INTRODUCTION:

Gout stands as the prevailing form of inflammatory arthritis, characterized by the deposition of monosodium urate (MSU) crystals in and around articular structures. These crystals lead to recurring bouts of intense inflammatory arthritis, marked by joint swelling, redness, warmth, pain, and stiffness¹.

Gout is caused by overproduction or under excretion of urate, leading to hyperuricemia. Interlukin-1ꞵ (IL-1ꞵ) plays a vital role in the inflammatory response associated with monosodium urate crystal deposition in gout patients².

Boswellia serrata, popularly known as Salai Guggul, is a Burseraceae plant. Its oleo gum resin contains triterpenoids known as Boswellic acids (BAs). Boswellia serrata has four primary Boswellic acids: 3-acetyl-11-keto-beta-Boswellic acid (AKBA)3 and alpha-beta-, and 11-keto-beta-Boswellic acid (KBA)³.

The main physiologically active ingredient in B. serrata is 3-acetyl-11-keto-beta-boswellic acid (AKBA), which has been the subject of much research due to its potential anti-inflammatory and anti-arthritic effects. AKBA acts by a non-redox, noncompetitive mechanism, demonstrating effective inhibition of 5-lipoxygenase with an IC50 value of 1.5mm⁴. This reduces toxicity and minimizes adverse effects when compared to other anti-inflammatory drugs. Although AKBA has therapeutic benefits, its oral absorption is poor; its biological half-life is 4.5±0.55 h⁵. With a molar mass of 512.7g/mol and a chemical formula of C32H48O5, AKBA is a white powder that is occasionally soluble in water, with isopropyl myristate exhibiting the highest solubility. Despite its potential, there is a substantial research gap in the development and validation of analytical methodologies for AKBA quantification in NLC formulations and medicinal dosage forms.

Nanostructured lipid carriers (NLC’s) are the next generation of solid lipid nanoparticles (SLN’s), with an average size of 10-500nm. They consist of a dual mixture of solid and liquid lipids, typically in ratio of 70:30 up to 99.9:0.1. NLC’s have a unique nanostructure that improves drug loading and enhances drug incorporation, leading to increased storage time. They can be administered through various routes, including oral, topical, intravenous (IV), and ocular routes⁶.

The review of literature indicates a scarcity of techniques for assessing AKBA in bulk, pharmaceutical preparations and nanoformulations. These methods include high performance thin layer chromatography (HPTLC), high-performance liquid chromatography (HPLC) as well as UV spectroscopy. Each of this techniques have been employed to analyze AKBA in different contexts, showcasing the versatility in assessing this compounds across diverse formulations and settings^7,8,9. Hence, there is a vision to establish a UV spectrophotometry method for estimating AKBA in NLC formulations and pharmaceutical dosage forms. This method involves a green solvent system with an economical percentage of organic solvent, aiming to provide a valuable, rapid, precise, and robust analytical solution considering the therapeutic significance of AKBA.

Therefore, the objective of the present study was to develop and validate a straightforward, quick, sensitive, precise, and accurate UV-spectrophotometric method for the quantification of 3-acetyl-11-keto-beta-boswellic acid.

MATERIALS AND METHOD:

Materials:

Bio-Med Ingredients Pvt Limited Verna, Salcete, Goa provided a gift sample of 3-acetyl-11-keto-beta-Boswellic acid (AKBA). Higher analytical grade lipids, reagents, and beneficial chemicals were acquired from KAHER'S KLE College of Pharmacy in Belagavi. For analysis, a Shimadzu UV-1900UV-Spectrophotometer with UV probe software was utilized. The investigation employed a calibrated weighing balance to weigh the medication sample and lipids.

Method Development:

Following the selection of an appropriate solvent combination and the identification of wavelengths in the literature, the development of the new UV Spectrophotometric method was proven. To test the samples' solubility, a variety of solvents were used, such as methanol, acetone, chloroform, acetonitrile, DMSO, etc. After taking into consideration their solubility conditions, methanol and water (1:1) were chosen as the preferred solvents and scanned in the UV area between 200 and 400nm. At 255nm, AKBA showed the maximum absorption.

Preparation of standard stock solution:

10 mg of AKBA was accurately weighed and transferred to a volumetric flask of 10mL, and the volume was made up to 10mL with methanol, obtaining a concentration of 1000μg/mL of analyte. 1 mL of AKBA solution has been pipette out from the volumetric flask and passed to a 10mL volumetric flask, and the volume was made up to 10mL with methanol and water (1:1) to achieve a concentration of 100μg/mL for AKBA. 1 mL of this solution has been pipette out from the volumetric flask and passed to a third 10mL volumetric flask and the volume was made up to 10mL with methanol and water (1:1) to achieve a concentration of 10μg/mL for AKBA.

Specificity and Selectivity:

The selective nature of the process is indicated by the fact that AKBA exhibits the maximum absorbance at 255nm. Additionally, the solvent’s spectra showed no absorbance at 255nm.Thus, this method was found to be selective at 255nm^10,11.

Linearity:

Linearity was carried out by weighing 10mg of AKBA into a clean and dried 10mL volumetric flask, the volume was made up to the mark by using the solvent methanol and water in the ratio of 1:1. From the above solution 1 mL was pipette out into the volumetric flask, which was then filled with solvent system to make up the volume. Further dilutions were prepared from this solution to assess the linearity. Linearity was tested in the 20-60µg/mL range^12,13.

Limit of Quantification (LOQ) and Limit of Detection (LOD):

The limit of detection (LOD) refers to the lowest analyte concentration that can be detected with statistical significance by means of given analytical procedure, (LOQ) refers to lowest amount of the analyte that can be accurately and precisely measure. To calculate these limits, the formula LOQ = 3.3× LOD and LOD = 3.3s/m is utilized, where ‘s’ represents the standard deviation of the response, and ‘m’ stands for the slope of the calibration curve associated with the analysis^14,15.

Accuracy:

The accuracy of the proposed ultraviolet-visible technique was validated through recovery trials involving the standard addition of the analyte. In triplicate, three distinct AKBA solutions were prepared, each at concentrations of 50%, 100%, and 150% relative to the standard AKBA concentration of 40 µg/mL. This approach aimed to assess the precision and reliability of the ultraviolet-visible method by confirming the recovery of AKBA across different concentrations in the prepared solutions. The accuracy was calculated using following equation^16,17.

%RC= (SPS-S/SP) × 100

Where, SPS = Amount obtained in the spiked sample, % RC = Percent recovery, SP = Amount added to the sample, and S = Amount found in the sample

Precision:

To evaluate the precision of the analytical method, three solutions were prepared containing AKBA at concentrations of 20μg/mL, 40μg/mL, and 60μg/mL. The absorbance of each solution was measured in triplicate at 255nm, and the relative standard deviation (%RSD) was calculated to determine system precision.

System precision was assessed in two categories: intraday and interday. For intraday precision, %RSD was calculated from three replicates of the AKBA solutions at the specified concentrations measured at three different time intervals within the same day. In the case of interday precision, %RSD was determined by analyzing three replicates of the AKBA solutions at 20 μg/mL, 40μg/mL, and 60μg/mL concentrations on three separate days^18,19

Ruggedness:

Ruggedness was assessed by applying a similar planned method to a different instrument and examining the reproducibility with different analyst. The results were depicted in terms of % RSD²⁰.

Robustness:

Three replicates of a solution containing 40 μg/mL AKBA were prepared to test the Robustness, at 253nm, 255nm and 257nm respectively. The absorbance of a solution was measured at three different wavelengths, and the relative standard deviation (%RSD) was computed^21,22.

Nanoformulationfor AKBA Estimation:

In this work, hot-melt emulsification and ultrasonication were used to create nanostructured lipid carriers. First, a 7:3 mixture of liquid and solid lipid was made, and the surfactant and co-surfactant were dissolved in 5 milliliters of distilled water to create the aqueous phase, which was kept at 75°C. The 30mg of AKBA was added to the melted lipid mixture after being dissolved in 2mL of methanol. A homogenous emulsion was achieved by progressively adding drops of the aqueous phase to the lipid phase while swirling at 600rpm after the methanol had evaporated. Later, the main emulsion was ultrasonically treated for 6 minutes (30 'ON' and 4 'OFF' cycles)²³.

Assay of Nanoformulation:

AKBA in nanoformulation was quantified using a newly developed UV spectrophotometry method. The optimized batch of NLC,s was centrifuged at 20,000 RPM under 4°C for 30 minutes, then 0.1mL of the supernatant was withdrawn and diluted with 10mL methanol: water (1:1) to achieve a concentration of 10 µg/mL. The measurement of the absorbance, coupled with the use of a calibration curve, facilitated the determination of the amount of AKBA present in the formulation^24,25.

Forced Degradation Studies:

Studies on forced degradation were carried out to evaluate the stability of the UV-Spectroscopic technique. Each study calculated the proportion of deterioration that occurred to the samples as a result of acid, base, oxidation, and photolytic degradation. The forced degradation study's limits are reasonable and fall inside the given range²⁶.

a) Acid Degradation Studies:

10 mg of AKBA was weighed and dissolved in a 10 mL of volumetric flask with methanol to achieve a concentration of 1000μg/mL. Secondary stock solutions of 100μg/mL were prepared using solvent system consisting of methanol and water. Subsequently, 1mL of the above secondary solution was transferred into 10mL volumetric flask, along with 1mL of 0.1 N HCl and the volume was adjusted with the solvent system to obtain a concentration of 10μg/mL. This 10μg/mL AKBA solution was then subjected to heating on a water bath at 80°C for 2 hours. Spectra were observed by scanning the samples in the UV range of 200-400nm^27,28.

b) Base Degradation Studies:

For the Base degradation study, AKBA (10μg/mL) was prepared using secondary stock solutions. To this solution, 1mL of 0.1M NaOH was added, and the volume was adjusted to the mark with solvent system. The sample was then subjected to a water bath for 2 hours at 80°C, followed by scanning of the spectra^29,30.

c) Oxidation Degradation Studies:

A solution of AKBA solution at a concentration of 10 μg/mL was created by combining secondary stock solutions. Then, 1mL of 30% hydrogen peroxide was added, and the volume was adjusted was adjusted with solvent system. The solutions were subjected to stress conditions at 80°C for 2 hours, after which their spectra were scanned³¹.

d) Photo Degradation Studies:

In photo degradation study 10μg/mL of AKBA sample solution was prepared from the secondary stock solution, after which it was exposed to sunlight for around 2hours then scanned to obtain appropriate spectra. Formula to calculate Percent degradation³².

(Initial concentration – Final concentration)

% degradation = ------------------------------------- × 100

Initial concentration

Particle Size and Zeta Potential Analysis:

Mean Particle size & Zeta Potential of AKBA loaded Nanoformulation were analyzed using Malvern Zetasizer. 1mL of AKBA nanoformulations were diluted with 10mL of mill-Q water.

RESULTS AND DISCUSSION:

Methanol and water (1:1) are the solvent system employed in the method development and validation, and AKBA reported a spectrum with 255 nm as the highest absorption. The parameters to be analyzed for method development and validation were presented in table1.

Table 1: Developed method parameters

Parameters	Specification
Analyte	3-acetyl-11-keto-ꞵ-Boswellic acid (AKBA)
Solvent	Methanol, water
Lamda max of AKBA	255 nm

Method Validation:

With a linearity range of 20–60 μg/mL, the method's development and validation adhered to the ICH criteria for evaluating linearity, specificity and selectivity, precision, robustness, stability, LOD, and LOQ for AKBA. Correlating absorbance with concentration led to the creation of the calibration curve. System, intraday, and interday studies were used to determine precision; findings were reported as a percentage of RSD. By switching analysts and recording the instrument's percent RSD, ruggedness was assessed. The linearity slope was used to determine the LOD and LOQ.

Specificityand Selectivity:

3-acetyl-11-keto-ꞵ-Boswellic acid (AKBA) showed highest absorbance at 255nm, indicating the specificity and selectivity of the method.

Figure 1. UV-Spectrum of AKBA

Linearity:

According to the abovementioned technique, dilutions were prepared for the linearity range of 20–60μg/mL. The linearity graph is shown in Figure 2, and the linearity and range are shown in Table 2.

Table 2: Linearity and range data of AKBA

Concentration (µg/mL)	Absorbance
0	0
20	0.277
30	0.384
40	0.509
50	0.627
60	0.715

Figure 2. Linearity curve for AKBA

LOD and LOQ:

LOD and LOQ values were found to be 3.297 and 9.992 µg/mL, respectively by statistical calculations.

Table 4: System precision data of AKBA

Concentration (µg/mL)	Absorbance
20a	0.259
20b	0.258
20c	0.257
Mean	0.25-8
SD	0.001
%RSD	0.387597
40a	0.516
40b	0.511
40c	0.518
Mean	0.515
SD	0.003606
%RSD	0.700107
60a	0.735
60b	0.737
60c	0.736
Mean	0.736
SD	0.001
%RSD	0.13587

Accuracy:

Precision was evaluated through recovery experiments, showing a mean recovery ranging from 99.5% to 100.9444%. This underscores the accuracy of the proposed method for estimating AKBA, as demonstrated by the data provided in Table 3.

System Precision:

As mentioned, three replicates of the solution containing 20μg/mL, 40μg/mL, and 60μg/mL of AKBA were prepared, and the absorbance of each solution was measured at 255nm to determine system precision. Calculating the percent RSD revealed that it was less than 2%as presented in Table 4.

Intraday Precision:

Three duplicates of a solution with concentrations of 20μg/mL, 40μg /mL, and 60μg /mL of AKBA were analyzed for intraday precision. %RSD was measured at various time intervals on the same day; and %RSD was shown to be less than 2% as presented in Table 5.

Table 5: Intraday precision data of AKBA

Concentration	Intraday Morning	Intraday Afternoon	Intraday Evening
20 μg/mL	0.26	0.257	0.259
20 μg/mL	0.261	0.259	0.255
20 μg/mL	0.259	0.257	0.258
% RSD	0.384615	0.448137	0.808938
40 μg/mL	0.511	0.511	0.511
40 μg/mL	0.515	0.511	0.515
40 μg/mL	0.516	0.515	0.515
% RSD	0.514738	0.450761	0.449591
60 μg/mL	0.733	0.735	0.733
60 μg/mL	0.735	0.733	0.733
60 μg/mL	0.733	0.733	0.735
% RSD	0.157388	0.157388	0.157388

Interday Precision:

Three duplicates of a solution containing AKBA concentrations of 20μg/mL, 40μg/mL, and 60μg/mL were assessed for interday precision, and %RSD was calculated on three separate days. Furthermore, it was found that the %RSD was less than 2% as shown in Table 6.

Table 6: Interday precision data of AKBA

concentration	1^STday	2^ndday	3^rdday
20 μg/mL	0.258	0.257	0.255
20 μg/mL	0.258	0.258	0.258
20 μg/mL	0.259	0.258	0.258
% RSD	0.22349	0.224069	0.67395
40 μg/mL	0.511	0.513	0.511
40 μg/mL	0.511	0.515	0.513
40 μg/mL	0.515	0.515	0.513
% RSD	0.449591	0.224504	0.225381
60μg/mL	0.733	0.511	0.733
60μg/mL	0.735	0.513	0.735
60μg/mL	0.735	0.513	0.735
% RSD	0.157245	0.225381	0.157245

Ruggedness:

The suggested approach was repeated on two distinct instruments (UV-1800 and UV-1900), and the repeatability was checked by different analyzers. This resulted with a percent RSD of less than 2%, showing that the method developed is rugged.

Robustness:

Robustness was evaluated by making a slight modification to the optimal value. The parameter, such as, wavelength (±2 nm), was employed to assess the AKBA absorbance and the percentage of relative standard deviation (% RSD) during the robustness testing. As shown in table 8

Table 8: Robustness data of AKBA

Analyte - Boswellic acid	Change made	Absorbance	%RSD
Change in wavelength	253 nm	0.5	0.34641
	255 nm	0.496667	0.23249
	257 nm	0.496	0.285124

Assay of Nanoformulation:

The Developed UV method was successfully applied for the estimation of AKBA content in Nanostructured lipid carriers (NLC’s). Average percent assay of AKBA nanoformulation was found to be 99.5 %.

Table 9: Assay data of AKBA nanoformulation

Drug	Amount of Drug	Amount of drug Estimated	Assay
AKBA	10mg	9.95	99.5 %

Particle Size and Zeta Potential Analysis:

Particle Size of the optimized batch was found to be 145.1nm indicating the nano-size of the particles and zeta potential was found to be -38.09mV, indicating that particles are strongly anionic in nature and the formulation is stable enough and without any aggregate formation.

Figure 3. Particle size of optimized nanoformulation.

Figure 4. Zeta potential of optimized nanoformulation.

Forced Degradation Studies:

(A)

(B)

(C)

(D)

Figure 5. UV spectra of AKBA (10μg/mL) obtained in the stress degradation assays using Acidic (A), Basic (B), Oxidative (C), Photolytic (D)

The above graphic provides a summary of the findings from forced degradation experiments that were carried out using a 1:1 ratio of methanol to water. The spectra at 255nm showed no signs of degradation after two hours of exposure to acid degradation, which was accomplished with 0.1N HCl heated at 80°C. In comparison to other situations, base and photolytic deterioration exhibited noticeably less degradation. Nonetheless, the medication was discovered to have undergone significant degradation after being exposed to 30% hydrogen peroxide for two hours at 80°C, as the distorted spectra clearly proved.

CONCLUSION:

The UV spectrophotometric approach that was developed was effectively evaluated for AKBA measurement in a novel nanoformulation as well as a medication, exhibiting stability, simplicity, precision, and ruggedness. It quickly produced encouraging results, demonstrating its suitability for large-scale analysis of AKBA and novel nanoformulations, and ultimately helping in quality control. The results of investigations on forced degradation indicated that AKBA was stable in circumstances that were acidic, basic, and photolytic. Nevertheless, AKBA breakdown was noted when oxidative stress conditions were present. For routine quality control examination, the simple UV spectrophotometric approach, which indicates stability, therefore proves useful. It has the ability to estimate AKBA in vivo as well as in vitro.

ABBREVIATION:

1. UV- Ultraviolet

2. AKBA- 3-acetyl-11-keto-ꞵ-Boswellic acid

3. NLC’s- Nanostructured lipid carriers

4. ICH- International Council for Harmonization

5. LOD- Limit of Detection

6. LOQ- Limit of Quantification

7. R2- Correlation coefficient

8. RSD- Relative Standard Deviation

9. SD- Standard Deviation

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

The authors are thankful toBio-med Ingredients verna, Goa for providing us with API,and also KLE college of pharmacy Belagavi, Karnataka for providing us with facilities, necessary for carrying out research work.

REFERENCES:

1. Yang Y, Li Z, Huang P, Lin J, Li J, Shi K, Lin J, Hu J, Zhao Z, Yu Y, Chen H. Rapidly separating dissolving microneedles with sustained-release colchicine and stabilized uricase for simplified long-term gout management. Acta Pharmaceutica Sinica B. 2023; 13(8): 3454-70. https://doi.org/10.1016/j.apsb.2023.02.011

2. Yadav S, Bhosale M, Sattigeri B, Vimal S. Pharmacological overview for therapy of gout and hyperuricemia. International Journal of Health Sciences. 2022: 7772-85. https://doi.org/10.53730/ijhs.v6nS2.6926

3. Mehta M, Dureja H, Garg M. Development and optimization of boswellic acid-loaded proniosomal gel. Drug delivery. 2016; 23(8): 3072-81. https://doi.org/10.3109/10717544.2016.1149744

4. Sabina EP, Indu H, Rasool M. Efficacy of boswellic acid on lysosomal acid hydrolases, lipid peroxidation and anti–oxidant status in gouty arthritic mice. Asian Pacific Journal of Tropical Biomedicine. 2012; 2(2): 128-33. doi: 10.1016/S2221-1691(11)60206-2

5. Goel A, Ahmad FJ, Singh RM, Singh GN. 3-Acetyl-11-keto-β-boswellic acid loaded-polymeric nanomicelles for topical anti-inflammatory and anti-arthritic activity. Journal of Pharmacy and Pharmacology. 2010; 62(2): 273-8. DOI 10.1211/jpp/62.02.0016

6. Javed S, Mangla B, Almoshari Y, Sultan MH, Ahsan W. Nanostructured lipid carrier system: A compendium of their formulation development approaches, optimization strategies by quality by design, and recent applications in drug delivery. Nanotechnology Reviews. 2022; 11(1): 1744-77. https://doi.org/10.1515/ntrev-2022-0109

7. Shah SA, Rathod IS, Suhagia BN, Patel DA, Parmar VK, Shah BK, Vaishnavi VM. Estimation of boswellic acids from market formulations of Boswellia serrata extract and 11-keto β-boswellic acid in human plasma by high-performance thin-layer chromatography. Journal of Chromatography B. 2007; 848(2): 232-8. https://doi.org/10.1016/j.jchromb.2006.10.026

8. Katragunta K, Siva B, Kondepudi N, Vadaparthi PR, Rao NR, Tiwari AK, Babu KS. Estimation of boswellic acids in herbal formulations containing Boswellia serrata extract and comprehensive characterization of secondary metabolites using UPLC-Q-Tof-MSe. Journal of Pharmaceutical Analysis. 2019; 9(6): 414-22. https://doi.org/10.1016/j.jpha.2019.09.007

9. Katragunta K, Siva B, Kondepudi N, Vadaparthi PR, Rao NR, Tiwari AK, Babu KS. Estimation of boswellic acids in herbal formulations containing Boswellia serrata extract and comprehensive characterization of secondary metabolites using UPLC-Q-Tof-MSe. Journal of Pharmaceutical Analysis. 2019; 9(6): 414-22. https://doi.org/10.1016/j.jpha.2019.09.007

10. Shinde GS, Jadhav RS, Tambe VB, Kote RB, Argade VP. Bioanalytical Method Development and Validation of UV Spectrophotometric Method for Estimation of Sitagliptin in Urine sample. Research Journal of Science and Technology. 2024; 16(1): 19-23. DOI : 10.52711/2349-2988.2024.00004

11. Rathod RD, Nangare SN, Patil VR, Rathod HS, Shitole MM, Tade RS. Zero-order derivative and area under curve uv-spectrophotometric methods for determination of calcium levofolinate in bulk and pharmaceutical dosage form. Asian Journal of Pharmaceutical Analysis. 2020; 10(3): 129-33. DOI: 10.5958/2231-5675.2020. 00023.X

12. Matole V, Birajdar A, Ingle S, Adlinge S, Nangare G, Madur S, Patil S, Shegaonkar A. UV spectrophotometric method development and validation of acotiamide in bulk and solid doage form. Asian Journal of Pharmaceutical Analysis. 2020; 10(3): 147-9. DOI : 10.5958/2231-5675.2020.00026.5

13. Benazir SB, Archana J, Sumakanth M. Method development and validation of empagliflozin in bulk and pharmaceutical dosage form using UV spectroscopy. Asian Journal of Pharmaceutical Analysis. 2021; 11(2): 123-6. DOI: 10.52711/2231-5675.2021.00021

14. Kudatarkar N, Jalalpure S, Singadi R, Gurao R, Dinnimath B. Development And Validation Of UV-Visible Spectrophotometric Method For Estimation Of Chrysin In Bulk Form. Journal of Pharmaceutical Negative Results. 2022: 4758-63. DOI:10.47750/pnr.2022.13.S08.621

15. Sharma S, Goyal S, Chauhan K. A review on analytical method development and validation. International Journal of Applied Pharmaceutics. 2018; 10(6): 8-15. http://dx.doi.org/10.22159/ijap.2018v10i6.28279

16. Chethankumar H B et.al., Development and Validation of UV-Spectrophotometric Method for Estimation of Vinpocetine International Journal of Ayurvedic Medicine, 2023; Vol 14 (3), 717-723. DOI: https://doi.org/10.47552/ijam.v14i3.3585

17. Begum S, Jahnavi B, Jabeen N, Umema NT, Sandhya M. Method development and validation of evening primrose oil and its pharmaceutical dosage form by simple UV-spectrophotometric method. Asian Journal of Pharmaceutical Analysis. 2021; 11(2): 133-8. DOI : 10.52711/2231-5675.2021.00023

18. Taleuzzaman M. Limit of blank (LOB), limit of detection (LOD), and limit of quantification (LOQ). Org. Med. Chem. Int. J. 2018; 7(5). DOI: 10.19080/OMCIJ.2018.07.555722

19. Salunke PA, Barhate SD, Wagh RS. Environment safe Method development and Validation of Dolutegravir in Bulk and Tablet dosage form by UV-Visible spectroscopy. Asian Journal of Pharmaceutical Analysis. 2021; 11(2): 139-44. Salunke PA, Barhate SD, Wagh RS. Environment safe Method development and Validation of Dolutegravir in Bulk and Tablet dosage form by UV-Visible spectroscopy. Asian Journal of Pharmaceutical Analysis. 2021; 11(2): 139-44. DOI: 10.52711/2231-5675.2021.00024

20. Jha DK, Shah DS, Talele SR, Amin PD. Correlation of two validated methods for the quantification of naringenin in its solid dispersion: HPLC and UV spectrophotometric methods. SN Applied Sciences. 2020;2:1-1.https://doi.org/10.1007/s42452-020-2536-3

21. Mittha J, Habib B. UV spectrophotometric method development and validation of dasatinib in bulk and formulation. Asian Journal of Pharmaceutical Analysis. 2021; 11(3): 203-6. DOI: 10.52711/2231-5675.2021.00036

22. Kapatrala ST, Kondreddy VK, Kandlapalli S, Male T. Method development and validation for the estimation of etizolam and propranolol hydrochloride in bulk and combined dosage forms by simultaneous and derivative spectroscopic methods. Asian Journal of Pharmaceutical Analysis. 2021; 11(4): 263-9. DOI: 10.52711/2231-5675.2021.00045

23. Shetti P, Jalalpure SS. Optimization of a validated UV-spectrophotometric methodology for assessment of apigenin in bulk powder. IJPER. 2022; 56(1): 281-6. DOI: 10.5530/ijper.56.1.33

24. Ware AL, Pekamwar SS. Development and validation of bioanalytical uv-spectrophotometric method for determination of candesartan and development and validation of uv-spectrophotometric method for determination of candesartan in bulk drug and formulation. Asian Journal of Pharmaceutical Analysis. 2021; 11(2): 79-83. DOI: 10.52711/2231-5675.2021.00015

25. Sri RS, Sri KB, Sumakanth M. Stability indicating UV-Spectrophotometric method development and its validation for the determination of imatinib mesylate in bulk and formulation. Asian Journal of Pharmaceutical Analysis. 2022; 12(2): 83-6. DOI: 10.52711/2231-5675.2022.00015

26. Waghule T, Rapalli VK, Singhvi G, Manchanda P, Hans N, Dubey SK, Hasnain MS, Nayak AK. Voriconazole loaded nanostructured lipid carriers based topical delivery system: QbD based designing, characterization, in-vitro and ex-vivo evaluation. Journal of drug delivery science and technology. 2019; 52: 303-15. https://doi.org/10.1016/j.jddst.2019.04.026

27. Kempwade A, Taranalli A, Jadhav K. Development and validation of UV Spectrophotometric method to study stress degradation behaviour of Rizatriptan Benzoate. Spectroscopy and Spectral Analysis. 2015; 35(1): 137-40. https://doi.org/10.3964/j.issn.1000-0593(2015)01-0137-04

28. Kurangi B, Jalalpure S, Jagwani S. A validated stability-indicating HPLC method for simultaneous estimation of resveratrol and piperine in cubosome and human plasma. Journal of Chromatography B. 2019; 1122: 39-48. https://doi.org/10.1016/j.jchromb.2019.05.017

29. Jadhav KV, Dhamecha DL, Asnani GP, Patil PR, Patil MB. Stability-indicating stress degradation studies of lafutidine using UV spectrophotometric method. Pharmaceutical Methods. 2013; 4(1): 21-5. https://doi.org/10.1016/j.phme.2013.03.001

30. Agrawal YP, Agrawal MY, Jadhav SB, Shinde RJ. Development and validation of novel UV spectrophotometric method for the determination of evogliptin tartarate in pharmaceutical dosage form. Indian Journal of Pharmaceutical Education and Research. 2020; 54(4): 1174-9. DOI: 10.5530/ijper.54.4.214

31. Kurangi B, Jalalpure S. A validated stability-indicating RP-HPLC method for piperine estimation in black pepper, marketed formulation and nanoparticles. Indian J Pharm Educ Res. 2020; 54(3s): s677-86. DOI: 10.5530/ijper.54.3s.168

32. Chimagave SS, Jalalpure SS, Patil AK, Kurangi BK. Development and validation of stability indicating UV-spectrophotometric method for the estimation of hesperidin in bulk drugs, plant extract, Ayurveda formulation and nanoformulation. Indian J Pharm Educ Res. 2022; 56(3): 865-72. DOI: 10.5530/ijper.56.3.139

Received on 10.04.2024 Revised on 09.09.2024

Accepted on 02.12.2024 Published on 12.06.2025

Available online from June 14, 2025

Research J. Pharmacy and Technology. 2025;18(6):2493-2500.

DOI: 10.52711/0974-360X.2025.00356

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.

Replicates	Concentration (µg/mL)	Absorbance Analyst-1	Absorbance Analyst-2	Absorbance Instrument-2
1	40	0.49	0.489	0.499
2	40	0.494	0.485	0.51
3	40	0.486	0.489	0.5
Average		0.49	0.487667	0.503
SD		0.004	0.002309	0.006083
% RSD		0.816327	0.473561	1.209297

Concentration added μg/mL	Level	Concentration (μg/mL) standard	Concentration (μg/mL) Formulation	Absorbance	Mean	Concentration found	% Recovery
60	50%	20	40	0.731	0.736	59.7	99.5
		20	40	0.732
		20	40	0.745
80	100%	40	40	0.98	0.988667	80.75556	100.9444
		40	40	0.991
		40	40	0.995
100	150%	60	40	1.215	1.214333	99.56111	99.56111
		60	40	1.216
		60	40	1.212