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Ashok Gorja1
, Santhoshi Abbineni1, Durga Panikumar Anumolu1and Jahnavi Bandla2*
1Faculty of Pharmacy, Gokaraju Rangaraju College of Pharmacy, Bachupally-500090, Hyderabad, Telangana, India
2Faculty of Pharmacy, Vishnu Institute of Pharmaceutical Education and Research, Narsapur, Medak, Telangana, India.
Coresponding Author E-mail:jahnavi.bandla@gmail.com
Article Publishing History
Article Received on : 14 Oct 2024
Article Accepted on :
Article Published : 29 Jan 2025
The current investigation sought to create and approve specific spectrofluorimetric technique to cabotegravir estimate in dose formats for medications. The relative fluorescence intensity for the cabotegravir was measured at a wavelength of excitation of 395nm and wavelength of emission at 484nm. There was a linear response with a correlation coefficient of 0.999 between 5 and 25µg/ml. Developed technique was supported with the various validation parameters such as accuracy, sensitivity, specificity, and precision in keeping with the ICH recommendations. The devised approach was shown to be sensitive, the corresponding quantification and detection limits of 0.6 µg/ml and 0.21 µg/ml. The found results indicates the method developed for the estimation of cabotegravir were specific, accurate, precise and reproducible. Finally, it can be concluded that this spectrofluorimetric method could be used in routine analysis for cabotegravir estimate in dose formats for medications.
KEYWORDS:Accuracy; Cabotegravir; Method development; Precision; Spectrofluorimetry; Validation
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Copy the following to cite this article:Gorja A, Abbineni S, Anumolu D. P, Bandla J. Spectrofluorimetric Technique Creation and Approval for Cabotegravir Estimate in Dose Formats for Medications. Orient J Chem 2025;41(1).
Gorja A, Abbineni S, Anumolu D. P, Bandla J. Spectrofluorimetric Technique Creation and Approval for Cabotegravir Estimate in Dose Formats for Medications. Orient J Chem 2025;41(1). Available from: https://bit.ly/4jBLwU0
Introduction
Chemically, cabotegravir (Fig. 1) was identified as (3S,11aR).2,4-difluorobenzyl) -N-3,2-a] hexahydrooxazolo (6-hydroxy-3-methyl-5,7-dioxo-2,3,5,7,11,11a) Pyrido[1,2-d]1-Pyrazine-8-carboxamide1, 2. Its molecular weight is 405.36 g/mol and its chemical formula is C19H17F2N3O5. It is a crystalline powder that ranges in color from white to off-white. It dissolves in DMSO3-6 but is essentially insoluble in aqueous solutions like methanol and water. An antiviral drug called cabotegravir is used to treat and prevent HIV (Human Immunodeficiency Virus). It is a member of the integrase strand transfer inhibitor (INSTI) medication class7,8. In order to stop the virus from replicating, cabotegravir blocks the HIV integrase enzyme, which is necessary for the virus to incorporate its genetic material into the DNA of the host cell9.
According to the literature review, there aren’t many analytical approaches available such as UV spectrophotometry10, HPLC11 and LC-MS12 methods were developed, but no spectrofluorimetric technique was created for cabotegravir estimate in marketed dose formats for medications. Spectrophotometry’s outstanding sensitivity and specificity have led to its remarkable prominence in drug analysis. In contrast to spectrophotometry, spectrofluorimetry allows for examination at both emission and excitation wavelengths13. Hence the current investigation sought to create and approve new spectrofluorimetric technique to cabotegravir estimate in dose formats for medications.
Material and Approaches
Chemicals and reagents
Hetero Drugs Pvt. Ltd., Hyd., Telangana, India, provided a complimentary sample of cabotegravir as part of their operating standard. Vocabria, the commercial version of cabotegravir, was bought from a nearby pharmacy. All of the solvents and chemicals that were employed were AR grade and were acquired from Merck in Mumbai, India.
Equipment
The current study made use of a number of instruments, including a spectrofluorometer (Shimadzu, RF 5301 PC, Japan), an ultrasonic cleaner (Sonica Ultrasonic Cleaner, Italy), a pH meter (Elico L120, Hyderabad, India), and a digital balance (Shimadzu, AUX 220D, Japan).
Preparation of cabotegravir solutions
Ten milliliters of dimethyl formamide (DMF) and ten milligrams of carefully weighed medication working standard were combined to make an initial solution with an amount of one milligram per milliliter. Later 10 µg/ml cabotegravir standard solution was prepared from the above stock solution using DMF as diluent.
Preparation of 2% ethyl acetoacetate (EAA) reagent
0.2 ml of EAA was diluted with ethanol up to 10ml.
Analytical method development
The development of the cabotegravir spectrofluorimetric method involved dissolving the analyte in a variety of solvents. It was discovered that DMF was chosen as the solvent because it yields clear spectra. Utilizing a standard solution including 10 µg/ml of cabotegravir in DMF, 2.5mlofsulfuric acid, and 0.1mlof 2% EAA reagent, excitation and emission wavelengths were determined through scanning within the 300-700 nm wavelength range. Emission spectra were obtained by scanning solutions at a fixed excitation wavelength. After being excited at 395 nm, cabotegravir exhibited fluorescence at an emission wavelength of 484 nm.
Analytical method validation
The devised procedure was validated for a number of criteria in compliance with the ICH guidelines13,14.
Linearity
Aliquots of cabotegravir in the 5–25 µg/ml range were produced in several volumetric flasks with a capacity of 10 ml containing 2.5 ml sulfuric acid, 0.1 ml 2% EAA reagent, and DMF. The fluorescence intensities of the previously stated solutions have been evaluated at an emission wavelength of 484 nm using an appropriate blank. The calibration curve’s characteristics, including its slope, intercept, and correlation coefficient, were calculated16.
Accuracy
By calculating the percentage recoveries of cabotegravir using the conventional addition method, the correctness of the results was ascertained. From the tablet powder stored in volumetric flasks, A predetermined concentration of cabotegravir was achieved by adding cabotegravir to standard solutions at 80, 100, and 120%levels. At emission wavelength of 484 nm, the produced solutions’ fluorescence intensities were determined. Analyte preparations were examined in triplicate at each concentration level in order to confirm the recovery.
Precision
The current method’s repeatability (intra-day precision) was determined by measuring the corresponding response for three different cabotegravir concentrations (5, 15 and 25 µg/ml) three times in a single day. Throughout a week, three separate days were used to estimate the response in triplicate for three distinct concentrations (5, 15 and 25 µg/ml) in order to discuss the intermediate(inter-day) precision. The percentage relative standard deviation (% RSD) was used to express the findings of both investigations.
Sensitivity
According to ICH criteria, samples with extremely low concentrations of cabotegravir were used in order to identify the lowest quantitative quantity (LOQ) and the lowest detected amount (LOD), which demonstrated the analytical method’s sensitivity. The formulas were used to compute LOD and LOQ, respectively. To demonstrate the analytical method’s robustness, by slightly altering the emission wavelength, the fluorescence intensity of the analyte solutions was also evaluated17.
Assay of cabotegravir
Average weight of 20tablets of Vocabria of 30mg of cabotegravir were taken and crushed into powder. To get a concentration of 1 mg/ml, 10mg of precisely weighed cabotegravir was dissolved in 10 ml of DMF. Whatmann filter paper was used to filter the aforementioned solution, and an aliquot of the clear filtrate was removed. This was diluted to make 10 ml with diluent, then 2.5mlof sulfuric acid and 0.1 ml of 2% EAA were added. Additionally, the assay percentage was determined by evaluating fluorescence intensity of the final solution at an emission wavelength of 484 nm.
Results and Discussion
The current investigation was developed for estimating cabotegravir using spectrofluorimetric method in pharmaceutical dosage form.
Analytical method development
The standard cabotegravir solution was prepared by dissolving the working standard in DMF solvent. For the selection of solvent, a variety of solvents such as methanol, water, DMSO and DMF were tested for obtaining spectrofluorimetric spectrum. Out of which DMF was chosen as the solvent because it yielded a clean spectrum. The standard solution for analysis was prepared by 2.5 ml sulfuric acid, 0.1 ml of 2% EAA reagent addition. When the standard solution was scanned in the range of 300-700 nm, it has shown excitation wavelength at 395 nm and emission wavelength at 484 nm. The spectrum was shown in Fig. 2.
 
Analytical method validation
The created technique was approved to meet the ICH criteria
Linearity
The linearity of the calibration curve (emission intensity vs. concentration) was investigated between the concentration 5 and 25 µg/ml. The cabotegravir overlay spectrum for the linearity range, as displayed in Fig. 3, is listed below. The correlation coefficient value (R) for captopril in the linear regression analysis was 0.9994, and the regression equation was y = 0.4141x + 0.014. Here are the cabotegravir linearity data from Table 1 and the calibration graph from Fig. 4, which demonstrated an increase in intensity at 484 nm emission wavelength with increasing medication concentration.
Table 1: Linearity data of cabotegravir
S. No. Concentration (µg/ml) Intensity (n=5) 1 0 0 2 5 2.22 3 10 4.084 4 15 6.166 5 20 8.157 6 25 10.515Accuracy
The accuracy of the standard addition process was assessed using the computation of cabotegravir recoveries. A standard cabotegravir solution at concentrations of 80%, 100%, and 120% was added to pre-qualified sample solutions containing 30 mg of cabotegravir. The concentration was determined by measuring the intensities of the spiking solutions at 484 nm [λems]. At each of the designated concentration levels, the recovery was found to be between 98.44 and 102%, which was excellent. The % recovery data for cabotegravir were summarized in the Table 2.
Table 2: Accuracy results for cabotegravir
Drug Level of accuracy, % Theoretical Concentration, (µg/ml) Concentration found (µg/ml) (AM±SD)(n=3) % Recovery % RSD Cabotegravir 80% 18 16.54±0.017 98.02% 0.25 100% 20 20.63±0.042 100.04% 0.55 120% 22 22.64±0.040 101.90% 0.41Arithmetic Mean (AM); Standard Deviation (SD), Relative Standard Deviation (RSD), and number of samples (n)
Precision
The precision within and between days of the fluorometric technique was achieved by computing the responses to the three different concentrations of cabotegravir (5,15, and 25 µg/ml), and the responses are recorded three times on various days, including the same day. The results showed the % RSD for both within-day-precision, and between-days-precision. The created technique was deemed accurate as the percentage RSD values were found to be less than 2.0. Table 3 presented a summary of the precision data.
Table 3: Precision data for cabotegravir
Drug Concentration (µg/ml) Inter-Day Precision (AM±SD)(n=3) % RSD Inter-Day Precision (AM±SD)(n=3) % RSD Cabotegravir 5 2.663±0.05 0.46 2.623±0.0196 0.13 15 6.583±0.038 0.32 6.621±0.0317 0.17 25 10.624±0.0594 0.43 10.57±0.0316 0.21Arithmetic Mean (AM); Standard Deviation (SD), Relative Standard Deviation (RSD), and number of samples (n).
Sensitivity
Spectrofluorimetric method’s LOD and LOQ values were presented in Table 4.
Table 4: Cabotegravir LOD and LOQ data
Parameters for Validation Values (µg/ml) LOD 0.21 LOQ 0.60Assay of marketed formulation
The evaluation of the proposed approach was done using the assay of 30 mg cabotegravir tablets. The cabotegravir test results were shared and contrasted with the recommended dosages on the package. 29.4 mg of cabotegravir (30 mg) was obtained. The assay result percentage for 30 mg was 98%. The result obtained fell between reasonable boundaries and two percent RSD was discovered. The assay data for commercial tablets of cabotegravir were presented in Table 5.
Table 5: Assay of cabotegravir tablets [30mg] by spectrofluorimetric method
Drug name Label claim [mg] AM±SD[mg] [n=3] %Assay %RSD Cabotegravir 30 mg 29.4 ± 2 98 % 1.018Arithmetic Mean (AM); Standard Deviation (SD), Relative Standard Deviation (RSD), and number of samples (n).
Conclusion
A brand-new, very accurate spectrofluorimetric technique was created to estimate the amount of cabotegravir present in pharmaceutical dosage forms. The drug solutions were prepared using the developed method, which employed dimethyl formamide as the solvent. The devised procedure was validated for a number of criteria in compliance with ICH guidelines. It was discovered that the procedure was sensitive, linear, accurate, and precise. Ultimately, it can be said that cabotegravir in medication dosage forms can be routinely analyzed using the established spectrofluorimetric approach.
Acknowledgement
We express our sincere gratitude to Gokaraju Rangaraju College of Pharmacy’s administration for providing the space needed to complete the current study.
Conflict of Interest
No conflicts of interest are disclosed by the writers.
Funding Sources
The author(s) received no financial support for the research, authorship, and/or publication of this article.
References
De Cock, K.M.; Jafe, H.W.; Curran, J.W. AIDS 2012, 26(10), 1205-1213.
This work is licensed under a Creative Commons Attribution 4.0 International License.
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