INTRODUCTION:

Dolutegravir is an integrase strand transfer inhibitors (INSTIs) and acts by binding to the active site and blocking the strand transfer step of retroviral DNA integration in the host cell¹. Antiretroviral agents are usually administered in combination which has considerably lifted the average life span of HIV-infected individuals.

Currently dolutegravir is formulated singly or combined with other antiretroviral drugs like either abacavir–lamivudine or tenofovir–emtricitabine². Dolutegravir is (4R,12aS)-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-carboxylic acid³. Stability studies and impurity profiling are most crucial parts in quality control of pharmaceuticals and for the detection of the presence of degradants which helps in maintaining the safety, efficacy and potency of the drug product^4-7. The main aim of the study is to identify the unknown degradants present in Dolutegravir, for which the drug was subjected to forced peroxide degradation condition. The study was carried out on Agilent Infinity 1200 series instrument (Agilent Technologies, Santa Clara, California, USA) coupled to quadruple time-of-flight (Q-TOF LC–MS 2695) equipped with an electrospray ionization (ESI) source and software used was Mass lynx. The method developed was validated as per ICH guidelines^8,9. By identifying the degradation products of the dolutegravir the degradation pathways of the molecule as well as information about the degradants that could be formed either during storage, transportation or manufacture of API and drug products are obtained. Review of literature showed methods by RP-HPLC for the detection of degradants in Dolutegravir and few in serum plasma^10-12 and other chromatographic methods were reported using RP-HPLC and UPLC^13-18. A stability indicating reverse phase HPLC method was developed for simultaneous estimation of Lamivudine and Dolutegravir in bulk and tablet dosage form under stress conditions and the separation was achieved by injecting 10μL of the standard solution into Xbridge Phenyl (250 × 4.6mm, 5μ,100 A0) column, using a mobile phase composition of methanol: buffer (0.1% v/v trifluoroacetic acid in water) (85:15 v/v) and isocratic elution programming have been done at a flow rate of 0.8mL/min. The eluted analytes detected at 258nm wavelength¹⁶. Structure of Dolutegravir is given in (figure 1)¹⁹.

Fig. 1: Structure of Dolutegravir

MATERIALS AND METHODS:

Solvents and Reagents:

All solvents used were of LC grade. HPLC grade water procured from Martinsynge Pharma sciences Pt.Ltd, Aleap Industrial Estate, Hyderabad. HPLC grade Acetonitrile, trifluoroacetic acid were Procured from Finar Limited, Gujarat. Dolutegravir was obtained as a gift sample from Mylan Laboratories Limited (R and D Centre), Bollaram, Hyderabad, Telangana, India.

Instrumentation and chromatographic conditions:

Prominence preparative high performance liquid chromatograph (Shimadzu, Kyoto, Japan) equipped with a reverse phase C₁₈Inertsil ODS (300mm×19mm ×7µ) column. LCMS Agilent Infinity 1260 series instrument (Agilent Technologies, Santa Clara, California, USA) coupled to quadruple time-of-flight (Q-TOF LC–MS 2695) equipped with an electrospray ionization (ESI) source and software used was Mass lynx. The ESI source conditions were optimized as follows: fragmentor voltage, 150V; capillary voltage, 4000V; skimmer, 65 V; nitrogen was used as drying (350^◦C; 10L/min) and nebulizing (40 psi) gas. For full scan MS mode, the mass range was set at m/z 50–2000. Ultra-high purity nitrogen was used as collision gas. The separation of degradation products was achieved using a Prominence series liquid chromatograph (LC) Shimadzu, LC-MS 8030 spectrometer equipped with a photodiode array detector (Shimadzu, Kyoto, Japan) and a Pursuit column C18 Hypersyl BDS (250×4.6)mm, 5µ, Varian, North America)

Experimental Procedure:

The objective of the experiment was to identify the unknown degradants present in Dolutegravir for which the drug was subjected to forced Peroxide stress condition.

Preparation of Dolutegravir standard solution:

Stock solution of Dolutegravir was prepared by transfering accurately weighed quantity of 100mg of Dolutegravir into a 100ml of volumetric flask to which 70ml of diluent was added and sonicated for 30 minutes with intermittent shaking and made upto the mark with HPLC grade water. 10ml of this solution was transferred to a centrifuge tube and centrifuged at 5000 RPM for 10 minutes and then filtered through 0.45µm nylon syringe filter. Diluent used was a mixture of acetonitrile and water in ratio 50:50 v/v respectively.

Sample preparation for forced degradation study:

Oxidative degradation:

An accurately weighed amount of 100mg of Dolutegravir was transferred into 100ml of volumetric flask to which 10ml of 3% H₂O₂was addedand kept aside on a bench for 2 hrs. After which 70ml of diluent was added and sonicated for 30minutes with intermittent shaking and then diluted to volume with diluent and mixed well and the resultant solutions were used to perform the degradation studies.

Isolation of peroxide degradant by preparative chromatography:

The oxidative degradation sample was isolated using Prominence preparative high performance liquid chromatograph (Shimadzu, Kyoto, Japan) equipped with a reverse phase C₁₈Inertsil ODS (300mm × 19mm × 7µ) column. Mobile phase was 0.1% trifluro acetic acid in water (A) and acetonitrile (B) (50:50), optimized conditions are mentioned in (Table 1). The resolution was obtained in a gradient mode with a flow rate 18 ml/min. The peaks were monitored at 210nm. The peak RT at 24 is found to be degradant under peroxide degradation condition and it was isolated for further structural identification of characteristics. 100mg of peroxide degraded sample was taken in a 25ml volumetric flask and made up the volume with diluent acetonitrile and kept in a water bath for 30 min about 50-60^oC and injected 5ml/inj. Required fraction collected and distilled under rotary evaporator for removing the organic part and the aqueous layer extracted with ethyl acetate. Organic part is (acetonitrile and tri fluro accetic acid ) removed by rotary evaporator. Aqueous layer is extracted with ethyl acetate and it is distilled. The collected fractions were evaporated to dryness for LCMS studies.

Table 1: Preparative HPLC optimized conditions

Parameters	Optimized conditions
Mobile phase A	0.1% Trifluoro acetic acid (TFA) in water
Mobile phase B	Acetonitrile
Method	Gradient method (0/30, 15/90, 20/90, 21/30, 25/30)
Column	Symmetry C 18 (300×19 mm×7µ)
Column temperature	30^oC
Flow rate	18 ml/ min
ƛ_max	210 nm
Injection volume	5000µl
Diluent	Acetonitrile
Run time	25 min

LCMS studies on the degradation products:

The degraded samples were subjected to LC–MS analysis in positive ESI mode. A satisfactory separation of degradation products was achieved by using C₁₈ column.

LCMS studies were carried out on oxidative degradation sample. The exact masses of the molecular ion peaks and fragments of the degradation products were determined by parameters optimized for the drug.

Preparation of sample solution for LCMS:

Accurately weighed 1 mg of oxidative degradation sample was dissolved in DMSO in a 10 ml volumetric flask and the solution was made upto 10 ml with methanol (1mg/ml). From the above solution, 1 ml of solution was pipette out and volume was made up to mark with diluent to give a solution containing 1μg/ml concentration of the solution and injected into the LCMS system. Optimized conditions for LCMS are specified ( tables 2 and 3).

Table 2: LC Parameters

Parameters	Optimized conditions
Mobile phase A	0.1% Trifluro acetic acid (TFA)
Mobile phase B	Acetonitrile
Method	Gradient (95/5,80/20,10/90,0/100,95/5)
Column	Eclipse Plus C18 4.6×150mm, 3.5µm
Temperature	30^o C
ƛmax	210 nm
Flow	0.5 ml/min
Injection volume	20µl

Table 3: MS parameters

Parameters	Optimized conditions
Model	QT of LCMS
Probe	ESI +Ve
Scan Range	700-2000 m/Z
Capillary (V)	3500 V
Collision energy	40
Nebulizer gas (psig)	40
Drying gas temp	300^o C
Dry gas flow rate	10 l/min

RESULTS AND DISCUSSION:

The presence of API related impurities affects the safety, efficacy and quality of the drug product. Therefore, detection and quantification of degradants in active pharmaceutical ingredients is being made mandatory and acceptable limits are being set as guidelines by regulatory bodies. This study performed on dolutegravir provided useful data on the degradation behaviour of the drug under conditions of peroxide oxidation and the degradation products formed were identified by HPLC, LCMS analytical techniques. The LC-MS spectrum of Dolutegravir Impurity was recorded by Shimadzu LCMS spectrometer in the electron spray ionization mode and exhibits an intense protonated molecular ion peak at m/z 304.27 and 249.06 in the positive ionization mode, scan range 120–560 amu and the other possible degradant structures with m/z 320.32, m/z 207.18, m/z 185.17, m/z 385.41, m/z 155.14 also identified and shown (figure 2 and tables 4 and 5) respectively. The probable fragmentation pattern and shown in (figure 3). The degradation pathway of dolutegravir was delineated and structures of all the degradation products by comparison of the accurate mass and fragmentation pattern.

Table 4: Observed m/z values and % peak intensity of the major probable degradants

Sl No	Structures of probable degradants	m/z	Peak intensity (%)
1		304.27	100
2		249.22	100

Table 5: Observed m/z values and % peak intensity of the minor probable degradants

S. No.	Structures of degradants	m/z	Peak intensity (%)
1		320.32	20
2		207.18	15
3		185.17	40
4		385.41	30
5		155.14	55

Fig. 2: LC-MS spectra of peroxide degradant sample of Dolutegravir

CONCLUSION:

The stress study of antiretroviral drug confirmed that degradation occurs under peroxide degradation and provides useful information to keep in mind while development of formulation and the degradation chemistry of drug substances. Method can also be utilized for evaluation of stability that is physical and chemical when they are in the crystal form and point out degradants that could appear in the final product thereby under mining the efficacy. This understanding helps in development of analytical method, improvement in formulation and packaging development and the design of end products with increased shelf life. Information of degradation products of dolutegravir can be employed for qualification of impurities during manufacturing and stability studies of dolutegravir.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGEMENT:

The authors are grateful to NGSM Institute of Pharmaceutical Sciences and Nitte (Deemed to be University), Mangalore, Karnataka State, India for their support.

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Received on 19.02.2021 Modified on 06.03.2022

Research J. Pharm. and Tech 2023; 16(3):1012-1016.

DOI: 10.52711/0974-360X.2023.00169