Method Development and Validation of Efavirenz as A Dosage Form by UV Spectrophotometer

Efavirenz (EFV) is an antiviral drug used in HIV/AIDS treatment and prevention. It is marketed under several trade names, including Sustiva. Usually, it is recommended to be taken in combination with other antiretrovirals. It can be used as a prophylactic after a needlestick injury or other potential exposure. You can buy Efavirenz, Emtricitabine and Tenofovir alone or in combination. It is taken orally.

STRUCTURE

Table 1: Efavirenz (Monograph)

History

On September 21, 1998, the FDA approved efavirenz. The FDA authorised Mylan's generic tablet formulation on February 17, 2016. Thailand's Government Pharmaceutical Organisation (GPO) announced in late 2018 that it would start manufacturing Efavirenz following WHO clearance. Du Pont Pharma, also known as DMP 266 (Efavirenz), discovered the discovery. In May 1999, European countries are expected to receive the production  licence for Efavirenz.

Chemical properties

(4S)-6-chloro-4-(2-cyclopropylethynyl)-4-(trifluoromethyl) is the chemical formula for Efavirenz. 2-one-benzoxazin -1H-3,1-. C14H9ClF3NO2 is the empirical formula for it. Efavirenz has 315.68 g/mol is the molecular mass. And is a crystalline powder that ranges from white to faintly pink.  It has a water solubility of less than 10μg/ml.

Medical uses

The US The Antiretroviral Guidelines Panel of the Health and Human Services Department suggests  Efavirenz utilising Truvada's Tenofovir/Emtricitabine is one of theNNRTI-based regimens for adults, adolescents and children with untreated HIV infection. Additionally, efavirenz is used in conjunction with other antiretroviral medications as part of a more thorough post-exposure preventive routine to lower the chance of contracting HIV in individuals who have been exposed to a high risk (such as injuries from needlesticks, some forms of unprotected intercourse, etc.).  Efavirenz is safe to use in the first trimester of pregnancy. Since efavirenz is a part of breast milk, it can be administered to breastfed babies

Contraindications

Those who have previously taken this drug and had an allergic response should refrain from taking  additional dosages of Efavirenz. Toxic skin eruptions, erythema multiforme and Stevens-Johnson syndrome are examples of hypersensitivity reactions.

Adverse effects

Headaches, vertigo, dizziness, anxiety, and cognitive impairment (fatigue, disorientation, and problems with memory and concentration), depression, including suicidal thoughts, and disturbed sleep (nightmares, insomnia, and daytime fatigue) are among the most common side effects. Euphoria is experienced by some people. There may be rashes and nausea. When Efavirenz is administered, some marijuana urine tests may produce a false-positive result. Because it may extend the QT interval, Efavirenz should not be administered to individuals who have torsades de pointes or are at risk for developing one. Using Convulsions may be brought on by Efavirenz in both adult and paediatric patients with seizures in the past.

Brands

Currently, Efavirenz is sold under several brand names including  Sulfina V, Filginase, Stocrin, Sustiva, Virorrever, Zuletel, Erige, Estiva, Evirenz, Avifanz, Efamat, Efatec, Efavir, Efavirenz, Efcure, Eferven, Efrin, and Filginase. 2016 saw Efavirenz, Tenofovir, and Emtricitabine are marketed under the brands Viraday, Citenvir, Oditec, Teevir, Trustiva, Vonavir, Atripla, and Atroiza  in several  jurisdictions¹.

MATERIALS AND METHOD

Materials: Sodium hydroxide, distilled water, Efavirenz.

Instrument: UV double beam spectrophotometer (Shimadzu model 1900i) was employed with a spectral band width of 1nm and a wavelength accuracy of 0.3 nm (with automatic wavelength correction with a pair of 1 cm matched quartz cells) 2025.

Method

Preparation of Standard Stock Solution (Stock-A)

Standard stock solutions were prepared by dissolving separately 50 mg of every medication in 50mL 0.1 N NaOH in 100 ml volumetric flask. Then shake for 20 minutes and the volume was up to par 100ml with 0.1 N NaOH to get a concentration of 1000 μg/ml (Stock-A) for Efavirenz.

Preparation of Sub Stock Solution (Stock-B)

Aliquots of 2.5 ml withdrawn with help of pipette from standard stock solution A of EFV and transferred into 25 ml volumetric flask separately and diluted up to 25 ml with 0.1 N NaOH that gave concentration of 100 μg/ml (Stock-B).

Preparation of Working Standard Solution

1) 0.5 ml, 1.0ml, 1.5 ml, 2.0 ml,  2.5 ml, 3.0 ml and 3.5 ml from sub stock solution (Stock-B) were taken separately 10 millilitres in a volumetric flask and volume was composed to 10 ml with 0.1 N NaOH. This gave the solutions of 5μg/ml, 10μg/ml, 15μg/ml, 20μg/ml, 25μg/ml, 30μg/ml and 35μg/ml respectively for EFV.

Selection of wavelength for linearity

Solution of5μg/ml of EFV was prepared separately. The solution was scanned in the spectrum mode from 200 nm to 400 nm. The maximum absorbance of EFV was observed at 265 nm. EFV showed linearity in the concentration range of 5-35μg/ml. Calibration curve was plotted, absorbance versus concentration².

Figure 1. Spectra of 5μg/mL solution of efavirenz in  0.1 N Sodium hydroxide.

RESULTS AND DISCUSSION

Validation of the Method

Linearity:

Linearity was established by least squares linear regression analysis of the calibration curve. The calibration curves were linear over the concentration range of 5-35μg/ml for efavirenz. The resulting curves were subjected to linear regression analysis after absorbances were plotted against the corresponding concentrations. The linearity study was carried out from 5 to 35μg/ml and the observed values were presented in Table 2. Correlation coefficient was found to be 0.999 for efavirenz (Fig. 2). The results are given in Table 3.

Table 2: Linearity profile of Efavirenz.

Figure 2: Linearity profile of EFA.

Table 3: Linearity and Correlation coefficient.

Precision:

To check the degree of repeatability of the method, suitable statistical evaluation was carried out. The concentrations  of drug was measured three times on the same day at intervals of 1hr and on three different days for intra and inter day study, respectively. The Relative Standard Deviation (RSD) and Standard Deviation (SD), respectively were calculated. The results are listed in Table 4.

Table 4: Precision studies.

Accuracy:

Studies on recovery were conducted by applying the method to drug sample to which known amount of efavirenz corresponding to 50, 100 and 150% of label claim had been added. At each level of the amount three determinations were performed. The findings are displayed in Table 5. The method requires by calculating the absorption of sample solution at the chosen wavelength followed by simple calculations. Hence, it was further employed for our study³.

Table 5: Accuracy.

LOD and LOQ:

The lowest analyte concentration at which an analytical procedure can consistently distinguish it from background levels is known as the LOD, or limit of detection. In this study, LOQ and LOD were according to the response's standard deviation and The incline of the corresponding curve using the following equations- LOD = 3σ/S; LOQ is 10σ/S. The standard deviation is represented by σ of the blank or low-concentration samples (noise) and S= slope regarding the calibration curve (sensitivity). The limit of quantification (LOQ) is described as the standard curve's lowest concentration that can be determined using an acceptable accuracy, precision and variability. The LOD of efavirenz was discovered to be 5.735μg/ml and the LOQ was discovered to be 19.12μg/ml. The results are listed in the Table 6⁴.

Table 6: LOD and LOQ  studies.

Ruggedness:

Ruggedness is estimated for another analyst.The results are listed in the Table 7⁵.

Table 7: Table showing the ruggedness  study.

DISCUSSION

The created technique for figuring out Efavirenz as a dosage form was discovered to be straight forward, sensitive, precise, selective, quick and affordable. The greatest absorption of efavirenz was seen at 265nm and complied with Beer's law within the range of 5-35 μg/ml. The proposed technique for figuring out linear regression was demonstrated by Efavirenz y=0.147x+0.132 having a r2 correlation value of 0.9992. The analytical method's linearity was tested to see if it could produce test findings that were proportionate to the analyte concentration within the sample of certain range. Different levels of standard solutions were prepared and estimate into the UV and the results was recorded. The validity and reliability of proposed methods were assessed by recovery studies. Three levels of enhanced standards recovery were observed: 50%, 100%, and 150% replicate and three concentrations level. The percentage number is very nearly equal to 100 and % RSD are Less than two indicates that the procedure is accurate. Repeatability and the drug's intermediate precision were used to calculate precision. The repeatability result shows the accuracy over a brief period of time in the same operational circumstances.Variation between laboratories on various days and between analysts is shown in the intermediate precision research by different analyst. The percentage RSD and SD values are less then 2 denotes the method's accuracy.

Table 8: Summary of the Validation Parameters.