Investigation of anti-Hepatitis C virus, sofosbuvir and daclatasvir, in pure form, human plasma and human urine using micellar monolithic HPLC-UV method and application to pharmacokinetic study

Accepted Manuscript Investigation of anti-Hepatitis C virus, sofosbuvir and daclatasvir, in pure form, human plasma and human urine using micellar monolithic HPLC-UV method and application to pharmacokinetic study

Dalia W. Zidan, Wafaa S. Hassan, Manal S. Elmasry, Abdalla A. Shalaby PII: DOI: Reference:

S1570-0232(18)30176-4 doi:10.1016/j.jchromb.2018.04.011 CHROMB 21126

To appear in: Received date: Revised date: Accepted date:

30 January 2018 29 March 2018 6 April 2018

Please cite this article as: Dalia W. Zidan, Wafaa S. Hassan, Manal S. Elmasry, Abdalla A. Shalaby , Investigation of anti-Hepatitis C virus, sofosbuvir and daclatasvir, in pure form, human plasma and human urine using micellar monolithic HPLC-UV method and application to pharmacokinetic study. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Chromb(2017), doi:10.1016/j.jchromb.2018.04.011

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ACCEPTED MANUSCRIPT Investigation of anti-Hepatitis C virus, sofosbuvir and daclatasvir, in pure form, human plasma and human urine using micellar monolithic HPLC-UV method and application to pharmacokinetic study

*Aga Health Insurance Hospital, Dakahlia

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Dalia W. Zidan*, Wafaa S. Hassan**, Manal S. Elmasry**, Abdalla A. Shalaby**

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** Department of Analytical Chemistry, Faculty of Pharmacy, Zagazig University

Abstract

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Simultaneous determination of sofosbuvir (SOF), and daclatasvir (DAC) in their dosage forms, human urine and human plasma using simple and rapid micellar high performance liquid

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chromatographic method coupled with UV detection (HPLC-UV) had been developed and validated. These drugs are described as co-administered for treatment of Hepatitis C virus (HCV). HCV is the cause of hepatitis C and some cancers such as liver cancer (hepatocellular

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carcinoma) and lymphomas in humans. Separation and quantitation were carried out on

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anonyxTM C8 monolithic (100 × 4.6 mm (i.d.) analytical column maintained at 25 ºC. The mobile phase consisted of 0.1M sodium dodecyl sulfate (SDS) solution containing 20% (V/V) n-

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propanolol and 0.3% (V/V) triethylamine and pH was adjusted to 6.5 using 0.02 M phosphoric acid, respectively. The retention times of SOF and DAC were 4.8 min, and 6.5 min.,

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respectively. Measurements were made at flow rate of 0.5 mL/min with injection volume of 20 µL and ultraviolet (UV) detection at 226 nm. Linearity of SOF and DAC was obtained over

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concentration ranges of 50-400, and 40-400 ng/mL, respectively in pure form, 60-300 and 50300 ng/mL, respectively for human plasma and over 50-400, and 40-400 ng/mL, respectively for human urine with correlation coefficient > 0.999. The proposed method demonstrated excellent intra- and inter-day precision and accuracy. The suggested method was applied for determination of the drugs in pure, dosage form, and in real human plasma, real human urine and drugdissolution test of their tablets. The obtained results have been statistically compared to reported method to give a conclusion that there is no significant differences. Key words: HPLC-UV; human urine; human plasma; dissolution test; sofosbuvir; daclatasvir

ACCEPTED MANUSCRIPT 1 Introduction Hepatitis C is an infection caused by the hepatitis C virus (HCV) that attacks the liver and leads to inflammation. The World Health Organization (WHO) estimates about 71 million people globally have chronic hepatitis C, with approximately 399,000 dying from this infection as primarily due to cirrhosis and hepatocellular carcinoma [1].

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Sofosbuvir,isopropyl(2S)‐2‐[[[(2R,3R,4R,5R)‐5‐(2,4‐dioxopyrimidin‐1‐yl)‐4‐fluoro‐3‐hyd

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roxyl‐4‐methyl‐tetrahydrofuran‐2‐yl]methoxy‐ phenoxy‐phosphoryl] amino] propanoate (Figure 1), is a prodrug (nucleotide analog) used for the treatment of hepatitis C virus (HCV) [2]. SOF

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metabolized to the analog uridine triphosphate. This metabolite is pharmacologically active can be joined to the ribonucleic acid (RNA) of the HCV leading to chain termination and so

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prevention to replication of the virus [3]. SOF can be used alone or co-administered with others like ribavirin and daclatasvir [4]. Co-administration of SOF with other drugs will result in

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increasing their activity against HCV virus. There is not many reported literature review for the estimation of SOF either alone or in combination with others as spectrophotometry [5], RP-

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HPLC methods [6-8], and LC-MS/MS methods [9, 10]. However, these methods had some drawbacks such as inadequate sensitivity, tedious and time-consuming and require sophisticated

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sample preparation. Micelles have been used in various separation techniques; for example ultrafiltration, cloud point extraction [11], and electrokinetic chromatography[12, 13]. The

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micellar liquid chromatographic method is one mode of green analytical chemistry. USA’s Environmental Protection Agency has evolved green chemistry to minimize chemistry

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undesirable effects. The field of analytical chemistry has been made many efforts to reduce analysis costs and to reduce the analytical methodologies hazards [14].

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Chemically; daclatasvir dihydrochloride is methyl ((1S)- 1-(((2S)-2-(5-(4’-(2-((2S)-1-((2S)-2((methoxycarbonyl)amino)-3- methylbutanoyl)-2-pyrrolidinyl)-1H-imidazol-5-yl)-4-biphenylyl)1H-imidazol-2-yl)-1-pyrrolidinyl)carbonyl)-2-ethylpropyl)carbamatedihydrochloride[15](Figure 1). DAC used for the treatment of HCV infection [16] through binding to HCV protein NS5A which is critical for HCV viral transcription and translation and so inhibition to its function [16]. From the literature survey, it is obvious that few methods are available for DAC such as spectroscopic methods [17] and chromatographic methods [18-22]. Since there were no methods reported for the analysis of these two drugs in combination, so the aim of the present work is to develop simple, accurate and precise HPLC-UV method for

ACCEPTED MANUSCRIPT the determination of DAC, and SOF in pure, dosage forms, and human plasma. In view of the fact that urine is a way of excretion of these drugs unchanged [23, 24], so the determination of these drugs in human urine samples may be a useful analytical tool for monitoring these drugs and accurate clinical decisions. The aim of this work was to develop an HPLC-UV method for simultaneous determination of these drugs in human urine. This work investigated a superior

< Figure 1>

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2 Experimental

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< Scheme 1>

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urine. Fast resolution to a complex system was obtained (Scheme 1).

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plan for dissolution test of these drugs in their tablets and environment of human’s plasma, and

2.1 Apparatus

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The HPLC system consists of a Perkin ElmerTM Series 200 Chromatograph with a 20 µL loop and UV/VIS detector, supplied with injector valve of Rheodyne. The analytical column

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wasC8 monolithic (100 mm × 4.6 mm (i.d.). 0.45 μm membrane filter (Millipore, Ireland) used for mobile phase filtration. pH –Meter purchased from Belgium of model NV P-901. Ultrasonic

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bath, model SS 101 H 230, USA, was used for sonication. A Prominence degasser DGU-20A5 used for mobile phase degassing. An automatic tablet dissolution tester (Abbott Laboratories

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Corp., Princeton, NJ, USA) used for in-vitro dissolution test.

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2.2 Chemicals and Reagents

A pure sample of sofosbuvir was kindly supplied by the Egyptian Pharmaceutical and

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Chemical Industry (EPCI); EPCI pharmaceutical company which is a part of Hikma group, Egypt with claimed purity of 99.8% according to manufacturer certificates of analysis. Pure daclatasvir was supplied from BDR life science private limited. Daklanork® (60 mg) tablet and Gratosovir® (400 mg) tablet were purchased from local market. Methanol (HPLC grade) was supplied by Sigma Aldrich, St.Louis, USA. Sodium dodecyl sulphate (SDS), triethylamine (TEA) and orthophosphoric acid were purchased from Winlab (UK).Phosphate buffer 0.05M (pH 6.8) were purchased from El- Nasr Pharmaceutical Chemicals Company (ADWIC) (Cairo, Egypt). Urine and plasma were collected from healthy volunteers (n=3).

ACCEPTED MANUSCRIPT 2.3 Chromatographic conditions RP-HPLC assays were carried out using an isocratic system with a flow rate of 0.5 mL/min at 25◦C. The mobile phase consisted of 0.1 M sodium dodecyl sulfate (SDS) solution containing 20% n-propanolol and 0.3% triethylamine and pH was adjusted to 6.5 using 0.02 M phosphoric acid was used. The detection was achieved at 226 nm. Samples were also filtered through 0.45

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µm disposable filters. The injection volume was 20µL. All determinations were performed at

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25ºC. The analysis was complete within 8 min with a flow rate of 0.5 mL/min.

2.4 General procedures

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2.4.1 Stock and working solutions

DAC and SOF stock solution (1 mg/mL) was prepared by dissolving 100 mg of DAC and

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SOF pure form into 100 mL water in a 100 mL volumetric flask then the stock solution was more diluted with distilled water to make a working standard solution of 1µg/mL. 2.4.2 Construction of the calibration graph

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2.4.2.1 Pure form calibrator

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The standard solutions of the bulk drug were prepared by dilution of the stock standard solution with mobile phase to reach concentration ranges of 50-400, and 40-400 ng/mL for SOF

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and DAC, respectively in pure form. Triplicate 20 µL injections were made for each concentration and chromatographed under the conditions described above. The calibration

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graph was constructed by plotting the corresponding peak area versus the final drug concentration in ng/mL. The regression equation was then calculated for the data.

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2.4.2.2 Human plasma calibrator Plasma samples were maintained at -20 oC and then thaw at room temperature before processing. 0.5 mL of drug-free individual plasma was spiked with different concentrations of the standard solutions prepared in 10 mL volumetric flasks and mixed well for 60 seconds then the samples were then completed to the volume with the mobile phase to reach concentration ranges 60-300 and 50-300 ng/mL. Triplicate 20 µL injections were made for each concentration and chromatographed under the conditions described above. The calibration graph was

ACCEPTED MANUSCRIPT constructed by plotting the corresponding peak area versus the final drug concentration in ng/mL. The regression equation was then calculated for the data. 2.4.2.3 Human urine calibrator The urine sample was diluted tenfold with bidistilled water then removing the cell debris and the particulate matter from the urine by means of low-speed centrifugation for 60 sec at 1500

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rpm [25] and filtered through 0.45 µm disposable filters then suitable amount of the stock

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solutions of DAC, and SOF were added to 0.5 mL of the final prepared urine and completed in a

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10 mL volumetric flasks to the volume with mobile phase and appropriately mixed for 60 seconds to provide final concentrations ranges from 50-400, and 40-400 ng/mL for SOF, and

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DAC, respectively. Triplicate 20 µL injections were made for each concentration and chromatographed under the conditions described above the calibration graph was constructed by

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plotting the corresponding peak area versus the final drug concentration in ng/mL. The

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regression equation was then calculated for the data.

2.4.3 Quality control samples preparation

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Low, medium and high-quality control samples of the bulk drug were prepared in methanol at (60, 200 and 300 ng/mL) for SOF, and at (50, 200, and 300 ng/mL) for DAC,

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respectively while. Quality control samples for human plasma analysis were prepared at (70, 100, and 200 ng/mL) for SOF, and at (60, 100 and 200 ng/mL) for DAC. Quality control samples

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for human urine analysis were prepared at (60, 200 and 300 ng/mL) for SOF, and at (50, 200, and 300 ng/mL), for DAC respectively. All samples were protected from light as previously

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recommended and stored at -20ºC until being used for analysis. 2.4.4 Samples preparation for commercial tablets Ten commercial tablets of Daklanork® (60mg) tablet and Gratosovir® (400 mg) tablet were accurately weighed and powdered. An amount of powder equivalent to 100 mg of DAC and SOF was taken in 100 mL conical flask then dissolved in 30 ml of methanol and vortex mixed for 10 min. This solution was filtered through Whatman filter paper into a 100 mL volumetric flask. The volume was then made up to the mark with the methanol then further dilution was performed with the mobile phase to get a working standard solution.

ACCEPTED MANUSCRIPT 2.4.5 Dissolution Test (In-vitro Test) Dissolution test performance was applied on Gratosovir® 400 mg tablet and Daklanork® (60mg) tablet. Dissolution medium volume used was 900 mL of 0.05 M phosphate buffer (pH 6.8) for SOF while the Dissolution medium volume for DAC used was 1000 mL of 0.05 M Phosphate Buffer ( pH 6.8). Dissolution apparatus was used USP Type II (Paddle) [26]. Dissolution speed was 75 rpm. Dissolution time was 45 min. Dissolution temperature was 37 

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0.5°C. 5 mL sample was introverted using a 0.45 mm syringe filter and then replaced with

release was calculated using the process mentioned above.

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another 5 mL of a suitable fresh dissolution medium at intervals up to 45 min. The % of drug

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2.4.6 Analysis of SOF and DAC in Samples of Real Plasma

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2.4.6.1 Subject

Two groups of three healthy (one male and two females) non-smokers or no-users of

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tobacco products volunteers aged between 25 and 34 years able to communicate clearly with the study personnel and able to give written consent for participation in the study. One group of

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three volunteers were given one tablet of Gratosovir® (each tablet contains 400 mg of SOF) and the other group of three volunteers were received one tablet of Daklanork® for at least 24 hours.

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After fasting overnight for 12h before dosing, the tablets were administered orally with 240 mL of boiled water (at room temperature). Neither more food nor fluid was allowed until 4 h after

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drug administration.

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2.4.6.2 Study design

This study was prepared according to the Note for Guidance on the Investigation of Bioavailability and Bioequivalence [27] and approved by the Ethics Committee of Zagzig University School of pharmacy after giving informed written consent to all volunteers. All subjects provided written informed consent to participate, were free to withdraw from the study at any time without any obligation.

ACCEPTED MANUSCRIPT 2.4.6.3 Blood sample collection The volunteers were given one tablet of Gratosovir® and Daklanork® for at least 24 hours. Blood samples were collected using cannula, introduced into the vein, after 0 (pre-dose) then after 12, and 20 min, and 0.5, 1, 2.0, 3.0, 4.0, , 6.0, 8.0, 10,and 12 hours for Gratosovir® ,and after 0.0, 12 min, and 0.5, 1.5, 2.5, 3.0, 4.0, 6.0, 8.0, 10.0, 12.0, 24.0 hours for Daklanork® from

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veins into the tubes containing anticoagulant. After collection, the blood samples were

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immediately cooled in an ice bath and then were centrifuged at 3500 rpm for 15 min at the

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temperature of the room and stored at –20°C until analysis. Plasma samples were then treated as mentioned above using the pre-dose as a reagent blank that had been treated similarly.

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2.4.6.4 Urine sample collection

Blank urine, free from SOF and DAC, was collected from the previous three healthy

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volunteers agreed to participate in this study after given informed written consent, and stored at 20 °C. Urine samples were collected from volunteers at 0 to 0.25, 0.25 to 0.5, 0.5 to 1, 1 to 2 to

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4, 4 to 8, 8 to 12 h, and 12 to 24 h for SOF and at 0 to 2, 2 to 4, 4 to 6, 6 to 12, 12 to 18, and 18 to 24 h. For each collection period, 10 mL aliquot urine were frozen at -20°C in urine collection

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tubes to keep collected samples stable. Aliquot of 1 mL of each urine sample was centrifuged at 3500 rpm for 10 min then filtered with syringe filter. Urine samples were then treated as

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described above using the pre-dose as a reagent blank that had been treated similarly

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3 Results and discussion

An HPLC method coupled with UV detection was developed and fully validated for the

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simultaneous determination of SOF, and DAC with satisfactory accuracy and precision. This method was applied to for the analysis of the studied drugs in plasma, urine samples and tablets using a rapid and simple procedure. The proposed analytical method was validated according to ICH and FDA guidelines with respect to certain parameters such as selectivity, linearity, precision, accuracy, and system suitability [27, 28]

ACCEPTED MANUSCRIPT 3.1 Chromatographic separation Chromatographic conditions, including the composition of the mobile phase, were optimized through several trials to achieve optimum resolution, high sensitivity, and symmetrical peak shape for SOF, and DAC. Different percentages of 0.1 M SDS and n-propanolol were tested. An isocratic elution using a mixture of 0.1 M sodium dodecyl sulfate (SDS) solution

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containing 20% n-propanolol and 0.3% triethylamine and pH was adjusted to 6.5 using 0.02 M

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phosphoric acid. The optimized conditions showed excellent peak shape and chromatographic

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resolutions from endogenous matrix component peaks; repeated injections showed no co-elution of any interfering peak with target analytic peak. The elution was achieved in 4.8 min for SOF, and 6.5 min for DAC, the run time was 8 min (Figure 2, 3, and 4). The precision of retention

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time was examined to evaluate the system suitability. Intra-day repeatability (n=5) and inter-day

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precision (n= 11) were evaluated.

< Figure 2, 3, and 4>

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3.2 Analytical characteristics and method validation 3.2.1 Linearity

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The linearity of the response for estimation of the studied drugs was determined via analysis to the sequence of different concentrations of each compound. Different volumes from

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DAC and SOF standard stock solution was transferred to 10 mL volumetric flask capacity. The volume was adjusted up to the mark with methanol to give a solution containing SOF and DAC

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over concentration ranges of 50-400, and 40-400 ng/mL, respectively in pure form, 60-300 and 50-300 ng/mL, respectively for human plasma and over 50-400, and 40-400 ng/mL, respectively

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for human urine. Characteristic parameters for regression equations are given in Table 1.

3.2.2 Limits of Detection and Quantification The limit of detection (LOD) and limit of quantification (LOQ) were calculated as LOD = 3.3 (SDi/S) and LOQ = 10 (SDi/S), where ‘SDi’ represents the standard deviation of the intercept and ‘S’ is the slope of the calibration line, as shown in Table 1.

< Table 1>

ACCEPTED MANUSCRIPT 3.2.3 System suitability test (SST) Evaluation of SST parameters was performed during the development and optimization of the method (Table 2). Moreover, to ascertain the effectiveness of the final operating system it was subjected to suitability testing. The test was performed by injecting the standard mixture in triplicate and the parameters were calculated as reported by the USP [29]. SST parameters

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include capacity factor (k`), selectivity factor (α), resolution factor (Rs) and column efficiency

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(number of theoretical plates, N). The final SST parameters under the optimum chromatographic

< Table 2>

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3.2.4 Precision and accuracy

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conditions are abridged in Table 2.

The precision and accuracy were performed at three concentration levels for each drug.

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The peak areas of all three drugs were calculated. For intra-day precision, the experiment was repeated five times in a day while for inter-day precision, the experiment was repeated on three different days (five times in the first day and three times in the next two days). To check the

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accuracy of the proposed method at three concentration levels for each drug was repeated three

< Table 3>

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drugs were given in Table 3.

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times. The precision (% RSD) and accuracy of measurements for determination of the studied

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3.2.5 Stability investigation of plasma and urine samples The stability of SOF and DAC in plasma samples was investigated by preparing spiked

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samples at the concentration levels of low and high-quality control samples. 3.2.5.1 Freeze/thaw stability Freeze/thaw stability was determined by analyzing low and high QC samples over three freeze/thaw cycles (n = 3). Samples were frozen at −20◦C for at least 24 hours for the first cycle and then frozen at −20◦C for at least 12 hours for each freeze/thaw cycle.

ACCEPTED MANUSCRIPT 3.2.5.2 Thawed matrix stability Stability in the thawed matrix was evaluated by analyzing low and high QC samples (n=3) from −20◦C storage. Thawed matrix was allowed to sit at room temperature for 4 hours and 24 hours before analysis.

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3.2.5.3 Long-term stability

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Long-term stability was investigated at low and high-quality control sample levels after

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storing for 30 days at −20 ºC.

Stability values demonstrated that SOF and DAC could be thawed without affecting the stability

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of these drugs in human plasma and human urine. The values were outlined in Table 4.

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3.2.6 Dilution integrity

Spiked human plasma samples prepared at concentrations above the upper maximum value of the calibration extent and need to be diluted for SOF and DAC to ensure the validity of

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drugs in samples. A fresh stock of SOF and DAC solution was prepared in plasma to give a

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concentration level of 100 times of the low and high-quality control sample levels then diluted 2 times then three aliquots of both dilutions were analyzed and the dilution integrity data of the

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samples were shown in (Table 4).

< Table 4>

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3.2.7 Investigation of variable endogenous matrix components (inter-subject variability)

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Quality control samples (n = 3) at a concentration of 300 ng/mL of DAC, and SOR were spiked into six different human blank urine and 200 ng/mL into six different human blank plasma samples from six different individuals, extracted and analyzed as previously described to investigate the effect of variable endogenous matrix components from multiple individuals. Acceptable accuracy and precision were obtained as shown in Table 5. < Table 5>

3.2.8 Robustness

ACCEPTED MANUSCRIPT The proposed procedures robustness was established by the constancy of the peak area with the deliberated small changes in the experimental criteria as shown in Table 6. The modification involved pH of the mobile phase (6.5 ± 0.1), n-propanol concentration (20 ± 1.0% (V/V)) and concentration of SDS (0.1 M ± 0.01). The values were shown in Table 6. It was obvious from Table 6 that small changes which may occur all the way through the experiment

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did not have an effect on the peak area.

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< Table 6>

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3.2.9 Selectivity

The selectivity of the proposed method was assisted by the analysis of SOF and DAC in

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different biological matrices such as plasma and urine. It was clear from Table 5 that the proposed method is selective enough for SOF and DAC determination in these matrices (as

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indicated by the small values of SD for SOF and DAC analysis in plasma and urine), and there were no interferences from urine or plasma endogenous components. Also, the selectivity of the

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proposed HPLC method established by its ability to determine the studied drugs in commercial tablets was investigated by observing any interference encountered from common tablet

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excipients. It was found that these excipients did not interfere with the results of the proposed

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method (Table 7).

3.3 Applications

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3.3.1 Applications on Commercial pharmaceutical tablets The results obtained from proposed methods were found to be satisfactory for the

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quantitative analysis of commercial tablets in agreement with the reported methods [5, 30] where no significant variance between both was observed, as presented in Table 7. < Table 7>

3.3.2 Application of the suggested method to the analysis of SOF, and DAC in real human plasma The preceding data showed that the suggested method is responsible for estimation of SOF and DAC in human plasma. The maximum concentration of plasma (Cmax) of SOF is about

ACCEPTED MANUSCRIPT 920 ng/mL [31] while for DAC is about 820ng/mL[32]. The dilution integrity showed the validity of drugs in samples prepared at concentrations above the upper maximum value of the calibration extent and need to be diluted for SOF and DAC. The application of this suggested method in vivo by receiving human plasma samples from two groups of three human volunteers taken one tablet of Gratosovir® and Daklanork®. Plasma concentration of drug against time was shown in Figure 5.The plasma pharmacokinetic parameters of SOF and DAC were shown in

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Table 8.

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< Table 8>

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< Figure 5>

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3.3.3 Application of the suggested method to the analysis of SOF, and DAC in real human urine About 3.5 % of SOF and 6.6% of DAC usual adult dose is excreted in the urine

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unchanged [23, 24]. Therefore, the drug level in urine is above our working range. The suggested method was useful for monitoring the unchanged SOF and DAC in human urine after giving one

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tablet of Gratosovir® and Daklanork® tablets (400 mg and 60 mg, respectively) orally (Figure 6).

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< Figure 6>

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3.3.4 Dissolution test for SOF and DAC.

The dissolution test was prepared on Gratosovir® and Daklanork® tablets (400 mg and 60

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mg, respectively). The percentages of drug released were estimated using the calibration curve.

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The results showed in Figure 7. < Figure 7>

4 Conclusion

Rapid, accurate, and simple micellar HPLC-UV method was suggested and effectively applied for the determination of DAC, and SOF in their pure, commercial tablet products. Satisfactory results were obtained upon comparing the results obtained for the proposed methods with those of the reported methods. The proposed methods were also applied for the determination of these drugs in human urine and plasma. The method was also extended for the

ACCEPTED MANUSCRIPT dissolution profiles for each of the drug in their commercial tablet product under the dissolution guidelines of FDA also the proposed method was extended for determination of the three drugs in real human plasma.

References [1] W.H. Organization, Global hepatitis report 2017: World Health Organization; 2017.

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[2] T. Asselah, Expert opinion on pharmacotherapy. 15 (2014) 121-30.

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[3] D.A. Herbst Jr, K.R. Reddy, Expert opinion on investigational drugs. 22 (2013) 527-36. [4] E.J. Gane, C.A. Stedman, R.H. Hyland, X. Ding, E. Svarovskaia, W.T. Symonds, R.G.

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Hindes, M.M. Berrey, New England Journal of Medicine. 368 (2013) 34-44. [5] P.M. Khedkar, M.P. Mahajan, S.D. Sawant, infection. 1 2.

US

[6] R. Vejendla, C. Subramanyam, G. Veerabhadram, International Journal of Pharmacy. 6 (2016) 121-7.

(2016)

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[8] R. Nemade, M. Dole, S. Sawant, (2017)

AN

[7] P.M. Vikas, T. Satyanarayana, D.V. Kumar, E. Mounika, M.S. Latha, R. Anusha, Y. Sathish,

[9] J.E. Rower, L.C. Jimmerson, X. Chen, J.-H. Zheng, A. Hodara, L.R. Bushman, P.L.

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Anderson, J.J. Kiser, Antimicrobial Agents and Chemotherapy. (2015) AAC. 01693-15. [10] E.F. Elkady, A.A. Aboelwafa, Journal of AOAC International. 99 (2016) 1252-9.

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[11] E. Pramauro, A. Prevot, SURFACTANT SCIENCE SERIES. 128 (2005) 305. [12] U. Pyell, Electrokinetic chromatography: theory, instrumentation and applications: John

CE

Wiley & Sons; 2007.

[13] S. Terabe, Annual Review of Analytical Chemistry. 2 (2009) 99-120.

2010.

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[14] M. De La Guardia, S. Armenta, Green analytical chemistry: theory and practice: Elsevier;

[15] I. Gentile, F. Borgia, N. Coppola, A. Buonomo, G. Castaldo, G. Borgia, Current medicinal chemistry. 21 (2014) 1391-404. [16] D.A. Herbst, K.R. Reddy, Expert opinion on investigational drugs. 22 (2013) 1337-46. [17] S.M. Azab, A.M. Fekry, RSC Advances. 7 (2017) 1118-26. [18] G. Nannetti, L. Messa, M. Celegato, S. Pagni, M. Basso, S.G. Parisi, G. Palu, A. Loregian, J Pharm Biomed Anal. 134 (2017) 275-81.

ACCEPTED MANUSCRIPT [19] M. Baker, D. El-Kafrawy, M. Mahrous, T. Belal. Validated stability-indicating HPLC-DAD method for determination of the recently approved hepatitis C antiviral agent daclatasvir. Annales Pharmaceutiques Françaises: Elsevier; 2017. p. 176-84. [20] V.A. Chakravarthy, B. Sailaja, European Journal of Pharmaceutical and Medical Research. 3 (2016) 356-64. [21] L. Yuan, H. Jiang, N. Zheng, Y.-Q. Xia, Z. Ouyang, J. Zeng, B. Akinsanya, J.L. Valentine,

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J.D. Moehlenkamp, Y. Deng, Journal of Chromatography B. 973 (2014) 1-8.

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[22] H. Jiang, H. Kandoussi, J. Zeng, J. Wang, R. Demers, T. Eley, B. He, R. Burrell, J. Easter,

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P. Kadiyala, Journal of pharmaceutical and biomedical analysis. 107 (2015) 409-18. [23] B.J. Kirby, W.T. Symonds, B.P. Kearney, A.A. Mathias, Clin Pharmacokinet. 54 (2015)

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677-90. [24] B.-M. Squibb, Princeton, NJ. (2016)

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[25] H. Khajehsharifi, Z. Eskandari, A. Asadipour, Drug testing and analysis. 2 (2010) 162-7. [26] M. Rockville, Electronic version. (2007)

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[27] I.H.T. Guideline, Q2 (R1). 1 (2005)

[28] Food, D. Administration, Food and Drug Administration, Center for Drug Evaluation and

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Research. 1 (2001) 124-9.

[29] U. Pharmacopeia. USP 29. General; 2011.

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[30] V. Kekan, S. Gholve, O. Bhusnure, Int J ChemTech Res. 10 (2017) 281-7. [31] M.R. Rezk, E.R. Bendas, E.B. Basalious, I.A. Karim, J Chromatogr B Analyt Technol

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Biomed Life Sci. 15 (2016) 63-70.

6.

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[32] M.R. Rezk, E.R. Bendas, E.B. Basalious, I.A. Karim, J Pharm Biomed Anal. 128 (2016) 61-

ACCEPTED MANUSCRIPT Figure caption Scheme 1

Schematic representation of the developed methodology in this work.

Figure 1 Chemical and molecular structure of DAC and SOF Figure 2 A typical chromatogram for a synthetic mixture under described chromatographic conditions: 0.3% (V/V) triethylamine, 20% (V/V) n-propanolol in a solution of 0.1 M SDS was min-1.

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(S): solvent front; (a): SOF (200 ng/mL); (b) DAC (200 ng/mL).

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adjusted to pH 6.8 utilizing 0.02 M orthophosphoric acid was pumped at a flow rate of 0.5 mL

Figure 3 A typical chromatogram for a synthetic mixture under described chromatographic

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conditions in spiked human plasma. (a): SOF (150 ng/mL); (b)DAC (150 ng/mL).

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Figure 4 A typical chromatogram for a synthetic mixture under described chromatographic conditions in spiked human urine. (a): SOF (200 ng/mL); (b)DAC (200 ng/mL).

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Figure 5 Mean plasma concentration after a single 60 mg and 400 of daclatasvir and sofosbuvir

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oral dose administered to 3healthy subjects.

Figure 6 Urine concentration-time curve of three human volunteers after oral administration of a

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single 60 mg and 400 of daclatasvir and sofosbuvir oral dose administered.

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Figure 7 dissolution profile of one tablet of Gratosovir® and Daklanork®.

ACCEPTED MANUSCRIPT

Table 1 Analytical performance data for the determination of SOF and DAC by the proposed method Plasma SOF (60-300) -0.0546 0.0313 0.9997 0.0881 0.0663 0.0004 6.3600 21.200

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CE

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ED

M

AN

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Linearity range (ng/mL) (50-400) (40-400) Intercept (a) -0.2555 0.4249 Slope (b) 0.0254 0.0305 Correlation coefficient (r) 0.9999 0.9999 S.D. of residuals(Sy/x) 0.0533 0.0800 S.D. of intercept (Sa) 0.0379 0.0550 S.D. of slope (Sb) 0.0002 0.0002 Limit of detection, LOD (ng/mL) 4.4800 5.4200 Limit of quantitation, LOQ (ng/mL) 14.920 18.060

Urine DAC

SOF

DAC

(50-300) -0.9076 0.0522 0.9999 0.0900 0.0620 0.0004 3.5500 11.8300

(50-400) -0.0459 0.0315 0.9999 0.0746 0.0531 0.0002 5.0600 16.8700

(40-400) 1.1550 0.0604 0.9999 0.1700 0.1150 0.0005 5.7200 19.070

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DAC

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SOF

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Pure

parameter

ACCEPTED MANUSCRIPT Table 2 System suitability test parameters of the developed HPLC method for the determination of SOF and DAC

SOF

DAC

No. of theoretical plates, N High equivalent theoritical plates, HETP Retention factor, k` Tailing factor, T Asymmetry factor, Af Selectivity factor, α Resolution, Rs

1474 0.068 0.655 1.087 1.174

2704 0.037 1.241 1.136 1.273

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CR US AN M

ED PT CE AC

T

Parameter

3.400 1.895

ACCEPTED MANUSCRIPT Table 3 Intraday and inter-day precision and accuracy calculated from quality control (QC) samples for SOF and DAC.

Pure drug Inter-day(n=11) SOF

Conc (ng/mL) 60 200 300

DAC

DAC Conc Conc Conc Meana ± SD precision Accuracy Meana ± SD precision Accuracy Meana ± SD precision Accuracy Meana ± SD precision Accuracy (ng/mL) (ng/mL) (ng/mL) 1.82 1.89 99.82 99.57 59.54±1.02 1.71 99.23 50 49.91±0.94 60 59.74±1.09 50 50.20±0.84 100.4 1.67 0.65 0.84 100.1 99.92 199.6±1.29 0.64 99.81 200 200.3±1.68 200 199.8±1.30 200 199.8±1.74 99.92 0.90 0.65 0.59 99.82 99.89 300.5±1.44 0.48 100.2 300 299.5±1.75 300 299.7±1.95 300 299.7±1.95 99.89 0.65

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U N

plasma

A

Inter-day(n=11) SOF Conc (ng/mL) 70 100 200

DAC

Meana ± SD precision Accuracy 69.36±1.12 99.94±1.50 200.3±1.74

1.61

99.09

1.50 0.87

99.94 100.1

SOF

M

Intra-day(n=5) SOF

DAC

Conc Conc Conc Meana ± SD precision Accuracy Meana ± SD precision Accuracy Meana ± SD precision Accuracy (ng/mL) (ng/mL) (ng/mL) 100.3 60 59.91±0.94 70 69.20±1.10 60 60.20±0.84 1.58 99.85 1.58 98.86 1.39 100.1 100 100.4±1.51 100 100.4±1.61 100 100.1±1.13 1.51 100.4 1.61 100.4 1.13 100.1 200 200.2±1.54 200 199.8±1.92 200 200.2±1.79 0.77 100.1 0.96 99.92 0.89

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E C

Inter-day(n=11)

Conc (ng/mL) 60 200 300

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I R

SOF

Intra-day(n=5)

urine Intra-day(n=5)

DAC SOF DAC Conc Conc Conc Meana ± SD precision Accuracy Meana ± SD precision Accuracy Meana ± SD precision Accuracy Meana ± SD precision Accuracy (ng/mL) (ng/mL) (ng/mL) 59.99±0.76 50 49.55±0.93 60 59.94±0.72 50 49.40±0.98 1.26 99.98 1.89 99.09 1.20 99.90 1.81 98.80 199.5±1.37 200 200.3±1.42 200 199.7±1.34 200 199.7±1.52 0.69 99.77 0.71 100.1 0.67 99.83 0.76 99.83 299.7±1.01 300 299.0±1.84 300 299.5±1.14 300 299.3±1.92 0.34 99.91 0.62 99.67 0.38 99.83 0.64 99.78 a

Mean of three determinations

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Table 4 Precision and accuracy calculated from stability experiments for SOF and DAC

Plasma

Urine

SOF Parameter

DAC

a

T P

SOF

a

a

DAC a

Conc Mean Conc Mean Conc Mean Conc Mean precision Accuracy precision Accuracy precision Accuracy precision Accuracy (ng/mL) ± SD (ng/mL) ± SD (ng/mL) ± SD (ng/mL) ± SD Freeze/thaw stability 70 69.33±0.58 0.83 60 59.00±1.00 1.69 60 59.83±0.64 1.07 50 50.30±0.20 0.40 99.05 98.33 99.72 100.6 (3cycle,- 20 ºC ) 200 198.8±0.72 0.36 200 198.7±1.53 0.77 300 299.8±1.53 0.51 300 298.6±1.40 0.47 99.90 99.33 99.92 99.52 Thawed matrix stability 70 69.83±0.76 1.09 60 59.33±1.15 1.95 60 59.50±0.44 0.73 50 49.17±0.21 0.42 99.76 98.89 99.17 98.33 200 199.0±1.15 0.58 200 198.8±1.53 0.77 300 299.3±0.97 0.32 300 299.1±1.59 0.53 (4 hours at RT) 99.50 99.38 99.79 99.70 Thawed matrix stability 70 69.67±1.15 1.66 60 59.47±0.50 0.85 60 60.17±0.15 0.25 50 49.53±0.57 1.15 99.52 99.11 100.3 99.07 200 199.3±0.96 0.48 200 199.5±0.58 0.29 300 299.7±1.11 0.37 300 299.0±1.41 0.47 (24 hours at RT) 99.63 99.75 99.93 99.67 Long term stability 70 69.41±1.65 2.38 60 59.77±1.08 1.80 60 59.70±0.20 0.34 50 49.57±1.17 2.36 99.16 99.61 99.50 99.13 200 199.3±1.73 0.87 200 198.5±1.73 0.87 300 300.3±1.65 0.55 300 300.1±0.72 0.24 (30 days at - 20 ºC ) 99.63 99.25 100.1 100.0 70 69.90±0.79 1.14 99.86 60 60.33±0.47 0.78 100.56 60 60.63±0.35 0.58 50 49.87±0.99 1.98 101.1 99.73 Dilution integrety 200 200.3±0.71 0.85 100.17 200 200.7±1.29 0.64 100.33 300 299.6 ±1.25 0.42 300 299.3±1.73 0.58 99.88 99.78

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a

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Mean of three determinations

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ACCEPTED MANUSCRIPT Table 5 Inter-subject variability experiment results of six different plasma samples fortified with SOF and DAC

Plasma DAC

SOF 3

4

5

6

1

200

200

200

200

200

200

200

198.7 198.0 198.7 199.0 198.3 1.528 2.646 1.528 1.732 1.155 0.769 1.336 0.769 0.870 0.582

198.9 1.914 0.962

199.9 1.852 0.926

199.5 1.290 0.647

199.1 199.4 199.5 200.4 1.825 0.702 1.852 1.762 0.917 0.352 0.928 0.879

-0.667 -1.000 -0.667 -0.500 -0.833

-0.533

-0.050

-0.267

-0.450 -0.317 -0.250 0.183

Urine SOF 4

300

300

300

300

299.3 0.961 0.321 -0.244

298.9 1.095 0.366 -0.366

298.4 1.185 0.397 -0.522

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300

300.5 299.1 0.503 1.447 0.168 0.484 0.156 -0.311

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Mean of three determinations

5

3

4

5

6

200

200

200

200

IP 200

DAC

6

1

2

3

4

5

6

300

300

300

300

300

300

300

299.3 1.563 0.522 -0.222

299.5 0.693 0.231 -0.167

299.3 1.264 0.422 -0.243

299.4 0.872 0.291 -0.200

299.9 1.206 0.402 -0.044

298.8 1.966 0.658 -0.411

299.0 1.054 0.352 -0.333

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3

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a

2

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Concentration (ngmL-1) Meana ±SD(ngmL-1) Precision (%RSD) Accuracy(% DFN)

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Individual source

2

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2

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Concentration (ngmL-1) Meana ±SD(ngmL-1) Precision (%RSD) Accuracy(% DFN)

1

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Individual source

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Table 6 Robustness of the proposed method

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n-propanol concentration, (V/V) SDS concentration (M) Mean of three determinations

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a

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6.4 6.6 19 21 0.09 0.11

pH of the mobile phase

%recoverya±SD 99.56±1.39 100.05±0.52 99.41±0.34 100.2±0.56 99.02±0.93 99.93±0.12

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Experimental parameter variation

ACCEPTED MANUSCRIPT Table 7 Determination of DAC and SOF in pharmaceutical dosage forms.

Mean

M ED PT CE AC

102.00 100.00 99.75 100.58 1.23 3.00 0.55 1.63

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50 200 400

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± S.D. n t-test F-ratio

101.67 99.00 99.50 100.06 1.42 3.00 0.58 1.68

SOF DAC Reference Method [5, 30] % recovery

101.00 98.00 97.67 98.89 1.84 3.00

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60 200 400

DAC Daklanork® 60mg tablet Taken % (ng mL-1) recovery

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parameters

SOF Gratosovir® 400 mg tablet Taken % (ng mL-1) recovery

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Pharmaceutical dosage forms

100.50 100.75 103.33 101.53 1.57 3.00

ACCEPTED MANUSCRIPT Table 8 Pharmacokinetic parameters of SOF and DAC after oral administration of Gratosovir® (400 mg tablet), and Daklanork® (60 mg tablet)

M ED PT CE AC

T

DAC

0.500

CR

1198 0.231 0.100 0.547

820.0

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920.0

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Volume of distribution, Vd

SOF

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Parameter Cmax (ngmL-1) Tmax (h) AUC0–24h (ngmL-1 h) Absorption rate constant, Ka(1/h) Absorption half life, t1/2a(h) Elimination rate constant, K (1/h) biological half life, t1/2 (h)

2.500 8980 2.310 0.300 0.178

1.268

3.883

0.009

0.001

ACCEPTED MANUSCRIPT Highlights

o We determine two anti-Hepatitis C Virus drugs in pure, human plasma and human urine by micellar liquid chromatography.

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o One-step dilution required for sample pretreatment and direct injection.

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o The method was validated by the guidelines of the ICH and FDA.

o It was inexpensive, rapid, eco-friendly, safe, and useful for routine analysis.

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o The method was successfully applied to pharmacokinetic study and dissolution test.

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Figure 7