A Stability Indicating Method Development and Validation for The Simultaneous Estimation of Emtricitabine, Tenofovir Alafenamide and Doultegravir in Pharmaceutical Formulation by Ultra Performance Liquid Chromatography

Kishore Konam¹*, Somasekhar Reddy Kanala²

¹Research Scholar, JNTUA, Ananthapuramu, Andhra Pradesh, India.

²Department of Pharmacology, Raghavendra Institute of Pharmaceutical Education and Research,

Chiyyedu Post, Ananthapuramu, Andhra Pradesh, India.

*Corresponding Author E-mail: kishorekonam671@gmail.com

ABSTRACT:

A Precise, specific, linear, accurate and robust technique used to be developed for the simultaneous estimation of the, Emtricitabine, Tenofovir alafenamide and Doultegravir in pill dosage structure and Validated as per ICH Validation guidelines. Method was once optimised with the useful resource of Acquity BEH C18 125A° (100 × 2.1 mm, 1.7μ) column at a flow rate of 0.6ml/min, Mobile part was as soon as pH 3.8 Buffer, Methanol and Acetonitrile (50:40:10 respectively). The Column Oven temperature used to be maintained at 35°C and working wave dimension used to be selected at 274nm. The retention times of Emtricitabine, Tenofovir Alafenamide and Doultegravir have been determined to be 3.77, 6.34 and 7.67minutes respectively.  The % RSD of the Emtricitabine, Tenofovir Alafenamide and Doultegravir had been and located to be 0.27%, 1.24% and 0.11% respectivelyIn Method precision Parameter, % Assay had been determined ninety five to 105.0% and p.c Recovery had been acquired as 98.9%, 100.4% and 100.0% for Emtricitabine, Tenofovir Alafenamide and Doultegravir respectively. Linearity was as soon as acquired as 0.999, 0.999 and 0.999 for Emtricitabine, Tenofovir Alafenamide and Doultegravir respectively. Analytical Range used to be determined from the linearity and accuracy for Emitricitabine used to be 50µg/mL to 150µg/mL, Tenofovir Alafenamide used to be as soon as 6.25µg/mL to 18.75µg/mL and Doultegravir was as soon as 12.5µg/mL to 37.5µg/mL..

KEYWORDS: Emtricitabine; Doultegravir; Tenofovir Alafenamide and ICH Validation; RP-HPLC; PDA Detection.

INTRODUCTION:

Emtricitabine (Fig.1) is a reverse-transcriptase nucleoside inhibitor (NRTI) for the
prevention and treatment of HIV illness in younger human beings and adults. Emtricitabine is a renowned scientific challenge in opposition to the hepatitis B virus (HBV), however is no longer general for the remedy of HBV illness by using the FDA.

Emtricitabine treatment has widespread implications for human beings with power HBV Infection with widespread advances in histology, virology and biochemistry

In remedy the security profile of emtricitabine is shut to that of a placebo. Emtricitabine, like all different medicinal drugs authorised by means of FDA, does now not deal with HIV or HBV infection. In a find out about involving human beings with HBV infection, signs and symptoms and signs and symptoms of publicity in 23 percentage of human beings handled with Emtricitabine used to be decrease returned.

Fig. 1. Structure of Emitricitabine¹

Tenofovir alafenamide² (Fig.2) is a prodrug of tenofovir. It was once developed through Gilead Sciences primarily based on the protide science for use in the therapy of HIV/AIDS and continual hepatitis B, and is utilized in the structure of tenofovir alafenamide fumarate (TAF). Closely associated to the normally used reverse-transcriptase inhibitor tenofovir disoproxil fumarate (TDF), TAF has increased antiviral pastime and higher distribution into lymphoid tissues than that agent.

Fig.2 Structure of Tenofovir alafenamide²

Dolutegravir (DTG) (Fig.3) is an antiretroviral medicine used, collectively with different medication, to deal with HIV/AIDS. It may additionally additionally be used, as section of publish publicity prophylaxis, to stop HIV contamination following manageable exposure. It is taken through mouth³.

Dolutegravir is authorized for use in a large populace of HIV-infected patients. It can be used to deal with HIV-infected adults who have in no way taken HIV remedy and HIV-infected adults who have until now taken HIV remedy (treatment-experienced), which include these who have been dealt with different integrate strand switch inhibitors³.

Fig.3 Structure of Doultegravir

The literature survey reveals that few of analytical method available for the estimation of Emtricitabine, Tenofovir alafenamide and Doultegravir in pharmaceutical dosage forms. The reported methods available for the estimation of Emtricitabine, Doultegravir and Tenofovir alafenamide are RP-HPLC^4-9 reverse phase high performance liquid chromatography method] Tenofovir Disoproxil Fumarate and Emtricitabine RPHPLC^9-11 reverse phase high performance liquid chromatography Method], Application of UV Spectrophotometric Methods for Simultaneous Estimation of Emtricitabine and Tenofovir Alafenamide Fumarate in Bulk¹², spectrofluorimetric analysis¹³, reverse phase high performance liquid chromatography^14-17. Since there are no official reported methods on Ultra-Performance Liquid Chromatographic (UPLC) methods for the simultaneous estimation of Emtricitabine, Bictegravir and Tenofovir alafenamide in the public domain, we have planned to develop a simple, precise, economic and accurate stability indicating Ultra-Performance Liquid Chromatographic (UPLC) method development and validation for the estimation of emtricitabine, bictegravir and tenofovir alafenamide in pharmaceutical dosage form.

MATERIALS AND METHODS:

Instruments was Used:

Shimadzu Balances, Thermo pH Meter, Agilent 1290 UPLC with PDA equipped with quaternary pump and Auto sampler integrated with Open lab software, Shimadzu UV-Visible spectrophotometer with Lab solution software, Ultasonicator

Drug Samples: 

Emtricitabine, Tenofovir Alafenamide and Doultegravir Active pharma ingradients and Marketed samples of Emtricitabine, Tenofovir Alafenamide and Doultegravir Tablet.

Chemicals and Reagents:

Triethylamine (Make: Rankem and Grade: AR), Trifluoro aceticacid (Make: Merck and Grade: Emparta ACS), Methanol (Make: Rankem and Grade: HPLC) and Acetonitrile (Make: Rankem and Grade: HPLC),

Methadology of Analysis:

Working wavelength optimization:

Preparation of Standard solutions:

Weighed precisely 5mg of every Emtricitabine in a volumetric flask of up to 50mL and brought 70mL of Methanol via sonication to dissolve the product supplies and Volume made up of Methanol and properly mixed. Take 5mL above inventory answer and dilute this answer similarly with methanol (10μg/mL of Emtricitabine) to 50mL. 10μg/mL of Tenofovir alafenamide and Doultegravir solutions are prepared in the above preparation manner. 200 to 400nm were scanned above normal Emtricitabine solutions, Tenofovir Alafenamide and Doultegravir. The UV graphs of the Emtricitabine, Doultegravir and Tenofovir have been shown at 258 nm ,257nm and 280 nm and graphs had been shown in Fig 4,5 and Fig 6 respectively

Fig-4. UV graph of Emtrictabine

Fig-5. UV graph of Doultegravir

Fig-6.  UV graph of Tenofovir alafenamide

Emitricitabine showed maximum absorption at 258.2nm and Doultegravir showed maximum absorption at 257.2 nm, but Tenofovir Alafenamide showed maximum absorption at 280.4, Chosen 274nm as working wavelength due to Tenofovir having low intensity at 250 nm to 260nm.

Method Development:

Following several trials by adjusting the column, Buffers and Mobile Phase Ratios, the conditions below were optimized, Blank and Placebo interference was tested in optimized condition and percent Assay was verified as well. Finally, these conditions of diluent, mobile phase and chromatography were taken as optimized conditions.

Preparation of pH 3.8 Buffer:

Accurately taken 1mL of Triethylamine and transferred to 1000mL of water, well blended with diluted Trifluoro aceticacid and balanced upto pH 3.8 and filtered through Nylon membrane filter 0.45μm.

Diluted Trifluoro aceticacid Preparation:

Diluted 3mL of Trifluoro aceticacid 25mL with water and mixed well.

Diluent:

Mixed pH 3.8 Buffer and Acetonitrile in the ratio of 50:50

Preparation of Standard stock:

Weighed Accurately and transferred 200mg of Emitricitabine, 50mg of Doultegravir and 25mg of Tenofovir Alafenamide into 200mL Volumetric flask, inserted 140mL of Diluent then held to dissolve for sonicator, Final volume was made up to diluent mark and well balanced. (Emtricitabine 1000μg/mL, Doultegravir 25μg/mL, and Tenofovir Alafenamide 125μg/mL).

Preparation of Standard working solution:

Take 5mL of the above regular stock solution into 50mL volumetric flask then dilute and mix well and mark upto the label with diluents. (Emtricitabine 100μg/mL, Alafenamide Tenofovir 12.5μg/mL, and 25μg/mL Doultegravir)

Preparation of Sample stock solution

Taken 20 Tablets correctly and measured, taking average weight from the twenty tablets and crushed into fine powder using mortar and pestle. Weighed accurately crushed powder equal to 200mg of Emitricitabine, 50mg of Doultegravir and 25mg of Tenofovir Alafenamide in a 200mL volumetric flask, added 150mL of Diluent, then held on a sonicator for up to 35min with occasional shaking by maintaintain by the sonicator temperature 23±2°C, After the sonication has placed the flask on the top of the bench to reach room temperature, the volume is diluent and well balanced. This solution was centrifuged for 5min at 10000RPM

Preparation of Sample solution:

Clear supernatant of sample stock solution correctly taken 5mL and transferred in to 50mL volumetric flask, diluted to label with diluents and blended well. These solution was filtered through 0.45μ PVDF Syringe filter by discarding the filtrate 3mL.

Preparation of Mobile Phase:

Mixed Accurately 500mL of pH 3.8 Buffer, 400mL of Methanol and 100mL of Acetonitrile. Degassed by soncation 10min

Optimised Conditions:

The most excellent chromatographic prerequisites had been acquired with a number of path and error strategies and the most excellent chromatographic stipulations have been shown in table no. 1 and the optimum chromatography used to be shown in Fig.7

Table No-1 : Optimised Chromatogaphic conditions:

Column	Acquity BEH C8 125A° (100 × 2.1 mm, 1.7μ)
Mobile Phase and Composition	pH 3.8 Buffer:Methanol:Acetonitrile(50:40:10)
Flowrate	0.6mL/min
Column ovenTemperature	35°C
Injection volume	10µL
Detection wavelength	274nm
Autosampler Temperature	25°C
Retention Times	3.770min of Emitricitabine, 6.345min for Tenofovir 7.676min for Doultegravir (Total Run time 10.0min)

Fig-7. Optimised Chromatogram

Method Validation

By using Optimised condition Analytical Method of Assay carried out by ICH Guideline Q2B^4-5:  The objective of validation of an analytical procedure is to demonstrate that it is suitable for its intended purpose.

System Suitability and System Precision

The suitability screening out of the device is an important part of many analytical procedures according to ICH guidelines4-5.The assessments are based on the assumption that the to be analyzed equipment, analytical operations and samples are an integral structure that can be tested as such. Unit suitability test parameters to be set for a given procedure depends on the type of procedure depending upon the procedure.

According to ICH Specifications: Theoretical Plates should not be less than 2000, Tailing factor should not be 0.95 to 2.0 and Resolution should not be less than 2.0 between Emitricitabine, Tenofovir and Doultegravir

System Precision Specification: %RSD for Area and Retention time for the six replicate injections should not be more than 2.0 of each Analyte and the outcomes for the system suitability had been summarized in table no.2 and the results for the system precision have been summarized in table no.2

Table No: 2 Results of System suitability

S.No	Rt in min	Plate count	Tailing Factor	Rt in min	Plate count	Tailing Factor	Resolution	Rt in min	Plate count	Tailing Factor	Resolution
1	3.770	4658	1.35	6.345	6701	1.27	4.8	7.676	5041	1.31	2.7
2	3.769	4652	1.37	6.359	6841	1.24	4.8	7.672	5121	1.32	2.7
3	3.768	4589	1.34	6.373	6825	1.26	4.7	7.668	5021	1.32	2.7
4	3.768	4690	1.36	6.387	6842	1.24	4.8	7.666	5032	1.33	2.6
5	3.767	4671	1.32	6.399	6895	1.26	4.7	7.663	5047	1.31	2.7
6	3.767	4682	1.33	6.410	6741	1.25	4.8	7.661	5066	1.30	2.7

Table 3.Results of System Precision

S.No	Rt in min	Area	Rt in min	Area	Rt in min	Area
1	3.770	1700.78	6.345	415.79	7.676	1198.56
2	3.769	1700.59	6.359	414.24	7.672	1201.8
3	3.768	1709.59	6.373	415.74	7.668	1201.53
4	3.768	1709.92	6.387	424.59	7.666	1200.95
5	3.767	1709.95	6.399	424.7	7.663	1199.94
6	3.767	1709.59	6.410	424.82	7.661	1198.83
AVG	0.001	4.691	0.025	5.205	0.006	1.378
%RSD	0.03	0.27	0.39	1.24	0.07	0.11

Observation:

%Relative standard deviation of Six Replications of Area and Retention time for Emitricitabine and Tenofovir, Tenofovir and Doultegravir was less than 2.0%.

SPECIFICITY;

Specificity is the ability to assess unequivocally the analyte in the presence of components which may be expected to be present. Typically these might include impurities, degradants, matrix, etc.^4-5

Procedure for Specificity: Blank (Diluent uised as a Blank) and Placebo solutions were injected into HPLC system.

The blank and placebo chromatograms have been shown in fig.8-9 respectively.

FORCED DEAGRADATION STUDY:

Forced Degradation learn about additionally as a phase of Specificity Parameter, It is a procedure in which exceptional stress stipulations are utilized over drug Substance or drug product and it will transformed in to distinctive degradant. These research are mostly used for the dedication of steadiness of molecule below accelerated conditions. Samples have been dealt with with Acid (5N Hydrochloric acid/4Hrs/50°C), Base (5N Sodium Hydroxide/4hrs/50°C), Peroxide (10% Hydrogen Peroxide for 4Hrs at Bench top), Thermal (60°C/48Hrs) and Photolytic condition. In base Sample remedy emitricitabine used to be degraded and degradation was once befell 11.8% for Emitricitabine, In ultimate all prerequisites Remaining all conditions, handled pattern has proven much less degradation (below 5%). All essential analytes have been Peak purityindex proven inside the specification. (Specification: All Main Analytes top purity index must be morethan Single factor threshold value).

The consequences for the degradation research had been performed in exclusive parameters and the outcomes for the all samples had been shown in table-4 and the chromatographic graphs of the control, acid treated, base treated, Peroxide treated, Photolytic handled and thermal handled samples have been shown in fig -10, fig-11, fig-12, fig-13 and Fig-14 and Fig-15 respectively

Fig-8. Blank Chromatogram

Fig-9. Placebo Chromatogram

Table No: 4 Degradation of the Sample

Degradation Condition	% Assay after Degradation
	Emitricitabine	Tenofovir	Doultegravir
Control Sample	99.5	99.7	98.5
Acid 5N HCl/50°C/4Hrs	99.4	99.4	99.6
Base NaOH/50°C/4Hrs	78.2	99.1	99.4
Peroxide (4Hrs/10%Hydrogen Peroxide/Bench top)	98.3	100.1	98.9
Photolytic (1.2 million lx h)	99.3	100.4	99.3
Thermal(60°C/48Hrs)	99.4	98.5	99.2

Fig-10. Control Sample

Fig-11. Acid Treated Sample(5N Hcl/50°C/4Hrs)

Fig-12. Base Treated Sample(5N NaOH/4Hrs/50°C)

Fig-13. Peroxide Treated Sample(10% H2O2/4Hrs Bench top)

Fig-14. Photolytic Treated Sample

Fig-15. Thermal Treated Sample

METHOD PRECISION:

Closeness of agreement between a series of measurements obtained from multiple sampling of the same homogeneous sample^4-5.

Sample solutions were six prepared individually and each injected in to HPLC System, Calculated %Assay by using average area of Six Standards.

The percent RSD for percent Assay of all peak area and retension time of Emitricitabine, Tenofovir and Doultegravir had been calculated and summarized in the table no -5.

Table No. 5 Method Precission

Name of the Sample	%Assay of Emitric itabine	%Assay of Tenofovir	%Assay of Doultegravir
Method Precsion-01	100.6	100.9	99.6
Method Precsion-02	100.9	100.4	99.5
Method Precsion-03	100.5	100.0	99.3
Method Precsion-04	100.3	99.7	98.8
Method Precsion-05	99.8	99.8	98.9
Method Precsion-06	99.9	100.0	98.9
Standard Deviation	0.427	0.423	0.357
Average	100.328	100.137	99.162
%RSD	0.43	0.42	0.36

ACCURACY:

Expresses the closeness of agreement between the value which is accepted either as a conventional true value and the value found^4-5 Three levels(50%, 100% and 150%) of accuracy sample were prepared in triplicate by Standard API addition method to the Placebo, At each level API taken 50%, 100% and 150% respectively in the presence of Placebo. The Accuracy, Recovery and %RSD of Emitricitabine, Tenofovir and Doultegravir had been calculated and summarized in the table -6.

Table No: 6 Accuracy Studies

Name of the Level	Emitric itabine	Tenofovir	Doultegravir
50% Accuracy	98.2	99.1	99.3
100% Accuracy	99.4	100.5	98.8
150% Accuracy	98.2	101.6	100.1
Mean	98.9	100.4	99.4
%RSD	0.74	1.28	1.04

LINEARIT AND RANGE:

Five linearity solutions (50%, 80% 100%, 120% and 150%) were prepared from standard stock solution i.e., 50µg/mL to 150µg/mL for Emitricitabine, 6.25µg/mL to 18.75µg/mL for Tenofovir and 12.5µg/mL to 37.5µg/mL for Doultegravir.

The linearity of one of a kind concentrations of the Emitricitabine,Tenofovir and Doultegravir have been calculated and summarized in the table no -7 and graphs had been shown in Fig.16-18 respectively .

Observation:

The Correlation coeffiecient was found 0.999 for Emitricitabine, Tenofovir Alafenamide and Doultegravir.

Table No:7 Linearity data

Emitricitabine		Tenofovir		Doultegravir
Conc. in µg/mL	Area	Conc. in µg/mL	Area	Conc. in µg/mL	Area
50.00	660.080	6.250	161.110	12.50	480.680
75.00	1177.040	9.375	291.650	18.75	835.820
100.00	1651.880	12.500	420.270	25.00	1206.160
125.00	2149.500	15.625	536.440	31.25	1522.510
150.00	2611.230	18.75	652.140	37.50	1851.690
Correlation coefficient	0.999	Correlation coefficient	0.999	Correlation coefficient	0.999

Fig-16. Calibration Curve of Emitricitabine

Fig-17. Calibration Curve of Tenofovir

Fig-18. Calibration Curve of Doultegravir

ROBUSTNESS:

Robustness conditions like flow (0.6mL/min±0.1mL) and Wavelength (274nm±5nm) was maintained in the HPLC System and injected six standards replicate injections in each condition. system suitability parameters were within the acceptance criteria, %RSD for Area of six standard injections for within the limit.

The Robustness of the distinct stipulations (Flow rate and wavelength) have been carried out, calculated and summarized in the table-8.

Table No: 8 Robustness data

S. No	Condition	%RSD for Emitri citabine	%RSD for Tenofovir	%RSD for Doultegravir
01	Flow Rate_0.4mL/min	0.47	0.26	0.37
02	Flow Rate_0.6mL/min	0.36	0.48	0.24
03	Wavelength (275nm)	0.41	0.28	0.49
04	Wavelength (285nm)	0.34	0.66	0.29

CONCLUSION:

A Basic, Reliable, Precise and Robust Stability indicating Assay Method was developed for simultaneous estimation of, in tablet dosage form, Emtricitabine, Tenofovir Alafenamide and Doultegravir, For the Optimized Assay Method Conditions, Use of ICH Q2B guidelines was validated. Precise, precise, reliable, linear and robust results have been shown for the system.  Test accuracy in system has demonstrated correct percentage of assay results. That was 95.0 to 105.0 percent. Recovery for Emtricitabine, Tenofovir Alafenamide and Doultegravir was 98.9 percent, 100.4 and 99.4 percent respectively. Linearity for Emtricitabine, Tenofovir Alafenamide and Doultegravir was obtained as 0.999, 0.999 and 0.999, respectively.This approach is also commonly used in routine research as in quality control.So this method very use full to Routine analysis like in Quality control to reduce the time and cost.

ACKNOWLEDGEMENTS:

Authors are thankful to the Chandra Lab-Hyderabad, Telanagana (state) and the administration,Principal of Raghavendra Institute of Pharmaceutical Education and Research, Ananthapuramu, A.P, for presenting the imperative services to elevate out the lookup work.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 02.08.2020            Modified on 01.12.2020

Accepted on 05.02.2021           © RJPT All right reserved

Research J. Pharm.and Tech 2021; 14(11):6017-6024.

DOI: 10.52711/0974-360X.2021.01046