Int J App Pharm, Vol 12, Issue 6, 2020, 41-50Review Article

A REVIEW OF ANALYTICAL TECHNIQUES FOR DETERMINATION OF ANTI-HIV DRUGS

RAJEEV KUMAR MISHRA1*, NEELESH CHAUBEY1, JAY RAM PATEL2, SATISH MISHRA3, ROHIT SINGH4

1Sri Satya Sai University of Technology and Medical Science sehore M. P. India, 1Dean Pharmacy, Sri Satya Sai University of Technology and Medical Science Sehore M. P., 2Rungta Institute of Pharmaceutical Science and Research, Bhilai Durg C. G. India, 3Gulabkali Memorial College of Pharmacy Chakghat Rewa M. P India, 4Aditya College of Pharmacy Satna M. P. India
Email: [email protected]

Received: 14 Jul 2020, Revised and Accepted: 23 Sep 2020


ABSTRACT

Pharmaceutical analysis plays a very prominent role in quality assurance as well as quality control of bulk drugs and pharmaceutical formulations. Rapid increase in pharmaceutical industries and production of drug in various parts of the world has brought a rise in demand for new analytical techniques in the pharmaceutical industries. As a consequence, analytical method development has become the basic activity of analysis. From the times of yore, people were trying to find safe and sound ways to treat viral infections. In the current scenario, due to the emerging of new viruses, the development of drugs for their treatment is also gaining equal importance. Before launching to the market, these drugs should undergo a validation process. High-performance liquid chromatography (HPLC) coupled with ultraviolet (UV), Photodiode array detectors (PDA), Mass spectrophotometer (MS) detectors etc. is one of the fastest, safe and precise technologies used for determination and separation of pharmaceutical drugs, impurities and biological samples. HPLC is versatile and it takes less time for quantification of drugs as compared to old liquid chromatography techniques. Tenofovir disoproxil fumarate (TDF), Emtricitabine (FTC) and Efavirenz (EFV) is antiretroviral medicine used treat AIDS as well as chronic Hepatitis-B. It is used alone or with other HIV medications to help control HIV infection. The present review article assesses the published analytical methods and a variety of approach for investigation of TDF, FTC and EFV in bulk drug as well as pharmaceutical formulations including combinations. The present studies revealed that HPLC technique along with the spectroscopic have been most widely explored for the analysis. The investigatory review may provide the comprehensive details to the researchers who are working in the area of analytical research of TDF, FTC and EFV.

Keywords: Pharmaceutical analysis, High-performance liquid chromatography, Tenofovir disoproxil fumarate, Emtricitabine, Efavirenz


INTRODUCTION

The main goal of the pharmaceutical industry is to provide drug products with sufficient quality, efficacy and safety. The development of a new drug product and its production consist of many pharmaceutical processes, including analytical testing. The analytical data generated support further decisions on how development should be pursued or provide information on whether a drug product should be released. It is important that each such development or production process provide credible results with constant quality and therefore, it needs to be controlled and, if necessary, continually improved. By improvement of the quality of a pharmaceutical process, the quality of a drug product is also improved. Analytical methods are among the most critical processes in drug product development and production. They play a key role in supporting other development and production processes throughout all stages of a drug product’s life cycle. It is essential that an analytical method be precise, accurate and reliable, making it suitable for its intended purpose [1, 2]. In most cases, the main working principle of an analytical method is separation of the analytes present in the sample. Liquid chromatography (LC) techniques are most commonly employed, such as HPLC or ultraperformance liquid chromatography (UPLC), often in reversed-phase mode with UV absorbance detection. The purposes of analysis differ depending on the number, importance and relation of analytes that are required to be determined. Analytical methods for the assay of an active pharmaceutical ingredient (API) or determination of its related substances and degradation products are most commonly applied [2]. Development of a specific and robust stability-indicating LC method for the determination of related substances and degradation products is a complex process. It requires a deliberate forced degradation of a drug substance and/or a drug product under various stress conditions, such as hydrolytic, oxidative, photolytic, or thermal conditions, to provide stressed samples containing the analyte and its degradation products. The stress conditions are more severe than the accelerated and long-term stability conditions prescribed in the ICH guidelines for stability testing. An analytical method for determination of degradation products should be capable of detecting their increase during the product’s shelf life and the method for the assay should be capable of detecting any decrease in the drug substance’s content during its shelf life. Such methods are stability indicating [3-6].

Recent estimates indicate that 34 million people are currently living with HIV/AIDS worldwide, with approximately 2.5 million new infections occurring annually [7]. The virus is transmitted through the exchange of virus containing fluids, including blood, breast milk, semen and genital secretions [8-10]. Routes of viral infection include sexual contact, injection drug use, from mother to child during pregnancy, childbirth, or breast-feeding, and exposure of infected body fluids to exposed membranes or tissue [10, 11]. Antiretroviral therapy (ART) is the primary modality for the treatment and management of the disease and can substantially reduce HIV-related morbidity and mortality [12-14]. ART is strongly recommended for all HIV-infected individuals, regardless of pretreatment CD41 T cell count. Furthermore, ART has shown efficacy not only in disease management but also in viral prevention as pre-exposure prophylaxis in high-risk populations [15-18]. There are currently more than 25 antiretroviral (ARV) agents approved for HIV treatment by the U. S. Food and Drug Administration (FDA) in both single-and multi-drug formulations [19]. Combinatorial ART regimens are typically required for the sustained suppression of viral replication and clinical benefit [20]. Currently, more than 100 regimens exist for the treatment of HIV [21]. ARVs elicit their therapeutic effects through the targeted inhibition of various stages of the viral infection cycle. Thus, drug classes are stratified as CCR5 antagonists, viral fusion inhibitors, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs/NtRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), integrase strand transfer inhibitors (INSTIs), and protease inhibitors (PIs). Many combinatorial ART regimens incorporate drugs from more than one ARV class, and the U. S. Department of Health and Human Services (DHHS) has indicated recommended and alternative regimens for disease management [22]. In addition, new therapies are continually being sought that exploit new viral targets, have activity against resistant viral strains, have a lower incidence of adverse effects, and offer convenient dosing. New agents of existing classes are currently in advanced stages of clinical development [23]. The growing demand for these agents stimulates a search for new even more effective drugs, but also calls for higher level of quality control of these therapeutic substances and preparations, so that they are in the highest possible degree free from any impurities that may come from the production process, as well as from decompositions products of active or auxiliary substances. Therefore, it seems appropriate to develop new analytical methods regarding their qualitative and quantitative analysis. For this aim, different analytical methods are used for determining anti-HIV drugs. Anti-HIV drugs are the recent developments of drugs and there is a great need to review the analytical work reported so far in the literatures. Efforts have been made to collect the literature from 2000 up to the present. Analytical methods allowing the determination of TDF, FTC and EFV drugs in various media, such as pharmaceutical formulations, biological matrices and environmental samples, is discussed. At present, there are five major classes of ARV drugs viz. nucleoside reverse transcriptase inhibitor [NRTI], non-nucleoside reverse transcriptase inhibitor [NNRTI], Protease inhibitors [PI], fusion inhibitor and integrase inhibitor [IIs].

The first single-tablet fixed-dose combination (FDC) antiretroviral (ARV) has been commercially available since 2006 and is marketed as Atripla® [24]. A generic product has been commercially available in South Africa since April 2013 [25, 26] and consists of efavirenz (EFV), emtricitabine (FTC) and tenofovir disoproxil fumarate (TDF) in a ratio of 600 mg/200 mg/300 mg. TDF in this quantity is equivalent to 245 mg tenofovir disoproxil (TNF) and 136 mg of tenofovir [27]. The tablet is taken once daily for the treatment of HIV-1 infection [28, 29]. Once-daily FDC tablets are the simplest antiretroviral therapy available [30]. FDC ARV therapy is convenient for patients as it reduces the “pill burden” which in turn improves adherence to therapy [28-30]. FDC were initially indicated for treating HIV-positive antiretroviral naïve patients and HIV-positive pregnant women and those who are breastfeeding. It is now available to all patients on the recommendation of a physician [26]. Treatment with EFV, FTC and TNF is the preferred first-line therapy for antiretroviral naïve HIV-1-infected persons [30]. Bioequivalence between the dosage form containing a single molecule and the FDC in addition to favourable pharmacokinetics facilitates once daily dosing of EFV, FTC and TNF [24, 30].

Nucleoside reverses transcriptase inhibitors (NRTIs)

The first generation of ARV drugs is NRTIs permitted to treat HIV [31]. Reverse transcriptase is an HIV enzyme that converts viral RNA into DNA in host CD4 cells and the process is known as reverse transcription. NRTIs inhibit the enzyme reverse transcriptase and prevent the synthesis of DNA. Without reverse transcriptase, HIV cannot replicate and infection cannot spread. Nucleoside analogues possess structural similarity with the natural building blocks of DNA and have to undergo phosphorylation to become active in the body. NRTIs are falsely chosen by reverse transcriptase to build the faulty DNA that denies further addition of natural nucleotides. Thus, the new DNA built incorrectly led to halt HIV replication [32]. Following are some NRTIs used for HIV therapy: Zidovudine, Didanosine, Stavudine, Lamivudine, Abacavir, Adefovir, Emtricitabine (FTC), Tenofovir disoproxil fumarate (TDF).

Emtricitabine (FTC)

FTC is a synthetic fluoro derivative of thiacytidine with potent antiviral activity approved in 2003. Chemically it is a 4-amino-5-fluoro-1-[[2R, 5S]-2-[hydroxymethyl]-1, 3-oxathiolan-5-yl] pyrimidin-2-one, the solubility of which in water is 112 mg/ml with logP value of-1.4. FTC is a white to off white, crystalline powder [33, 35]. FTC has an empirical formula of C8H10FN3O3S and a relative molecular mass of 247.2 g/mol [34, 36]. FTC contains no less than 99.0 percent and not more than 101.0 percent of emtricitabine (C8H10FN3O3S), calculated with reference to the anhydrous reference material [34]. FTC, when combined with TDF, has shown together greater HIV RNA suppression compared to the combination of Zidovudine and Lamivudine [37, 38]. Co administration of FTC/TDF with antiviral drugs that eliminate through kidney by means of active tubular discharge may enhance plasma TDF or FTC concentrations and/or those of simultaneously given drugs [39]. FTC undergoes phosphorylation to form active FTC triphosphates metabolite using cellular kinase enzymes. FTC and phosphorylated metabolite give varying pharmacokinetic results [40]. The molecules mimic normal nucleos(t)ides that are incorporated into DNA at the 3’ terminus. However FTC and TNF lack the 3’-OH and their incorporation at the 3’ terminus of the DNA therefore terminates chain elongation by preventing incorporation of additional nucleotides [28, 41, 42]. TDF diphosphate and FTC 5’-triphosphate are weak inhibitors of α, β, ϒ cellular DNA polymerases and FTC 5’-triphosphate weakly inhibits DNA polymerase ε [28]. The combination of TNF and FTC has been the preferred NRTI regimen since 2003 since approval by FDA [30]. A single-tablet combination has been approved by the FDA and more recently by the Medicines Control Council (MCC) of South Africa for pre-exposure prophylaxis (PrEP) although it is not widely used clinically [43, 44]. The HIV-1 reverse transcriptase (RT) mutation K65R is a common multi-drug resistance mutation that confers resistance to NRTI including TNF and FTC [11, 42, 45, 46] and this mutation may be responsible for cross-resistance between different NRTI [42, 46]. Cases of acute renal failure and Fanconi syndrome (FS) have been reported in patients treated with TNF [47] although clinically important renal toxicity is rare [48]. FTC and TNF undergo limited systemic metabolism [49].

Tenofovir disoproxil fumarate (TDF)

TDF is the acyclic nucleotide analogue of adenosine monophosphate approved for HIV treatment in 2004 [50]. Chemically, it is an [[2R]-1-[6-aminopurin-9-yl]propan-2-yl]oxymethyl phosphonic acid, the solubility of which in water is 13.4 mg/ml at 25 °C with a logP value of-1.6. TDF has an empirical formula of C19H30N5O10P, C4H4O4 and a relative molar mass of 635.5 g/mol. It occurs as a white to almost white crystalline powder [51-53]. TDF contains no less than 98.5 percent and not more than 101.0 percent of TDF (C19H30N5O10P, C4H4O4), calculated with reference to an anhydrous reference material [52]. TDF refers to the solid/raw material whereas TNF refers to TDF in solution and tenofovir peaks in chromatograms. TDF is phosphorylated twice to the active diphosphate form. High fat meal increases the bioavailability of TDF and remains unaffected by normal meal [54]. As TDF is eliminated through the kidney and is not a substrate for CYP45, its dosage regimen needs to be modified in renal complications [55, 56]. Gervasoni et al. showed that HIV-infected females with diminished body weight are in danger to be exposed to elevated TDF plasma trough concentrations, eventually bringing about a huge threat to produce long-term TDF complications [55].

Non-nucleoside reverse transcriptase inhibitor [NNRTI]

NNRTIs restrain the process of viral DNA synthesis by directly binding to the hydrophobic pocket of reverse transcriptase enzyme [31]. Unlike NRTIs, which must be phosphorylated to prevent HIV from infecting the cell, NNRTIs are active in the form administered. NNRTIs are classified as 1st generation and 2nd generation NNRTIs. 1st generation NNRTIs include Nevirapine and Efavirenz [EFV] and 2nd generation NNRTIs are Etravirine and Rilpivirine. HIV-2 is naturally resistant to NNRTIs.

Efavirenz (EFV)

EFV is a benzoxazin analogue approved by FDA in 1998 for the treatment of patients infected with HIV [57]. Chemically, it is an [4S]-6-chloro4-[2-cyclopropylethynyl]-4-[trifluoromethyl]-1H3,1-benzoxazin-2-one and occurs as a white to slightly pink crystalline powder [58-60]. The empirical formula for EFV is C14H9ClF3NO2 and the relative molar mass is 315.7g/mol [58, 60, 61]. EFV contains no less than 97.0 percent and not more than 103.0 percent of C14H9ClF3NO2 calculated with reference to the anhydrous reference material [58]. The solubility of which in water is 0.093 mg/l at 25 °C with logP value of 4.6. The dosing of EFV is once-daily due to its long half-life. EFV is usually preferred to treat HIV patients co-infected with tuberculosis [TB]. Both the diseases are life-threatening and treatment becomes very difficult due to drug-drug interactions between EFV and rifampicin [62, 63]. Side effects of EFV are found to be associated with the EFV plasma concentration. Various side effects are associated with high and low plasma levels of EFV particularly in HIV-TB co-infected patients for which TDM studies become necessary. EFV levels are directly correlated with optimum therapeutic output and central nervous system side effects. Therefore, TDM of EFV in clinical practice is essential for optimum therapeutic output, especially in HIV-TB co-infected patients who are under treatment with the combination of EFV and rifampicin. On the other hand, EFV possesses high protein binding property [>99%] and thus gets penetrated into male genital tract through blood. High penetration in male genital tract makes it an important candidate to study its concentration for prophylaxis use. HIV replication took place inside the cell, so ARV drugs have to enter the cells at an adequate concentration to restrain viral replication. Subsequently, studying intracellular drug concentration is a valuable tool to ascertain effective levels of ARVs in target cells mainly in virological failure regardless of efficient plasma level concentrations.

Dissociation constant (pKa)

EFV is a weak acid with a pKa of 10.2. It is therefore ionised at high pH, at which the carbonate moiety undergoes deprotonation to form a negatively charged species. The trifluoromethyl and ethylene moieties are most likely responsible for the lowering of the pKa [64, 65]. The pKa of FTC and TNF are 2.65 and 3.75 respectively [35, 36, 66]. EFV is a weak acid whereas FTC and TNF are weak bases.

Solubility

EFV is practically insoluble in water (9.0μg/ml) but is freely soluble in methanol [14, 31, 38, 39]. FTC is freely soluble in methanol and water (112 mg/ml) and is practically insoluble in dichloromethane R [34-36, 58]. TNF has a solubility of 13.4 mg/ml in distilled water at 25 °C [51].

Biopharmaceutical classification system (BCS)

The BCS provides a framework to classify molecules into categories based on their aqueous solubility and membrane permeability. Class 1 drugs have high solubility and high permeability, class 2 drugs low solubility and high permeability, class 3 drugs high solubility and low permeability and class 4 drugs low solubility and low permeability. EFV has low aqueous solubility and high intestinal permeability and is classified as a Class 2 molecule. FTC has high aqueous solubility and high intestinal permeability and is classified as a Class 1 molecule. TNF, the form of tenofovir that is aborbed, has high aqueous solubility and low intestinal permeability and is classified as a Class 3 molecule [67, 68].

Melting range

EFV melts within the range of 139-141 °C [69]. FTC melts within the range of 136-140 °C [70]. TNF melts within the range of 276-280 °C [71].

(A) (B) (C)

Fig. 1: Chemical structure of (A) Emtricitabine, (B) Tenofovir, (C) Efavirenz, reported analytical methods for TDF, FTC and EFV

Spectrophotometric methods

Many analytical methods involving spectroscopic analysis of the drug individually and as multicomponent samples have been reported. These methods include a simultaneous equation method, derivative spectrophotometric method, absorption ratio and a method based on Q analysis.

Chromatographic method

Liquid chromatographic analysis for the determination of TDF, FTC and EFV individually and in combination has been reported covering different phases of analytical research viz; profiling of impurities, stability indicating analytical methods, bioanalytical method development in different biological fluids to determine the concentration of TDF, FTC and EFV in human serum and to determine simultaneously in synthetic mixture or combination dosage form.

Stability indicating method

Stability indicating method is used to check drug stability under different conditions. Here, TDF, FTC and EFV are studied by RP-HPLC and UPLC for stability studies.

Table 1: RP-HPLC/UPLC methods for determination of TDF, FTC and EFV

S. No. Name of drug/formulation /biological fluid Column Mobile phase composition Detection (nm) Ref.
1 FTC-Tablet Peerless basic C18 (50 mm x 4.6 mm, 3μm) Buffer (pH 3.0): methanol-90:10% (v/v) 280 nm 72
2 FTC-Nanoparticles Phenomenex C18 (250 mm × 4.6 mm, 5𝜇m) 40 mmol phosphate buffer (pH 6.8), methanol and 2% acetonitrile (83: 15: 2, v/v/v) 280 nm 73
3 FTC-Tablet Phenomenex C18 (250 mm × 4.6 mm, 5𝜇m) 10 mmol phosphate buffer (pH 6.8) methanol-2% acetic acid (73: 25: 2, v/v/v) 280 nm 74
4 FTC-Capsule Luna RP-18(2),250X4.6 mm, 5 µm Buffer: acetonitrile (85:15 %v/v) 280 nm 75
5 FTC-Capsule Phenomenex (Torrance, CA) C8 250× 4.6 mm 0.03M Phosphate buffer (pH 4.86±0.02): acetonitrile: methanol (40:20:40 v/v/v) 280 nm 76
6 FTC/TDF/Elvitegravir/Cobicistat-Tablet Inertsil ODS 3V C18 (250 mm×4.6 mm, 5 μm, 100Å) A =KH2PO4 (0.02M) pH 2.5, B= acetonitrile 240 nm 77
7 FTC/TDF-Tablet Hypersil, 250 X 4.6 mm, 5µ Buffer (pH 3.7): acetonitrile 60:40 (v/v) - 78
8 FTC/TDF/Rilpivirine-Tablet Inertsil C18 (150x4.6 mm, 5 µm) 0.1N Phosphate buffer(pH: 4): acetonitrile (40:60v/v) 275 nm 79
9 FTC/TDF-Tablet Inspire C18 (150×4.6 mm) 5.0 µm Buffer (pH 2.5): methanol (30:70 v/v) 272 nm 80
10 FTC/TDF-Tablet Inspire C18 (4.6×250 mm) 5 µm Mixed buffer (KH2PO4 and K2HPO4) pH 3: ACN (30:70v/v) 273 nm 81
11 FTC/TDF/Rilpivirine–Tablet Kromasil C18 (250 mm × 4.6 mm, 5µ) 0.01N Potassium dihydrogen phosphate and acetonitrile 65:35 (v/v/) 279 nm 82
12 FTC/TDF/Elvitegravir/Cobicistat-Tablet Atlantis C18 (100×4.6 mm, 5 μm) Gradient mixture of 0.1% trifluoroacetic acid and acetonitrile 240 nm 83
13 FTC/TDF–Plasma Hypersil C18 (250 mm×4.0 mm, 5 μm) sodium dihydrogen orthophosphate buffer (pH 6.9) and methanol (96:4) 259, 265,280 nm 84
14 FTC/TDF/Rilpivirine Thermo Hypersil ODS C18 (150×4.6 mm, 5µ) Acetonitrile and Phosphate buffer pH 3 (60:40) 260 nm 85
15 FTC/TDF-Tablet DIKMA (50 x 2.1 mm, 1.7µ) Phosphate buffer (pH 5.6) and methanol 60:40 240 nm 86
16 FTC/TDF-Tablet Hypersil TM BDS C18 120A (250 × 4.60 mm, 5µ) Methonal and phosphate buffer pH 2.5 (65:35 % v/v) 261 nm 87
17 FTC/TDF/Bictegravir-Tablet Zodiac C18 150x4.6 mm, 5µ Buffer and Acetonitrie (55:45 v/v) 272 nm 88
18 FTC/TDF-Tablet Phenomenex-Luna C18 (25 cm x 4.60 mm, 5 µm) 10 mmol phosphate buffer (pH 6.8): acetonitrile; 40: 60 (v/v) 260 nm 89
19 FTC/TDF/Elvitegravir/Cobicistat-Tablet ODS (250 × 4.6 mm, 5 µm)

A= (potassium dihydrogen orthophosphate, pH 2.5)

B= (acetonitrile) 55:45% v/v

250 nm 90
20 FTC/TDF-Tablet Phenomenax Luna C18 (150 mm x 4.6 mm, 5 µm) Acetonitrile: methanol: water 30:50:20 (v/v) 258 nm 91
21 FTC/TDF-Tablet BEH C18 (100 mm × 2.1, 1.8 µm) 0.68% potassium dihydrogen orthophosphate buffer of pH = 6 and methanol 45:55 v/v 261 nm 92
22 FTC/TDF-Tablet Luna C18 (25 cm x 4.60 mm, 5 µm) Acetonitrile: potassium dihydrogen phosphate buffer (pH 3.0±0.05): triethylamine 70:30:0.5(v/v) 260 nm 93
23 FTC/TDF-Tablet Promosil C18, (250 mm, 4.6 mm, 5 µm) Methanol: Phosphate cushion 68:32 % v/v. 259 nm 94
24 FTC/TDF-Tablet Inertsil ODS C18 (250 mm x 4.6 mm, 5 µm) 0.1% triflouro acetic acid (TFA) buffer and methanol 39:61 (v/v) 261 nm 95
25 FTC/TDF/Cobicistat/Elvitegravir-Tablet Kromasil C18 (250×4.6 mm, 5 µm) Orthophosphoric acid buffer: acetonitrile (55:45 %v/v) 240 nm 96
26 FTC/TDF-Tablet Phenomenax Luna C18 (250 mm x 4.6 mm, 5 µm) Methanol: phosphate buffer pH-3 (70:30 v/v) 258 nm 97
27 FTC/TDF/Dolutegravir-Tablet Phenomenex kinetex Biphenyl 250x4.6 mm, 5 µm A= ammonium acetate (10 mmol) pH 3.0, B= Acetonitrile, ammonium acetate (10 mmol) pH 3.0 and methanol 70:15:15%v/v/v 260 nm 98
28 FTC/TDF/Cobicistat, Elvitegravir-Tablet Kromasil C18 (250 mm x 4.6 mm x5 µm) 0.01N KH2PO4(pH 2.5) and acetonitrile (43:57v/v) 254 nm 99
29 FTC/TDF/Cobicistat, Elvitegravir-Tablet Hypersil BDS C18 250x4.6 mm, 5 µ, 100A. 0.05M Phosphate buffer pH 3.0 and acetonitrile 95:5 240 nm 100
30 FTC/TDF/Rilpivirine-Tablet Agilent C18 (250 × 4.6 mm, 5 μm) 0.1%Formic acid: acetonitrile (65:35%, v/v) 250 nm 101
31 TDF-Tablet Phenomenex Luna C18 (250 x 4.6 mm x 5 μm) Orthophosphoric acid (pH 3.0): acetonitrile: aethanol (40:50:10% v/v) 254 nm 102
32 TDF/lamivudine-Tablet HypersilTM BDS C18 120A (250×4.60 mm, 5µ) Acetonitrile and phosphate buffer pH 3.5 (80:20% v/v) 260 nm 103
33 TDF/lamivudine-Tablet Inertsil C18 column (15 cm x 4.6 mm, 5 µm) 6.5 mmol Phosphate buffer pH 2.5 and acetonitrile (50:50 v/v) 260 nm 104
34 TDF-Tablet Acquity UPLC BEH C18 (1.7 µm, 1 mm X 50 mm) 0.1% Formic acid and acetonitrile - 105
35 TDF/lamivudine Waters X-terra RPC18 (150 x 4.6 mm, 3.5 µm) A= (ammonium acetate buffer, pH 5.0±0.05) and B= (methanol and ammonium acetate buffer 20:80) 260 nm 106
36 TDF/lamivudine-Tablet C18 (Inertsil ODS 3V, 250 mm x 4.6 mm; 5µ) Phosphate buffer (pH3.5) and acetonitrile 55:45 v/v 264 nm 107
37 TDF-Tablet HiQ Sil C18HS (250 mm×4.6 mm, 5.0 µm) Methanol: water (60:40, v/v) 260 nm 108
38 EFV-Tablet Welchrom C18 (4.6 X 250 mm, 5 µm) 10 mmol Phosphate buffer (pH3.0): acetonitrile (50:50 v/v) 246 nm 109
39 EFV-Plasma Waters X-Terra Shield, C18 50 x 4.6 mm, 3.5 μm Phosphate buffer pH 3.5 and acetonitrile 260 nm 110
40 EFV/Lamivudine/Stavudine-Tablet Inertsil ODS C18(4.6 X 250 mm, 5.0μm) Phosphate Buffer (pH 4): methanol 30:70 v/v 254 nm 111
41 EFV-Tablet C18 250 x 4 mm (10 µm) ACN: water: 85% H3PO4 (70:30:0.1) 252 nm 112
42 EFV-Tablet Develosil ODS HG-5 RP 150 mm × 4.6 mm 5 µm Phosphate buffer (pH 3.1) and acetronitrile 40:60. 249 nm 113
43 EFV-Tablet Waters XBridge (4.6× 250 mm, 5 µm) Ammonium format buffer (pH 5): ACN (28:72 v/v) 247 nm 114
44 EFV/Lamivudine-Tablet Acquity UPLC BEH Shield RP18 (50 × 3 mm, 1.7 µm) 10% acetonitrile in methanol and 10 mmol phosphate buffer (pH 4.0) 254 nm 115
45 EFV/TDF/lamivudine-Tablet Kromasil C18 analytical column (150 × 4.6 mm, 5 μm) 10 mmol phosphate buffer (pH 5.0) and methanol (30:70) 254 nm 116
46 EFV/TDF/lamivudine-Plasma Luna C18 (250 × 4.6 mm, 5µ) A: Water with 0.1% (THF); B: acetonitrile with 0.1% THF 254 nm 117
47 EFV/TDF/lamivudine-Tablet SHISEIDO C18 (250 x 4.6 mm, 5µ) Acetonitrile: 50 mmol phosphate buffer (pH 5.0) 256 nm 118
48 FTC/TDF/EFV-Tablet Zorbax SB-Phenyl, (250 mm X 4.6 mm), 5 µm A: Buffer pH 3.7 B: methanol, acetonitrile and tetrahydrofuran (50: 50: 2 v/v/v) 265 nm 119
49 FTC/TDF/EFV-Tablet Inertsil ODS 3V (250 x 4.6 mm, 5µ) A: 0.02M Sodium dihydrogen orthophosphate monohydrate B: methanol and water (85:15) 265 nm 120
50 FTC/TDF/EFV-Plasma Chromolith Performance RP-18e (100 × 4.6 mm) A: (0.1% formic acid), B: acetonitrile - 121
51 FTC/TDF/EFV Acquity UPLC BEH m) cµPhenyl (2.1×100 mm, 1.7 A: (Buffer, 1.0 ml of TEA in 1000 ml of water pH 4.0±0.05) and B: (buffer: acetonitrile (20:80, v/v) 265 nm 122
52 FTC/TDF/EFV-Tablet Phenomenex C8 (250 mm ×4.6 mm, 5 µm) Buffer pH: 7.0 acetonitrile and methanol (40:40:20 v/v) 262 nm 123
53 FTC/TDF/EFV-Tablet Kromosil C18 100 x 4.6,3.5µ 0.1M ortho phosphoric acid buffer and acetonitrile in 60:40 v/v 265 nm 124
54 FTC/TDF/EFV-Tablet XTerra symmetry C18 (4.6 x 150 mm, 3.5 µm) Di hydrogen sulphate: methanol 270 nm 125
55 FTC/TDF/EFV-Tablet Zorbax C8 column (150 mm x 4.6 mm, 5 µm) Buffer pH 2.4±0.02 and acetonitrile 70:30 v/v 252 nm 126
56 FTC/TDF/EFV-Tablet Acquity 50 mm×m 2.1 µUPLC BEH Phenyl 1.7 A: 0.2% TEA in water pH 6.5±0.05, B: methanol and acetonitrile 50: 50 (v/v) 260 nm 127
57 FTC/TDF/EFV-Tablet Hypersil BDS C18 column (25 cm × 4.6 mm 5 µm) Acetonitrile, phosphate buffer and water 280 nm 128
58 FTC/TDF/EFV-Tablet Thermosil C18 (100*4.6 mm, 5µ) Methanol and Triethylamine (pH 7) (70:30 V/V 260 nm 129
59 FTC/TDF/EFV-Tablet Inertsil ODS 3 V C18 (150 mm × 4.6 mm 5 𝜇m) Phosphate buffer (pH 3.5): acetonitrile (70: 30 v/v) 256 nm 130
60 FTC/TDF/EFV-Tablet Zorbax SB CN, (250 × 4.6 mm, 5 µm) A: methanol, B: buffer at pH 4.5 260 nm 131
61 FTC/TDF/EFV-Tablet HSS C18 (100 × 3 mm, 1.7 µ) 0.01 N Phosphate buffer (pH 4.5) and acetonitrile (40:60, v/v) 265 nm 132
62 FTC/TDF/EFV-Tablet Inerstil ODS C18 250x4.6 mm, 5 µm A: Buffer (0.05% Trifluro acetic acid in water) B: methanol 262 nm 133
63 FTC/TDF-Tablet C18 (250 x 4.6 mm) Methanol: distill water 60:40 v/v (pH-3) 260 nm 134
64 FTC/TDF/Rilpivirine-Tablet Inertsil ODS 3V C18 (250 mm×4.6 mm, 5 µm), 0.01M phosphate buffer (pH 4) and acetonitrile (30:70 v/v) 265 nm 135
65 FTC/TDF-Tablet Hi Q C18 W (150 mm: 4.6 mm, 5 μ) Buffer, methanol and acetonitrile (40: 50: 10) 265, 278 nm 136
66 FTC/TDF/Rilpivirine-Tablet Acquity BEH C18 (50 x 2.1 mm, 1.7 µm) Acetonitrile and phosphate buffer (pH 3)55:45(v/v). 261 nm 137
67 FTC/TDF/Lamivudine-Plasma ACE 5 CN (150 mm × 4.6 mm, 5 µm) 0.5% Formic acid in water and acetonitrile (55:45, v/v) - 138
68 FTC/TDF-Tablet Premsil C18 (250 mm×4.6 mm, 5 μm) Methanol: water (70:30 v/v) pH 3 273 nm 139
69 FTC/TDF-Tablet Zorbax SB-C8 5 μm, 4.6 × 250 mm 50 mmol Phosphate buffer (pH 6.0)-acetonitrile (50:50, v/v) 260, 280 nm 140
70 FTC RP C18 (25 cm×4.6 mm), 5 μm ACN, phosphate buffer (pH 4.4), and water 280 nm 141
71 FTC/TDF-Plasma Synergi Polar-RP, 2.0 mm×150 mm, 3% Acetonitrile/1% acetic acid, aq.) - 142
72 FTC/TDF-Plasma Chromolith Speed Rod RPC18 (50 mm×4.6 mm) Methanol,acetonitrile and ammonium acetate (pH 3.0, 40 mmol) (20:80, v/v) - 143
73 FTC/TDF-seminal plasma Atlantis T3 C18 (2.1 × 100 mm, 3 µm) A: Deionized water with 0.05% formic acid, B: methanol with 0.05% formic acid - 144
74 FTC/TDF-Tablet Agilent TC-C18 5 mm, 4.6´250 mm Methanol and phosphate buffer (30:70 v/v, pH 4) 261 nm 145
75 FTC/TDF-Tablet Inertsil ODS C18, (4.6 × 250 mm, 5 µm) Buffer: Acetonitrile (80:20) 259 nm 146
76 FTC/TDF/EFV-Tablet Hypersil BDS C18, 250x4.6 mm, 5 μm Acetonitrile and 0.03M KH2PO4 (pH 3.2) 60:40 v/v  260 nm 147

Table 2: Spectrophotometric methods used for determination of TDF, FTC and EFV alone and in combined dosage form

S. No. Name of drug Sample matrix Method Detection (nm) Ref.
1 EFV Tablet Zero order 247 148
2 TDF Tablet Zero order 260 149
3 EFV/TDF/Lamivudine Tablet Simultaneous equation 247, 260, 272 nm 150
4 FTC Tablet Zero order and area under curve 280 nm, 272-286 nm 151
5 TDF Tablet Zero order 260 nm 152
6 EFV/TDF/Lamivudine Tablet Zero order 247, 260, 272 nm 153
7 FTC/TDF Bulk Simultaneous equation, Q–absorbance ratio method (1)280 nm, 260 nm (2) 251,237 nm 154
8 FTC/TDF Tablet Simultaneous equation eethod 282,261 nm 155
9 FTC/TDF Tablet Simultaneous Equation Method 281, 210 nm 156
10 FTC/TDF/EFV Tablet Simultaneous equation method 260, 241, 240 nm 157
11 FTC/TDF/Cobicistat/Elvitegravir Tablet Simultaneous equation method 283, 259, 240, 258 nm 158
12 FTC Tablet Zero order, first order derivative 241.1 and 232.7 nm 159
13 FTC/TDF Tablet Least square, first order, area under curve 281,260.5 nm; 234.5, 281 nm; 278-283 nm,258-262 nm 160
14 TDF Tablet Zero order, first order 260 nm, 273 nm 161
15 EFV/TDF/Lamivudine Tablet Simultaneous equation method, multicomponent analysis and derivative spectroscopy method 247, 259 and 272 nm 162
16 EFV/TDF/Rilpivirine Tablet Simultaneous equation method 240.8, 257.6, and 305.6 nm 163
17 EFV/TDF Tablet Simultaneous equation and Absorbance ratio method 250, 274 nm; 255, 274 nm; 164
18 EFV/TDF Tablet Ratio derivative spectra, first-order, absorption corrected method 271.07 and 302.17 nm; 224.38 and 306.88 nm 165
19 EFV/TDF Tablet Zero order 298 nm 166
20 TDF/Lamivudine Tablet Simultaneous equation method 271and 261 nm 167

Table 3: HPTLC methods for determination of TDF, FTC and EFV

S. No. Name of drug Formulation Stationary phase plates Mobile phase composition Rf Ref.
1 EFV/Lamivudine Tablet silica gel 60G F254 Ethyl acetate: methanol: formic acid 7.0:2.5:0.5 (v/v) Lamivudine= 0.57±0.02 EFV= 0.72±0.01 168
2 FTC/TDF/EFV Tablet silica gel 60F 254 Chloroform: methanol (90:10) FTC =0.15, TDF =0.34, EFV =0.55 169
3 FTC/TDF/Rilpivirin Tablet silica gel 60 F254 Methanol: toluene: ethylacetate: ammonia (1.5:5.5:1.5:0.1 v/v/v/v) Rilpivirin= 0.59, FTC= =0.29, TDF= 0.41 170
4 TDF/Lamivudine Tablet silica gel 60 F254, (20 × 10 cm) Chloroform: methanol: toluene (8: 2: 2, v/v/v) TDF= 0.51, Lamivudine= 0.27 171
5 EFV Tablet silica gel 60 F 254 Toluene: ethyl acetate: formic acid (10: 3: 1 v/v) 0.41±0.01 172
6 EFV Plasma silica gel 60F254 Dichloromethane: methanol (5:0.3 v/v) 0.69±0.01 173
7 TDF Tablet silica gel GF aluminum Ethyl acetate: methanol: formic acid(7:2.5:0.5 %v/v) 0.78 174
8 FTC/TDF/Rilpivirin Tablet silica gel 60 F254 Chloroform: ethyl acetate: methanol: glacial acetic acid (5:2:1:0.1 v/v/v/v) FTC =0.28, TDF =0.52, Rilpivirin= 0.70 175

CONCLUSION

The present review discussed about different analytical approach employed for the assessment of TDF, FTC and EFV. Profuse examinations have been accomplished including, Bio-analytical, HPLC, UPLC, HPTLC, UV/Vis-Spectroscopy, LC-MS, LC-ESI-MS etc. for evaluation of TDF, FTC and EFV in bulk and in its combination with other drugs from pharmaceutical formulations and also biological fluids. Liquid chromatography with UV detection has been found to be most studied for estimation of TDF, FTC and EFV in bulk as well as pharmaceutical dosage forms, while hyphenated LS-MS, LSMS/MS methods reported for determination of TDF, FTC and EFV and its metabolite in plasma and other biological fluids. Few chromatography approaches like HPTLC and Stability-indicating HPLC, UPLC and HPTLC are also reported. Few simple UV-Spectrophometric methods may be used for routine analysis of TDF, FTC and EFV alone and in combination with other drugs. These compiled data may of use for research for further studies in analysis of TDF, FTC and EFV.

FUNDING

Nil

AUTHORS CONTRIBUTIONS

All the authors have contributed equally.

CONFLICT OF INTERESTS

Declared none

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