Analytical method development and validation of Etravirine by

UV Spectroscopy

 

Barath M.¹, Chandan R. S.*, Maruthi R., N. Paramakrishnan

Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Mysuru, JSS Academy of Higher Education and Research, Sri Shivarathreeshwara Nagara, Mysuru- Karnataka, India 570015

 

ABSTRACT:

In the current work, a simple and extraction free UV spectrophotometric technique was developed and validated for the estimation of ETR as per ICH guidelines. ETR approach has been created using the pure drug. The drug demonstrates effective absorption in the Concentration Range of 1-5μg/ml at 311nm and linearity. The technique relay on the response of the drug with MeOH to get the colourless solution. Absorbance was measured at its absorption maximum (λ max) 311nm. Beer's regulation has been obeyed in the concentration vary of 1-5μg/ml. The calibration graph was determined to be linear, and the Coefficient of correlation (r2) was found to be 0.9956%. The recommended method's analytical activity parameters LOD and LOQ were calculated respectively as 1.91 and 2.67μg/mL. Recovery for ETR was discovered to be 100.71 percent. To optimize the response situation, all variables had been examined.

 

 

 

INTRODUCTION:

ETR is the 2nd-generation reverse transcriptase non-nucleoside (NNRTI) inhibitor, developed to be effective against HIV with mutations that impart sensitivity to the two NNRTIs, Efavirenz K103N mutation and Y181C mutation of first-gen, most commonly administered. The flexibility of ETR as a molecule seems connected to this efficiency. ETR is a pyrimidine diary (DAPY), a kind of natural isomeric molecule which can bind an enzyme of reverse transcriptase in several configurations, which allows a stronger relationship between ETR and the enzyme, even with mutations. Currently, another pyrimidine diary, especially rilpivirine, is used as anti-HIV analogs. ETR is a synthetic by-product of diaryl pyrimidine, human immunodeficiency virus (HIV). It is a secondary blocker of the reverse transcriptase non-Nucleoside, which activates directly into the reverse transcriptase enzyme and inhibits the catalytic elements of the enzyme to alter genetic material replication of HIV¹. ETR acts against wild HIV-1 and is allowed by the use of the Food and Drug Administration as an anti-AIDS agent.

 

Chemically, 4-[6-Amino-5-bromo-2-[(4-cyanophenyl) amino] pyrimidin-4-yl] oxy-3,5-dimethylbenzonitrile¹.

 

Figure 1. Chemical structure of Etravirine²

 

MATERIALS AND METHODS:

Instrumentation:

For the current study, UV Spectrophotometer 1700AD a make of Shimadzu was used³.

 

Chemicals and reagents:

Etravirine standard was procured from Apotex Ltd, Bengaluru. Etravirine marketed formulation manufactured by Kavya Pharma was procured from Gujarat. All chemicals used were analytical grade purchased from Merck pharmaceuticals HPLC grade MeOH was used as the diluent for preparation of the solutions.

 

Method Development:

Selection of wavelength:

The λ max of Etravirine was determined by using UV-visible spectrophotometer 1700. UV spectrum for Etravirine is shown in figure 2.

 

Figure 2: UV spectrum

 

Preparation of standard stock:

ETR 100mg was drawn into a volumetric flask of 100 mL. Add 50mL of diluent and sonicate to dissolve, and the quantity with diluent (1000μg/ml) has been produced up to the mark. Pipette 1ml of the above solution into a volumetric flask of 10ml and use diluent (100μg/ml) to form the volume. Pipette 1ml of the above solution into a volumetric flask of 10ml and use diluent (10μg/ml) to form the volume. The linearity solutions were prepared by diluting with diluent from the inventory solution. ETR has been prepared for concentration ranging from 1, 2, 3, 4, 5μg/mL. In 10ml volumetric flasks, pipette 1, 2, 3, 4, 5mL, and form the quantity using diluent to acquire the above levels.

 

Preparation of Calibration Curve:

Aliquots of Etravirine are made from the stock solution (100μg/mL) in a 10ml volumetric flask sequence. The HPLC grade diluent has been used to dilute the label to achieve a concentration range of 1-5μg/mL. Solutions were observed for the absorbance and reported. The calibration curve has been set. The law on beer applies to 1-5μg/mL range.

 

Figure 3: Overlay Complexity of Etravirine

 

METHOD VALIDATION^3-11:

Calibration curve:

It consisted of matrix sample processed without analyte and matrix sample with calibration standards. It is showing good linearity over the range of 1 to 5µg/ml with coefficient of correlation 0.9956.

 

Table 1: Calibration data of Etravirine

Sl. No	Conc. (µg/ml)	ABS Of Etravirine
1	0	0
2	10	0.146
3	20	0.24
4	30	0.356
5	40	0.431
6	50	0.52

 

Figure 4: Linearity graph of Etravirine.

 

Linearity:

The linearity of this approach for ETR ranged from 1-5 μg/ml. The regression equation was Y=0.0117x+0.2209, R2=0.9952.

 

Accuracy:

The precision of ETR's analytical approach was decided at preferred solution concentrations of 30 percent, 60 percent, and 90 percent. Absorbance was measured at 311nm, and proportion recovery effects were demonstrated.

 

Table 4: Accuracy results for ETR by UV spectroscopy

Sl. No	Level of % Recovery	Drug added	Drug recovered	% Recovery
1	0	0	0
2	50	2.56	2.58	102.14
3	100	5.05	5.01	98.20
4	150	7.52	7.59	101.80

 

Precision:

The accuracy (the measurement of intraday, interday, repeatability) findings showed excellent reproducibility with the current relative normal deviation (RSD percentage) below 2.0%. This stated an extremely accurate technique.

 

Table 5: Results for Intraday precision of ETR by UV spectroscopy

Intraday morning precision:

Sl. No	Concentration (μg/ml)	Absorbance	STDV	% RSD
1	3	0.304	0.0053 44156	1.7475 98328
2	3	0.297
3	3	0.306
4	3	0.309
5	3	0.313
	Mean	0.3058

 

Intraday evening precision:

Sl. No	Concentration (μg/ml)	Absorbance	STDV	% RSD
1	3	0.298	0.0045 86938	1.55 1738152
2	3	0.297
3	3	0.302
4	3	0.292
5	3	0.289
	Mean	0.2956

 

Table 6: Results for Interday Precision of ETR by UV spectroscopy

Interday morning precision:

Sl. No	Concentration (μg/ml)	Absorbance	STDV	% RSD
1	3	0.297	0.0051 22499	1.709 779503
2	3	0.309
3	3	0.296
4	3	0.295
5	3	0.301
	Mean	0.2996

 

Interday evening precision:

Sl. No	Concentration (μg/ml)	Absorbance	STDV	% RSD
1	3	0.308	0.006 0663	1.969 578037
2	3	0.316
3	3	0.306
4	3	0.298
5	3	0.312
	Mean	0.308

 

Repeatability:

Table 7: Results for Repeatability of ETR by UV spectroscopy

Sl. No	Concentration (μg/ml)	Absorbance	STDV	% RSD
1	3	0.301	0.0050 83306	1.6743 43218
2	3	0.297
3	3	0.312
4	3	0.306
5	3	0.302
	Mean	0.3036

 

Table 8: Results for LOD and LOQ of ETR by UV spectroscopy

LOD (μg/ml)	1.91 μg/ml
LOQ (μg/ml)	2.67 μg/ml

 

Robustness:

An analytical method's robustness is a measure of its ability to stay unaffected by tiny but intentional differences in method parameters and gives an indication of its reliability during ordinary use.

 

Table 9: Results for Robustness of ETR by UV spectroscopy

Sl. No	Wavelength (nm)	Absorbance	STDV	% RSD
1	306	0.564	0.06714 2833	13.89 54538
2	307	0.557
3	308	0.548
4	309	0.537
5	310	0.512
6	311	0.475
7	312	0.456
8	313	0.413
9	314	0.394
10	315	0.376
	Mean	0.4832

 

Ruggedness:

Ruggedness was determined by two distinct analysts performing research, and the corresponding absorption was observed, and the outcomes were shown as a percentage of RSD

 

Table 10: Results for Ruggedness of ETR by UV spectroscopy

Analyst 1
Concentration (μg/ml)	Absorbance	Statistical analysis
3	0.349	STDV = 0.003 %RSD = 0.8450 70423
3	0.355
3	0.356
3	0.359
3	0.355
3	0.356
AVG	0.355
Analyst 2
3	0.351	STDV = 0.0047 40906   %RSD = 1.334 213719
3	0.357
3	0.362
3	0.361
3	0.348
3	0.353
AVG	0.355333333

 

CONCLUSION:

While different techniques for the development and validation of ETR were reported with UV-Visible spectrophotometric techniques, efforts were made to produce simple and reliable techniques for growth and validation of ETR. This research is associated with the development and verification of ETR's spectrophotometric UV process. The developed methodology has been described as linear, exact, reliable, detailed, and all proved subject to pure medicine, convenient, and effective.

 

CONFLICT OF INTEREST:

The authors have no conflict of interest.

 

ACKNOWLEDGEMENT:

We would like to thank the Principal, JSS College of Pharmacy, Mysuru and JSS Academy of Higher Education and Research, Mysuru for providing the facilities in successfully completion of the research work.

 

REFERENCE:

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4.      https://www.gmp-compliance.org/guidelines/gmp-guideline/ich-q2r1-validation-of-analytical-procedures-text-and-methodology

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Received on 21.12.2019          Modified on 05.02.2020

Accepted on 20.03.2020         © RJPT All right reserved