Research Article Open Access Development and validation of ... · available. This study compared the relative bioavailability and pharmacokinetics of test formulation (fixed dose

Kiran kumar.A et al, IJRPLS, 2017, 5(2): 40–53 CODEN (USA): IJRPKL | ISSN: 2321–5038

International Journal of Research in Pharmacy and Life Sciences 40

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Research Article Open Access

Development and validation of a LC-MS/MS assay for bioequivalence study ofdolutegravir, emtricitabine, Tenofovir alafenamide in human plasma

Kiran Kumar.A1*, Komal Lata Nagpal2 and Jaswanth Kumar.I3

1Research scholar, Department of life sciences, OPJS University, Churu, Rajasthan -313301, India.2Associate Professor, Department of life sciences, OPJS University, Churu, Rajasthan -313301, India.3Jaswanth Kumar.I, Head-Operations, Clinse labs private limited, Balanagar, Hyderabad, TG- 500037, India.

A B S T R A C TCombination antiretroviral (cARV) treatment is more common in human immunodeficiency virus (HIV) infection. In manyinstances, treatment regimen includes two or more combination of drugs from six different classes. Some of the antiretroviralcombination medications are under study at preclinical and clinical stages. A precise method is required to quantify the drugconcentration in biological matrices to study pharmacokinetic behavior and tissue distribution profile in animals and/orhumans. We have developed and validated a sensitive and precise liquid chromatography–tandem mass spectrometry methodfor simultaneous quantification of selected antiretroviral drugs, Dolutegravir, Emtricitabine, Tenofovir Alafenamide inhuman plasma samples. This method involves a solid phase extraction, simple isocratic mobile phase Methanol: 5Mmammonium acetate (75:25), chromatographic separation using ZODIAC CN, 5µ, 100 X 4.6mm column and massspectrometric detection by an API 4500 instrument. The total run time for each sample was 3 min.. The results of intra andinter-run assay precision and accuracy were within acceptance limits for all the three analytes. This study was designed toevaluate the bioequivalence of fixed dose combination of Dolutegravir, Emtricitabine and Tenofovir Alafenamide Tablets50mg/200mg/25mg (Test formulation) with Dolutegravir, Emtricitabine and Tenofovir Alafenamide Tablets 50mg/ 200mg/25mg (Reference formulation) in 12 healthy male volunteers under fed conditions.Keywords: Dolutegravir, Emtricitabine, Tenofovir Alafenamide, LC-MS/MS, Bioequivalence; Human Plasma, Pharmacokinetic parameters.

A R T I C L E I N F O

CONTENTS1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .412. Materials and Methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 413. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434. Acknowledgement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .455. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 466. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49

Article History: Received 30 September 2017, Accepted 09 October 2017, Available Online 24 November 2017

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Citation: Kiran kumar.A, et al. Development and validation of a LC-MS/MS assay for bioeqivalence studies of dolugravir, emtricitabine,Tenofovir alafenamide in human plasma. Int. J. Res. Pharm, L. Sci., 2017, 5(1): 40-53.

*Corresponding AuthorKiran kumar.AResearch scholar, Department of life sciences,OPJS University, Churu, Rajasthan -313301, India.Manuscript ID: IJRPLS3317



Copyright © 2017 Kiran kumar.A, et al. This is an open-access article distributed under the terms of the Creative CommonsAttribution License, which permits unrestricted use, distribution and reproduction in any medium, provided the original workis properly cited.

1. IntroductionHIV‐associated neurocognitive disorder (HAND) is acommon disease complication due to neurotoxic eventsresulting from HIV replication in perivascular macrophagesand microglia as well as immune and glial activation.Antiretroviral drugs vary substantially in their distributioninto protected compartments such as the central nervoussystem (CNS). While integrase inhibitors, specificallyraltegravir and dolutegravir, have low concentrations incerebrospinal fluid (CSF) compared to those in plasma,they are in the therapeutic range exceeding the 50%inhibitory concentration for wild‐type HIV‐1.[1-3].

Dolutegravir (Fig:1.0) an HIV INSTI is Sodium (4R,12aS)-9-{[(2,4-difluorophenyl)methyl]carbamoyl}-4-methyl 6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido [1',2':4,5]pyrazino [2,1-b] [1,3] oxazin-7-olate. Dolutegravir (DTG;S/GSK1349572) is an integrase inhibitor for the treatmentof HIV infection, does not require boosting with ritonavir,and possesses activity against raltegravir-resistant strains.The chemical name of dolutegravir sodium is sodium(4R,12aS)-9-{[(2,4-difluorophenyl) methyl] carbamoyl}-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5] pyrazino[2,1-b][1,3]oxazin-7-olate. The empiricalformula is C20H18F2N3NaO5 and the molecular weight is419.26 g/mol[1-5].

Emtricitabine (Fig:1.0) is the most important nucleosidereverse transcriptase inhibitor (NRTI) used for thetreatment of HIV infection in adults. Emtricitabine acts asan analogue of cytidine. By using this drug to inhibiting thereverse transcriptase and the enzyme that copies Humanimmune deficiency virus RNA into novel viralDeoxyribonucleic acid. Emtricitabine (EC) is chemically 4-amino-5-fluoro-1-(2R-hydroxymethyl-1,3 oxathiolan-5S-yl)-(1H)-pyrimidin-2-one. Emtricitabine (EC) is the (-)enantiomer of a thio analog of cytidine, which differs fromother cytidine analogs in that it has a fluorine in the 5position. It has a molecular formula of C8H10FN3O3S anda molecular weight of 247.240 [6-9].

Tenofovir alafenamide (TA) (Fig:1.0) is oral pro-drug ofTenofovir. Tenofovir alafenamide fumarate (TAF) is morestable in plasma than Tenofovir diphosphate (TDP),provides higher intracellular levels of the activephosphorylated metabolite Tenofovir diphosphate (TDP),and approximately 90% lower circulating levels ofTenofovir alafenamide relative to Tenofovir alafenamidefumarate. Tenofovir Alafenamide (TA) is chemicallypropan-2-yl (2S)-2-{[(S)-({[(2R)-1-(6-amino-9H-purin-9-yl) propan-2-yl]oxy}methyl)(phenoxy) phosphoryl] amino}propanoate. Tenofovir alafenamide has an empiricalformula of 476.474 C21H29N6O5P and a formula weight of476.474. [9-11]

Figure.1.0: Chemical structures of A) Dolutegravir (DG)B) Emtricitabine (EC) C) Tenofovir Alafenamide (TA) D)Dolutegravir-D3 (DGIS) E) Emtricitabine-D2-15N (ECIS)F) Tenofovir Alafenamide-D5 Fumarate (TAIS).

There are several analytical methods that have beenreported for the determination of single and in combinationof these selected drugs like Dolutegravir, tenofovir andemtrcitabine by UV- Spectroscopy [19-25], HPTLC [22-25], HPLC in pharmaceutical formulations and biologicalsamples [26-60], UPLC [61] and LC-MS/MS [62-83] inbiological samples techniques. From the literature it isevident that there is no LC-MS/MS method available yetfor the quantitative determination of Dolutegravir (DG),Tenofovir (TA) and Emtrcitabine (EC) in human plasmasamples using deuteredted internal standards likeDolutegravir-D3 (DGIS), Emtricitabine-D2-15N (ECIS),Tenofovir Alafenamide-D5 Fumarate (TAIS). Hence thisLC-MS/MS method is novel.

Bioequivalence can be established through variousmethods, including in vivo and in vitro methods; however,the pharmacokinetic approach is the most commonly usedmethod. The reason might be that the primary aim ofbioequivalence studies is to assess the rate and extent ofdrug absorption, which can be readily assessed by keypharmacokinetic parameters such as Cmax, Tmax, AUC,mean residence time (MRT), area under moment curve(AUMC), and t1/2. Currently pharmacokinetic andbioequivalence data on fixed dose combination ofDolutegravir, Emtricitabine and Tenofovir AlafenamideTablets 50 mg/200 mg/25 mg in healthy volunteers is notavailable. This study compared the relative bioavailabilityand pharmacokinetics of test formulation (fixed dosecombination of Dolutegravir, Emtricitabine and TenofovirAlafenamide Tablets 50mg/200mg/25mg) with that ofreference formulation (Dolutegravir, Emtricitabine andTenofovir Alafenamide Tablets 50mg/200mg/25mg) in 12healthy male volunteers under fed conditions. To access thereproducibility and wide applicability of the developedmethod, it was validated as per FDA guidelines.[84-88]

2. Materials and MethodsChemicals and ReagentsDolutegravir (DG), Tenofovir Alafenamide (TA) andEmtrcitabine (EC) (Fig.1.0) reference standards were



purchased from commercial sources. Dolutegravir-D3(DGIS), Emtricitabine-D2-15N (ECIS), TenofovirAlafenamide-D5 Fumarate (TAIS) were obtained fromToronto Research chemicals, Canada (Fig.1.0). Milli-Qwater was obtained from in-house Milli-Q waterpurification system, Millipore. All other chemicals andsolvents were purchased from S.D fine chemicals Mumbai.Human plasma was obtained from Deccan pathologicallabs, Hyderabad.InstrumentationSchimadzu Prominence HPLC system connected with ABSciex API 4500 triple quard mass spectrometer was usedfor the present study. Compound ionization was performedusing electro spray ionization (ESI) probe in positive ionmode and the data processing was performed with Analyst1.6.2 software package (SCIEX). An aliquot of 5µL of theprocessed samples were chromatographed on a Zodiac CN(100 X 4.6mm, 5 µ) column using methanol and 5mMammonium acetate (75:25, v/v) as mobile phase with 1.0mL/min flow-rate in isocratic mode. DG, EC and TA alongwith their ISs were eluted at 2.12 min, 1.36 min, 1.49 min,respectively allowing the run time of 3 min.Chromatographic conditionsChromatography was performed on ZODIAC CN, 5 µ, 100X 4.6mm. Mobile phase composed of Methanol: 5Mmammonium acetate (75:25), with 1.0 mL/min flow-rate inisocratic mode. DGIS, ECIS and TAIS were used asinternal standards in terms of chromatography andextractability. The drugs and internal standards were elutedat 2.12 min., 1.36 min., 1.49 min. ± 0.01min. window with3.00 min. total run time.Preparation of Standards and Quality Control (QC)Samples: Standard stock solutions of DG, EC, TA, DGIS,ECIS and TAIS (1mg/mL) were prepared in methanol. Thedrug standards spiking solutions prepared from DG, EC andTA in 50 % methanol. The internal standards spikingsolutions (500.0 ng/mL) were prepared in 50% methanolfrom DGIS, ECIS, TAIS standard stock solution. Standardstock solutions and internal standard spiking solutions werestored in refrigerator conditions (2-8 °C) until analysis.Standard stock solutions were added to screened drug-freehuman plasma to obtain calibration standards of 50.336,100.671, 201.343, 402.685, 805.370, 1505.365, 3010.730,6021.459, 8028.613 ng/mL for DG, 25.589, 51.178,102.356, 204.711, 602.093, 1204.185, 1802.672, 2403.563,3004.454 ng/mL for EC, 5.012, 10.024, 30.011, 60.021,120.043, 240.086, 359.948, 479.931, 599.914 ng/mL forTA and Quality control samples like LLOQ (Lower limit ofquality control), LQC (Low quality control), MQC(Medium quality control), HQC (High quality control) of50.498, 1004.937, 4019.748, 6699.580 ng/mL for DG,25.969, 310.408, 1443.757, 2555.322 ng/mL for EC, 5.026,91.193, 303.976, 467.655 ng/mL for TA and stored in a -70°C freezer until analysis. Respective aqueous standardswere prepared in reconstitution solution Methanol: 5Mmammonium acetate (75:25) and stored at room temperatureuntil analysis.Sample preparationA 100 μL aliquot of plasma was mixed with 25 μL of IS(500.00ng/mL) spiking solution followed by mixing with

500 uL water and then samples were vortexed for 30 sec.The Orochochem Panthera Deluxe (30 mg, 1 ml) SPEcartridges were equilibrated with 1mL of methanolfollowed by water and then samples were loaded.Cartridges were washed 2 times with water each 1 ml,followed by eluted with 1 mL of mobile phase (Methanol:5Mm ammonium acetate (75:25)). 5.0 uL of Eluent wasinjected into the LC-MS/MS instrument.Selectivity and specificityThe selectivity of the method was determined by sixdifferent human blank plasma samples including lipemicand haemolytic samples, which were pretreated andanalyzed to test the potential interferences of endogenouscompounds co-eluting with analyte and IS.Chromatographic peaks of analyte and IS were identifiedbased on their retention times and MRM responses. Thepeak areas of DG, EC, TA at the respective retention timein blank samples should not be more than 20% of the meanpeak area of LOQ of DG, EC, TA. Similarly, the peak areasof DGIS, ECIS, TAIS at the respective retention time inblank samples should not be more than 5% of the meanpeak area of ISs in LOQ of DGIS, ECIS, TAIS.RecoveryThe extraction recovery of DG, EC, TA and DGIS, ECIS,TAIS from human plasma was determined by analyzingquality control samples. Recovery at three concentrations(150.741, 4019.748, 6699.580ng/mL for DG, 75.274,1443.757, 2555.322 ng/mL for EC, 15.229, 303.976,467.655 ng/mL for TA) was determined by comparing peakareas obtained from the plasma sample and the standardsolution spiked with the blank plasma residue. Aconsistsent and reproducable recovery was consideredadequate to obtain required recovery.Limit of Detection (LOD) and Lower Limit ofquantification (LOQ):The limit of detection (LOD) is a parameter that providesthe lowest concentration in a sample that can be detectedfrom background noise but not quantitated. LOD wasdetermined using the signal-to-noise ratio (s/n) of 3:1 bycomparing test results from samples with knownconcentrations of analytes with blank samples.

The Lower limit of quantitation (LOQ) is defined as thelowest concentration of analyte that can be determined withacceptable precision and accuracy. The LLOQ was foundby analyzing a set of mobile phase and plasma standardswith a known concentrations of DG, ET and TA.Matrix effectTo predict the variability of matrix effects in samples fromindividual subjects, matrix effect was quantified bydetermining the matrix factor, which was calculated asfollows:Matrix Factor = Peak response ratio in presence ofextracted matrix (post extracted)/ Peak response ratio inaqueous standards. Eight lots of blank biological matricesincluding lipemic and haemolytic were extracted each in sixreplicates and post spiked with the aqueous standard at theLQC and HQC QC level, and compared with aqueousstandards of same concentration. The overall precision of



the matrix factor is expressed as coefficient of variation(CV %) and %CV should be ≤15%.Calibration curve, Precission and AccuracyThe calibration curves were constructed using valuesranging from 50.336 to 8028.613 ng/mL for DG, 25.589 to3004.454 ng/mL for EC, 5.012 to 599.914 ng/mL for TA inhuman plasma. Calibration curve was obtained by quadraticmodel with weighted 1/x2 regression analysis. The ratio ofDG/DGIS, EC/ECIS and TA/TAIS peak areas were plottedagainst the ratios of DG, EC and TA concentration inng/mL. Calibration curve standard samples and qualitycontrol samples were prepared in replicates (n=6) foranalysis. Precision and Accuracy for the back calculatedconcentrations of the calibration points, should be within≤15 and ± 15% of their nominal values. However, forLLOQ the Precision and Accuracy should be within ≤20and ± 20%.Stability studies: Bench top stability (11h), processedsamples stability (39h), freeze–thaw stability (Six cycles),autosampler stability for 36 h, long-term stability (85 days)were performed at LQC and HQC levels using sixreplicates at each level. Stability samples were processedand quantified along with the freshly spiked calibrationcurve standards. Samples were considered to be stable ifassay values were within the acceptable limits of accuracy(±15% ) and precision (≤15%).Analysis of Bioequivalence study samplesThe bio analytical method described above was used todetermine DG, EC and TA concentrations in plasmafollowing oral administration healthy human volunteers.These volunteers/subjects were contracted in Clinse LabsPrivate Limited, Hyderabad, India. The reference and testproducts containing DG, EC and TA dose of 50mg, 200mgand 25mg respectively was used for the study. Studyprotocol was approved by IEC (Institutional Ethicscommittee) as per ICMR (Indian council of medicalresearch) and each subject was administered with 50mg,200mg and 25mg of single oral dose of DG, EC and TAwith 250 mL of drinking water. Blood samples werecollected as the predose (0) hr 5 minutes prior to dosingfollowed by further samples at 0.25, 0.50, 0.75, 1.00, 1.25,1.50, 1.75, 2.00, 2.25, 2.50,2.75, 3.00, 3.33, 3.67, 4.00,4.50, 5.00, 6.00, 8.00, 10.00, 12.00, 16.00, 24.00,36.00,48.00 and 72.00 hours. After dosing 5 mL blood wascollected each time in vaccutainers containing K2EDTA. Atotal of 54 (27 time points for Reference, 27 time points forTest) time points were collected by performingcentrifugation at 3200 rpm, 10°C, 10 min. and then storedat below -70°C until sample analysis. Test and Referencetablets were administered to same human volunteers underfed conditions separately with proper washing periods asper protocol.Pharmacokinetics and statistical analysisPharmacokinetics parameters from the human plasmasamples were calculated by a non compartmental statisticmodel using Win Non-Lin 5.0. Software (Pharsight, USA).Blood samples were taken for a period of 3 to 5 times theterminal elimination half-life (t1/2) and it was considered asthe area under the concentration time curve (AUC) ratiohigher than 80% as per FDA guidelines. Plasma DG, EC,

TA concentration-time profiles were visually inspected, andCmax and Tmax values were etermined. The AUC0–t wasobtained by the trapezoidal method. AUC0–∞ wascalculated up to the last measureable concentration andextrapolations were obtained using the last measureableconcentration and the terminal elimination rate constant(Kel) it was estimated from the slope of the terminalexponential phase of the plasma of the DG, EC and TAconcentration-time curve (by means of the linear regressionmethod). The terminal elimination half-life (t1/2), was thencalculated as 0.693/Kel. Regarding AUC0–t, AUC0–∞ andCmax bioequivalence were assessed by means of analysisof variance (ANOVA) and calculating the standard 90%confidence intervals (90% CIs) of the ratio's test/reference(logarithmically transformed data). The bioequivalence wasconsidered when the ratio of averages of log transformeddata was within 80-125% for AUC0–t, AUC0–∞ andCmax.

3. Results and DiscussionMethod development and validationLC-MS/MS has been used as one of the most powerfulanalytical tools in clinical pharmacokinetics for itsselectivity, sensitivity and reproducibility. The goal of thiswork is to develop and validate a simple, sensitive, rapid,rugged and reproducible assay method for the quantitativedetermination of DG, EC, TA from human plasma samples.Chromatographic conditions, especially the compositionand nature of the mobile phase, usage of different columns,different extraction methods such as solid phase,Precipitation, Liquid-liquid extraction methods wereoptimized through several trials to achieve the bestresolution and increase the signal of analyte and internalstandard. The MS optimization was performed by directinfusion of solutions of both analyte and internal standardinto the ESI source of the mass spectrometer.

The critical parameters in the ESI source include the needle(ESI) voltage, Capillary voltage, source temperature andother parameters such as nebulizer gas, heater gas anddesolvation gases were optimized to obtain a better sprayshape, resulting in better ionization of the protonated ionicanalyte and internal standard molecules (Fig.2.0).Production spectrum for analyte and internal standard yielded high-abundance fragment ions of 277.20, 130.00, 270.00,277.30, 130.80 and 470.00 of DG, EC, TA, DGIS, ECISand TAIS respectively (Figure. 2.0). After massspectrometer parameters optimized, chromatographicconditions such as mobile phase optimization, columnoptimization, extraction method optimization wasperformed to obtain a fast and selective LC method. A goodseparation and elution were achieved using Methanol: 5Mmammonium acetate (75:25), as the mobile phase, at a flow-rate of 1.0 mL/min and injection volume of 5 μL. ZODIACCN, 5 µ 100 X 4.6mm column and SPE method wasoptimized for the best chromatography.Selectivity and SpecificityThe analysis of DG, EC, TA, DGIS, ECIS and TAIS usingMRM (Multiple reaction monitoring) function was highlyselective with no interfering compounds (Fig: 3.0).



Figure.2.0. Mass Spectra of Q1→Q3 A) DG, B) EC, C) TA, D) DGIS, E) ECIS, F) TAIS.

Figure: 3.0: Blank plasma chromatograms of A) Dolutegravir (DG) and Dolutegravir-D3 (DGIS), B) Emtricitabine (EC) andEmtricitabine 15N-D2 (ECIS), C) Tenofovir Alafenamide (TA) and Tenofovir Alafenamide-D5 Fumarate (TAIS). Blankplasma spiked internal standard chromatograms of D) Dolutegravir-D3 (DGIS), E) Emtricitabine 15N-D2 (ECIS), F)Tenofovir Alafenamide-D5 Fumarate (TAIS).

Limit of Detection (LOD) and Quantification (LLOQ)The limit of detection was used to determine the instrumentdetection levels for DG, EC, TA even at lowconcentrations. 5µL of 50.498, 25.969, 5.026 ng/mLsolutions of DG, EC, TA were injected and estimated LOD

was 50.498, 25.969, 5.026 ng/mL of DG, EC, TA with S/Nvalues ≥5:1. The lower limit of quantification for thismethod was proved as lowest concentration of thecalibration curve which was proved as 50.336, 25.589,5.012 ng/mL for DG, EC, TA.



Matrix effectEight lots of blank biological matrices (including lipemicand Haemolytic) were extracted each in six replicates andpost spiked with the aqueous standard at the LQC, HQClevel, and compared with neat standards of sameconcentration in alternate injections. The overall precisionof the matrix factor is 1.043, 1.009 for DG, 0.908, 0.943 forEC and 1.005, 0.992 for TA. There was no ion- suppressionand ion- Enhancement effect observed due to IS and analyteat respective retention time.Calibration curve standardsCalibration curves (Fig.4.0) were plotted as the peak arearatio (DG/DGIS, EC/ECIS and TA/TAIS) versus (DG, EC,TA) concentration. Calibration was found to be linear overthe concentration range of 50.336 to 8028.613 ng/mL ofDG, 25.589 to 3004.454 ng/mL of EC, 5.012 to 599.914ng/mL of TA. The CV% was less than 3.75 % and theAccuracy ranged from 95.23 to 106.11%. Thedetermination coefficients (r) were greater than 0.99 for allcurves (Table 2). Chromatograms obtained from plasmaspiked with LLOQs and ULOQs concentrations of 50.336ng/mL, 25.589 ng/mL, 5.012 ng/mL and 8028.613 ng/mL,3004.454 ng/mL, 599.914 ng/mL of DG, EC, TA alongwith DGIS, ECIS TAIS (500.0 ng/mL) are shown in Fig. 5.

A

B

CFigure: 4.0: Calibration curve details of A) Dolutegravir(DG) B) Emtricitabine (EC) and C) Tenofovir Alafenamide(TA).

Precision and accuracy:As shown in Table 2.0, the intra-batch CV% was less than10.16 and the overall accuracy ranged from 90.48 to 109.13%. The inter-batch CV% was less than 13.10 % and theoverall accuracy ranged from 91.45 to 108.48 %. Theseresults indicate the adequate reliability and reproducibilityof this method within the analytical range.Recovery:The % recovery following the sample preparation usingsolid phase extraction was calculated by comparing thepeak area of DG, EC, TA in plasma samples with the peakarea of solvent samples and was estimated at control levelsof DG, EC, TA. The %recovery of DG, EC, TA wasdetermined at three different concentrations of 150.741,4019.748, 6699.580 ng/mL for DG, 75.274, 1443.757,2555.322ng/mL for EC, 15.229, 303.976, 467.655 ng/mLfor TA, were found as 72.24, 58.61 and 43.82 %respectively. The overall average recovery of drug (DG,EC, TA) and internal standard (DGIS, ECIS, TAIS) werefound to be 79.22%, 65.08 % and 36.06 % respectively.Stability (Freeze - thaw, Auto sampler, Bench top, Longterm):The results of various stability studies were listed in Table4. No significant degradation of the DG, EC, TA wasobserved even after 36 h storage in the autosampler andafter 11h storage at room temperature. Freezthaw stabilityestablished for 6 freezthaw cycles.The overall % C.Vranged from 0.52 to 9.29 % of the theoretical values. Inaddition, the long-term stability of DG, EC, TA samplesafter 85 days of storage at -70 oC was also evaluated. Theoverall %C.V ranged from 1.51 to 5.48 % of the theoreticalvalues. These results confirmed the stability of DG, EC, TAin human plasma for at least 85 days at -70 oC (Table-4).Application to biological samplesThe above validated method was used in the determinationof DG, EC, TA in plasma samples for establishing thebioequivalence of a single tablet (one tablet contains 50mg,200mg, 25mg dose) in 12 healthy male volunteers. All theplasma concentrations of DG, EC, TA were within thestandard curve region and retained above the LLOQ levelsfor the entire sampling period. The chromatograms ofsubject samples and typical plasma concentration versustime profiles of DG, EC, TA were depicted in Fig. 6 andFig. 7. The mean and Test/Reference ratio ofpharmacokinetic parameters of 12 healthy human malevolumteers were depicted in Table.5.0 and 6.0.

4. ConclusionsThe method described here is novel, fast, robust, sensitive,selective, and rugged with high recovery and has significantadvantages over other techniques previously described formeasuring Dolutegravir (DG), Emtricitabine (EC) &Tenofovir Alafenamide (TA) in biological fluids and wasquantified by LC-MS/MS. The validated method wassuccessfully applied in Bioequivalence study of twoformulations (Test and Reference) by oral administrationsingle tablet (one tablet contains 50mg, 200mg, 25mg dose)in 12 healthy human volunteers under fed conditions.



5. AcknowledgementsAuthors wish to thank the support received from Clinse labsprivate limited, Hyderabad, India, Telangana-500018 forconducting of clinical study and IICT (Indian institute ofchemical technology) Hyderabad India for providing

Literature survey for successful completion of this Researchwork.

Conflict of Interest: Authors declare that, there is noconflict of interest.

Table.2.0: Calibration curve details of Dolutegravir (DG), Emtricitabine (EC) and Tenofovir Alafenamide (TA).

STDLEVL

Dolutegravir (DG) Emtricitabine (EC) Tenofovir Alafenamide (TA)Conc (ng/mL)(Mean±S.D)

%CV

%Accuracy Conc (ng/mL)(Mean±S.D)

%CV

%Accuracy Conc (ng/mL)(Mean±S.D)

%CV

%Accuracy

STD-A

49.2384±0.25 0.51 97.82 25.9660±0.37 1.41 101.47 5.0260±0.04 0.78 100.28

STD-B

106.8240±1.79 1.68 106.11 50.2484±1.88 3.75 98.18 9.8780±0.19 1.89 98.54

STD-C

195.5790±3.57 1.83 97.14 101.1564±2.98 2.95 98.83 29.8186±0.26 0.88 99.36

STD-D

386.4462±3.62 0.94 95.97 199.1676±6.18 3.10 97.29 63.5284±1.35 0.88 105.84

STD-E

831.1920±6.32 0.76 103.21 599.9736±5.43 0.91 99.65 124.5396±1.35 1.09 103.75

STD-F

1575.4920±17.92 1.14 104.66 1236.6386±16.68 1.35 102.70 238.2822±2.04 0.85 99.25

STD-G

3094.1810±35.83 1.16 102.77 1824.4708±36.86 2.02 101.21 359.1488±2.92 0.81 99.78

STD-H

5734.0532±19.49 0.34 95.23 2418.0586±33.21 1.37 100.60 457.9252±3.92 0.86 95.41

STD-I 7795.8322±87.97 1.13 97.10 3006.4882±30.94 1.03 100.07 586.6530±14.34 2.44 97.79

Figure: 5.0: Chromatograms of LLOQs A) DG, B) EC and C) TA and ULOQs D) DG, E) EC and F) TA.



Table.3.0: Precision and accuracy (Analysis with spiked samples at Four different concentrations).

Spiked PlasmaConcentration

(ng/ml)

Dolutegravir (DG)Within-run (Intra-day) Between-run (Inter-Day)

Concentrationmeasured

(n=12; ng/ml;mean±S.D)

%CV%

Accuracy



%CV %Accuracy

LLOQ (50.498) 49.7411±3.14 6.30 98.50 50.5408±6.62 13.10 100.08LQC (150.741) 156.2986±3.80 2.43 103.69 156.7199±4.99 3.19 103.97

MQC(4019.748) 4386.7948±42.52 0.97 109.13 4362.3038±42.99 0.99 108.52HQC

(6699.580)6265.9543±189.57 3.03 93.53 6401.7781±424.67 6.63 95.55


(ng/ml)

Emtricitabine (EC)

Within-run (Intra-day) Between-run (Inter-Day)



%CV %AccuracyConcentration

measured(n= 30; ng/ml;

mean±S.D)

%CV %Accuracy

LLOQ (25.969) 27.0864±2.75 10.16 104.30 27.4815±2.76 10.06 105.82LQC (75.274) 78.6643±3.91 4.97 104.50 78.0750±5.41 6.93 103.72

MQC(1443.757) 1321.8304±41.38 3.13 91.55 1323.7748±38.80 2.93 91.69HQC

(2555.322)2312.1608±124.26 5.37 90.48 2372.5545±121.11 5.10 92.85


(ng/ml)

Tenofovir Alafenamide (TA)

Within-run (Intra-day) Between-run (Inter-Day)



%CV %AccuracyConcentration

measured(n= 30; ng/ml;

mean±S.D)

%CV %Accuracy

LLOQ (5.026) 4.5656±0.10 2.13 90.84 4.9453±0.43 8.60 98.40LQC (15.229) 14.5777±0.90 6.19 95.72 14.6268±0.59 4.02 96.05

MQC(303.976) 280.1659±12.29 4.39 92.17 277.9837±10.37 3.73 91.45HQC (467.655) 426.6820±10.74 2.52 91.24 428.1972±16.53 3.86 91.56

Table.4.0: Stability studies of Dolutegravir (DG), Emtricitabine (EC) and Tenofovir Alafenamide (TA) in plasma samples.Dolutegravir (DG)

SpikedPlasma

concentration

(ng/mL)

Roomtemperature

Stability

Processed sampleStability

Long term stabilityFreeze and thaw

stability

Auto SamplerStability

11 h 39h 85 days Cycle (6) 36hConcentrati

onmeasured

(n=6;ng/ml;mean±S.D)

%CV

(n=6)

Concentration

measured(n=6;ng/ml;mean±S.D)

%CV

(n=6)

Concentration measured(n=6;ng/ml;mean±S.D)

%CV

(n=6)

Concentration


%CV

(n=6)

Concentration


%CV

(n=6)

150.741157.3522

±6.053.84

159.1950±4.81

3.02159.0013±4.2

22.66

157.3865±8.49

5.40156.7392±4.

162.66

6699.5806994.8367

±45.250.65

7095.9345±64.58

0.917018.5375±1

13.971.62

7018.4512±48.94

0.707053.8623±

47.500.67

Emtricitabine (EC)SpikedPlasma

concentration

Roomtemperature

Stability


Long term stabilityFreeze and thaw

stability


11 h 39h 85 days Cycle (6) 36h



(ng/mL) Concentration


%CV

(n=6)

Concentration


%CV

(n=6)


%CV

(n=6)

Concentration


%CV

(n=6)

Concentration


%CV

(n=6)

75.27477.8533±7.2

39.29

77.6290±2.87

3.69 75.4695±4.14 5.4873.9012±2.7

93.77

74.3838±4.27

5.74

2555.3222470.5543±

32.891.33

2456.4647±46.01

1.872452.9645±5

7.702.35

2416.1513±52.83

2.192458.1702

±26.301.07


SpikedPlasma

concentration

(ng/mL)

Roomtemperature

Stability


Long term stability Freeze and thawstability


11 h 39h 85 days Cycle (6) 36hConcentrati

onmeasured

(n=6;ng/ml;mean±S.D)

%CV

(n=6)

Concentration


%CV

(n=6)


%CV

(n=6)

Concentration


%CV

(n=6)

Concentration


%CV

(n=6)

15.22914.4798±0.1

81.21

14.7777±0.18

1.19 14.6593±0.32 2.1614.7710±0.1

30.86

14.5292±0.33

2.27

467.655426.1015±3.

840.90

422.3497±4.76

1.13427.8358±6.4

81.51

424.8822±4.30

1.01427.1995±2.

220.52

Figure. 6.0: Chromatograms of healthy human male Volumteers/Subject samples after administration of 50mg, 200mg,25mg single oral dose containg A) Dolutegravir (DG) and Dolutegravir-D3 (DGIS), B) Emtricitabine (EC) and Emtricitabine15N-D2 (ECIS), C) Tenofovir Alafenamide (TA) and Tenofovir Alafenamide-D5 Fumarate (TAIS).

Figure.7.0: Mean plasma concentration of TestVs Reference after administration of 50mg, 200mg, 25mg single oral dosecontaing A) Dolutegravir (DG), B) Emtricitabine (EC) and C) Tenofovir Alafenamide (TA) in 12 healthy human malevolumteers.



Table.5.0: Mean pharmacokinetic parameters of in 12 healthy human male volumteers after oral admisntration of 50mg,200mg, 25mg single dose of Test and Reference product containg Dolutegravir (DG), Emtricitabine (EC) and Tenofovir

Alafenamide (TA).Dolutegravir (DG)

Productname

Pharmacokinetic ParameterCmax

(ng/mL)Tmax(Hr)

AUC0-t(ng/mL)

AUC0-∞t(ng/mL)

Test 3164.725 2.882 63780.762 67438.14

Reference 2668.527 2.604 52350.391 55510.637

Emtricitabine (EC)

Productname


(ng/mL)Tmax(Hr)

AUC0-t(ng/mL)

AUC0-∞t(ng/mL)

Test 2371.325 1.333 12547.291 12868.316

Reference 2430.108 1.042 12327.008 12640.387


Productname


(ng/mL)Tmax(Hr)

AUC0-t(ng/mL)

AUC0-∞t(ng/mL)

Test 435.276 0.563 296.918 300.333

Reference 428.527 0.604 282.906 286.566Cmax: Maximum concentration observed, Tmax: Time at which the maximum concentration, AUC – Area under theConcentration – time curve, AUC0-t: AUC up to the last measurable concentration, AUC0-∞: AUC curve to infinite time.

Table.6.0. Pharmacokinetic parameters Test/Reference ratio of 50mg, 200mg, 25mg single dose after oral administration ofof Test and Reference product containg Dolutegravir (DG), Emtricitabine (EC) and Tenofovir Alafenamide (TA) in 12

healthy human male volumteers.Dolutegravir (DG)

PK Parameters

CmaxAUC0-t(ng/mL)

AUC0-∞t (ng/mL)

Test/Reference 118.59 121.83 121.49Emtricitabine (EC)

PK ParametersTest/Reference 97.58 101.79 101.80

Tenofovir Alafenamide (TA)PK Parameters

Test/Reference 101.57 104.95 104.80

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Research Article Open Access Development and validation of ... · available. This study compared the relative bioavailability and pharmacokinetics of test formulation (fixed dose