Development of Visible Spectrophotometric Method and its Validation for Dolutegravir in Tablet Dosage Form

 

Vijaykumar T. Pawar¹, Mrunalee D. Magadum²*

¹Assistant Professor, Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy, Kolhapur - 416013 Maharashtra, India.

²Student, Department of Pharmaceutical Chemistry, Bharati Vidyapeeth College of Pharmacy,

Kolhapur - 416013 Maharashtra, India.

 

ABSTRACT:

Objective: There are several methods for analyzing the same, however, they are time-consuming and costly. We created a new spectrophotometric method for determining Dolutegravir (DLT) in tablet dosage forms that is simple, accurate, and precise. We developed and validated a simple, accurate, and precise colorimetric method for the quantitative analysis of Dolutegravir in bulk and dosage form by ICH recommendations in this study. Methodology: There are several methods for analyzing the same, however, they are time-consuming and costly. We created a new spectrophotometric method for determining Dolutegravir (DLT) in tablet dosage forms that is simple, accurate, and precise. In methanol, the initial stock solution of Dolutegravir was prepared. The method is based on the formation of a blue color chromogen complex from Dolutegravir oxidation-reduction with Ferric chloride in the presence of potassium ferricyanide. Result: The color complex was measured at 710nm. Beers law was observedin the concentration range of 3.5-6.5μg/ml witha coefficient of correlation (R²) was 0.998. The system suitability criteria were found to be within the limits. The LOD and LOQ were found to be 0.91 and 2.47, indicating that the method is sensitive. Conclusion: The relative standard deviation (RSD) and percent recovery values were found to be satisfactory, indicating that the proposed method is suitable, accurate, and precise and that it can be used in routine analysis of Dolutegravir in tablet dosage forms, with relatively low-cost solvents.

 

 

 

INTRODUCTION:

The human immunodeficiency virus (HIV) is a blood-borne lentivirus and retrovirus that causes acquired immune deficiency syndrome.6,80,000 (4, 80, 000–1.0 million) people died from HIV-related causes and 1.5 million (1.0–2.0million) people acquired HIV in 2020¹.

 

Medications for HIV treatment work at many stages of the viral life cycle, including entrance, reverse transcription, integration, and maturation. Antiretroviral drugs treat HIV infection, reduce morbidity, and slow the development of the disease. Co-receptor inhibitors, nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), fusion inhibitors, non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors, and integrase strand transfer inhibitors (INSTIs) are one of them¹. human immunodeficiency virus (HIV) integrase inhibitors have a potent new class of drugs for HIV-infected individuals².

 

Dolutegravir (Tivicay) is a second-generation integrase strand transfer inhibitor (INSTI) that was approved by the Food and Drug Administration on August 12, 2013, for the treatment of HIV in both treatment-naive and treatment-experienced patients¹. Whose integrase potential and binding half-life are considerably superior to those of raltegravir and elvitegravir, conferring its distinctive characteristics regardingits genetic barrier to resistance and action against viruses with one or more integrase mutations³. Dolutegravir was chemically (RS)(4R, 12aS)-N-(2, 4- difluorobenzyl)-7-hydroxy-4-methyl-6, 8-dioxo-3, 4, 6, 8, 12, 12a-hexahydro-2H-pyrido 11, 21, 4, 5pyrazino 2, 1- b1, 3oxazine-9-caboxamide.The molecular formula is C₂₀H₁₉F₂N₃O₅ and the molecular weight is 419.4g/mol. It is slightly soluble in water and methanol⁴

 

The pharmacokinetic features of Dolutegravir allow once-daily dosing (50 mg), with or without food, maintaining concentrations far above those effective against wild-type viruses³. Dolutegravir binds to the viral enzyme integrase's active site and inhibits it from activating¹. This inhibits HIV from incorporating its DNA into the DNA of the host cell, effectively inhibiting the retroviral DNA integration strand transfer phase¹.

 

The author intended to develop a simple, economical, selective, accurate, and precise UV spectrophotometric method for the determination of Dolutegravir in bulk and pharmaceutical dosage forms using the hydrotropic solubilization technique and validated according to ICH guidelines⁴. Considering the availability of several analytical techniques, spectrophotometry continues to play a significant role in pharmaceutical micro/nanogram analysis ⁴. Using an ultraviolet-visible spectrophotometer and chromatographic analysis methods is simple, time-consuming, and labor-intensive⁴.

 

To this extent, no simple colorimetric technique for Dolutegravir (using Fe3+/potassium ferricyanide) has been developed. Considering the above, a simple, sensitive, and extraction-free colorimetric technique for Dolutegravir was attempted utilizing Fe3+/ potassium ferricyanide as the chromogenic reagent. The same method worked well for determining Dolutegravir in pharmaceutical formulations⁵.

 

Spectrophotometric measurement of several medications relies significantly on iron [III] salts. The ferric form of iron (Fe3+) functions as anoxidizing agent, causing the analyte under investigation to oxidize before being reduced to the ferrous (Fe2+) form. The later ions generate a chromophoric complex with the reagent, which has a maximum in the visible range. Because of its capacity to form complexes with various metal ions, potassium ferricyanide is an inorganic compound that is used as a redox indicator⁵. The proposed method is based on the oxidizing property of Dolutegravir which is found to quantitatively reduce the ferric (III) form of iron to ferrous (II) form, which is then made to interact with potassium ferricyanide to give blue-colored chromogen complex, whose absorbance is measured as its max of 710nm. Fe(II) and/or ruthenium (II)-1, 10-phenanthroline complex determination is widely documented in literature⁵.

 

A literature survey revealed that there are several methods available for determining Dolutegravir individually and in combined dosage forms such as the Uv-visible spectrophotometric technique⁵, UV spectroscopic method using hydrotropic solubilizing agents⁶, HPLC method for itsstereoisomers^1,7, HPLC and HPTLC methods for its salt analysis^8,9, UPLC method¹⁰ and bioanalytical methods using HPLC¹¹ and HPLC- mass spectroscopy¹². However, some of these methods are costlier and time-consuming.

 

Fig. 1: Chemical structure of Dolutegravir.

 

Fig 2: probable redox reaction between Dolutegravir and Fe³⁺/potassium ferricyanide

 

MATERIAL AND METHOD:

Instrument:

A. Shimadzu UV-Vis Spectrophotometer Model V-1900,

B. Ultrasonic cleaner,

C. Shimadzu ATX- 124 Electronic balance.

 

Chemicals and Reagent:

All the chemicals and reagents used in the spectrophotometric analysis were of AR grade. A pharmaceutically pure sample of Dolutegravirwas obtained as gift samples from Strides Pharma Science Ltd, (Banglore, India). Methanol, 0.2% w/v Ferric chloride, 0.3% w/v potassium ferricyanide was of analytical grade. Tablets – Instagram, manufactured by Emcure Pharma LTD. Containing 50 mg per tablet was purchased from the market.

 

EXPERIMENTAL:

Preparation of standard stock solution of DLT:

The standard stock solution of DLT was prepared by accurately weighing 50mg of pure DLT drug was transferred into a 50ml volumetric flask and dissolved in AR grade methanol solvent was diluted with Methanol to make the final 50ml. This solution was kept at temp 60-70ºcfor 10minutes in an ultrasonic cleaner for sonication after the sonication volume was made up to the mark with the same solvent asthe concentration of a stock solution (1000μg/ml). 1ml of this stock solution was taken and then diluted up to 10ml by using AR grade methanol to produce a concentration of 100μg/ml.

 

Preparation of 2% w/v Ferric chloride solution:

Accurately weighed 0.20gm of ferric chloride was added in 10ml volumetric flask distilled water added in same and the volumetric flask was kept in ultrasonic cleanerat temp 50-60ºC for 10 minutes. The flask was removed, and the distilled water was used to make the final volume up to the mark.

 

Preparation of 3% w/v potassium ferricyanide reagent solution:

Accurately weighed 0.30gm of potassium ferricyanide was added in a 10ml volumetric flask. The volumetric flask was kept in bath ultrasonic cleanerat temp 50-60ºC for 10minutes. The flask was removed, and the distilled water was used to make the final volume up to the mark.

 

Determination of λmax:

The standard stock solution of DLT was prepared by dissolving 5mg of DLT in 50ml of AR grade methanol to produce a concentration of 100μg/ml which is the standard stock solution scanned at different concentrations in the range of 400-800nm and the λmax was determined against reagent blank. The maximum absorbance for the colored solution was found to be 710 nm.

 

Preparation of standard calibration curve:

From the stock solution of (100μg/ml), aliquots were transferred to a 10ml volumetric flask to get aconcentration of 3.5-6.5μg/ml. To each volumetric flask, 0.2ml offerric chloride solution was added and the contents of the flask were mixed for 2-3 minutes. After mixing of contents 0.2ml of potassium ferricyanide reagent solution was added to each volumetric flask. The final volume up to the mark was maintained by adding waterto it. The flask was kept in ultrasonic cleaner at temp50-60⁰C for 10 minutes. The blank solution was prepared in the same manner asdescribed above but omitting standard DLT.

 

Fig 3: Absorption spectrum of Dolutegravir with Fe³⁺ and potassium ferricyanide

 

The absorbance of the resulting blue-colored solution was measured at 710nm by using a spectrophotometer. The calibration curve of the drug was then plotted by taking the absorbance obtained on the y-axis and the concentration of the solutiononthe x-axis. (Fig.4). The curve showed linearityin the concentration range of 3.5-6.5μg/ml with a correlation coefficientof 0.998.

 

 

Fig. 4: Standard calibration curve of DLT.

 

 

 

Determination of DLT in marketed tablet dosage formulation:

The method was extended for the determination of DLT from tablets that were purchased from the local market. Tablets are weighed individually and triturated in mortar and pestle. Powder equivalent to 50mg of DLT was weighed accurately and dissolved in 50ml methanol. Whatman no. 42 paper was used to filter the resultant solution. Then the filtrate was diluted to 10ml with distilled water. The procedure given for the standard calibration curve was then followed for the development of color.

 

Optimization of reagent volumes and conditions:

The volume of reagent concentrations needed to achieve maximumabsorbance for the solutions has been optimized.

 

Method Validation:

The method was validated according to International Council for Harmonization guidelines for parameters like linearity, accuracy, specificity, precision, the limit of detection (LOD), and the limit of quantification (LOQ) of the analyte.

 

Table 1.

 

RESULT AND DISCUSSION:

Linearity:

Standard calibration curve for DLT, covering the range 3.8-7.1μg/ml, prepared by serial dilution with methanol and its reduction reaction with ferric chloride and potassium ferricyanide for pure drug and tablet formulation was developed and validated. The procedure was adopted as per desired protocol, based on ICH guidelines. The calibration curve was obtained by plotting absorbance Vs analyte concentration. The slope and intercept of the calibration line weredetermined by linear regression; Shown in table no. 2.

 

Table 2: Evaluation data of Linearity.

 

Fig 5: Linearity overlay of DLT

 

Accuracy (Recovery studies):

Calculating DLT recovery using the standard addition method was used to determine the method's accuracy. To the sample solutions, known concentration of was added in different levels viz., 80, 100 and 120% levels. The amounts of DLT were recorded and calculated as per the ICH guidelines. This concentration was repeated three times. The results are shown in (Table 3).

 

 

Table 3: Recovery study of DLT from tablet samples.

 

 

Table 4: Result of specificity study Precision.

 

Specificity:

Calculating the recovery of Dolutegravir was used to determine the method's specificity. The 20 Dolutegravir tablets were triturated, and an amount was added to the volumetric flask that was equivalent to a concentration of 1000 (g/ml). From this stock solution of 1000(μg/ml), the solution was pipette out 3, 6, 9 (μg/ml) to make the concentration added in 10 ml volumetric flask, and the final volume was made up to the mark with the methanol and the absorbance was consideredas 710 nm.

 

Precision:

Six samples of the same concentration (15g/ml) were taken for the repeatability study, the absorbances were examined and the percent RSD was calculated. The acceptable limit should be less than 2%. The results are shown in Table 5.

 

Table5: Repeatability study of DLT.

 

Intraday precision:

Six different solutions with the same concentration of 5ug/ml were analyzed three times a day i.e in the morning, afternoon, and evening and the absorbances were noted for the intraday precision study. From the absorbance, result mean, standard deviation and %RSD were calculated. The acceptable limit for intraday variation should be within 1%. Results were shown in Table 6.

 

Table 6: Intraday precision for DLT.

 

Inter Day Precision:

For inter-day precision studies, solutions of the same concentration of 5μg/ml were analyzed three times for the three consecutive days and the absorbance result wasobserved. Mean, standard deviation, and %RSD was calculated. The acceptable limit for interday variation should be within 2%. Results are shown in (Table 7).

 

Table 7: Interday precision for DLT.

 

LOD:

Based on the standard deviation of the blank: Measurement of the amount of analytical background response was performed by analyzing the six replicates of blank samples and calculating the standard deviation of these responses by using the formula,

LOD = 3.3 σ/S .

Where, σ = Standard deviation of the response and S = Slope of the corresponding calibration curve.

LOD=3.3*2.42/0.122

        =0.91

LOQ:

Based on the standard deviation of the blank: Measurement of the amount of analytical background response was performed by analyzing the six replicates of blank samples and calculating the standard deviation of these responses by using the formula,

LOQ = 10 σ/S.

Where, σ = Standard deviation of the response and S = Slope of the corresponding calibration curve.

LOQ =10*2.42/0.122

          =2.74

 

Ruggedness:

The ruggedness of the proposed method was evaluated by applying the developed procedure the concentration of 5μg/ml of DLT by using the same instrument by two different analysts under optimized conditions for two days. The observed result was found to be reproducible since there was no difference showing in the result by different analysts so the method could be considered rugged.

 

Table 8: Ruggedness study.

DISCUSSION:

The proposed method for analyzing DLT using UV-Visible spectrophotometry is simple, inexpensive, accurate, and convenient. The use of ferric chloride instead of other ferric salts like citrate or sulfate is recommended as the solubility of chloride salts is generally satisfactory in an aqueous medium and resulting solutions are stable for a longer duration of time. The Beers law was followed in the concentration range of 3.5-7.5μg/ml, with a correlation coefficient of 0.998.

 

CONCLUSION:

The present work describes a simple, accurate, precise, and economic method for selective determination of DLT in formulation based on the reduction of DLT in the presence of potassium ferricyanide by Ferric chloride. The colored complex was measured at 710nm. Beers law was observed in the concentration range of 3.5-7.5μg/ml with a correlation coefficient of 0.998. Ferric chloride can suitably replace the commonly used ferric citrate and ferric sulfate salts used in such methods of analysis.

 

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Received on 27.08.2022       Modified on 17.10.2022

Accepted on 09.12.2022   ©Asian Pharma Press All Right Reserved

Asian J. Pharm. Ana. 2023; 13(4):249-254.