INTRODUCTION:

Tenofovir Disoproxil Fumarate belongs to the class of antiretroviral drugs as nucleotide reverse transcriptase inhibitors which block reverse transcriptase an enzyme crucial to viral production in HIV infected people. After oral absorption, Tenofovir Disoproxil Fumarate is rapidly converted to Tenofovir and then undergoes subsequent phosphorylation by cellular enzymes to the active Tenofovir Diphosphate, which inhibits the activity of HIV-1 reverse transcriptase. Chemically it is 9-[(R)-2-[[bis[[(isopropoxycarbonyl) oxy] methoxy] phosphinyl] methoxy] propyl] adenine fumarate (1:1).It is white to light-yellow crystalline powder and it is soluble in water and in Dimethyl Sulfooxide (DMSO).

There are very few reported methods for analysis of degradation products and impurities of Tenofovir Disoproxil Fumarate have been reported including Liquid chromatography–tandem mass spectrometric¹, Validated liquid chromatographic², LC/MS/MS^3,4, LC-positive ESI-MS/MS⁵, Simultaneous estimation in Tablet dosage forms⁶ and Reverse phase liquid chromatography ⁷ .Hence the main purpose of this investigation is to develop a precise, accurate, simple, reliable and less time consuming RP-HPLC method for estimation of Tenofovir Disoproxil Fumarate drug from plasma.

MATERIAL AND METHOD:

Drug and Chemicals:

Tenofovir Disoproxil Fumarate as a gift sample from Cipla ltd. Patalganga. HCl (AR grade), Sodium Hydroxide (NaOH), Orthophosphoric acid and Acetonitrile was purchased from Loba chemie Pvt. Ltd. Mumbai and Finar chemical Ltd. Ahemedabad.

Instrument Used:

UV visible double beam spectrophotometer (Jasco V530), High Performance Liquid Chromatography with UV detector (LC-2000Plus Series), Ultrasonicator (Spectralab) was used.

Method:

Preparation of standard drug solution:

To the 5 ml of blood sample 0.2 ml of 10% trichloroacetic acid added. RBC allowed to settle. Sample was centrifuged at 3000 RPM for 35 min. The supernatant was collected as plasma. 10 mg of drug TDF was added in 8 ml of mobile phase separately. To this solution 2 ml of plasma was added to get the solution 1000 μg ml ^-1 of drug. This solution was filtered through syringe filter of size 0.45 μm. These solutions were diluted with mobile phase to get 100 μg ml ^-1 stock solutions of drug. Chromatographic separation was shown in Table No.1.

Table No. 01. Separation Conditions

Chromatographic Mode	Chromatographic Conditions
Stationary Phase	HIQ Sil C₁₈column(250 mm x 4.6 mm i.d.,5µm)
Mobile phase	Acetonitrile: Water(70:30) pH 4.5 adjusted by OPA
Detection Wavelength	260 nm.
Flow Rate	1 ml min^-1.
Sample Size	20 μL.

Selection of Chromatographic Conditions:

The selection of HPLC method depends upon the nature of the sample, its molecular weight and solubility. RP-HPLC method was selected for the initial separations because of its simplicity and suitability. The chromatographic variables such as mobile phase, flow rate and solvent ratio were studied. The resulting chromatograms were recorded and the chromatographic parameters such as selectivity, asymmetric factor, and peak resolution were calculated. The condition that gave the best resolution, symmetry and selectivity was selected for estimation.

Optimization of Chromatographic Parameters:

Optimization in HPLC is the process of finding a set of conditions that adequately analyze the quantification of the analyte with acceptable accuracy, precision, sensitivity, specificity, cost, ease, and speed of analysis.

Optimization of Mobile Phase Strength:

For selection of mobile phase, various mobile phase compositions containing Acetonitrile: water in different ratios was tried but the resolution was not found to be satisfactory. Finally, mobile phase containing ACN: Water: (70:30) pH adjusted 4.5 by OPA was found to give best resolution. Before analysis, the mobile phase was filtered through a 0.45µ nylon filter, and then degassed ultrasonically for 15 min.

Optimization of Detection Wavelength:

An ideal wavelength is one that gives good response for the drugs to be detected. Optimization of wavelength was done at different wavelengths for detection to get good response. In the present study, a drug solution of 25 µg/ml of TDF was prepared in mobile phase. After observing UV spectra of both the drug and the internal standard, wavelength of 260 nm was selected for further study.

Selection of Internal Standard:

Ezetimibe was selected as an internal standard after observing retention behavior of several drugs.

Preparation of Internal Standard Solution:

Standard stock solution containing 1000 µg/ml of Ezetimibe was prepared by dissolving 50 mg of Ezetimibe in 20 ml of mobile phase. It was then sonicated for 10 minutes and the final volume of solution was made up to 50 ml with mobile phase to get 1000 μg/ml of Ezetimibe in a 50 ml volumetric flask.

Linearity Study of Drug at Selected Wavelength:

In to a series of 10 ml volumetric flasks, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2ml of drug stock solution (100μg/ml) was pipette and final volume of the solutions was made up to 10 ml with mobile phase. These working standard solutions were of concentration 0.5, 1, 2, 4, 8, 16, 32 µg ml ^-1 of TDF. To each of this standard solution 0.5 ml of internal standard solution (50 µg ml ^-1) was added and volume was made up to mark with mobile phase. A 20 μL of sample solution was injected into the injection port of chromatographic system having fixed volume loop injector.

Chromatograms were noted and response factor was plotted against concentration to get calibration curve. The data for calibration curve were given in Table No. 02. while corresponding calibration curves were shown in Fig. No. 01. and chromatogram of physical mixture was shown in Fig. No. 02.

Table No. 02. Linearity of TDF at 260 nm

Sr. No.	Concentration (μg ml^-1)	Response Factor
1.	0.5	0.0108
2.	1	0.0240
3.	2	0.0436
4.	4	0.0879
5.	8	0.1869
6.	16	0.3738
7.,	32	0.7476

Figure No. 01. Calibration curve for TDF at 260 nm

Fig. No. 02. Overlain Chromatogram of Physical Mixture

Interpretation of Linearity study:

Correlation coefficient and slope and intercept values are given to interpret linearity.

System Suitability Parameters:

System suitability parameters were analyzed on freshly prepared standard stock solution of Tenofovir disoproxil fumarate. Parameters that were studied to evaluate the suitability of the system were shown in Table No. 3.

Table No. 03. System Suitability Parameters

Sr. No.	Parameters	TDF
1.	Theoretical Plates	4183.15
2.	Asymmetry	1.11
3.	Retention Time in minutes	3.11
4.	Calibration Range (µg ml ^-1)	0.5-32
5.	Limit of Detection (µg ml ^-1)	0.0314
6.	Lower Limit of Quantification (µg ml^-1)	0.0951

Specificity/ selectivity:

Analysis of blank samples of the appropriate biological matrix (plasma) should be obtained from at least 6 sources. Each blank should be tested for interference and selectivity should be ensured at LLQ.

Accuracy:

It should be measured using a minimum of 6 determinations per concentration. Minimum of 3 concentrations in the range of expected concentration is recommended for determination of accuracy mean should be ± 15 % of the actual value except at LLQ where it should not deviate by ± 20 %.this deviation of mean from the true serve as the measure of accuracy.

Precision:

It should be measured using minimum of 5 determinations per concentration. Minimum of 3 concentrations in the range of expected concentration is recommended. The precision determined at each concentration level should not exceed 15 % of CV except for LLQ where it should not exceed 20 % of CV. Results of accuracy and precision study were shown in Table No. 4.

Table No.04. Results of Accuracy and Precision study

Analyte		Concentration level			*% concentration found (Accuracy) (Mean ± S.D.)**	R.S.D.
TDF	T1	LLOQ	1	93.794 ± 1.2943		1.3790
		Other than LLOQ	2	94.174 ± 0.0041		0.0043
			3	99.118 ± 1.6242		1.6387
	T2	LLOQ	1	93.324 ± 0.9010		0.9654
		Other than LLOQ	2	97.290 ± 1.8206		1.8706
			3	98.276 ± 1.1034		1.1227

Accuracy has been calculated as the % of nominal concentration,

S.D.: standard deviation; R.S.D.: relative standard deviation.

* Average of five determinations.

Recovery:

Recovery experiments should be at 3 concentrations (low, medium and high) with unextracted standards that represent 100 % recovery were shown in Table No. 5.

Table No.05. Result of Extraction Recovery

Level	Concentration µg ml^-1	*Mean Recovery ± S.D. (%)**	R.S.D.
LLOQ	0.5	95.80 ± 1.271	1.326
MQC	16	97.32 ± 1.140	1.171
HQC	28	98.78 ± 0.921	0.932

LLOQ: Lower Limit of Quantification, MQC: medium Quality Control

concentration, HQC: High Quality Control concentration.

* Average of four determinations.

Limit of Detection (LOD):

LOD is the lowest concentration of analyte in the sample that can be detected but not quantified under the stated experimental conditions. LOD is also defined as lowest concentrated that con be distinguished from the background noise with a certain degree of confidence. There is an overall agreement that the LOD should represent the smallest detectable amount or concentrations of analyte of interest were shown in Table No. 6.

Table No. 06. Limit of Detection and Lower Limit of Quantification

LOD (µg ml ^{- 1}) *	LLOQ (µg ml ^{- 1}) *
0.0314	0.0951

* Average of six determinations

Short-term stability:

Aliquots each of the low and high concentrations should be thawed at room temperature and kept at this temperature for 4-24 hours and analyzed. %S.D. should be less than 15 % were shown in Table No. 7.

Table No. 07. Result of Short term stability

Analyte

Level

Concentration

(µg ml^-1)

% stability*

(Mean ± S.D.)

TDF

Low

100.0733 ± 1.1036

High

99.15333 ± 1.4718

*Average of three readings.

RESULT AND DISCUSSION:

The proposed method describes a RP-HPLC procedure employing a C₁₈ column and a mobile phase containing acetonitrile and water in 70:30 proportions (pH 4.5). To develop the method with good resolution the change in proportion of organic solvents were studied. Acetonitrile, methanol and water in various ratios were tested to get an appropriate mobile phase composition. Best resolution of drug was achieved with the mobile phase having composition of acetonitrile and water in the ratio70:30v/v (pH 4.5). The linearity response of the HPLC system for TDF was obtained over the range of 0.5-32 µg ml^-1.

Various drugs were checked for use as an internal standard to obtain well resolved peaks along with the TDF. Retention time of both the drugs were studied with flow rate of mobile phase at 0.5, 1, 1.2 ml min^-1. Optimum retention time with greater resolution of the TDF and internal standard eluting within 5.0 minutes was achieved with a flow rate of 1 ml min^-1. The drug solution having concentration of 10 µg ml¹was scanned in the UV-Vis. range of 200 nm to 400 nm on a UV-Visible spectrophotometer for selection of sampling wavelength. After recording the spectra of the drugs 260 nm was selected as suitable wavelength for estimation.

Linearity of the method was determined from Correlation coefficient which is 0.999 which shows that method is linear. Precision and accuracy determined from minimum of five determinations per concentration (three concentrations representing the entire range of the standard curve studied for intraday. The results for precision were R.S.D. ≤ 20% at LLOQ and R.S.D. ≤ 15% other than LLOQ. This shows that the method is accurate and precise. Result of short term stability study performed by thawing samples at low and high concentration for 6 hours and % stability calculated which is between 85-115%. Extraction recovery was determined by spiking the pure drug sample in previously analyzed sample which is more than 50 %.

CONCLUSION:

A convenient and rapid RP- HPLC method has been developed for estimation of Tenofovir Disoproxil Fumarate in bulk form. The proposed method is simple, fast, accurate and precise for RP-HPLC estimation of Tenofovir Disoproxil Fumarate in plasma. The proposed method could be applied for routine analysis in quality control laboratories.

ACKNOWLEDGEMENTS:

Authors are grateful to Cipla Ltd, Patalganga for providing the gift sample of Tenofovir Disoproxil Fumarate. We are also thankful to the Principal and management of Bharati Vidyapeeth College of Pharmacy, Kolhapur for providing necessary facilities to carry out this work.

REFERENCES:

1. Tracy King, Lane Bushman, Jennifer Kiser, Peter L. Anderson, Michelle Ray, Thomas Delahunty and Courtney V. Fletcher, Liquid chromatography–tandem mass spectrometric determination of tenofovir-diphosphate in human peripheral blood mononuclear cells, Journal of Chromatography B, 843 (2), 2006, 147-156.

2. Dunge Ashenafi, veralaxmi chintam, Daisy Van Veghel ,Sanja Dragovic, Jos Hoog martens, Ervin Adams, Development of Validated liquid chromatographic method for Determination of related substances and assay of Tenofovir disoproxil fumarate, Journal sep.science, 33, 2010, 1708-1716

3. Tom Delahunt, Lane Bushman, Courtney V. Fletcher, Sensitive assay for determinination of Plasma Tenofovir concentrations by LC/MS/MS, Journal of chromatography B, 830 ( 1), 2006, 6-12.

4. Delahunty T, Bushman L, Robbins B, Fletcher CV, Simultaneous assay of tenofovir and emtricitabine in plasma using LC/MS/MS and isotopically labeled internal standards, Journal of Chromatography B, Analyt Technol Biomed Life Sci.;877(20- 21), 2009, 1907-14, .

5. Pruvost A, Théodoro F, Agrofoglio L, Negredo E, Bénech H, Specificity enhancement with LC-positive ESI-MS/MS for the measurement of nucleotides: application to the quantitative determination of carbovir triphosphate, lamivudine triphosphate and tenofovir diphosphate in human peripheral blood mononuclear cells, Mass Spectrum , 43(2), 2008, 224-33.

6. F Rajesh Sharma, Pooja Gupta, Simulataneous Estimation of Emtricitabine and Tenofovir Disoproxil Fumarate in a Tablet Dosage Form, Eurasian J. Anal. Chem, 4(3), 2009, 276-284.

7. S. Sentenac, C. Fernandez, A. Thuillier P. Lechat G. Aymard, Sensitive determination of tenofovir in human plasma samples using reversed-phase liquid chromatography, Journal of chromatography B ,793(15),2003,317-324.

Received on 25.07.2011 Modified on 01.08.2011

Research J. Pharm. and Tech. 4(10): Oct. 2011; Page 1626-1629