HPLC Method Development and Validation for Efavirenz in Pharmaceutical Dosage form

Effective monitoring and control of the assay and contamination frequently ensure a drug's quality and safety. Contamination will determine The medication's safety, whereas assay determines the drug's potency. Pharmaceutical product testing are essential to the medication's efficiency in healing patients.  Most of medicines in multicomponent levels form might be investigated with the HPLC technique has many benefits. a number of the advantages, including its speed, specificity, accuracy, precision, and clarity of automation.  The HPLC Method gets away with lengthy extraction and isolation methods. In HPLC, there are multiple ways of separation. They involve size exclusion chromatography, affinity chromatography, reversed phase mode, reverse phase ion phase chromatography, and normal phase mode.

A. Mode of Phase Normal: Polarity characterizes the mobile phase, as the stationary It is a non-polar phase. If using these techniques, compounds, elute first and migrate quicker. This comes from reduce in the affection between the non-polar components along with their phase which is stationary.

B. Phase Mode Reversed: elements including their stationary phase is a non-polar hydrophobic packing it is bounded to silica gel with an octyl or octa decyl function group polar solvent as a mobile phase. The use of secondary solute chemical equilibrium can be made accessible by an aqueous mobile phase to control retention Along with selectivity. The polar compounds is the initial ejection in this mode, and Compounds are stored for an extended period of time As the majority of pharmaceutical medications Since they are naturally polar, they elute more quickly since they are not held for a long period of time. Several columns that are used are C₁₈ Octa Decyl Silane, C₈ Octasilane, C₄ Tetrasilane. Ion Exchange Chromatography: Ionic groups such as NR3 + or SO3 - + in the The ionic groups of the sample molecules interact with the stationary phase. This is sufficient in order to be separated only molecules which can be charged.

1. Affinity Chromatography: The technique utilizes very particular biological reactions to separate materials. If particular steric and charge-related conditions are met, a certain set of molecules in the stationary phase can adsorb the sample.
2. Exclusion of Size Chromatography is a method of separate molecules based on their molecular mass. The smallest molecules elute last, then following the largest molecules last^1,2

A Drug Profile

Described:

A An inhibitor to reverse transcriptase is not a nucleoside called efavirenz It is used to treat HIV infection or stop it to spread.

Structure:

Fig. Structure of Efavirenz

MATERIALS & INSTRUMENTS:

MATERIALS:

Efavirenz pure medicines (API), Efavirenz (Estiva 600 Tablet Methanol, potassium dihydrogen, phosphate buffer, distilled water, or acetonitrile  ortho phosphate orthophosphoric acid with buffer. The solvents or chemicals The following are all The Rankem products.

Instruments:

• Denver Electronics Balance
• p^H BVK Enterprises, India p H meter
• Ultrasonicator- BVK Enterprises
• Quaternary pumps, a photo diode array detector, and an auto sampler integrated with Empower 3 software are all features of the Waters HPLC 2695 System.

• The absorbance of UV-VIS was detected using a PG Instruments T60 UV-VIS spectrophotometer with a unique bandwidth of 2 and 10 mm and that corresponds quartz cells integrated with UV Win 6 software Efavirenz solution.

Eluite Preparation

Diluting substance: Diluent selected According to the drugs' solubility Opa With water taken in a 50:50 ratio, the ph is adjusted to 3.4 with NaOh solution.

Buffer preparation:

OPA Buffer at 0.1%: 1000 ml of Water of HPLC quality used to dilute 1 ml of for orthophosphoric acid.

0.01N KH₂PO₄ Buffer: 1.36% of orthophosphate potassium dihydrogen that has been carefully measured, in a One thousand milliliter volumetric flask, containing roughly 900 milliliters of The milli-Q water was introduced, dehydrated to sonicate, as well as then filled with water to complete the volume r (4.8-pH).

NA2HPO4 0.01N Buffer: A 1000 milliliter volumetric flask, add 1.41 g of precisely measured Sodium hydrogen phosphate, 900 ml of milli-Q water, sonicate by degassing, and then top off the volume of solution with water (4.0 pH).

0.1%TFA Buffer: 1ml of TFA was with water of HPLC quality to 1000 ml.

Standard stock solution preparation: precisely weighed 15mg of Efavirenz It moves into a 50 ml volumetric flask. A third of the After adding diluents, the flask was sonicated for ten minutes. The container was labeled "Standard stock solution" and included diluents (300µg/ml of Efavirenz)

Standard working solutions (100%) are prepared as follows One milliliter of the stock solution was put into a 10-milliliter volumetric flask, it was removed using a pipette and diluted using diluent. (30 µg/ml of Efavirenz).

Getting the sample stock solutions ready: After weighing ten tablets and calculating their average weight, the weight of one pill was determined (1054.8 Avg.mg) had been moved into a 500ml ,50ml volumetric flask was sonicated while diluents had been added for 25 minutes, after which the volume was adjusted using diluent and filtered using HPLC filters. (1200µg/ml of Efavirenz)

Sample working solutions (100 % solution) preparation: 0.5ml A stock solution of the filtered sample was transferred to 20ml volumetric flask, then diluent was added. (30 µg/ml of Efavirenz)

RESULTS AND DISCUSSION

Method development: To be able to develop the method, different buffers, mobile phase ratios, etc.

Authentication and identification of received API 

Authentication by UV-VIS spectra

After using a UV-VIS spectrophotometer to scan from 400 to 200 nm, Efavirenz Absorption maxima were observed at 249 nm in diluent solution Drug's UV spectra are shown in the figure.

Spectra of Efavirenz

Fig 2.0 optimized Chromatogram

Observation: Efavirenz peak was eluted at 2.922 min, each with high levels of resolution. Due to very good plate count and tailing factor, this approach was optimized and validated.

Method Validation:

When the ongoing security as well as efficiency of each batch processed depends entirely on the assessment of quality, it is critical for pharmaceutical analysis that analytical methods been correctly verified. The ability to regulate this attribute depend on the analytical methodologies' capacity to offer a reliable illustration of any departure from the target criteria whenever applied under certain circumstances and at a certain degree of sensitivity. Analytical methods must be developed in compliance with the principles established by the International Conference on Harmonization (ICH) and applied in environments which conform to GLP stands for good laboratory practice and GMP stands for good manufacturing procedures. (GMP). Along with Q2B). Both the US Pharmacopoeia (USP) and the US Food and Drug Administration (FDA) makes a reference to ICH guidelines. Specificity, detection limit, quantitation limit, linearity, range, robustness, stability, accuracy, precision (repeatability and intermediate precision), and specificity of analytical solutions are the validation characteristics that are frequently used. Before being used, a documented and authorized process for method validation is necessary.

1.System Suitability Parameter

According to the ICH criteria, every system suitability metric was within the acceptable range.

2. Linearity: 

 A linearity of method is a measurement how well a response is calibrated in relation to concentration graphic It resembles a straight line. Single measurements at various the concentrations of analytes can be used to evaluate linearity.

Table 2 Linearity table for Efavirenz.

Fig No. 6.14 Calibration curve of Efavirenz

One definition of precision is “The degree of agreement among individual test results when the procedure is applied repeatedly to multiple samplings of a homogenous sample”. An additional thorough an explanation

The International Conference's proposal

Three different kinds of accuracy are distinguished by the Institute for Harmonization (ICH): repeatability, intermediate precision, and. The ability to reproduce.

4. Accuracy-

The extent to which the value being measured closely resembles The measurement accuracy is the actual value. An example whose "true value" is recognized and analyzed in a technique with exceptional accuracy, and The measured value and the true value are exactly the same. Usually, recovery studies are used to illustrate and evaluate accuracy.

5. Sensitivity:

Sensitivity table of Table 7 Efavirenz

single injection of sample stock solution of 0.3 & 0.9 were injected to detect LOD &LOQ value n final values were calculated using respected formulas.

6. Robustness:

The International Committee of Harmonization ( ICH) defined robustness as "a measure of its capacity to be unaffected by minor but intentional modifications to the technique

 7. Assay:

Assay: Soranib bearing the label claim Efavirenz 600mg. The aforementioned formulation was used for the assay. Average Assay % for Efavirenz obtained was 100.10%.

Preparation of Standard working solutions (100% solution): After being pipetted out, the stock solution was moved then diluted in a 10 ml volumetric flask.. (30µg/ml of Efavirenz).

Preparation of Sample stock solutions: 10 After measuring the tablets and determining their median weight, the weight equivalent one tablet (1054.8 Avg.mg) had recently transferred into a a 500ml volumetric flask, was added, and after 25 minutes of the sonication the volume was altered with diluent and filtrated with HPLC filters (1200µg/ml of Efavirenz).

Assay was calculated by: -

Table 9 Assay Data of Efavirenz

Stability Indicating Method

The analytical techniques developed for estimating purity and impurities must be sufficient to separate all desired and undesirable components and free from formulation interference mErtugliflozinix. Analytical techniques are referred to as stability indicating methods when they can precisely and accurately quantify without missing any impurities, without underestimating or overestimating, and detect all potential impurities and degradants that may form during stability studies with sufficient sensitivity, accurately reflecting the quality of drug substances and drug products (formulated products of drugs).The active components should be precisely measured using a stability-indicating test technique, without experiencing interference by excipients, the degradation products, contaminants in the process, or additional possible contaminants. if an industry conducts release testing using a non-stability indicating analytical approach, then it should be augmented by a technique that can track the contaminants, including degradation products, both qualitatively and quantitatively. Methods of analysis for stability research of assay should be stability indicating. Following stability testing, there is a retest period for the active substance or a period to be utilized for the drugs can be determined, and suggestions for storage conditions can be made.The ICH (International Harmonization Conference) guideline QIA on Testing for Stability in Innovative Drug Substances and Products emphasizes the characteristics that can alter while being stored and may influence quality, safety, and/or efficacy must be checked by confirmed stability-indicating methods for testing. Additionally, it is stated that the material should undergo forced decomposition tests (stress testing) at temperatures in increments of 10 °C above the accelerated temperatures, pH extremes, and oxidative and photolytic conditions. consequence of drugs in order to establish the innate stability traits and processes of degradation that will support the appropriateness of the proposed analytical procedures.

SUMMARY and CONCLUSION –