RP-HPLC method development and validation for the estimation of Emtricitabine, Bictegravir and Tenofovir alafenamide in bulk and pharmaceutical dosage form

Figures & data

Figure 1. Chemical structures of Emtricitabine, Bictegravir & Tenofovir alafenamide.

Figure 2. Optimized Chromatogram of Emtricitabine, Bictegravir and Tenofovir alafenamide.

Table 1. System suitability parameters for Emtricitabine, Bictegravir, and Tenofovir alafenamide.

S. No.	Emtricitabine			Bictegravir			Tenofovir alfanamide
Inj	RT (min)	USP Plate Count	Tailing	RT (min)	USP Plate Count	Tailing	Resolution	RT (min)	USP Plate count	Tailing	Resolution
1	2.302	4236	1.36	3.216	6872	1.19	5.9	3.742	6306	1.15	3.0
2	2.303	3828	1.37	3.217	5893	1.25	5.8	3.742	6805	1.16	3.0
3	2.303	4014	1.35	3.218	5740	1.23	5.7	3.745	6127	1.18	2.9
4	2.305	4031	1.35	3.219	6660	1.21	5.7	3.746	6063	1.16	3.0
5	2.305	4193	1.32	3.221	5703	1.24	5.8	3.748	6190	1.19	2.9
6	2.31	4484	1.31	3.226	5605	1.22	5.8	3.751	6531	1.19	2.9

Table 2. Linearity table for Emtricitabine, Bictegravir and Tenofovir alafenamide.

Emtricitabine		Bictegravir		Tenofovir alafenamide
Conc (µg/mL)	Peak area	Conc (µg/mL)	Peak area	Conc (µg/mL)	Peak area
50	1142190	12.5	124908	6.25	90956
100	2300130	25	252095	12.5	165918
150	3459688	37.5	375283	18.75	246203
200	4577306	50	498726	25	321916
250	5750094	62.5	621513	31.25	398323
300	6787410	75	730387	37.5	476649

Table 3. Determination of repeatability and intermediate precision.

	Repeatability			Intermediate
Drug Name	Peak Area	Std Dev	%RSD	Peak Area	Std Dev	%RSD
Emtricitabine	4444663	41245.5	0.93	4586663	29843.4	0.65
Bictegravir	496262	4265.2	0.86	504185	4070.9	0.81
Tenofovir alafenamide	314654.5	818.6	0.3	323931	1255.4	0.4

Table 4. Accuracy results of Emtricitabine, Bictegravir and Tenofovir alafenamide.

	Emtricitabine			Bictegravir			Tenofovir alfanamide
Conc.	Amount added (µg/ml)	Amount recovered (µg/ml)	% Recovery	Amount added (µg/ml)	Amount recovered (µg/ml)	% Recovery	Amount added (µg/ml)	Amount recovered (µg/ml)	% Recovery
50%	100	99.66	99.66	25	25.05	100.20	12.5	12.59	100.73
	100	99.65	99.65	25	25.08	100.33	12.5	12.55	100.42
	100	100.57	100.57	25	25.09	100.37	12.5	12.55	100.40
100%	200	196.30	98.15	50	50.52	101.04	25	25.11	100.42
	200	199.36	99.68	50	50.49	100.97	25	25.09	100.36
	200	200.11	100.06	50	50.28	100.57	25	25.15	100.60
150%	300	302.20	100.73	75	76.01	101.35	37.5	37.53	100.07
	300	301.33	100.44	75	75.49	100.65	37.5	37.55	100.12
	300	300.26	100.09	75	75.30	100.40	37.5	37.62	100.31
Mean % Recovery			99.89%	Mean % Recovery		100.13%	Mean % Recovery		100.38%

Table 5. Sensitivity table of Emtricitabine, Bictegravir and Tenofovir alafenamide.

Molecule	LOD (µg/ml)	LOQ (µg/ml)
Emtricitabine	1.04	3.14
Bictegravir	0.62	1.89
Tenofovir alfanamide	0.70	2.13

Table 6. Robustness data for Emtricitabine, Bictegravir and Tenofovir alafenamide.

S. No.	Condition	%RSD of Emtricitabin	%RSD of Bictegravir	%RSD of tenofovir
1	Flow rate (−) 0.9 ml/min	0.5	0.5	0.8
2	Flow rate (+) 1.1 ml/min	0.4	0.2	0.9
3	Mobile phase (−) 55B:45A	0.8	0.6	0.9
4	Mobile phase (+) 45B:55A	1.0	1.2	1.1
5	Temperature (−) 25°C	0.3	0.5	0.6
6	Temperature (+) 35°C	0.4	0.8	1.0

Table 7. Assay results of Emtricitabine, Bictegravir and Tenofovir alfanamide.

	Emtricitabine			Bictegravir			Tenofovir alfanamide
S. No.	Standard area	Sample area	% of Assay	Standard area	Sample area	% of assay	Standard area	Sample area	% of assay
1.	4593842	4540828	98.80	501126	510494	101.14	322508	325670	100.89
2.	4591629	4614989	100.41	500085	506540	100.35	323688	323292	100.15
3.	4591716	4559513	99.20	502775	505497	100.15	323902	324864	100.64
4.	4586871	4598084	100.04	509261	501652	99.38	321335	323974	100.36
5.	4589341	4595219	99.98	501801	501384	99.33	322962	323737	100.29
6.	4574811	4611344	100.33	507477	499545	98.97	320506	322050	99.77
Mean	4586874	4586663	99.80	503754	504185	99.89	322484	323931	100.35
S.D	6901.1	29843.4	0.649	3723.7	4070.9	0.81	1335.8	1255.4	0.389
%RSD	0.2	0.7	0.7	0.7	0.8	0.8	0.4	0.4	0.4

Figure 3. Forced degradation study under acidic conditions.

Figure 4. Forced degradation study under basic conditions.

Figure 5. Forced degradation study under peroxide conditions.

Figure 6. An acid purity plot of Emtricitabine, Bictegravir and Tenofovir alafenamide.

Figure 7. A base purity plot of Emtricitabine, Bictegravir and Tenofovir alafenamide.

Figure 8. A peroxide purity plot of Emtricitabine, Bictegravir and Tenofovir alafenamide.

Table 8. Degradation data of Emtricitabine, Bictegravir and Tenofovir alfanamide

Drug substance	Degradation condition	% Drug degraded	Purity angle	Purity threshold
Emtricitabine	Acid	4.62	1.290	1.315
	Alkali	2.83	0.483	0.534
	Oxidation	5.95	0.928	1.299
	Thermal	0.60	0.260	0.327
	UV	3.11	0.293	0.312
	Water	0.77	0.269	0.324
Bictegravir	Acid	4.59	0.181	0.275
	Alkali	2.70	0.150	0.271
	Oxidation	4.48	0.169	0.279
	Thermal	0.65	0.158	0.269
	UV	3.53	0.186	0.272
	Water	0.57	0.195	0.274
Tenofovir alfanamide	Acid	4.93	1.336	1.680
	Alkali	2.85	1.297	1.570
	Oxidation	4.99	1.514	1.945
	Thermal	0.94	0.391	0.557
	UV	4.11	0.342	0.568
	Water	0.67	0.344	0.546