Trends in Bioanalytical Method Development for Antiretroviral Drugs: A Review

INTRODUCTION:

Antiretroviral drugs are a key component of HIV treatment, and their development has seen significant advancements. Currently, more than 35-40 antiretroviral drugs are available, including various fixed-dose combinations. These drugs work by targeting different stages of HIV life cycle. Arts & Hazuda (2012), . The antiretroviral drug development has been marked by the introduction of novel formulations and new drug classes to address issues like drug toxicities and resistance Saag et al (2020). The early regimens of antiretroviral therapy were complex, requiring multiple drugs to be taken at different intervals throughout the day, leading to challenges in long-term adherence. However, advancements in drug development have led to the simplification of treatment regimens, with many patients now being able to control their HIV with a Single pill once a day. Bioanalytical methods are vital for measuring antiretroviral drug levels in biological samples, supporting drug monitoring, adherence checks, and treatment evaluation Charbe et al (2019),Mohammad et al (2016). HPLC is widely used to quantify antiretroviral drugs in biological matrices for therapeutic monitoring. Method development and validation are crucial to ensure accurate, reliable measurements, supporting better patient care and treatment outcomes Deshpande et al (2019).

This review highlights key bioanalytical methods, including HPLC-UV, LC-MS/MS, and UPLC, for the estimation of anti-HIV drugs. These techniques are essential for pharmacokinetic evaluation in HIV treatment.

The diagram below FDA approved anti-HIV drugs over the years as depicted in Figure 1.

Fig.1 Approved Antiretroviral drugs

MATERIALS AND METHODS:

2.Overview of Antiretroviral Drugs

Antiretroviral drugs represent a cornerstone in the treatment of AIDS. These medications, classified based on their mechanism of action, play a pivotal role in suppressing viral replication. Understanding the classification and mechanisms of action of these drugs is essential for optimizing treatment strategies effectively Pau and George (2014).

2.1 Classification of Antiretroviral Drugs

Antiretroviral drugs are categorized into several classes, each targeting specific steps in the HIV life cycle.

a) Nucleoside/Nucleotide Reverse Transcriptase Inhibitors (NRTIs): NRTIs mimic natural nucleosides and, once incorporated into viral DNA, block further synthesis, halting HIV replication.

b) Non-Nucleoside Reverse Transcriptase Inhibitors (NNRTIs): NNRTIs bind directly to the reverse transcriptase enzyme, inhibiting its function and preventing the conversion of viral RNA into DNA.

c) Protease Inhibitors (PIs): PIs block the activity of the HIV protease enzyme. PIs prevent the formation of infectious viral particles.

d) Integrase Strand Transfer Inhibitors (INSTIs): INSTIs block the integration of viral DNA into the host genome by inhibiting the HIV integrase enzyme and prevent the formation of a persistent viral reservoir replication cycle.

e) Entry Inhibitors: Entry inhibitors interfere with viral entry into host cells by targeting either the viral envelope glycoprotein which is the co-receptor for HIV entry Holec et al (2017).

2.2 Mechanism of Antiretroviral Drugs

Fig.2 Mechanism of ARV Drugs

RESULT:

3.Bioanalytical Techniques for Estimation of Anti-HIV

3.1 Chromatographic methods

Ultra-rapid chromatographic techniques like HPLC and UPLC are widely used for analyzing antiviral drugs, enabling simultaneous quantification with high sensitivity and specificity. Mass spectrometry (MS) and tandem mass spectrometry (MS/MS) are the primary detectors used, offering excellent sensitivity and selectivity. Among these, LC-MS and LC-MS/MS have become the leading methods for determining ARV drugs Meng (2013). Fluorescence detection (FLD) offers a selective, robust, and cost-effective alternative to MS/MS in chromatographic analysis. Fast and stability-indicating HPLC methods are essential for analyzing antiviral drugs and metabolites. Though liquid-liquid and solid-phase extraction work well for plasma and urine, complex matrices like animal tissues require specialized preparation. A stationary phase in chromatographic systems is vital for the separation process, as the elution or retention of different substances depends on stationary phase properties. Column selection is important for sensitive and rapid analytical results Zuvela (2019).

The type of column packaging materials greatly impacts separation efficiency. Current trends focus on developing durable, high-performance, and environment friendly columns compatible with a wide range of mobile phases. With growing environmental concerns, green analytical chemistry is gaining prominence. Hassana et al. (2020) proposed a micellar HPLC-UV method for simultaneous estimation of Sofosbuvir and Daclatasvir in plasma and urine. HPTLC, an advanced form of TLC, offers several benefits for compound analysis Tuzimski and Sherma (2016). Samia M.et al.(2018) developed and validated a simple, efficient, and cost-effective HPTLC method for the simultaneous determination of Sofosbuvir and Ledipasvir in rabbit plasma. This method provided a straightforward, rapid, and economical alternative compared to reported LC-MS/MS methods.

3.2 Spectroscopic methods for Anti-HIV drugs

Spectrometric methods, based on light absorption or emission, such as UV, VIS, IR, fluorescence, and Raman spectroscopy, are widely used in pharmaceutical research for drug analysis and quality assessment. These techniques are especially valuable in developing regions due to their simplicity, cost-effectiveness, minimal sample requirements and reliable structural insights Mishra et al (2020). In HIV therapy, ARV combinations are vital, but genetic differences can impact drug bioavailability. Monitoring levels using HPLC with UV, MS detection is necessary for optimizing treatment. Tuzimski and Petruczynik (2020). Mansour (2017) introduced a spectrometric method to determine the concentration of Sofosbuvir and Ledipasvir in binary mixtures by identifying the intersection wavelength, both drugs were simultaneously quantified, with specific linear ranges and limits of detection (LODs).

Zaman et al. (2017) establish a simple spectrophotometric Synchronous fluorescence spectroscopy (SFS) method for determining sofosbuvir in vitro dissolution media. SFS offers a simple, selective, and sharp method for the simultaneous quantification of multiple analytes. Fourier transform infrared (FTIR) spectroscopy offers a non-destructive, solvent-free method for directly analyzing Daclatasvir dihydrochloride and Sofosbuvir in tablet formulations Mohamed A.et al., (2018).

Table No.1. Chromatographic methods for determination of anti-HIV drugs

Drugs	Method	Matrix	Stationary Phase (Column)	Mobile Phase	Flow rate	Ref
3TC/DTG	HPTLC	Tablet	Hypurity Advance (4.6x 50mm,5µm)	0.1% formic acid buffer–acetonitrile (20:80%v/v)	0.25ml/min	(Venkatesh and Daggumati 2012)
3TC/TDF	RP-HPLC	Tablet	Phenomenax Luna C18 (150mm x 4.6 mm,5µm)	Acetonitrile: Methanol: Water (30:50:20v/v)	1.0ml/min	(Karunakaran et al 2012)
DRV	HPLC	Blood mononuclear cells (PBMCs)	YMC-Pack Pro C18 (250×4.6mm,5µm)	Potassium phosphate buffer: Acetonitrile (57:43v/v)	1.0mL/min	(Nagano et al 2013)
ZDV	RP-HPLC	Human Plasma	Phenomenex C18 (250×4.6 mm, 5 μm	methanol and 0.1% orthophosphoric acid (40:60 %v/v),	1.0mL/min	Nalanda R. Baby (2016)
EFV	HPLC	Human Plasma	BDS hypersil C18 (15cm, 4.6mm x 5μm)	Acetonitrile: 50mM sodium dihydrogen orthophosphate (pH 5.5,48:52v/v)	0.8mL/min	(Deepakumari et al 2017)
ABC/3TC	HPLC	Tablet	Symmetry Premsil C18 (250mm × 4.6mm,5μm)	Methanol: Water (0.05% OPA, (pH 3,83:17v/v)	1.0ml/min	(Sufiyan Ahmad et al 2018)
FTC/TDF	RP-HPLC	Tablet	Inspire C18 (150×4.6mm 5.0μm)	OPA Buffer (pH 2.5 with NaOH): Methanol (30:70v/v)	1.0ml/min	(Sattar and Achanta 2018)
3TC/ABC/DTG	RP-UPLC	Tablet	Zodiac sil RP C18 (4.6 mm × 250 mm, 3.0μm)	Phosphate buffer: Methanol (30:70%v/v)	0.25ml/min	(Dubey and Duggirala 2018)
PAR/RTV/OMB	HPLC	Tablet	RP-C18 Kinetix (150mm×4.6mm,5μm)	MP-A: 0.15 M SLS, 0.01 M of sodium dihydrogen phosphate dihydrate (pH 6.2) MP-B: Ethanol	1ml/min.	(Ibrahim et al 2019)
PAR/RTV/OMB	HPTLC	Tablet	Aluminum silica gel 60F254 pre-coated plates (20 ×20 cm)	Methylene chloride: Methanol: Ethyl acetate: (5:1:3:1v/v/v/v) Ammonia (25%)		(Ibrahim et al 2019)
DTG	RP-HPLC	Tablet	Kromasil C8(150 × 4.6 mm 5μm)	MP-A: 0.1% of trifluoroacetic acid MP-B: Methanol (Gradient elution)	1.0mL/min	(Venkatnarayana and Jyothi 2020)
LPV	HPLC‐UV	Rat Plasma	Phenomenex Gemini (C18, 150 mm × 2.0 mm, 5 μm)	water and acetonitrile	0.3 mL/min	Chaolong Qin (2020)

Table No. 2. UPLC-MS/MS methods for determination of anti-HIV drugs

Analyte	Method	Matrix	Stationary phase (column)	Mobile phase	Flow rate	Ref
ATV/DRV/RTV	UPLC-MS/MS	Human Plasma	Waters Acquity UPLC C18 (50 × 2.1mm, 1.7μm)	10mM Ammonium Formate: acetonitrile (50:50 v/v)	0.300mL/min	(Mishra and Shrivastav 2014)
3TC/ZDV/LPV/RTV	UPLC-MS/MS	Breast milk	ACQUITY UPLC1BEH C18 (2.1 ×50mm,1.7μm)	MP-A:0.1% formic acid in water MP-B: ACN (90:10% v/v)	0.50mL/min	(Ramirez et al 2018)
DAC/ELB/GZR/ LDV/SIM/SOF/VEL	UPLC-MS/MS	Human Plasma	Acquity UPLC BEH C18 (2.1 x 50mm,1.7μm)	10mM ammonium formate with MP-A: 0.005% formic acid in water (pH 4.5) MP-B: 0.005 % formic acid in Methanol MP-C: MP B 0.005 % formic acid in a mixture of methanol and acetonitrile (10:90)	0.4ml/min.	(Seyen et al 2018)
DTG/EVG/RAL/NVP/ETV	UPLC-MS/MS	Human Plasma	Acquity UPLC BEH C18(2.1 x 50mm1,7μm)	MP-A:0.1% formic acid in water MP-B:0.1% formic acid in acetonitrile (Gradient elution)	0.475mL/min	(Pauline et al 2018)
ABC/ATV/BIC/DTG/ EFV/EVG/FTC/ 3TC/NVP/RAL/RPV/RTV/TFV/ETV	UPLC-MS/MS	Human Plasma	Acclaim TMRSLC 120 C18 (2.1 × 100 mm, 2.2μm)	0.1% (v/v) formic acid in water: acetonitrile	0.45mL/min	(Gouget et al 2020)

Table No. 3. LC-MS/MS methods for determination of anti-HIV drugs

Analyte	Matrix	Stationary phase (column)	Mobile phase	Flow rate	Ref
TFV/FTC/3TC	Human Plasma	ACE 5 CN column (150 mm × 4.6 mm, 5 μm)	0.5% formic acid in water and acetonitrile (55:45, v/v).	1 ml/min	Manish Yadav et al (2010)
ZDV/ABC/FTC/ 3TC/TFN/RBV	Human Plasma	Synergy Polar RP C18 column (150mm×2.0mm ID,4μm)	0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in methanol	300μL/min	(W. Kromdijk et al (2013)
LPV/RTV/TFV	Human Plasma	Synergi column (100 × 2.0 mm; 4-μm)	buffers A (water with 0.1% HAc) and B (acetonitrile with 0.1% HAc)	0.35 ml/min	Josefin Koehn (2014)
Maraviroc	Human Plasma	Waters BEH C8, 50 × 2.1 mm UPLC column, 1.7 μm	Mobile phase A: water with 0.1% formic acid Mobile phase B: 0.1% formic acid	1 ml/min	Emory J. F. (2014)
TFV	HPLC-MS/MS Human Plasma	C18 (Phenomenex Kinetex TM 30 mm × 2.1 mm, 2.6 µm)	10 mmol/L ammonium acetate in water and acetonitrile/methanol (50:50, v/v)	-	Mulubwa et al (2015)
EFV	Dried breast milk	Fortis TM C18 column: 3 mm, 100 mm×2.1 mm	1 mM ammonium acetate in water/acetonitrile	400 mL/min	Adeniyi Olagunju et al (2015)
BIC	Human Plasma	Kinetex EVO C18 column, 50 × 3.0 mm, 5 µm	80:20 acetonitrile/water with 0.1% formic acid	0.250 mL/min	Pavan Kumar Prathipati et al (2018)
EFV/3TC/TDF/FTC	Dried blood and dried breast milk spots	Synergi polar column (80A, 150 ×2.0 mm, 4μm	0.1% formic acid in water and 0.1% formic acid in acetonitrile	400 μL/min	Catriona Waitt et al (2017)
TFV/EFV	Mouse vaginal tissue, Vaginal lavage and Blood plasma	RP C18 column (3 m, 100 × 2.1 mm)	0.1% (v/v) formic acid in water and 0.1% (v/v) formic acid in acetonitrile	0.35 mL min−1	Luisa Barreiros et al (2017)
βLAP	Human Plasma	Phenomenex Kinetex C18(2.1×50mm, 1.7μm)	MP-A:0.1% formic acid in water MP-B:0.1% formic acid in acetonitrile (Gradient elution)	0.5mL/min.	(Putnam et al 2020)

CONCLUSION :

This review summarizes key analytical methods from 2011 to 2020 for detecting antiviral agents in biological, environmental and pharmaceutical samples. Techniques like, spectrophotometry, HPTLC, HPLC, UPLC, LC-MS, LC-MS/MS are widely used, especially in serum and plasma analysis. HPLC, particularly with reverse-phase C18/C8 columns and acidic mobile phases, remains the preferred method due to its reliability, cost-effectiveness, and broad accessibility. UV detection often lacks the sensitivity needed for antiviral analysis in biological samples. LC-MS/MS is the gold standard for pharmaceutical analysis in complex matrices due to its high sensitivity and specificity. The rising use of antivirals, developing robust, selective and sensitive analytical/Bioanalytical methods is crucial.

ACKNOWLEDGEMENT: Not applicable

SOURCE OF FUNDING: Not applicable

CONFLICT OF INTEREST: None.

ABBREVIATIONS:

OPA	Ortho Phosphoric acid	SOF	Sofosbuvir	TFV	Tenofovir
DRV	Darunavir	LDV	Ledipasvir	EFV	Efavirenz
DTG	Dolutegravir	3TC	Lamivudine	TDF	Tenofovir Disoproxil Fumarate
ZDV	Zidovudine	RTV	Ritonavir	TAF	Tenofovir Alafenamide
ABC	Abacavir	FTC	Emtricitabine	NVP	Nevirapine
DAC	Daclatasvir	βLAP	β-lapachone	BIC	Bictegravir
COBI	Cobicistat	EVG	Elvitegravir	RPV	Rilpivirine
LPV	Lopinavir	ELB	Elbasvir	GZR	Grazoprevir
SIM	Simeprevir	VEL	Velpatasvir	PAR	Paritaprevir
OMB	Ombitasvir	RAL	Raltegravir	ETV	Entecavir
ATV	Atazanavir	RBV	Ribavirin	EVG	Elvitegravir

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Trends in Bioanalytical Method Development for Antiretroviral Drugs: A Review

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