Analytical Profiling of The Dolutegravir–Lamivudine Antiretroviral Combination: A Systematic Review

Dolutegravir and Lamivudine is a combination antiviral medicine used to treat human immunodeficiency virus (HIV), the virus that can cause acquired immunodeficiency syndrome (AIDS). Dolutegravir and Lamivudine is not a cure for HIV or AIDS [1]. Sold under the brand name Dovato, it contains dolutegravir, an integrase inhibitor, and lamivudine, a nucleoside reverse-transcriptase inhibitor [2].

Dovato is a prescription medicine approved by the U.S. Food and Drug Administration (FDA) for the treatment of HIV infection in adults who meet certain requirements, as determined by a healthcare provider. Dovato is a complete HIV treatment regimen and should not be used with other HIV medicines [3].

Dolutegravir is chemically designated as Isopropyl (4R,12aS)-N-(2,4-difluorobenzyl)-7-hydroxy-4-methyl-6,8-dioxo-3,4,6,8,12,12a-hexahydro-2H-pyrido[1',2':4,5] pyrazino[2,1-b][1,3] oxazine-9-carboxamide. Its molecular formula is C₂₀H₁₉F₂N₃O₅, with a molecular weight of 419.38 g/mol [4].

Dolutegravir is an integrase strand transfer inhibitor active against HIV type 1, with some in vitro activity against HIV type 2. It blocks the strand transfer step of viral genome integration into the host cell, thereby preventing HIV replication and lowering the amount of HIV in the blood [5]. Its mechanism has no homology in human host cells, which gives it excellent tolerability and minimal toxicity. Common side effects in clinical trials included insomnia and headache; serious side effects included allergic reactions and abnormal liver function in patients co-infected with hepatitis B or C [6].

Lamivudine is chemically designated as 4-Amino-1-[(2R,5S)-2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2-dihydropyrimidin-2-one. Its molecular formula is C₈H₁₁N₃O₃S, and its molecular weight is 229.26 g/mol.

Lamivudine (commonly called 3TC) is an antiretroviral medication used to prevent and treat HIV/AIDS, and is also used to treat chronic hepatitis B when other options are not possible. It is effective against both HIV-1 and HIV-2 [7]. Common side effects include nausea, diarrhea, headaches, fatigue, and cough. Serious side effects include liver disease, lactic acidosis, and worsening hepatitis B among those already infected [8]. The dual regimen of lamivudine and dolutegravir, administered once daily, maintains virological efficacy up to 24 weeks and is associated with improvements in immunologic and metabolic status [9].

This review summarizes various qualitative and quantitative analytical methods used for the estimation of dolutegravir and lamivudine.

2. QUALITATIVE ANALYTICAL TECHNIQUES

Qualitative analysis is performed to establish the composition of natural or synthetic substances. These tests indicate whether a specific substance or compound is present in a sample. Various qualitative tests include detection of evolved gas, formation of precipitates, limit tests, colour change reactions, determination of melting and boiling points, solubility, refractive index, and chromatographic techniques using reference standards.

2.1. Thin-Layer Chromatography (TLC)

TLC has been applied for the transfer of a method for the antiviral drug lamivudine (as described in Minilab Volume II, 2006, Method 6.21, pp. 112–115) and for the development of HPTLC-densitometry methods for coformulated antiviral drugs dolutegravir and tenofovir disoproxil fumarate. Lamivudine and TDF are simultaneously analyzed on one plate by scanning their separated fluorescence quenching bands at 254 nm, while dolutegravir is analyzed on a second plate by scanning absorbance at 366 nm.

Lamivudine was analyzed simultaneously with TDF on Merck Premium Purity silica gel 60 F plates using the mobile phase ethyl acetate–methanol–acetone–concentrated ammonium hydroxide (30:7:3:1) and densitometric scanning at 254 nm. Dolutegravir was analyzed on a second plate by scanning at 366 nm after chromatography with the chloroform–methanol–formic acid (32:8:2) mobile phase. Mobile phases were developed in a mobile-phase-vapor-saturated CAMAG twin trough chamber. Automated HPTLC-densitometry was performed using a CAMAG Scanner 3 controlled by winCATS software, with 4.00 mm × 0.45 mm micro slit dimensions and a 20 mm/s scan rate. L and TDF quenched fluorescence of a phosphor indicator in the silica gel layer and were scanned under 254 nm UV light from a deuterium lamp, while absorbance of dolutegravir was scanned under 366 nm UV light from a mercury lamp [10].

2.2. Electrophoresis Techniques

Electrophoresis techniques are particularly applicable to dolutegravir and lamivudine. A simple, specific, and cost-effective micellar electrokinetic chromatographic (MEKC) method has been developed for the simultaneous separation and determination of the nucleoside reverse transcriptase inhibitors (NRTIs) lamivudine (3TC) and stavudine (d4T), along with the non-nucleoside reverse transcriptase inhibitor (NNRTI) nevirapine (NVP). The method used an uncoated fused-silica capillary (length 73.5 cm, effective length 62 cm) as the stationary phase, with 10 mM sodium tetraborate, 100 mM sodium dodecyl sulfate (SDS) and 15% (v/v) 2-propanol as solvent. All analytes were separated within 14 min at a voltage of +20 kV [11].

Electrokinetic chromatography methods have also been developed for the separation and quantitative analysis of anti-HIV drug mixtures containing lamivudine (3TC), didanosine (ddI), and saquinavir in human serum, using an uncoated fused-silica capillary (length 52 cm, internal diameter 50 µm) with diltiazem as an internal standard. Detection was at 210 nm with a voltage of +20 kV [12].

2.3. X-Ray Powder Diffractometry (XRPD)

X-ray powder diffraction (XRD) is a rapid analytical technique primarily used for phase identification of a crystalline material and can provide information on unit cell dimensions [13]. Kommavarapu et al. [14] published an X-ray procedure for the identification and determination of the degree of crystallinity in abacavir, lamivudine, and nevirapine drug products.

3. QUANTITATIVE ANALYTICAL TECHNIQUES

Analytical techniques for quantitative analysis help an analyst to accurately determine the concentration of individual components in a test sample. Classical quantitative analysis uses mass or volume changes to quantify amounts. Quantitative instrumental methods include chromatography, field-flow fractionation, and electrophoresis for separation; and physical properties such as absorption, fluorescence, conductivity, and light scattering for precise measurement of analytes [15].

3.1. UV Spectroscopy

A simple, rapid, precise, and accurate spectrophotometric method has been developed for quantitative analysis of dolutegravir and lamivudine in active pharmaceutical ingredients (API) or their dosage forms, either alone or in combination with other active components [16]. All spectroscopic methods for the determination of dolutegravir and lamivudine are summarized in Table 1.

A UV spectroscopic method for the estimation of dolutegravir in bulk and formulation was proposed by Vaishnavi et al. [17] using methanol as the solvent. Samples were scanned and analyzed at 254 nm, with linearity over 10–50 µg/mL. Similarly, Naveen et al. [18] reported a validated UV spectrophotometric method with methanol as solvent and absorbance measured at 260 nm, with linearity between 5–40 µg/mL.

A UV spectrophotometric method for lamivudine in API and tablet formulations was proposed by Anandakumar et al. [19] using distilled water as solvent at 271 nm (linearity: 5–40 µg/mL). Sonar et al. [20] estimated lamivudine in tablets at 268 nm with linearity at 6–14 µg/mL. Mohammed et al. [21] reported a UV spectrophotometric method for lamivudine and tenofovir in pharmaceutical dosage forms at 271 nm and 260 nm, respectively, both with linearity of 10–60 µg/mL.

Nagulwar et al. [22] simultaneously estimated lamivudine, nevirapine, and zidovudine in combined tablet dosage forms using UV derivative spectroscopy at 280.2 nm, 312 nm, and 266.8 nm, respectively, with linearity ranges of 5–25 µg/mL, 5–50 µg/mL, and 5–40 µg/mL. Elvis et al. [23] reported assay of lamivudine in API and tablet formulations based on UV absorbance at 270 nm, with linearity of 5–15 µg/mL.

Table 1: UV Spectroscopy Methods for Dolutegravir and Lamivudine

3.2. High-Performance Thin Layer Chromatography (HPTLC)

High-Performance Thin Layer Chromatography (HPTLC) is a simple, fast, and inexpensive chromatographic separation technique used to identify compounds based on Rf values, and is useful for monitoring reaction completion in synthetic laboratories.

Venkatesh et al. [24] published a study on lamivudine and zidovudine in combined tablet dosage forms using HPTLC. Chromatograms were developed using toluene:ethyl acetate:methanol (4:4:2 v/v/v) mobile phase on pre-coated silica gel GF aluminium TLC plates, quantified by densitometric absorbance at 276 nm. The Rf values were 0.41 ± 0.03 and 0.60 ± 0.04 for lamivudine and zidovudine, respectively. Similarly, Sudha et al. [25] reported simultaneous determination of lamivudine and abacavir sulphate in combined tablet dosage forms using acetone:chloroform:methanol (4:4:2 v/v/v) mobile phase on aluminium plates precoated with silica gel 60F254, detected at 265 nm.

3.3. High-Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) is one of the most accurate methods widely used for quantitative and qualitative analysis of drug products, and for determining drug product stability. HPLC is an analytical tool that can detect, separate, and quantify drugs, their impurities, and drug-related degradants that may form during synthesis or storage. All HPLC methods for the determination of dolutegravir and lamivudine are summarized in Table 2.

The RP-HPLC method for determination of dolutegravir was proposed by Venkatnarayana et al. [26] using 0.1% trifluoroacetic acid and water (50:50 v/v) mobile phase on a C8 column, UV-Vis detection at 240 nm, flow rate of 1.0 mL/min, and a retention time of 7.40 min. A stability-indicating RP-HPLC method for simultaneous quantification of dolutegravir and lamivudine was presented by Noorbasha et al. [27] using phosphate buffer pH 3.0, acetonitrile, and methanol (50:20:30 v/v/v) on an Inertsil ODS 3V column, detected at 257 nm. Retention times were 6.36 min for dolutegravir and 2.16 min for lamivudine.

Godela et al. [28] reported an RP-HPLC method for dolutegravir and lamivudine using methanol and buffer (85:15 v/v) on an Xbridge Phenyl column at 258 nm, with retention times of 5.0 min and 3.4 min, respectively. Dhanwate et al. [29] used buffer pH 3, acetonitrile, and methanol (55:35:10 v/v/v) on a C18 column at 260 nm, with retention times of 15 min for dolutegravir and 5 min for lamivudine. Bhavar et al. [30] developed an RP-HPLC method for dolutegravir sodium using acetonitrile and water (80:20 v/v) on an ODS C18 column at 260 nm.

Naveen et al. [31] presented an RP-HPLC method for dolutegravir and rilpivirine in bulk and plasma using 0.1% orthophosphoric acid and acetonitrile (60:40 v/v) on a Phenomenex C18 column at 262 nm. A stability-indicating method for the same combination was reported by Ismail et al. [32] using phosphate buffer pH 3.5 and acetonitrile (45:55 v/v) on a Thermosil C18 column at 260 nm, and by Kanchipogu et al. [33] using 0.1% OPA and acetonitrile (50:50 v/v) on a Waters X-Bridge C18 column at 245 nm.

Sindu Priya et al. [34] reported a stability-indicating method for simultaneous determination of abacavir, dolutegravir, and lamivudine by RP-HPLC on a Kinetex 5µ C18 100A column at 258 nm. Pal et al. [35] used buffer and acetonitrile (65:35 v/v) on a Non-Polar Kromasil column at 257 nm. Mallikarjuna Rao et al. [36] described a stability-indicating HPLC method for lamivudine, tenofovir, and dolutegravir on a C18 RP column at 260 nm. Kanawade et al. [37] estimated the same three-drug combination using Luna C8 column at 260 nm, and Saravanan et al. [38] used a Symmetry C18 column at 260 nm.

Additional HPLC methods have been reported for lamivudine alone and in combination. Hefnawy et al. [39] used a Monolithic Column at 285 nm. Singh et al. [40] estimated lamivudine in rabbit plasma using a Hypersil BDS C18 column at 256 nm. Uslu et al. [41] and Sivasubramanian et al. [42] reported methods for lamivudine and zidovudine. Singh et al. [43] simultaneously estimated lamivudine and raltegravir. Jayaseelan et al. [44] and Hariprasad et al. [45] reported methods for lamivudine and stavudine. Diwan et al. [46] and Gorja et al. [47] reported methods for lamivudine with efavirenz and tenofovir, respectively. Rajkumar et al. [48] reported a method for lamivudine, abacavir, and dolutegravir.

Table 2: RP-HPLC Methods for Dolutegravir and Lamivudine