanol can increase the retention of TFV. At the same
time, 20 mmol/l ammonium acetate in mobile phase can further in-
crease the retention of TFV to avoid being eluted early with other polar
endogenous substance. Contrarily, TAF is a kind of weak polar com-
pound which could be eluted only by high ratio of methanol. In the
following gradient elution 100% methanol and high flow rate were set
to ensure elution of more weakly polar species on chromatography
column which can avoid the carryover effect to the next analysis
sample.
4.3. Development of the sample preparing
Generally there are three methods for preparing biological specimen
which are liquid-liquid extraction (LLE), solid phase extraction (SPE)
and protein precipitation (PPT).

155
L. Zhao, et al. Journal of Chromatography B 1117 (2019) 148–157

Fig. 3. Mean plasma concentration-time profiles by treatment group following a single oral dose of TAF 25 mg (n = 8). Vertical error bars represent standard
deviation around the mean.

Table 5 extracts are not very clean. In order to get rid of the co-eluting matrix
TAF and TFV plasma PK following a single dose of TAF at 25 mg (n = 8). components, and avoid the ion source contamination, a six-port divert
Parameters TAF TFV valve was used in our method. The eluent was diverted to waste for the
first 1.6 mins and the last 5 mins. This process can also cut down the
Tmax(h) 0.593 ± 0.288 1.72 ± 0.653 noise caused by these undesirable components effectually. Matrix ef-
T1/2(h) 0.346 ± 0.124 31.3 ± 6.05 fects of the method were small and the extraction recoveries of all of the
Cmax(ng/ml) 161 ± 65.4 9.26 ± 2.98
AUC0→t(ng/ml·h) 113 ± 32.9 195 ± 48.85
analytes were stable. The LLOQ of TFV and TAF were 0.400 ng/ml and
AUC0→∞(ng/ml·h) 116 ± 33.9 243 ± 69.60 4.00 ng/ml, respectively, which are sensitive enough for measurement
requirements.

In the reported assay, Andrew J.Ocque et al. and Pamela Hummert
et al. [9,10] used the SPE method for sample processing. However, the
sample preparation process of SPE requires too many steps, which
consumes a lot of time and amount of reagent. PPT is the simplest,
fastest method among all sample preparation technique, but the final
5. Conclusion
A rapid, sensitive LC-MS/MS method for simultaneous determina-
tion of TAF and TFV in human plasma was developed and validated.
Compared with the published methods, the steps of sample preparation

156
L. Zhao, et al.
Table 6
Stability of TAF and TFV in different matrix after −80 °C storage for 75 days (n = 3).
TAF
sample within 2% ACE Unacidified sample
MCb Ac (%) MCb
Plasma within 8.000 ng/ml TAF 8.47 ± 0.263 105 7.51 ± 0.156
Plasma within 320.0 ng/ml TAF 314 ± 8.63 97.7 294 ± 10.1
a
ND:not detected.
b
MC: Measured concentration.
c
A: Accuracy.
were very simple and the volume of plasma used for sample preparing
was only 200 μl. The lower limits of quantification were 0.400 ng/ml
and 4.00 ng/ml, for TFV and TAF respectively. The method has been
successfully applied to the clinical pharmacokinetics study and sa-
tisfactory results have been obtained. It demonstrates that the method
is reproducible and suitable for high throughput sample analysis.
References
[1] J.J. Ott, G.A. Stevens, J. Groeger, S.T. Wiersma, Global epidemiology of hepatitis B
virus infection: new estimates of age-specific HBsAg seroprevalence and en-
demicity, Vaccine 30 (2012) 2212–2219, https://doi.org/10.1016/j.vaccine.2011.
12.116.
[2] E. Murakami, T. Wang, Y. Park, J. Hao, E. Lepist, D. Babusis, A.S. Ray, Implications
of efficient hepatic delivery by tenofovir alafenamide (GS-7340) for hepatitis B
virus therapy, 59 (2015) 3563–3569, https://doi.org/10.1128/AAC.00128-15.
[3] K. Agarwal, M. Brunetto, W.K. Seto, Y. Lim, S. Fung, P. Marcellin, S.H. Ahn,
N. Izumi, W.L. Chuang, H. Bae, M. Sharma, H.L.A. Janssen, C.Q. Pan, M.K. Çelen,
N. Furusyo, K.T. Yoon, H. Trinh, J.F. Flaherty, A. Gaggar, A.H. Lau, A.L. Cathcart,
L. Lin, N. Bhardwaj, V. Suri, G.M. Subramanian, E.J. Gane, M. Buti, H.L.Y. Chan, 96
weeks treatment of tenofovir alafenamide vs. tenofovir disoproxil fumarate for
hepatitis B virus infection, J. Hepatol. (2018), https://doi.org/10.1016/j.jhep.
2017.11.039 xxx.
[4] K. Agarwal, S.K. Fung, T.T. Nguyen, W. Cheng, E. Sicard, S.D. Ryder, J.F. Flaherty,
E. Lawson, S. Zhao, G.M. Subramanian, J.G. Mchutchison, E.J. Gane, G.R. Foster,
Twenty-eight day safety, antiviral activity, and pharmacokinetics of tenofovir ala-
fenamide for treatment of chronic hepatitis B infection, J. Hepatol. (2015) 1–8,
157
Journal of Chromatography B 1117 (2019) 148–157
TFV(ng/ml)
sample within 2% ACE Unacidified sample
Ac (%)
93.4 NDa 1.15 ± 0.373
91.4 NDa 27.9 ± 16.0
https://doi.org/10.1016/j.jhep.2014.10.035 (xxx).
[5] E. De Clercq, Tenofovir alafenamide (TAF) as the successor of tenofovir disoproxil
fumarate (TDF), Biochem. Pharmacol. (2016) 1–7, https://doi.org/10.1016/j.bcp.
2016.04.015.
[6] T.J. Cory, N.M. Midde, S. Kumar, Investigational Reverse Transcriptase Inhibitors
for the Treatment of HIV, (2015), pp. 1–10, https://doi.org/10.1517/13543784.
2015.1058357.
[7] J.M. Custodio, M. Fordyce, W. Garner, M. Vimal, K.H.J. Ling, B.P. Kearney,
S. Ramanathan, Pharmacokinetics and Safety of Tenofovir Alafenamide in HIV-
Uninfected Subjects with Severe Renal Impairment, vol. 60, (2016), pp. 5135–5140,
https://doi.org/10.1128/AAC.00005-16 (Address).
[8] A. Mills, J.R. Arribas, J. Andrade-villanueva, G. Diperri, J. Van Lunzen, E. Koenig,
R. Elion, M. Cavassini, J.V. Madruga, J. Brunetta, D. Shamblaw, E. Dejesus,
C. Orkin, D.A. Wohl, I. Brar, J.L. Stephens, Switching from Tenofovir Disoproxil
Fumarate to Tenofovir Alafenamide in Antiretroviral Regimens for Virologically
Suppressed Adults with HIV-1 Infection : A Randomised, Non-inferiority Study, vol.
3099, (2015), pp. 1–10, https://doi.org/10.1016/S1473-3099(15)00348-5.
[9] P. Hummert, T.L. Parsons, L.M. Ensign, T. Hoang, M.A. Marzinke, Journal of
pharmaceutical and biomedical analysis validation and implementation of liquid
chromatographic-mass spectrometric (LC – MS) methods for the quantification of
tenofovir prodrugs, J. Pharm. Biomed. Anal. 152 (2018) 248–256, https://doi.org/
10.1016/j.jpba.2018.02.011.
[10] A.J. Ocque, C.E. Hagler, G.D. Morse, S.L. Letendre, Q. Ma, Journal of pharmaceu-
tical and biomedical analysis development and validation of an LC–MS/MS assay
for tenofovir and tenofovir alafenamide in human plasma and cerebrospinal fluid, J.
Pharm. Biomed. Anal. 156 (2018) 163–169, https://doi.org/10.1016/j.jpba.2018.
04.035.
[11] Chinese Pharmacopoeia, 9012 Guideline, (2018).