A Precise Review on Tenofovir Disoproxil Fumarate: An Analytical Profile

Tenofovir Disoproxil Fumarate (TDF) Fig. 1 is an antiretroviral medicine used to treat HIV/AIDS and chronic hepatitis B (Goicoechea et al., 2008). TDF is a prodrug of Tenofovir (TNF) and exists as dominant form due to low oral bioavailability of TNF. The active substance TNF inhibits the Nucleotide Reverse Transcriptase. TDF is quickly hydrolysed into Tenofovir monophosphate in the body and gets converted into the active drug. The chemical reaction for this conversion in vivo is as represented in Fig. 2(Avihingsanon et al., 2015). TDF is available in the market as tablets; alone and in combination with other drugs. TDF is mostly expelled with Glomerular filtration and in that being transported into renal proximal tubule cells through organic anion transporter-1 (OAT-1). TDF usually considered as a safe drug, but renal toxicities are reported with its use. The reports are available which provides cause of proximal tubulopathy of kidney, Fanconi syndrome, kidney related other toxicities including insipidus calcium and phosphorus dysregulation with bone disease and reduction in Glomerular function (Patel et al., 2010). Besides the aforementioned effects, the antiretroviral therapy (ART) has transformed HIV infection into a manageable, lifelong disease. The first line regimens are critical to successful ART for its long-term treatment (Bygrave et al., 2011). The present review offers a critical account on analytical methods published during 2008 to 2016 for determination of TDF.


Introduction
Tenofovir Disoproxil Fumarate (TDF) Fig. 1 is an antiretroviral medicine used to treat HIV/AIDS and chronic hepatitis B (Goicoechea et al., 2008). TDF is a prodrug of Tenofovir (TNF) and exists as dominant form due to low oral bioavailability of TNF. The active substance TNF inhibits the Nucleotide Reverse Transcriptase. TDF is quickly hydrolysed into Tenofovir monophosphate in the body and gets converted into the active drug. The chemical reaction for this conversion in vivo is as represented in Fig. 2 (Avihingsanon et al., 2015). TDF is available in the market as tablets; alone and in combination with other drugs. TDF is mostly expelled with Glomerular filtration and in that being transported into renal proximal tubule cells through organic anion transporter-1 (OAT-1). TDF usually considered as a safe drug, but renal toxicities are reported with its use. The reports are available which provides cause of proximal tubulopathy of kidney, Fanconi syndrome, kidney related other toxicities including insipidus calcium and phosphorus dysregulation with bone disease and reduction in Glomerular function (Patel et al., 2010). Besides the aforementioned effects, the antiretroviral therapy (ART) has transformed HIV infection into a manageable, lifelong disease. The first line regimens are critical to successful ART for its long-term treatment (Bygrave et al., 2011).
The present review offers a critical account on analytical methods published during 2008 to 2016 for determination of TDF.
TDF is absorbed in the gut and quickly converted into TNF. After administration of TDF in the body, ester hydrolysis takes place and in the blood dissociation of two ester linkages occurs. The first cleavage of the ester group offers monoester of TDF then second group of ester gets TNF metabolized intracellularly and followed by its conversion into an anabolite; tenofovir diphosphate which inhibits HIV-1 reverse-transcriptase competitively and stops the DNA synthesis (Kearney et al., 2006).

Mechanism of Action
This drug inhibits HIV-1 transcriptase and HBV retrotranscriptase. Tenofovir diphosphate is a weak inhibitor of DNA polymerases α and β (mammalian) and DNA polymerase γ (mitochondrial) (James et al., 2004).

Dosage Forms and Recommended Dose
TNF is available only in oral tablet dosage forms having 245 mg of TNF corresponding to 300 mg of TDF. The recommended dose for the drug is a tablet per day (James et al., 2004).

Pharmacodynamics and Pharmacokinetics
The range for oral bioavailability is in-between 25 % and 40 % (depending on the amount of fat), and can be administered safely to patients having liver function impaired Cihlar et al., 2002).

Adverse Effects and Contraindications
Obesity and prolonged nucleoside therapy may be predisposing factors (Porche, 2002). Slight rise in serum creatinine and decrease in serum phosphate levels occurring 4-12 months after starting TDF is characterized by nephrotoxicity (Fontana, 2009).

Various Approaches for Pharmaceutical Analysis
The use of analytical methods for pharmaceutical analysis is an essential part of drug development and validation procedure. Analytical techniques like UV/Visible-Spectrophotometry, Atomic Absorption, Capillary-Electrophoresis, Liquid -Chromatography either alone or coupled with Mass Spectroscopy, Measurement of Luminescence, Voltammetry and Polarography have been explored for analysis of drugs in bulk as well as in various formulations and biologicals (Rajput et al., 2015). Amongst all these methods, Chromatographic methods HPLC, HPTLC and UV/Visible-Spectrophotometry have generally been studied and preferred over other methods due to sensitivity and simplicity of analysis (Siddiqui et al., 2013). TDF is available as individual or in combination with other drugs such Emtricitabine, Rilpivirine, Lamivudine, Nevirapine, Efavirenz, Elvitegravir and Cobicistat in various dosage forms.
Thorough literature survey revealed various analytical methods viz HPLC, HPTLC (simple and stability- indicating), UV-Spectrophotometry, bio-analytical methods for analysis in human plasma and biological fluids used for analysis of TDF alone or in combination with other drugs. Few pharmaceutical analytical methods such as Capillary-Electrophoresis and specified Electro-Analytical determinations are also critically been reviewed.

HPLC analysis
Total twenty seven methods have been reported for determination for TDF alone and or in combined dosage form. Reversed-Phase HPLC analyses for TDF (Sarala The identification and separation of the TDF was achieved using C18 column with dimension 250/150 x 4.6 mm; 5 µm and variable combination of mobile phases containing acetonitrile, Methanol, and Water. Whereas, in three developed methods combination of Acetontrile/ Methanol and Sodium Hydrogen phosphate, pH adjusted to 2.3 was chosen as mobile phase. UV/ Visible detector / Photo-Diode-Array detectors were used to perform the detection. The retention time for all these methods reported to be below 10 min. Small values of LOD and LOD in the reported methods indicate that these methods are sensitive. All these established methods are claimed to be simple, economical and less time consuming. In many studied analytical methods, the percentage amount of TDF estimated in pharmaceutical formulation was reported to be in the range of 97 -101, indicating superior conformity with the label claimed. . Total twenty RP-HPLC methods have been reported for analysis of TDF in combination with other drugs. RP-HPLC methods using Isocratic modes have been explored in around sixteen methods; whereas, only four methods were studied using gradient mode of HPLC. In most of these analytical methods C18 column was exclusively applied for separation of TDF and other drugs in its combination. The mobile phase chosen for analysis contains various proportions of Acetonitrile/Methanol and Sodium Hydrogen Phosphate buffer/ water and the pH adjusted below 7.0. The retention time below 11 min was reported for TDF and its combinations with other drugs. The coefficient correction value for linearity of both these drugs were reported to be greater than 0.99. The detailed account of mobile phases used for determination, sample matrix, wavelength maximum, linearity, retention time and the correlation coefficient is described in Table 1. Whereas, the comparison of the column(s) with the specification(s) for HPLC analysis along with the conditions of flow rate, temperature, detector and type of analysis are quoted in Table 2.    Table 3.  Table 4.

Spectrophotometric analysis
About sixteen UV-Spectrophotometry procedures have been studied for determination of TDF alone and in combined dosage forms. Table 6 depicts about spectrophotometry methods with respect to sample matrix, technique, solvent used, λmax, linearity range, and correlation coefficient.

A Typical Methods of Pharmaceutical Importance
A simple and reliable method of capillary electrophoresis (CE) combined with field-amplified sample stacking, using hydroxypropyl methyl cellulose as electro-osmotic flow suppressant has been reported for separation and identification of TNF and Adefovir, using phosphate buffer solution containing 0.3% of hydroxypropyl methylcellulose and measured at 18 kV or 214 nm. The method has been successfully established for determination of TNF and Adefovir in bovine serum and which is suitable for pharmacokinetic study. The prominent characteristic as claimed by author is to be highly sensitive, cost-effective, relatively simple, and time-saving than other methods such as LC/MS/MS, HPLC-UV, Fluorescent derivation HPLC, and SPME-HPLC (Liu and Duan 2015). The electrochemical reduction of (TNF) was studied in Britton-Robinson (BR) buffer at the dropping mercury drop electrode (HMDE). A method based on square-wave cathodic adsorptive stripping voltammetry (SWCAdSV) was studied and validated for assay of TNF in human plasma and a tablet formulation. Sample preparation of plasma involved protein precipitation with acetonitrile. The method reported to be linear in the concentration range 0.5−5.0µg/ml. The method was reported to quite inexpensive compare to other methods (Jain and Sharma 2013). Quantification of TDF in pharmaceutical formulations via chemical derivatization using p-chloranilic acid and sodium nitroprusside as a reagent have been studied as extractions free spectrophotometric methods determination of TDF in bulk drugs and pharmaceutical formulations (tablets). The first method reported to be based upon the charge transfer complexation reaction between drug as anelectron donor and p-chloranilic acid (p-CA) as a p-acceptor to form a violet chromogen measured at 531 nm. The colored product depicts linearity in the concentration range of 2-10 µg/mL. The second method was reported to be based on the formation of light green molecular complex with sodium nitroprusside in presence of hydroxylamine under alkaline conditions and exhibiting λ-max at 401 nm and showed linearity in theconcentration range of 2-10 µg/ml (Disha and Gurupadayya 2013). A solid-phase extraction (SPE) method has been studied and validated on a liquid chromatography coupled with a mass spectrometer for the determination of plasma concentrations of TDF and EMT in HIV patients. Separation was achieved with a gradient (Acetonitrile and water with formic acid 0.05%) on an Atlantis 4.6 mm×150 mm, reversed-phase analytical column. Detection of TNF, EMT, and internal standard (IS) was achieved by electrospray ionization mass spectrometry (ESI-MS) in the positive ion mode. Plasma was analyzed, and the limit of quantitation was 15.6 ng/mL for TNF and 11.7 ng/mL for EMT; limit of detection was 2 ng/mL for TNF and 1.

Conclusion
Review of literature reported during the period of 2008 -2016 described analytical methods for estimation of TDF. Different analytical methods implemented for analysis of TDF in bulk and in its combined dosage forms and in plasma include LC-MS/MS, HPLC, HPTLC and UV-Spectroscopy methods. Fig. 2 depicts method prevalence for pharmaceutical analysis of TDF and Fig. 3 give comparative account on various methods published using different analytical techniques for TDF. One of the most commonly used solvents for sample extraction of TDF from pharmaceutical formulation and also from biological fluids is methanol and acetonitrile.
The detection of HPLC analysis of most of methods was monitored at 260 nm with PDA/UV detector. The mobile phase composition for the separation of TDF includes methanol, acetonitrile and buffer or their combination in appropriate proportion. The present review article can provide wisdom to the reader about the various methods employed for analysis of TDF and update them with several options for analysis of TDF. The date compiled may be useful for the further analysis of TDF.