Development, Optimization and Validation of Analytical Procedures for The Simultaneous Determination of Selected Antiviral Drug Combinations

The number of newly launched pharmaceuticals is escalating daily, making the development of appropriate analytical methods crucial for the qualitative and quantitative assessment of drugs in analytical chemistry. Analytical method development involves establishing a series of experimental conditions for performing analytical operations on chemical samples. Established analytical methods can evaluate the identification, potency, purity, and stability of drug substances, drug products, and other pharmaceutical constituents [1]. Viral infections are of paramount importance in today's healthcare landscape for several reasons. Viral illnesses such as influenza, hepatitis, and HIV/AIDS can lead to widespread outbreaks and pandemics, causing significant morbidity, mortality, and strain on healthcare systems. Consequently, the clinical relevance of antiviral agents used in treating HIV and hepatitis infections is substantial. Combination drug therapy offers a convenient once-daily dosage regimen to enhance adherence, effectively combat viral infections, and minimize side effects. The aim of this project is to develop simple, precise, sensitive, and accurate methods for the quantification of certain antiviral medications, specifically Pibrentasvir - Glecaprevir and Lamivudine-Tenofovir in multicomponent dosage forms [2,3]. An Absorptivity Factor approach was employed using a UV-Visible spectrophotometer for the simultaneous quantification of PBR and GLE, as no prior UV-Visible spectrophotometric analytical techniques were identified in the literature. Additionally, an RP-HPLC method was established for the simultaneous quantification of LAM and TNF in multicomponent formulations, aiming for enhanced accuracy and sensitivity compared to existing methodologies [4-19]. Validation of the analytical methods was based on ICH guidelines. These guidelines recommend key performance criteria such as accuracy, precision, linearity, Limit of Detection (LOD), Limit of Quantification (LOQ), robustness, ruggedness, and system suitability tests [20].

Pibrentasvir (PBR) and Glecaprevir (GLE) were obtained as gift samples from Mylan Pharmaceuticals Private Limited, Karnataka. Tablet formulation Mavyret (Intas Pharmaceuticals Ltd., India) labelled to contain 100 mg Glecaprevir and 40 mg Pibrentasvir was procured from a local pharmacy.

Instruments

UV-Visible Spectrophotometer (Shimadzu-1800, software version - UV probe 2.32), Digital pH meter (Systronics), Electronic weighing balance (Shimadzu AY220), Ultrasonicator RC system-MU700.

Preparation of standard stock solution

50 mg of the reference standard of PBR and GLE were accurately weighed and transferred into two separate 50 ml volumetric flasks. Both drugs were dissolved in acetonitrile and volume was made up to mark with same to get 1000 µg/ml of standard solution. 1ml of the above standard solution was transferred separately into 10 ml volumetric flask and made up to the volume to get 100 µg/ml standard stock solution.

Selection of analytical wavelength

From the stock solutions 20 µg/ml of both drugs were prepared and scanned in the spectrum mode from 200 - 400 nm. PBR and GLE showed absorbance maxima at 252 nm and 246 nm respectively as shown in Fig 1.

Twenty tablets of Mavyret were accurately weighed and pulverized. A quantity of tablet powder equivalent to 100 mg of PBR was transferred into a 100 ml volumetric flask. To this, 50 ml of acetonitrile was added, and the mixture was sonicated to dissolve the contents. The volume was then made up to 100 ml with acetonitrile and filtered through a 0.45 ?m membrane filter, discarding the initial few milliliters of the filtrate. From this stock solution, suitable aliquots were diluted with acetonitrile to achieve a final concentration of 20 ?g/ml each for PBR and GLE, considering their amounts in the combined tablet formulation.

Absorptivity factor method

When it comes to the investigation of binary mixes, the absorptivity factor approach is applied, with the exception of circumstances in which there is a large variance in the absorptivity of both drugs. As a precautionary measure, this is taken to eliminate the possibility of an isoabsorptive point occurring. It is possible to employ the absorptivity factor approach in order to ascertain the concentration of drug x in a mixture that contains two medicines, x and y, where drug y can be determined by any of the well established spectrophotometric methods. The calculation of the absorptivity factor, which is the ratio between the two absorptivities (ax, ay) at the intersection point with the same absorbance value, is the foundation of this method [21]. Also known as the absorptivity factor, this particular point is referred to as the absorbtivity factor point (F), and calculation of it can be accomplished by utilizing the equation,

A_m  =    a_y (Fc_x + c_y)

A_m is absorbance of mixture

a_y is absorptivity of y at F

c_x and c_y are the concentrations of x and y respectively.

       
           
       
    Fig 3: Calibration curve of GLE (6 -16 µg/ml)

Validation of proposed methods

After the development of UV-spectrophotometric methods for the estimation of drugs in a combined dosage form, validation of the method was performed. Performance characteristics are expressed in terms of analytical parameters [20].

Simultaneous Estimation Of Lamivudine And Tenofovir In Combined Dosage Form Using RP-HPLC

Chemicals and reagents

Lamivudine (LAM) and Tenofovir (TNF) were obtained as gift sample from Mylan Pharmaceuticals Private Limited, Karnataka. Tenolam tablets labelled to contain Lamivudine 300 mg and Tenofovir 300 mg (Hetero Drugs Ltd., India) was procured from local pharmacy.

Instruments

Liquid chromatography (Shimadzu LC-20AD, UV-detector (Shimadzu SPD-20A), Analytical column (Phenomenex C18 (250×4.6 mm & 5 µm) Data processor (LC solution software (Schimadzu, Japan)), Injector (Rheodyne-7725 (Capacity loop of 20 µl)), Syringe (Hamilton, 25 µl), Pump (Shimadzu LC 20 AD), Electronic weighing balance (Shimadzu AY220).

Selection of analytical wavelength

The standard solutions of LAM (5 µg/ml) and TNF (5 µg/m) in acetonitrile were scanned in UV region and an overlain spectrum as shown in Fig 4 was recorded. It was observed that both drugs show absorbance at 264 nm. Therefore 264 nm was considered as optimum due to less interference and appreciable absorbance.

       
           
       
Fig 4: Overlain spectra of LAM and TNF (5 ?g/ml)

Preparation of standard stock solution and calibration curve

10 mg of reference standards of both LAM and TNF were accurately weighed and transferred into two separate 10 ml volumetric flasks. Both drugs were dissolved in acetonitrile and volume was made up to mark with same to get 1000 µg/ml of standard solution. 1 ml of the above standard solution was transferred separately into 10 ml volumetric flasks and made up to the volume to get 100 µg/ml standard stock solutions. Standards containing 2.0 - 12 ?g/ml of LAM and 0.5 – 3.0 ?g/ml TNF were prepared by transferring appropriate aliquots from the above solutions. The drugs were injected into HPLC system by using mobile phase consisting of phosphate buffer (pH 5.0) and acetonitrile in the ratio 60:40 and their chromatograms were recorded as in Fig 5, Fig 6 & Fig 7. Peak areas were recorded both for LAM and TNF.
       
           
       
    Fig 5: Chromatogram of LAM

       
           
       
Fig 6:  Chromatogram of TNF
       
           
       
    Fig 7: Chromatogram of drug combination

Preparation of sample stock solutions

Ten tablets of sample were accurately weighed, their average weight was calculated and ground to fine powder. An amount equivalent to 100 mg LAM was accurately weighed and transferred to 100 ml volumetric flask, add acetonitrile and then sonicated for 15 minutes. Adjust the volume with acetonitrile and filtered through 0.45 µm nylon filters. From this solution 1 ml was taken and made up to 10 ml with mobile phase. From the above solution 1 ml was taken in to 10 ml volumetric flask and made to final volume with acetonitrile. The solution injected in to HPLC system and chromatograms were recorded.

Validation of proposed method

The developed analytical method for LAM, TNF was validated as per ICH guideline in the terms of linearity, precision, accuracy, specificity, sensitivity, system suitability, robustness which are required to justify the purpose of the developed method [20].

RESULTS AND DISCUSSION

UV- Vis spectrophotometric method for Pibrentasvir and Glecaprevir

Linearity

The calibration plot of absorbance versus concentration was found to be linear over the concentration range selected as shown in Table 1. It indicate that test results were directly proportional to the concentration of the analyte in the sample as in Fig 8 and Fig 9.

Table 1: Linearity table

       
           
       
  Fig 8: Linearity graph (6.0 - 8.5 ?g/ml)                                  

       
           
       
Fig 9: Linearity graph (2.0 - 4.5 ?g/ml)

Recovery and precision studies were done to validate the accuracy and reproducibility of the developed method as shown in Table 2 and 3. The % recovery was found to be within 98.16 % -102.50 % which indicate the accuracy of the developed method.

Table 2: Result of recovery study

Table 3: Results of precision study

Limit of detection and quantitation

By using calibration curve, the standard deviation of the response (SD) and slope of the calibration curve (S) was determined. From this LOD and LOQ was calculated as shown in Table 4.

Table 4: Limit of detection and quantification

Result of sample analysis

The concentration of PBR estimated by simultaneous equation method is found to be 19.05 µg/ml. By using the above concentration, the concentration of GLE was determined by using absorptivity factor method was found to be 18.21 µg/ml. The percentage purity was reported in Table 5.

Table 5: Analysis of sample

RP- HPLC method for lamivudine and tenofovir

Accuracy and precision

Accuracy and precision studies were carried out and results were satisfactory. Accuracy determined by standard addition method shows % recoveries from 98.12 % - 100.87 % for LAM and 98.20 % - 101.98 % for TNF. Percentage RSD for the peak area of LAM and TNF for 3 replicate injections of standard solution was within the limit of < 2>

Table 6: Accuracy table for lamivudine

Table 7: Accuracy table for tenofovir

Table 8: Results of precision study

Linearity

The linearity of the drug response on the basis of variable concentration has been found to be within the range of 2.0 - 12.0 ?g/ml for LAM & 0.5 - 3.0 ?g/ml for TNF as shown in Table 9. The graph of peak response versus concentration shows the linear graded response within the same range of concentrations as shown in Fig 10 & Fig 11.

Table 9: Linearity table for concentration v/s peak area

       
           
       
 Fig 10: Linearity graph for lamivudine                                          

       
           
       
 Fig 11: Linearity graph for tenofovir

By using calibration curve, the standard deviation of the response (SD) and slope of the calibration curve (S) was determined. From this LOD and LOQ was calculated as shown in Table 10.

Table 10: LOD and LOQ

System suitability study

The system suitability parameters observed by using these optimized conditions were reported. The results of the system suitability test assure the adequacy of the proposed HPLC method for routine analysis of LAM and TNF alone or in combination discussed in Table 12.

Table 12: System suitability data

Robustness

Deliberated changes in chromatographic conditions like increasing or decreasing flow rate, pH, temperature of column, and wavelength. The data on the robustness of results were examined and found within the limit shown in Table 13.

Table 13: Results of robustness study