INTRODUCTION:

This research article provides important insight into development, validation and application of reverse phase high pressure liquid chromatographic method for the assay of tenofovir disoproxil fumarate, bulk drug and pharmaceutical dosage form. It is a 1-(6-Aminopurin-9-yl)-prapan-2-yl-oxymethylphosphonic acid, drug used in the treatment of human immunodeficiency virus (HIV) Type-1 patients.

According to the literature review several methods has been developed for drug, like spectroscopy methods^1-6. HPLC^7-25, and HPTLC^26,. The newly developed HPLC method can be used for the routine analysis due to retention time as 1.82 minutes hence large no. of sample can be analysed in short period. In the proposed methods optimization and validation of this method are reported.

Structure of tenofovir and tenofovir disoproxil fumarate

Tenofovir

Tenofovir disoproxil fumarate

EXPERIMENTAL:

Instrumentation:

The HPLC system Merck-Hitachi equipped with separation module and UV detector (L-7400) was used. The chromatogram was recorded and peaks are quantified by means EZChrom Elite software. A Shimadzu analytical balance with 0.01 mg was used.

Materials and reagents:

Reference standard of tenofovir disoproxil fumarate were obtained from reputed firms with certificate of analysis. Analytical grade of phosphoric acid and tri ethyl amine and HPLC grade of methanol were used from Merck and the HPLC grade water was obtained by using Millipore water system.

Procedures:

Standard stock solution:

About 10 mg of standard tenofovir disoproxil fumarate was weighed accurately and transferred in 10 ml volumetric flask. About 5 ml of diluent (buffer: methanol (90:10 % v/v)) was added and sonicated for 5 minutes. The volume was adjusted to the mark with diluents to give concentration as 1000 μg/ ml. The working standard solution was prepared by diluting 1 ml of 1000 μg/ ml solution to 10 ml with diluent to get concentration 100 μg/ ml.

Sample preparation:

Twenty tablets (each contains 300 mg of dosage) were weighed accurately and average weight of each tablet was determined. The powder equivalent to 10 mg of tenofovir disoproxil fumarate was weighed accurately and transferred in 10 ml volumetric flask. About 5 ml of diluent (buffer: methanol (90:10 % v/v) was added and sonicated for 5 minutes. The volume was adjusted up to mark with diluent to give concentration as 1000 μg /ml. The working sample solution was prepared by diluting 1 ml of 1000 μg/ ml solution to 10 ml with diluent to give 100 μg/ ml. A 10 µl was injected for analysis.

Method Development:

Chromatographic condition:

Different columns containing octyl and octadecyl silane stationary phase were tried for separation and resolutions. It was found that Hypersil BDS C18 (150mm x 4.6mm x 5μm) column offered more advantage over other columns. The mobile phase was a mixture of buffer and methanol (90:10 % v/v). The buffer was 1 ml of tri-ethyl amine dissolved in 1000 ml of HPLC grade water. The pH 5.0 was adjusted with dilute phosphoric acid. The flow rate of the mobile phase was adjusted to 1 ml /min. The detection was carried out at wavelength 260 nm. Fig 1. The injection volume of the standard and sample solution was set at 10.0 µl. The elution and resolution parameters of drug were recorded at the wavelength 260 nm and its response optimization was compared with adequate sensitivity. It produced well shaped peaks for the drug assay. A chromatogram of the drug assayed is depicted in fig. 2.

Method validation:

System suitability:

System performance parameters of developed HPLC method were determined by injecting standard solutions. Parameters such retention time, area, % area and asymmetry were shown in Table-1. It indicated good performance of the system.

Table 1: System performance parameters for alprazolam. (n= 6).

Retention time	symmetry factor	Area	% Area
1.823 minutes	1.47	462241	100.00

Linearity:

The linearity of the method was determined for tenofovir disoproxil fumarate six concentrations level ranging from 50 to 150 µg/ml. The calibration curve was constructed by plotting response factor against concentration of the drugs. The regression equation was given as y = 11267 x + 37546. The correlation coefficient (r²) was 0.9999 and concentration range indicated above. The results of the same are tabulated in the table 2.

Table 2: Linearity – regression analysis data

Parameters	Values
Correlation Coefficient (r)	0.9999
Intercept (y)	37546
Slope (m)	11267

Accuracy:

The accuracy of the method was determined by recovery experiments. The recovery studies were carried out and percentage recovery was calculated and presented in Table 3.

Precession:

The method precision was established by carrying out the analysis of tenofovir disoproxil fumarate. The assay was carried out of the drug using analytical method in six replicates. The value of relative standard deviation lies well with the limits (0.10 %). The results of the same are tabulated in the table 4.

Stability of solution:

The stability studies of the solutions under study were established by keeping the solutions at room temperature for 24 hours. The results indicated no significant change in the assay results of the same solutions. It confirmed the stability of the drug in the solvents used for the analysis.

Robustness:

Robustness study of the method was performed by making slight changes in the chromatographic conditions.

In flow rate, variation ± 0.2˚ C.

In wavelength, variation ± 5 units

In variation mobile phase composition ± 0.2 unit

The chromatograms demonstrated have no marked changes that developed HPLC method was robust.

Method Application:

The validated high performance liquid chromatographic method was applied for determination of tenofovir disoproxil fumarate its formulation. Twenty tablets of tenofovir disoproxil fumarate were used. A portion equivalent to 10 mg of tenofovir disoproxil fumarate was weighed accurately. It was dissolved in 10 ml of diluent to obtain final concentration 1000 µg/ml. The working sample solution was prepared by diluting 10 ml of 1000 μg/ ml solution to 100 ml with diluent to give 100 μg/ ml. 10 µl of this solution was injected under specified conditions. The analyte peaks were identified by comparison with respective standard and sample chromatogram were recorded. (Fig.3).

Table 3: Accuracy - %Recovery

level	test	Amount in mg	Area	Quantity added in μg /ml	Quantity recovered in μg /ml	% recovery	mean recovery
level	test	Amount in mg	Area	Quantity added in μg /ml	Quantity recovered in μg /ml	% recovery	mean recovery
80%	1	10.12	368646	83.28	81.89	98.33	98.24
	2	10.25	368626	83.28	81.89	98.33
	3	10.17	367634	83.28	81.67	98.06
100%	1	1.02	468882	104.1	104.16	100.05	100.17
	2	10.07	469490	104.1	104.29	100.18
	3	10.15	469958	104.1	104.39	100.28
150%	1	10.21	695087	156.15	154.40	98.88	98.53
	2	10.26	692674	156.15	153.87	98.54
	3	10.23	690110	156.15	153.30	98.17

* Average of triplicate analysis

Table 4: Precision – method precision

The assay results expressed as mg/tablets are shown in Table-3. It indicated the amount of each drug in the product meet the requirement.

RESULTS AND DISCUSSION:

In the proposed method, the retention time of tenofovir disoproxil fumarate was 1.823 min. The linearity was in the range of 50-150μg/ml. The regression equation of the linearity was given as Y= 11267x - 37546 where X is concentration of tenofovir disoproxil fumarate in μg/ml. and Y is corresponding peak area. The coefficient of co-relation was 0.9999. The result shows that an excellent correlation between peak area and concentration of tenofovir disoproxil fumarate in the range indicated. The relative standard deviation for method precision was 0.297 (limit % RSD < 2.0%). The mean recovery of the tenofovir disoproxil fumarate was 100.43%. The high percentage recovery indicates that the proposed method is highly accurate.

The use of 0.1 % tri ethyl amine (pH 5.0) and methanol 90:10% (v/v) gave peak with good resolution. The robustness studies indicated that there was no effect other parameters on the drug study. No interfering peaks were found in the chromatogram of the formulation within the run time indicted that excipents used in the formulation did not interfere the estimation of drug.

The reproducibility, repeatability and accuracy of the proposed method were found to be satisfactory which is evidenced by low values of standard deviation 0.297 and percent relative standard deviation 0.295. (Table no.4) The accuracy and reproducibility of the proposed method was confirmed by recovery experiments, performed by adding known amount of the drug to the pre-analyzed active pharmaceutical ingredient and reanalyzing the mixture by proposed method. (Table no.3) The percent recovery obtained indicates non- interference from the excipients used in the formulations. The methods reported in literature method indicate large retention time. Hence more time will be required for validation of drug and its formulation as well it requires more amount of organic solvent.

This can be successfully used for validation of drug as well as for determining stability of drug in various conditions as per ICH guidelines.

Thus the proposed RP-HPLC method is used for validation of tenofovir disoproxil fumarate from active pharmaceutical ingredient and marketed formulation due to its simplicity and non interference of other peaks and relatively short retention time as 1.823 min. It is more precise, accurate, linear, robust, simple and rapid method. Hence the proposed RP-HPLC method is strongly recommended for the quality control of the raw material, active pharmaceutical ingredient and pharmaceutical formulation per ICH guidelines.

ACKNOWLEDGEMENT:

Authors express sincere thanks to the Principal, Dr. T.M. Desai, D. G. Ruparel college for providing laboratory facilities.

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Received on 23.05.2019 Modified on 13.06.2019

Research J. Pharm. and Tech. 2019; 12(10):4752-4756.

DOI: 10.5958/0974-360X.2019.00819.9

Test	Weight of test sample used in mg	Area obtained	% assay
Solution-1	10.41	464954	100.47
Solution-2	10.45	462093	100.24
Solution-3	10.51	460530	100.47
Solution-4	10.4	467561	100.94
Solution-5	10.31	469078	100.39
Solution-6	10.27	469342	100.05
	Mean Assay		100.43
	SD		0.297
	RSD		0.295