MS and its application to pharmacokinetics study in clinic

Accepted Manuscript Simultaneous determination of tenofovir alafenamide and tenofovir in 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 PII: DOI: Reference:

S1570-0232(19)30400-3 https://doi.org/10.1016/j.jchromb.2019.04.011 CHROMB 21591

To appear in:

Journal of Chromatography B

Received date: Revised date: Accepted date:

8 March 2019 3 April 2019 4 April 2019

Please cite this article as: L. Zhao, Z. Li, Z. Zhou, et al., Simultaneous determination of tenofovir alafenamide and tenofovir in human plasma by LC-MS/MS and its application to pharmacokinetics study in clinic, Journal of Chromatography B, https://doi.org/10.1016/ j.jchromb.2019.04.011

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ACCEPTED MANUSCRIPT

Simultaneous determination of tenofovir alafenamide and tenofovir in human plasma by LC-MS/MS and its application to pharmacokinetics study in clinic

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Lizhi Zhao, Zhou Li*, Zhen Zhou, Xiuyuan Kang, Baihuan Fang, Huan Ma,

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Qinghua Ge

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1111 Halei Road, Zhangjiang Hi-Tech Park, Shanghai , China

ultra

performance

liquid

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Abstract An

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*Shanghai Institute of Pharmaceutical Industry, National Pharmaceutical Engineering and Research Center,

chromatography-tandem

mass

spectrometric

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(UHPLC-MS/MS) method has been developed for the simultaneous determination of

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tenofovir alafenamide (TAF) and it’s metabolite tenofovir (TFV) in human plasma. The analytes and inter standards, TAF-d5 and TFV-d6 were extracted from human

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plasma via protein precipitation (PPT) and only 200 μL plasma was needed.

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Chromatography separation was achieved on a Waters Acquity UHPLC HSS T3 column (100*2.1mm, 1.8 µm) with a total run time of 10 minutes. A tandem mass spectrometric detection was conducted using multiple reaction monitoring (MRM) mode under positive ionization 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

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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 pharmacokinetics study of treatment of 25mg

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TAF in 8 healthy volunteers under fasting condition.

Keywords: Tenofovir alafenaminde, Tenofovir, LC-MS/MS, Stability, Simultaneous

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determination, Pharmacokinetic

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1. Introduction

There are 257 million people infected with the hepatitis B virus (HBV) and over 350

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million affected by chronic hepatitis B (CHB) worldwide[1]. Nucleotide analogue

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(NA), as antiviral treatment drug, has shown to inhibit viral replication, halt the progression of HBV, even reverse cirrhosis and improve survival[2]. Tenofovir

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alafenamide (TAF, formerly GS-7340), a prodrug of tenofovir (TFV), is a potent

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nucleotide analogue inhibitor 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 hydrolyzed 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

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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 mineral density changes[3–7].

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Currently FDA had approved a few TAF-containing products including Venlidy®,

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Descovy®, Biktarvy®, Genvoya® in treatment of HIV and HBV infection[6,8].

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Previously a few assays have published for measurement TAF and TFV in human

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plasma[9,10], but none of them have focused on the stability of TAF and TFV in

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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

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simultaneous determination of these two drugs. In this article, we described a new

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method for the simultaneous determination of TAF and TFV in human plasma and its application to pharmacokinetics study.

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2. Experimental

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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,

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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 Industrial Corporation (Shanghai China). Purified water used throughout the study was commercially available (Wahaha®, Hangzhou Wahaha

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Co.,Ltd., China).

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2.2. UHPLC-MS/MS Conditions

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A UHPLC system (Shimadzu Co., Kyoto, Japan), Consisting of two LC-30AD pumps,

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a SIL-30ACMP autosampler, a CTO-20A column oven, DGU-20A5R Degassing Unit,

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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 TM

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BEH C18 1.7 m, Waters Co., Milford, MA, USA) was used for the chromatographic

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separation.

Mobile phase ‘A’ and ‘B’ consisted of water and methanol, respectively, both

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containing 20mmol/L ammonium acetate (pH:6.89) and 0.1% formic acid. A gradient

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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 4.7-8 min. An on-line motorized six-port divert valve was used to introduce the LC eluent flow into the mass spectrometer over the period of 1.6-5.0 min, while the other eluent flows were diverted to waste. The injection volume is

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10L.

The temperatures of the analytical column and auto sampler were maintained at 40℃ and 4℃, respectively. Under the current LC–MS/MS conditions, the two analytes and

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two IS analytes were well separated from interferences in the matrix. The retention times were approximately 2.54, 2.54, 4.14, 4.14 min for TFV, TFV-d6, TAF, TAF-d5,

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respectively.

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A LCMS-8060 triple quadrupole mass spectrometer (Shimadzu Co., Kyoto, Japan)

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equipped with an electrospray ionization (ESI) interface operated in positive ionization mode was used for the mass spectrometric detection. A multiple reaction

TAF-d5 and TFV-d6. The operation conditions were optimized by

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of TAF, TFV,

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monitoring (MRM) mode was operated to detect specific precursor and product ions

injecting diluted stock solutions of each analyte as follows: nebulizing gas flow was 3

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L/min, heating gas flow was 10 L/min, interface temperature was 300 ℃, DL

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temperature was 250℃, heat block temperature was 400℃, 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

ACCEPTED MANUSCRIPT methanol-water (20:80, v/v) at 200 g/mL, respectively. The independent 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

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TAF-d5 (IS) and 40 ng/mL for TFV-d6 (IS), respectively. All solutions described

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above were stored at 4℃.

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The calibration standards were freshly prepared by adding 10L working standard

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solutions into 204L acidified blank plasma (contained 200 L blank plasma and 4

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L acetic acid). The final calibration 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,

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respectively.

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QC samples were prepared at lower limit quantification, low, medium and high level (LLOQ, QCL, QCM, QCH) by spiking 204L acidified blank plasma (contained 200

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L blank plasma and 4 L acetic acid) with 10L QC working solutions. The final

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calibration concentrations 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 L plasma sample was transferred to a disposable tube. Then 10 L IS

ACCEPTED MANUSCRIPT working solution and 10 L working solutions was spiked into each sample, followed by precipitation with 800 L methanol. After vortexed-mixing for 5 min, centrifugation was applied at 15870 ×g for 10 min. 900 L upper supernatant was carefully transferred to a glass tube, and evaporated to dryness under a gentle stream

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of nitrogen in water bath at 40℃. The residue was reconstituted with 100 L of

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was injected into the LC-MS/MS system for analysis.

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methanol-water (20:80,v/v), which containing 2% acetic acid, and an aliquot of 10 L

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2.5. Method validation

The current method was validated for specificity, linearity, intra-day and inter-day

2.5.1. Specificity

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precision, accuracy, matrix effect, recovery, dilution integrity and stability.

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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

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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 quantification of method ).

ACCEPTED MANUSCRIPT 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 4.00-400 ng/mL for TAF and 0.400-40.0 ng/mL for TFV. The lower limit

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quantification of TAF and TFV were 4.00 ng/mL and 0.400 ng/mL, respectively.

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2.5.4. Accuracy and precision

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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

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separate days. The accuracy was expressed by percentage of measured concentration

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to nominal concentration and precision was expressed by relative standard deviation

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(RSD).

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2.5.5. Matrix effect and Recovery

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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% indicates no absolute matrix effect, if ME% >

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100%, the signal is enhanced, and accordingly, if ME% < 100%, the signal is suppressed.

Relative matrix effects was used to evaluate the variations of different lots of plasma

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suffered from the matrix effects, and was calculated by the coefficients of variation [CV, %] of peak areas of analytes added post-extraction from five different lots of

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blank plasma.

Recovery presents the extraction efficiency of a method. It was evaluated at each QC

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level by comparing peak areas of QC samples (C) with B and expressed as (C/B×

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2.5.6. Dilution integrity

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100%).

Dilution effect was investigated to ensure that plasma samples could be diluted with

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blank matrix without affecting the final concentration. One concentration plasma sample (1600 ng/mL for TAF and 160 ng/mL for TFV) were diluted with blank

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human plasma at dilution factors of 5 in 6 replicates and analyzed.

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 conditions that the analytes might experience during collection, storage and analysis, including three cycles of freeze

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and thaw, stored frozen at -80℃, stored in ice-water bath, and storage of extracted samples in an autosampler (4℃) and room temperature.

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2.6 Application of the analytical method in pharmacokinetic study The study protocol and informed consent forms were reviewed and approved by the

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Medicine Ethics Committee of Shanghai Public Health Center (Shanghai, China). All

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subjects provided written informed consent prior to participation in the study.

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These volunteers were administered a tablet of tenofovir alafenamide (25 mg) in the

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fasting state, an indwelling intravenous catheter was inserted following aseptic techniques and blood samples were drawn for the determination of pre-dose

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concentrations. Pharmacokinetics sampling was performed at 0h, 0.17h, 0.33h, 0.5h,

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0.75h, 1.0h, 1.33h, 1.67h, 2.0h, 2.5h, 3.0h, 4.0h, 6.0h, 8.0h, 12.0h, 24.0h, 48.0h and 72.0 h. Blood (4.0 mL) was collected in K2EDTA tubes each time and then

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centrifuged at 1300 ×g for 10 min. A volume of 800 L plasma was transferred to a

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cryogenic vials which had contained 16L acetic acid. The vials were capped and vortexed for 3 seconds. Then all samples were stored at -80 ℃ until assay.

ACCEPTED MANUSCRIPT 3. Results 3.1. Specificity and selectivity Abundant protonated molecules of TAF and TFV that formed the base peak of each mass spectrum were observed from Q1 scans during the infusion of the neat solution

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in positive mode. Two [M+H] + precursor ions, m/z 288.00 for tenofovir, m/z 477.20

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for tenofovir alafenamide, were subjected to collision induced dissociation (CID). The

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product ion tandem mass spectra of the protonated molecules of TAF and TFV are

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shown in Fig. 1. Mass transition patterns, m/z 477.20→176.15 and m/z 288.00→ 176.10 were selected to monitor TAF and TFV, respectively. A MS/MS channel of

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m/z 482.20→176.10 and m/z 294.15→182.05 were chosen to monitor the internal

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standard, TAF-d5 and TFV-d6 respectively.

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Under the current LC–MS/MS conditions, the two analytes were well separated from interferences in the matrix. Chromatograms of different lots of blank plasma were

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found to contain no endogenous peak co-eluted with any of the analytes or the internal

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standard. Representative chromatograms of blank samples with or without the presence of analytes and internal standard are shown in Fig. 2. In addition, the “cross-talk” between channels used for monitoring the analytes and IS was evaluated by analysis of their individual solution at high concentration. The responses in the MRM mass transition channels used for quantification were monitored. No “cross-talk” or interference between the analytes and IS was observed.

ACCEPTED MANUSCRIPT 3.2. Matrix Effects and Recovery 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

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results coule be obtained. And the result of recovery indicated that the extraction

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efficiency for all the analytes as well as IS was consistent and reproducible.

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3.3. Linearity and sensitivity

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Seven-point calibration curves were prepared ranging from 4.00-400 ng/mL for TAF and from 0.400-40.0 ng/mL for TFV, respectively. The curves were obtained by

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plotting the peak area ratio of the analytes to IS against the corresponding

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concentration of the analytes in the freshly prepared plasma calibrators. The

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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

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TAF in Labsolution 5.82 SP1 software used in Shimadzu LCMS 8060. Excellent linearity was achieved with correlation coefficients greater than 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 regression equation of the calibration

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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.

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3.4. Precision and accuracy The intra-day precision and accuracy were determined by the replicate analyses of QC

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samples (n=6) at four level concentrations (LLOQ, QCL, QCM and QCH) in three

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separate days. All replicate of the QC samples at each concentration level from three

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separate validation batches were used to evaluate the inter-day precision. The assay

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precision and accuracy results were shown in Table 4. The intra-day precision was within 7.00% and the inter-day precision was within 6.18%. The assay accuracy was

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3.5. Stability

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95.1–106.0% of the nominal values.

The stability experiments were aimed at testing the effects of possible conditions that

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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℃. 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℃) for 66 h.

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3.6. Dilution effect The results indicates that concentrations above the curve can be determined accurately

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3.7. The results of pharmacokinetics study

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and precisely when diluted 1:5 in appropriate blank plasma.

8 volunteers (18-45 years old) including six males and two non-pregnant,

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non-lactating females were enrolled our study. The pharmacokinetics parameters such

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as Cmax, AUC0→∞ were calculated for TAF and TFV. Average plasma concentration-time curves of TAF and TFV was shown in Fig 3. The

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4.Discussion

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pharmacokenitics parameters are presented in Table 5.

4.1. Study of stability

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At the beginning of method development, we found that the concentration of TFV in the normal plasma samples increased during the storage period. That’s because TAF is a kind of phosphoranidate compound and it transforms to TFV in weakly alkaline plasma. Through our 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

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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 ℃ 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

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significantly while that of TAF tend to decline. However in the acidified plasma

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samples no TFV was detected and there was no significant change in the

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concentration of TAF. It indicates that acidifying plasma with 2% volume of acetic

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acid can inhibit the conversion from TAF to TFV during storage.

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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

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this case, even if a small amount of TAF is converted to TFV, the concentration of

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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 described

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good stability of TAF and TFV in normal plasma. This may be dependent on the

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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 pharmacokinetic parameters in clinical studies, it is necessary to acidify all the plasma samples with 2% acetic

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acid.

4.2. Optimization of Chromatography Conditions TFV is strong polar compound and its’ retention behavior on reverse chromatography

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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

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mobile phase can further increase the retention of TFV to avoid being eluted early

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with other polar endogenous substance. Contrarily, TAF is a kind of weak polar

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compound which could be eluted only by high ratio of methanol. In the following

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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

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to the next analysis sample.

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4.3. Development of the Sample Preparing Generally there are three methods for preparing biological specimen which are

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(PPT).

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liquid-liquid extraction (LLE), solid phase extraction (SPE) and protein precipitation

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 extracts are not very clean. In order to get rid of the co-eluting matrix

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components, and avoid the ion source contamination, a six-port divert 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 noise caused by these undesirable components effectually. Matrix effects of the method were small and the extraction

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recoveries of all of the analytes were stable. The LLOQ of TFV and TAF were 0.400

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ng/mL and 4.00 ng/mL, respectively, which are sensitive enough for measurement

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requirements.

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5. Conclusion

A rapid, sensitive LC-MS/MS method for simultaneous determination of TAF and

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TFV in human plasma was developed and validated. Compared with the published

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methods, the steps of sample preparation were very simple and the volume of plasma used for sample preparing was only 200 L. The lower limits of quantification were

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0.400 ng/mL and 4.00 ng/mL, for TFV and TAF respectively. The method has been

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successfully applied to the clinical pharmacokinetics study and satisfactory 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 endemicity, Vaccine. 30 (2012)

ACCEPTED MANUSCRIPT 2212–2219. doi: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. doi:10.1128/AAC.00128-15.

K. Agarwal, M. Brunetto, W.K. Seto, Y. Lim, S. Fung, P. Marcellin, S.H. Ahn, N. Izumi, W.L. Chuang, H.

PT

[3]

Bae, M. Sharma, H.L.A. Janssen, C.Q. Pan, M.K. Çelen, N. Furusyo, K.T. Yoon, H. Trinh, J.F. Flaherty,

RI

A. Gaggar, A.H. Lau, A.L. Cathcart, L. Lin, N. Bhardwaj, V. Suri, G.M. Subramanian, E.J. Gane, M. Buti,

SC

H.L.Y. Chan, 96 weeks treatment of tenofovir alafenamide vs . tenofovir disoproxil fumarate for hepatitis B virus infection, J. Hepatol. xxx (2018). doi:10.1016/j.jhep.2017.11.039.

K. Agarwal, S.K. Fung, T.T. Nguyen, W. Cheng, E. Sicard, S.D. Ryder, J.F. Flaherty, E. Lawson, S. Zhao,

NU

[4]

G.M. Subramanian, J.G. Mchutchison, E.J. Gane, G.R. Foster, Twenty-eight day safety , antiviral

MA

activity , and pharmacokinetics of tenofovir alafenamide for treatment of chronic hepatitis B infection, J. Hepatol. xxx (2015) 1–8. doi:10.1016/j.jhep.2014.10.035.

E. De Clercq, Tenofovir alafenamide ( TAF ) as the successor of tenofovir disoproxil fumarate

D

[5]

[6]

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( TDF ), Biochem. Pharmacol. (2016) 1–7. doi:10.1016/j.bcp.2016.04.015.

T.J. Cory, N.M. Midde, S. Kumar, Investigational reverse transcriptase inhibitors for the treatment

J.M. Custodio, M. Fordyce, W. Garner, M. Vimal, K.H.J. Ling, B.P. Kearney, S. Ramanathan,

AC

[7]

CE

of HIV, (2015) 1–10. doi:10.1517/13543784.2015.1058357.

Pharmacokinetics and Safety of Tenofovir Alafenamide in HIV-Uninfected Subjects with Severe Renal Impairment, 60 (2016) 5135–5140. doi: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, 3099 (2015) 1–10. doi:10.1016/S1473-3099(15)00348-5.

ACCEPTED MANUSCRIPT [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. doi:10.1016/j.jpba.2018.02.011.

[10]

A.J. Ocque, C.E. Hagler, G.D. Morse, S.L. Letendre, Q. Ma, Journal of Pharmaceutical and

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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

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Chinese Pharmacopoeia,2018, 9012 Guideline.

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[11]

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(2018) 163–169. doi:10.1016/j.jpba.2018.04.035.

ACCEPTED MANUSCRIPT Table 1.Optimized mass parameters for analyte and IS MRM(m/z)

Dwell time(ms)

Q1 Pre Bias (V)

CV (V)

Q3 Pre Bias (V)

TFV

288.00→176.10

100

-14

-25

-19

TFV-d6 (IS)

294.15→182.05

100

-10

-27

-19

TAF

477.20→176.15

100

-10

-10

-19

TAF-d5 (IS)

482.20→176.10

100

-10

-30

-19

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Analyte

ACCEPTED MANUSCRIPT Table 2. Matrix effects and extraction recovery for TAF and TFV in six different lots of human plasma (n=6) Analyte concentration

Matrix effects (%)

Extraction recovery(%)

(ng/mL)

Mean±SD

CV(%)

Mean% and SD

CV(%)

8.00

95.9±2.06

2.15

78.6±3.13

3.98

40.0

96.8±1.28

1.32

80.5±2.77

3.44

320

96.7±0.753

0.78

TAF-d5

2.00

102±9.85

9.70

TFV

0.800

104±1.89

1.83

4.00

100±1.95

32.0

101±0.982

10.0

96.4±1.89

1.96

83.9±2.91

3.47

68.4±4.60

6.76

1.95

68.2±2.31

3.39

0.97

68.4±2.09

3.06

70.2±2.74

3.90

NU

SC

RI

84.0±1.65

1.97

MA

Matrix effects (%): (peak areas of analytes added post-extraction in six different lots of plasma)/(mean peak areas of standard)×100.

D

Extraction recovery (%): (peak areas of analytes added pre-extraction in

PT E

plasma)/(peak areas of analytes added post-extraction in plasma)×100. CV, (%): coefficient of variation of matrix effects.

CE

TFV-d6

AC

TAF

PT

Analyte

ACCEPTED MANUSCRIPT Table 3.Linearity for assay of TAF,and TFV in human plasma Ru Analyte

Linear range (ng/mL)

Calibration curve

r

n 0.9999

y=-3.54e-006x2+0.0109x+0.00118

0.9999

3

y=-3.58e-006x2+0.0111x+-8.12e-006

0.9999

1

y=0.116x+0.00258

0.9999

y=0.118x+0.000499

0.9999

y=0.119x+0.00175

0.9999

2

2

0.400-40.0

CE

PT E

D

MA

NU

SC

3

AC

TFV

4.00-400

PT

y=-2.24e-006x2+0.0115x+0.00461

1

RI

TAF

ACCEPTED MANUSCRIPT Table 4.Intra-day and inter-day precision and accuracy for assay of TAF and TFV in human plasma

Analyte

Intra-day (n=6) MCb

RSD

(ng/mL)

(%)

L) 1.50

101

8.00

8.03±0.0887

1.12

99.8

40.0

38.8±1.16

3.01

96.6

320

319±7.29

2.27 5.88

5.49

95.8

7.64±0.318

4.16

95.1

38.6±0.993

2.57

96.0

99.3

312±0.91

2.92

97.0

6.18

103

SC 106

0.812±0.0500

6.15

101

NU

106

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

PT E

AC : Analyte concentration.

D

4.00

MC: Measured concentration.

CE

A: Accuracy.

AC

c

Ac (%)

7.00

0.850±0.059 0.800

b

RSD

0.414±0.0256

0.400 1

a

3.85±0.212

RI

4.06±0.0611

5

MCb (ng/mL)

(%)

4.00

0.428±0.025 TFV

Ac (%)

MA

TAF

Inter-day (n=18)

PT

ACa(ng/m

ACCEPTED MANUSCRIPT Table 5.TAF and TFV plasma PK following a single dose of TAF at 25mg (n=8) TAF

TFV

Tmax(h)

0.593±0.288

1.72±0.653

T1/2(h)

0.346±0.124

31.3±6.05

Cmax(ng/ml )

161±65.4

9.26±2.98

AUC0→t(ng/ml·h)

113±32.9

195±48.85

AUC0→∞(ng/ml·h)

116±33.9

PT

parameters

AC

CE

PT E

D

MA

NU

SC

RI

243±69.60

ACCEPTED MANUSCRIPT Table 6.Stability of TAF and TFV in different matrix after -80℃ storage for 75 days (n=3) TAF

TFV(ng/ml)

sample within 2%

sample Unacidified sample

ACE Ac (%)

MCb

Ac (%)

8.47±0.263

105

7.51±0.156

93.4

314±8.63

97.7

294±10.1

PT

MCb

TAF

SC

Plasma within 320.0ng/ml TAF

b

MC: Measured concentration.

CE

PT E

D

MA

A: Accuracy.

AC

c

NU

ND:not detected

RI

plasma within 8.000ng/ml

a

Unacidified within 2%

91.4

sample ACE NDa

1.15±0.373

NDa

27.9±16.0

ACCEPTED MANUSCRIPT Highlight Validated UPLC-MS/MS method for quantification of tenofovir alafenamide (TAF) and it’s metabolite tenofovir (TFV) in human plasma

PT

The method overcome the lack of stability of analytes in plasma samples

AC

CE

PT E

D

MA

NU

SC

RI

The method is suitable for pharmacokinetics study

Figure 1

Figure 2a

Figure 2b

Figure 2c

Figure 3