INTRODUCTION:

Nevertheless, the classification of the material, the description of its nature, and its qualitative analysis are all aspects addressed by current analytical science, mainly perturbed with the estimation of the chemical mixture of the matter. The sample of matter should be divided, diagnosed, and defined by relative quantities of factors. Analytical chemistry may extract the structural factors found in a science or artwork to discover the fabric structure. The same division of analytical chemistry was drug analysis. The study of drugs through request relies on their concepts in the areas of chemistry, mechanics, microbiology, atomic sciences, and telecommunications, respectively. Analytical methods can be used to carry out qualitative and quantitative analyses. The quality analysis shows the sample species ' chemical identity. Quantitative definitions numerically define the quantitative magnitude of one or more of organisms or analytes.

Qualitative records are needed before a quantitative evaluation is carried out. Usually, a qualitative evaluation, as well as a quantity analysis, need a distinction. Two tests may compare the effects of a typical statistical assessment. The first is the weight and volume of the specimen to measure, and the second is to calculate those quantities which are proportional to the amount of analyte in this sequence. ETR is the replacement of the human immunodeficiency virus (HIV) produced by diaryl pyrimidine. It is a non-nucleoside reverse transcriptase secondary blocker which specifically reverses a transcriptase enzyme in opposition to ds and inhibits catalysts for the enzyme that alter genetic element replication. The wild type HIV-1 becomes active and with the aid of the Food and Drug Administration recognized as anti-AIDS. Chemically, pyrimidinyl-3,5-dimethylbenzonitrile 4[6-amino-5-bromo-2,(4-cyanophenyl)-amino] pyrimidine. The literature survey shows that only three techniques are used in the research of high-performance liquid chromatography (HPLC) in distinct formulations. Consequently, this research aimed to improve a simple and accurate HPLC Reverse Phase (RP) process for ETR evaluation activities.¹

Etravirine:

Molecular complex:

Figure 1: Complex of ETR ²

Diluent preparation:

MeOH was the dilutant used.

Formulation of Mobile Phase:

The mobile phase is MeOH, and ACN has been used in the interaction of (60:40) (v/v).

Typical standard Stock Solution Prep of ETR:

A 100mL volumetric flask of ETR 100mg was drawn. The diluent has been reduced by applying 50mL, and the diluent volume made up to mark (1000μg/ml). Use 1 ml of the above solution in a 10ml volumetric bottle and dilute (100μg/ml) to form the volume. Pipette 1ml in 10 ml volume flask with a dilution of 10μg/ml volume (10 μg/ml).

Chemical name/ IUPAC name	4-[6-Amino-5-bromo-2-[(4-cyanophenyl)amino] pyrimidin-4-yl]oxy-3,5-dimethylbenzonitrile
Synonyms	Intelence
Melting point	265℃
Solubility	Slightly water soluble, MeOH-soluble.
Physical appearance	Light yellow colour powder
Category	Antiretroviral
Dose and Administration: Oral

Response mechanism:

ETR is a 2^nd generation antagonist of non-nucleoside reverse transcriptase's (NNRTI) engineered to be useful as opposed to HIV by the addition of NNRTI, Efavirenz mutation (K103N), and Y181C nevirapine mutations in the first generation. This power seems to be related to the stability of ETR as a molecule. ETR is a pyrimidine diary (DAPY), a type of natural molecule that can bind the enzyme in many respects, making for extra-strong connections between ETR and the enzyme even though mutations occur. Some pyrimidine analogs, particularly rilpivirine, are widely used as anti-HIV agents. The stability of ETR seems to be related to this capacity.²

MATERIALS AND METHODS:

Table 1: Reagent and Chemicals

S. No	Name of chemical/drug	Make	Grade
1	Acetonitrile	Merck	HPLC
2	MeOH	Merck	HPLC
3	ETR	Apotex Ltd	API

Table 2: List of Instruments

S. No.	Name of the Equipment	Make	Model
1	UV spectrophotometer	Shimadzu	UV-1800
2	UFLC with PDA Detector	Shimadzu	LC-2010 AD
3	Analytical balance	Shimadzu	AY220

Table 3: Chromatographic Conditions

Optimized Chromatographic Conditions
Column	Phenomenex Kinetex C₁₈ (250 x 4.6 mm. 5μ)
Flow rate	1.0mL/min
Run time	10 min
Wavelength	311nm
Injection Volume	10μL
Detector	PDA Detector
Elution	Binary
Mobile Phase	MeOH: ACN (60:40) (v/v)
Column oven temperature	25 ± 5ºC

Preparation of solutions for linearity:

Linearity solutions are produced by diluting with inventory product diluents. ETR has been approved with levels of 1, 2, 3, 4, 5μg/mL. In 10ml volumetric flask pipette 1, 2, 3, 4, 5mL, and extract the quantity in the above grades.

Preparation of Calibration Curve:

In a 10mL series of volumetric flasks, stock solution (100μg/ml) ETR aliquots had been pipetted. The amount was processed to the limit and purified using an HPLC-grade diluent with a 0.45μ pore membrane filter to obtain concentration between 1 and 5μg/mL. Injecting and reporting 10μL maximum areas, the calibration curve is calculated. In the range of 1-5μg/mL, the beer rule is tested.

RESULTS AND DISCUSSION^3-16

A systematic analysis of the effects of different parameters was undertaken in the development of this method. The solubility of the ETR drug was initially developed. The RP-UFLC system for maintaining a high peak controlled chromatographing. The drug was initially evaluated at different formulations in flowing processes. The mobile phases and level of flow are chosen based on high altitude, efficiency, plate theory, asymmetry of the tailing variable, runtime, and resolution. The mobile-phase-A (MeOH) and mobile phase-B (ACN) with a flowing rate of 1.0ml/min at the ratio of 60:40 (v/v) was robust. The optimum wavelength for measurement was 311nm, in which the medication responded more strongly. ETR retention time was observed in 3.226 mins, blank chromatogram, and ETR, respectively, as shown in FIG 2 and 3.

Figure 2: Chromatogram of Blank (Diluent)

Figure 3: Chromatogram for ETR (Standard)

System suitability:

The suitability checking of the device is used to control the chromatographic approach to reproductive efficiency. Efficiency has been tested for newly produced solutions to drug applications.

Table 4: Results for System Suitability of ETR

Criteria	Acceptance criteria	Results
Tailing factor	Not More Than 2.0	1.315
Theoretical plates	Not Less Than 2000	4478.495

Data interpretation:

Table 4 shows the system fitness parameters to be within the acceptability requirements of the information, as mentioned above.

Linearity:

Table 5: Results for Linearity of ETR by UFLC Method

Sl. No	CONC (µg/mL)	Peak Area of ETR
1	1	333159
2	2	679754
3	3	926802
4	4	1287360
5	5	1501963
Regression EQN		Y = 294521x ₊ 62243
Correlation Coef (R²)		0.9942
Slope		294521
Intercept		62243

Figure 4: Calibration curve of ETR

Criteria of approval:

· Not Less Than 0.99 should be the regression factor.

Data evaluation:

Table 5 of ETR reveals that the reaction of ETR is constant between 15% and 50% of the function rate through numerical analysis of its Linearness tests.

Precision:

The accuracy of an analytical method is the degree to which the technique is used regularly for various samples of a homogeneous sample.

System Reliability (Intraday Precision):

System reliability means ensuring the proper functioning of the analytics unit.

Table 6: Results for System precision studies of ETR by UFLC Method

Conc (µg/mL)	Peak Area of ETR	Conc(µg/mL)	Peak Area of ETR	Conc (µg/mL)	Peak Area Of ETR
1	333159	3	926802	5	1501963
1	337164	3	925804	5	1515949
1	338147	3	927812	5	1507936
1	335170	3	936818	5	1512958
1	339162	3	939815	5	1501913
1	334155	3	929798	5	1516892
AVG	336159.5	AVG	931141.5	AVG	1509602
STDV	2159.255794	STDV	5286.192447	STDV	6122.933
% RSD	0.642330737	%RSD	0.567710971	%RSD	0.405599

Table 7: Method precision studies for ETR by UFLC Method

Concentration (µg/mL)	Peak Area of ETR	Concentration (µg/mL)	Peak Area of ETR	Concentration (µg/mL)	Peak Area of ETR
1	313519	3	916802	5	1401693
1	317614	3	918540	5	1415949
1	318417	3	918821	5	1407396
1	315710	3	918681	5	1412598
1	319612	3	919851	5	1401193
1	314515	3	917989	5	1416982
AVG	316564.5	AVG	918447.3333	AVG	1409301.833
STDV	2367.536842	STDV	1008.98814	STDV	6947.146505
% RSD	0.747884505	%RSD	0.109858029	%RSD	0.492949512

Criteria of approval:

RSD level should be less than 2.0 percent of the Etravirin maximum region reaction collected from 6 normal preparation injections.

Data evaluation:

Table 6 above shows a coherent area response, shown by a relative standard deviation

Method precision: Accuracy of technique shows whether or not a single-material technique produces coherent outcomes.

Criteria of approval:

In Table 7 assessments, the amount RSD determined should be greater than 2.0%.

Data evaluation:

It can be inferred from Table 7 above that the process is exact.

Limit of Detection and Limit of Quantitation:

The lowest level of analysis in the specimen that could be observed but not quantified under the experimental conditions defined is the detection limit (LOD). Quantitative limit (LOQ) implies the smallest amount of analyte in a specimen, quantifying with sufficient accurate and Precision in the experimental conditions specified. The pitch, the intercept and the coefficient of variance, and the relative average deviation from the linearity curve are based on LOD and LOQ.

Table 8: Results for LOD and LOQ results for ETR by UFLC Method

LOD	0.02μg/ml
LOQ	0.073μg/ml

Data evaluation:

Table 8 above shows that distinctly noticeable peaks at the LOD level have been observed. ETR LOD and LOQ were respectively 0.514 and 1.713μg/mL.

Accuracy:

The reliability of an experimental procedure is that the test results produced using this approach have a near true value (default value).

Amount of drug recovered

% Recovery = --------------------------------------× 100

Amount of drug added

Table 9: Recovery results for ETR by UFLC Method

Level of recovery	Amount of Pure drug	Amount of Formulation	The total amount of drug	Peak area	Difference	% recovery	Mean
50	2	1	3	958472	625313	91.99	98.36
	2	1	3	103749	704339	103.61
	2	1	3	100950	676343	99.41
100	2	2	4	134736	667608	98.21	98.11
	2	2	4	132929	649544	95.55
	2	2	4	136340	683647	100.57
150	2	3	5	219789	1271088	98.3	98.2
	2	3	5	225469	1327890	96.2
	2	3	5	216226	1235466	100.1

Table 10: Results for Robustness of ETR by UFLC method

Wavelength (nm)	Conc (µg/mL)	Abs	Wavelength (nm)	Conc (µg/mL)	Abs
309	3	0.116	313	3	0.116
	3	0.109		3	0.118
	3	0.112		3	0.11
	AVG	0.112333333		AVG	0.11466667
	STDV	0.003511885		STDV	0.00416333
	%RSD	3.126306752		%RSD	3.63081279

Criteria of approval:

The percentage recovery between individual and average should be between 98.0% and 102.0%.

Data evaluation:

Table 9 shows that the recovery is well within the limit. The technique is therefore correct

Robustness:

The robustness of an analytical process measures its ability to remain uninfluenced by small but intentional process parameter differences and shows its reliability while using the technique normally.

Data interpretation:

No important improvements could be found from Table 10 because of modifications in the circumstances mentioned above, and therefore the technique is robust.

Forced degradation studies:

The stress studies were performed ETR drug at 5μg/ml concentration. Here, acidic stress of 2.0mL of 0.1 Molar Hydrochloric acid is added to the solution, with 2.0mL of 0.1 Molar Sodium hydroxide, with 0min, 30min, 1hrs, 2hrs, 4hrs, 8hrs, 6hrs, and 32hrs respectively. Here the bulk medicine is exposed to acidic stress. In addition, 2.0 ml 0.1 Molar Sodium hydroxide and neutralized with 2.0 mL 0.1 Molar Hydrochloric acid were used for fundamental stress research. The addition of 1ml of 3% Hydrogen peroxide was used for oxidation experiments with bulk medicines. Sample heating heat studies were performed at 600C, and Ultra Violet studies were performed at the Ultra Violet -Lamp 450, respectively. Different volumetric flasks (10ml) were placed in complete samples and dissolved in the HPLC grade. MeOH had been injected into a chromatographic scheme at the initial drug concentration for testing. For all of these stability research, the development of the decayable product was confirmed in contrast to the chromatogram of the structure. An improved RP-UFLC technique has been used to analyse each strained sample. The degradation of data of ETR was presented below.

Acid Stress:

Add 2ml 0.1N Hydrochloric acid for 5 min, add 2.0ml 0.1N Sodium hydroxide, then inject 36 hours at a time of 30 min, one hour, and 1.30 min. In a 2ml Hydrochloric acid sample.

Base Stress:

For 2ml samples, add 2ml of 0.1N Sodium hydroxide, add 2ml of 0.1N Hydrochloric Acid, and inject the simple

Figure 5: Acid Stress chromatogram peak for ETR

Figure 6: Basic Stress chromatogram peak for ETR

Peroxide Stress:

For 2ml of the sample, add 1ml of 3% peroxide solution and inject this sample

Figure 7: Peroxide Stress chromatogram peak for ETR

Heat Stress:

Take 2ml of sample and thermal at 80.c for 1 hr.

Photolytic Stress:

Take samples of 2ml and position for 1 hour in a UV chamber. UV- Lamp 450 Inject the sample respectively.

Figure 8: Chromatogram for Heat stress of ETR

Figure 9: Photolytic stress Chromatogram peak for ETR

Table 11: Results for recovery studies of ETR after the stress conditions (% recovery of the drug)

Time	Ultra Violet	Thermal	0.1N Hydrochloric Acid	0.1N Sodium hydroxide	3% Hydrogen Peroxide
0 Min	85.23%	86.56%	87.79%	89.35%	85.34%
30 Min	82.34%	68.39%	84.14%	87.34%	76.33%
1 hr	75.43%	55.19%	78.86%	87.34%	69.63%
2 hr	69.34%	39.18%	74.78%	78.38%	66.87%
4 hr	69.34%	25.60%	67.27%	40.34%	49.34%
8 hr	59.23%	34.18%	59.65%	58.23%	82.62%
16 hr	49.87%	32.19%	44.64	49.24%	62.23%
32 hr	84.24%	---	--	---	--

CONCLUSION

A simple, quick, efficient RP-UFLC technique was effectively established to test ETR in its purest form. For various test conditions, the proposed procedure was tailored and tested. The results of pH, mobile stage proportion, and flow rate have been tested by ETR research. All analyses were well resolved and split in less than ten minutes. The design approach can be easily used in daily and stability tests utilizing quality control methods for evaluating the Etravirin material. This technique could be used in pharmaceutical preparations for drug assessment and routine laboratory tests. Overall, the proposed method provides excellent reliability, accuracy, selectivity, and reproducibility for deciding ETR.

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Received on 24.03.2020 Modified on 04.06.2020

Research J. Pharm. and Tech. 2021; 14(7):3537-3542.

DOI: 10.52711/0974-360X.2021.00613