Simultaneous Estimation of Lamivudine, Tenofovir Disoproxil Fumarate and Efavirenz in Bulk and Tablet Dosage Form by Cramer’s Rule

Tablet containing Lamivudine (LAM), Efavirenz (EFZ), Tenofovir Disoproxil Fumarate (TDF), is prescribed combination used to stop or slow down the progression of HIV infection; also used to treat chronic hepatitis B virus infection in adults. This combination shows promising results as it helps to increase the lifespan of patient by restricting the HIV growth in the body. Basically, it is an antiretroviral medicine which boost up the immunity to fight against the Acquired Immune Deficiency Syndrome (AIDS).

Lamivudine and Tenofovir are designated as synthetic nucleoside reverse transcriptase inhibitor while Efavirenz belong to class non-nucleoside reverse transcriptase inhibitor. Lamivudine is soluble in methanol and in water. Efavirenz is soluble in methanol, insoluble in water while Tenofovir disoproxil fumarate is soluble in methanol and distilled water.^1–4

Currently, the literature survey reveals few methods available for the simultaneous estimation of LAM, EFZ and TDF in a combined dosage form like UPLC,^5,6 RP-HPLC^7–12 and UV-vis- derivative spectroscopic methods.^12,13 The aim of present study is to develop a simple, accurate, effective and rapid method for the simultaneous estimation of LAM, EFZ and TDF in a combined dosage form.

Jasco V-730 double beam UV-vis-spectrophotometer with 1 cm matched quartz cells was used for study. The spectra in the presented study were recorded at spectral band width of 1.0 nm with the scanning speed 400 nm/min and data pitch 1nm. Range for scanning wavelength was 200-400 nm.

Trioday tablets manufactured in India by Cipla containing Lamivudine (300 mg) Efavirenz (600mg) and Tenofovir Disoproxil Fumarate The methanolwas obtained from multi-speciality hospital pharmacy. Methanol used was of AR grade (LOBA Chemie, India). Freshly prepared double distilled water was used in the experiment.

Stock solution of 100 µg/mL for LAM, TDF and EFZ was prepared by weighing accurately 10 mg of standard drugs and dissolving in 100 mL of methanol to get concentration of 100 µg/mL each.

The prepared dilution of LAM, EFZ and TDF having concentration of 30 μg/mL was scanned in the range of 200-400 nm. The wavelength of maximum i.e., lambda max for LAM, EFZ and TDF were found to be at 272, 247, 262 nm respectively. Overlain spectra of EVZ, TDF and LAM in methanol is shown in Figure 1.

Figure 1:
Overlain spectra of EVZ, TDF and LAM in methanol.

The stock solution of LAM and TDF was diluted appropriately with methanol and six dilutions were prepared in concentration range of 5-30 μg/mL and the dilutions for both drugs were measured for absorbance at 272, 247, 262 nm (Figures 2 and 3).

Figure 2:
Calibration curve of LAM at 262, 247, 272 nm.

Figure 3:
Calibration Curve TDF at 262, 247, 272 nm.

The stock solution of EFZ was diluted appropriately with methanol and dilutions was prepared in concentration range of 10-60 μg/mL and the absorbance of these six dilutions was measured at 272, 247, 262 nm (Figure 4).

Figure 4:
Calibration Curve EFZ at 262, 247, 272 nm.

Marketed tablets of LAM, TDF and EVZ, Trioday (300 mg, 300 mg, 600 mg) were weighed accurately and finely powdered. Tablet powder equivalent to 10 mg of TDF (10 mg of and LAM; 20 mg of EVZ) was taken and transferred to 10 mL volumetric flask and was diluted with 5 mL of methanol. The prepared solution was sonicated for 10 min, and after sonication volume made up to 10 mL. Whatman filter paper no. 41 was used to filter the sonicated solution. 5 mL of filtrate was taken in 50 mL of volumetric flask and diluted to 50 mL with methanol. The procedure was repeated 6 times for tablet formulation. Absorbance was measured at three selected wavelengths and concentrations were determinedby solving matrix by using a Cramer’s rule. The assay results are presented in Table 1.

The intraday and inter-day precision of the method was carried expressed as relative standard deviation. Intraday precision was determined at three concentration level in triplicate for all three drugs (n = 9). For LAM and TDF at 10, 15, 20 µg/mL and for EVZ at 10, 20, 30µg/mL. Inter-day precision was determined by analysing each drug on the next consecutive day with the same concentration as mentioned above.

The accuracy study was carried out by standard addition method. The standard solution was spiked into the sample solution at three levels 50%, 100% and 150% of assay concentration. The sample concentration used were 10 μg/mL for LAM and TDF and 20 μg/mL for EVZ. The prepared samples were scanned in the range of 200-400 nm. The amount of drugs was calculated by solving the Cramer’s matrix. Accuracy data is presented in Tables 2, 3 and 4.

To begin with, the overlain spectra of LAM, TDF and EVZ were studied in the different solvents. As a result, the methanol was found to be more suitable solvent for the analysis of these drugs in the UV spectrophotometry as it offered the advantages of ease of solubility over the other solvents. All the three drugs LAM, TDF and EVZ shows absorbance at the wavelength of maximum of each other. So, the absorbance of the mixture at one of the drug’s wavelength of maximum is the sum of absorbance all three drug. By applying the Beer- lambert’s equation (A= abc, where a = absorptivity, b = path length (1 cm), c= concentration)The absorbance of the mixture at the three wavelength represents as following:

Here, Am₁, Am₂, Am₃ are the absorbances of the mixture at 262 nm (λ₁), 247 nm (λ₂), 272 nm (λ₃), respectively where cx, cy and cy are the concentration of LAM, TDF and EVZ. In the above equation, ax1, ax2, and ax3 are the absorptivity’s of LAM; ay1, ay2, and ay3 are the absorptivity’s of TDF and az1, az2, and az3 are the absorptivity’s of EVZ at the three wavelength of maximum 262 nm, 247 nm, 272 nm; respectively.

The above matrix was further solved by calculating the absorptivity’s from the calibration curve and the equation was derived as follows.

So, in the above equation there are three unknown concentrations, to easily solve the above matrix Cramer’s rule method was applied. The result was the concentrations of the LAM, TDF, and EVZ in the mixture.

It was observed that the all drugs obey the Beer-Lambert’s law in the different concentration range. Precision was carried at intraday and inter-day level and result found that the method was precise, as RSD for proposed method was satisfactory (< 2%) as shown in Table 5.

Recovery study was also carried out on the developed method and result found to be in the range of 97.5-102.5%. The assay was performed on the marketed tablet Trioday with the label claim 300 mg LAM, 300 mg TDF and 600 mg EVZ. The amount of drug found within pharmacopeial limits. Assay was performed (n = 6) and results are given in Table 1.

Traditional method needs to separate the LAM, TDF and EVZ before analysis. Proposed method needs to prepare the sample solution as per given in the assay procedure and measure the absorbance at 262 nm, 247 nm and 272 nm. Matrix was drawn using the absorbances obtained at the entire three wavelengths and then the matrix was solved using Cramer’s rule. The final answer was the amount of each drug in the sample solution. The results for the validation parameters are given in Table 5.

The proposed UV-vis spectrophotometric method based on Cramer’s rule was found to be less time consuming and easy, as it involves very limited steps for analysis. Method was found to be simple, sensitive, precise and accurate. The developed method can be applied for the routine analysis of the LAM, TDF and EVZ.