Analytical Method Development and Validation of Efavirenz by Using RP- HPLC

INTRODUCTION

Efavirenz, an antiretroviral medication primarily used in the treatment of HIV/AIDS. It belongs to the class of drugs known as Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs). It works by inhibiting the activity of the enzyme reverse tran scriptase, which is essential for the replication of HIV. By blocking reverse transcriptase, Efavirenz prevents HIV from converting its RNA to DNA, thereby halting the virus’s ability to replicate and reducing the viral load in the body. Efavirenz is typically administered orally in the form of tablets or capsules. The recommended dosage for adults is usually 600 mg once daily, preferably taken on an empty stomach. Some formulations of efavirenz may be taken with food to reduce the risk of gastrointestinal side effects. Efavirenz can interact with a wide range of medications, including certain antibiotics, antifungals, antidepressants and anticonvulsants. It’s crucial for patients to inform their healthcare provider about all the medications, supplements and herbal remedies they are taking to avoid potential interactions that could affect the efficacy or safety of Efavirenz. Antiretroviral drugs from different classes, Efavirenz helps to control the progression of HIV infection and improve immune function.^1,2 Efavirenz may include dizziness, drowsiness, trouble sleeping, vivid dreams and rash. Some individuals may experience psychiatric symptoms such as mood changes, depression, anxiety, or hallucinations, particularly during the initial weeks of treatment. Most side effects tend to be temporary and diminish over time as the body adjusts to the medication. However, it’s essential for patients to report any severe or persistent side effects to their healthcare provider promptly.^3-5 Efavirenz is not recommended for use during pregnancy, particularly during the first trimester, due to concerns about potential adverse effects on fatal development. Pregnant individuals living with HIV should discuss the risks and benefits of Efavirenz with their healthcare provider to determine the most appropriate treatment regimen that minimizes the risk to the fetus. Similarly, caution is advised for breastfeeding individuals, as Efavirenz can pass into breast milk and potentially affect the infant. All antiretroviral drugs Strict adherence to the prescribed treatment regimen is crucial for preventing the development of resistance and maintaining viral suppression. Regular monitoring of viral load and CD4 cell count is necessary to assess treatment efficacy and detect any signs of virology failure or drug resistance.⁶ Effective treatment with Efavirenz can significantly prolong the life expectancy and improve the quality of life for individuals living with HIV/ AIDS. Efavirenz plays a vital role in the management of HIV/ AIDS by effectively suppressing viral replication and improving immune function. However, patients should be aware of their side effects, drug interactions and the importance of adherence to treatment to maximize its benefits while minimizing risks. Regular communication with healthcare providers is essential for individualized management and optimal outcomes. Efavirenz, chemically known as (4S)-6-chloro-4-(2-cyclopropylethynyl)- 4-(trifluoromethyl)-1,4-dihydro-2H-3,1-benzoxazin-2-one, is a synthetic compound with the chemical structure shown on Figure 1. Efavirenz molecular weight is 315.67 g/mol.^7-12

While looking through the literature, we only came across a handful of analytical spectrophotometry, high-performance thin-layer chromatography and liquid chromatography-mass spectrometry in order to test Efavirenz, all of the methods that are mentioned are used. In 2021, Quantification and validation of stability-indicating RP-HPLC Method for Efavirenz in bulk and tablet dosage form using Quality by Design (QBD) studied by Gurumukhi et al. (2021). In 2021, Saha et al. they optimised the chromatographic conditions, adjusting the level of acetonitrile in the mobile phase to 51. 4 and 17% v/v, the phosphate buffer had a pH of 4. 04, while the flow rate was at 1. 25 mL/min. Using these optimized parameters, it was possible to achieve a retention time of 11. 031 min in RP-HPLC.

Analytical Method Validation

Detailed experimental procedures for testing the efficacy of an analytical parameters are provided. These approaches are classified in accordance with the ICH Guidelines.

Specificity

Specificity in the development of an analytical method pertains to its capacity to precisely and selectively detect the desired analyte amid the presence of other elements like impurities, degradation byproducts, or interferences from the matrix. In simple terms, it guarantees that the method can differentiate and quantify the intended analyte without being affected by other substances.

Linearity

Linearity in the validation of an analytical method signifies the method’s capability to yield test outcomes that vary in direct accordance with the concentration of the substance being analysed within a predetermined range. Essentially, it implies that as the concentration of the substance rises or falls, the instrument’s response, such as peak area or signal intensity, changes in proportion.

Precision

Precision in the validation of an analytical method measures how closely the results agree when repeatedly analysing the same sample using the same method under specific conditions. It evaluates the consistency and reproducibility of results obtained through multiple analyses under identical conditions.

Accuracy

Accuracy in analytical method validation is about how closely the obtained values match the true or accepted reference values. It evaluates how accurately the method reflects the actual concentration or amount of the substance being analysed. In simple terms, accuracy shows how near the measured values are to the true values.

Detection Limit (LOD)

The detection limit, or LOD (Limit of Detection), in analytical method validation, is the minimum concentration or quantity of an analyte that can be detected with confidence under specified experimental conditions. It signifies the lowest level of analyte in a sample that can be differentiated from background noise or baseline signal with a reasonable degree of certainty.

Quantification Limit (LOQ)

The Limit of Quantification (LOQ) refers to the minimum concentration of an analyte within a sample that can be reliably determined with both acceptable precision and accuracy under the specified experimental conditions.

Robustness

Upon assessing the results, it is evident that alterations in the mobile phase had a notable impact on the method. This suggests that the method remained unaffected despite variations in the mobile phase. Furthermore, the system suitability parameters remained within acceptable limits.

Ruggedness

Ruggedness, in the context of flow rate, refers to the ability of a system or device to maintain consistent performance despite variations or changes in operating conditions. In other words, it describes how resistant a system is to fluctuations or disturbances in the flow rate.^13-16

MATERIALS AND METHODS

The API Efavirenz was gifted from KIET School of Pharmacy and a commercial tablet (Duovir-E Kit, Mfg- Cipla Pvt. Ltd.,) was purchase from the pharmacy. We used HPLC grade methanol and tert-Butyl Methyl Ether solvent and reagents during the research and was obtained from Thermo Fisher Scientific India Pvt. Ltd., in Mumbai, India, while the latter was procured from Central Drug House Pvt. Ltd., in New Delhi, India.

Instrumentation

The High-Performance Liquid Chromatography Analysis were performed using a water Shimadzu HPLC (model 2489) system which is manual, a column (X-Bridge® C8 5 μm 4.6×250 mm), Shimadzu UV Detection will be used in this step to identify, tell apart and determine exact amounts of particular components among mixtures and using a digital precision balance which was manufactured by Shimadzu Japan.

Chromatographic Condition

A reverse phase column [ X-Bridge® (C8 5 μm 4.6×250 mm)], maintained in the presence of mobile phase (methanol) we kept the mobile phase flow rate at 0.8 mL/min and the eluent were measured at 252 nm.

Method development

Preparation of standard solution

A standard solution of 1000ppm was prepared by dissolving 10 mg EFZ in Tertiary Butyl Methyl Ether (TBME). The EFZ was accurately weighed and transfer to a 100 mL volumetric flask that had been thoroughly cleaned and dried and fill up to the mark with TBME and prepared 100 μg/mL has been sonicated raised to a final volume of 10 mL by subjecting to sonication for 10 min and drug concentration was adjusted to a range of 2-64 μg/mL by repeatedly diluting a standard stock solution.

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Preparation Of Sample Solution

Twenty tablets of EFZ-600 were measured by weight and the average weight of each pill was determined and transfer into mortar pestle and crushed the tablet to make a powder. The powder was transferred into the centrifugal tube for 20 min. Take the EFZ solution was filtered through the Whatman filter paper. The Solution equal to 200 ppm further produce dilution to obtain final concentration of 2-64 ppm. These solutions were injected into under chromatographic condition and peak areas were measured and chromatogram was recorded 3.7 min respectively.

Selection of the Solvent

The selection of the solvent is based on the solubility examine by the maximum absorbance observed through UV spectroscopy. Tertiary butyl methyl ether showed the high solubility for EFZ. As a result, tertiary butyl methyl ether was selecting as the preferred solvent and diluent for this research.

Selection of wavelength

The selection of wavelength of efavirenz was assessed using a variety of solvents, such as water, ethanol, methanol, tertiary butyl methyl ether, n-hexane and a mixture of solvents. To quantify the solubility in each solvent, using a UV spectrophotometer, the absorbance of efavirenz solutions at specific wavelengths was measured. The drug profile of efavirenz is as follows: the solubility of the drug is higher in methanol and tertiary butyl methyl ether with its maximum wavelength (λ_max) at 250 nm and 252nm.

Analytical Method Validation of HPLC and Result Discussion

Linearity

Calibration curve of EFZ was prepared from the stock solution, in a series of (2, 4, 8, 16, 32 and 64 ppm) separately in 10 mL volumetric flask and diluted with tertiary butyl methyl ether as a diluent, a volume of 10 μL from each solution was injected several times at each concentration level, the peak area that resulted was recorded in Table 1 and Figure 2 for API.

Similarly, the result of market formulation is shown in Table 2 and Figure 3.

Accuracy

In order to determine how precise the developed method is, the non-exist sample was spiked with three distinct concentrations of the efavirenz standard, which are 80%, 100% and 120% of the precise concentration of 24 ppm, 32 ppm, 40 ppm. The outcome was examined in three different versions and the percent recoveries and the relative standard deviation of the concentration were calculated. The accuracy result of EFZ API was recorded in Table 3 and similarly the accuracy result of EFZ Market formulation was recorded in Table 4.

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Robustness

The robustness was measured by changing the flow rate (± 10%). The system approaches must be satisfied as part of the method considerations. The robustness was carried out at different flow rate. The robustness results of EFZ for API were shown in Table 5 and similarly the robustness result of EFZ for Market Formulation were shown in Table 6.

Limit of detection and limit of quantification

The HPLC method of Efavirenz detection serves as the basis for the limit of detection. The statistical equation is used to compute the LOD and LOQ values and the LOQ value is multiplied by 3x. The LOQ and LOD result of EFZ API were shown in Tables 7 and 8.

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Similarly, the LOQ and LOD result of EFZ Market formulation were shown in Tables 9 and 10 respectively.

Where, Ω =Standard deviation of the y-intercept of the calibration curve and

S=Slope of the calibration line.

Specificity

The specificity of the proposed method was assessed through chromatograms of Efavirenz API, the commercial product and blank. As expected, no interference was observed since the retention times of the sample and the standard were closely related. The blank chromatogram was shown in Figure 4 and standard chromatogram was shown in Figure 5 and sample chromatogram was shown in Figure 6 respectively.

Precision

Precision defines the random errors associated with reproducibility and repeatability of an experiment. The method to be emplaced is quantified using the terms %RSD (relative standard deviation). For percentage agreement, %RSD of less than 2% is considered acceptable. The precision studies associated with the developed analytical techniques also entailed determination of intra-day variability and inter-day variability. As a means to evaluate accuracy, the experiments were carried out based on repeatability and inter-day precision. The results were found to have %RSD in acceptable limit that is not more than 2%. The precision results for EFZ API were shown in Table 11 and for market formulation were shown in Table 12 respectively.

Ruggedness

The repeatability of the method validation was checked where the results of analyst A and analyst B did not differ significantly with the percentage of relative standard deviation being below 2% which means that this method is quite reliable. The outcomes referring to the ruggedness of the method for the Active Pharmaceutical Ingredient (API) at the levels of 32 ppm and 64 ppm. The ruggedness result for EFZ API for 32 ppm and 64 ppm were shown in Tables 13 and 14 respectively. Similarly, the result for EFZ market formulation for 32 ppm and 64 ppm were shown in Tables 15 and 16 respectively.

CONCLUSION

In conclusion, the present work has demonstrated that the analysis time of the HPLC method has been reduced thus making the technique more efficient. HPLC is one of the most commonly used analytical techniques for creating new techniques and in the separation of compounds in various samples. This technique mostly uses a C-8 stationary phase and a mobile phase that usually consists of methanol. The sample with a volume of 10 microliters is delivered by a mobile phase to a detector that determines the retention time 3.7 min and height and width of the peak and the area under the curve. In the previous literature review Rt was found to be 6- 11 min. These factors are important when evaluating the method for its recovery studies, such as repeatability, linearity, Limit of Detection (LOD) and Limit of Quantification (LOQ). Furthermore, the modifications made to this HPLC method are aimed to provide better accuracy and reproducibility in several analytical processes, making it even more valuable in the field of chemical analysis.