INTRODUCTION:

The advancement of self-nanoemulsifying drug delivery systems (SNEDDS) has attracted considerable attention in improving the solubility and bioavailability of medicines with low solubility^1,2. Rifampicin, a medication classified as BCS class II due to its limited solubility in water, poses difficulties when administered orally. In addition, Rifampicin is susceptible to the low pH level of the stomach, particularly when used with Isoniazid, another medicine used to treat tuberculosis^3,4. Multiple researches have concentrated on creating and assessing SNEDDS Rifampicin, showcasing enhanced pharmacokinetic characteristics and in vitro drug release. The in vivo and in vitro investigations have shown improved drug loading, rheological characteristics, and nanoscale globular structure of SNEDDS, underscoring the significance of effective drug delivery systems for medications such as Rifampicin^5,6.

The improved formulations of SNEDDS Rifampicin were analyzed for viscosity, optical clarity, particle size, zeta potential, morphological evaluation, and in vitro drug release^7,8.

The literature review identified several analytical techniques, including UV-visible spectrophotometry^9,10,11, RP-HPLC^12,13,14, and LC-MS/MS^15,16, for the quantitative analysis of Rifampicin. These methods were used to analyze Rifampicin alone or in combination with other important pharmaceutical drugs such as Isoniazid³ and Piperine¹⁶. However, there is a scarcity of material for the validation of Rifampicin alone in SNEEDS formulation using a UV spectrophotometer. Therefore, this work adds to the increasing amount of research on the creation of efficient drug delivery systems for pharmaceuticals that have low solubility, such as Rifampicin. The study specifically focuses on confirming the accuracy of analytical techniques used to evaluate the effectiveness of drug loading in SNEDDS formulations.

Figure 1 Two dimensional structure of Rifampicin

MATERIAL AND METHODS:

Chemicals and reagents:

Rifampicin bulk was acquired from PT Phapros Indonesia Tbk (Indonesia), whereas methanol, sodium phosphate dibasic (Merck), and potassium phosphate monobasic (Merck) were procured.

Instrumentation:

The instrument being used is a Thermo Scientific UV-Vis spectrophotometer (Genesys50), which has a single-beam detector configuration and is fitted with a quartz-matched cell that has a path length of 1 cm.

Preparation of Solvents:

The solvent used was phosphate buffer (PB) pH 7.4. PBsolution was formulated by dissolving 7.52g of sodium phosphate dibasic, 1.60g of potassium phosphate monobasic, and 1000g of filtered water. The components are homogenized using a magnetic stirrer in a 1000mL beaker. The pH of the solution is determined by using a pH meter.

Preparation of Rifampicin Stock Solutions:

The Rifampicin stock solution was made by accurately weighing 10.0mg of Rifampicin and transferring it into a 10.0 mL volumetric flask. Dissolve Rifampicin (1mg/mL) by adding 10.0mL of methanol.

Selectivity Rifampicin:

A 1mg/ml stock of Rifampicin was diluted using PB pH 7.4 solution to achieve a concentration of 30µg/ml for the Rifampicin solution. The solutions were subjected to a UV spectrophotometer scan to measure their absorbance within the wavelength range of 200-600nm.

Linearity Study:

The concentrated Rifampicin solutions were diluted to provide a range of solutions with concentrations of 10, 20, 30, 40, and 50µg/mL. The absorption of these solutions was evaluated at the wavelength that yielded the highest selectivity in the investigation. The calibration curve was derived by graphing the absorbance and concentration of the Rifampicin solution.

Validation of Proposed Method:

The Validation method used complies with the ICH requirements, ensuring compliance with accuracy, precision, LOD (Limit of Detection), and LOQ (Limit of Quantitation).

Accuracy:

The accuracy data is expressed as a percentage of the recovery rate (% recovery). The assessment of percentage recovery was conducted at three distinct concentrations: 20, 30, and 40µg/mL.

Precision:

Precision is achieved both within a single day and over several days. Three intraday precision tests were conducted on the same day with concentrations of 20, 30, and 40µg/mL. Inter-day precision was assessed by experimenting on three separate occasions using the same solution.

LOD and LOQ

The limit of detection (LOD) and limit of quantification (LOQ) are determined by using the standard deviation and slope derived from the calibration curve, employing the following formula:

3.3 x σ 10 x σ

LOD= ----------------- LOQ= ---------------

S S

The standard deviation is denoted by σ while the slope of the calibration curve is represented by S.

Determination of drug concentration of SNEDDS Rifampicin and Rifampicin tablet:

The formulation of SNEDDS Rifampicin consists of Miglyol, Tween 80, and PEG 400 at a ratio of 1,5: 6: 2.5, respectively. A solution of SNEDDS Rifampicin with a concentration of 14mg/ml was diluted with methanol to achieve a final solution concentration of 50µg/mL. The SNEDDS containing Rifampicin under went centrifugation at a speed of 10,000 ppm for 15 minutes, then the supernatant was extracted. The quantity of Rifampicin was quantified using a UV spectrophotometer at the wavelength of maximum absorption of Rifampicin.

Loading efficiency = (Concentration of measured drug/ Total amount of drug) x 100%

RESULT AND DISCUSSION:

Selectivity study:

The maximum wavelength of Rifampicin in PB pH 7.4 was determined by scanning in the region of 200-600nm, revealing several peaks. Although the absorbance is stronger at λ 219nm and 339nm, the peaks are not completely distinct. According to Benneton et al⁹, the wavelength of Rifampicin at a pH of 7.4 is precisely 474nm.

Fig.2: Spectra of Rifampicin at 474 nm wavelength

Linearity Study:

Linearity refers to the capacity of an analytical procedure to provide a response that is directly proportional to the concentration of the substance being analyzed within a certain range. A high level of linearity is achieved in the analytical procedure when the correlation coefficient (r) is equal to or greater than 0.999. The results of the linearity test, as seen in Figure 3 and Table 1, demonstrate that the generated standard curve exhibits linearity.

Table 1: Regression characteristics of Rifampicin calibration curve

Parameters	Value
Correlation Coefficient (r)	0.9999
Regression equation
Slope	0.0378
Intercept	0.0189

Fig. 3: Rifampicin calibration curve at 474 nm wavelength

Accuracy Study:

Accuracy refers to the degree of precision of the analytical process or how closely a result corresponds to the real cost. The percentage recovery number falls within the range of 99.138 - 101.349% (Table 2), satisfying the acceptance requirements of the Association of Analytical Chemists (AOAC).

Table 2: Recovery Study

Concentration of RIFAMPICIN (µg/mL)	%Recovery ± SD
20	99.138± 0.003
30	101.349 ± 0.001
40	99.771 ± 0.001

SD =Standard Deviation

Precision Study:

The precision is a methodological error measure expressed as the relative standard deviation (RSD) for both repeatability and intermediate precision. The RSD value, as determined by the Association of Analytical Chemists (AOAC), falls within the range of 5.3% to 7.3%. The obtained intraday and interday precision meet the specified criteria (Table 3). Nevertheless, the analysis of interday precision has shown that Rifampicinshows significant instability in the solvent employed, as seen in Table 4. On the third day, solid particles were discovered in the solution. This phenomenon occurred as a result of the limited solubility of Rifampicin in the phosphate buffer. The solubility of Rifampicin in methanol is 16 mg/ml, but in phosphate buffer at pH 7.4 it is only 9.9 mg/ml^18,19,20.The disparity in solubility between the solvents might result in the gradual formation of solid Rifampicin.

Table 3: Intraday Precision Study

Concentration (µg/mL)	Concentration (µg/mL)	S. D	% RSD
20	0.420	0.009	2.206
30	0.612	0.002	0.298
40	0.792	0.001	0.179

RSD = Relative standard deviation

LOD and LOQ Study:

The limit of detection (LOD) and limit of quantification (LOQ) for Rifampicin were determined to be 0.934 µg/mL and 2.830 µg/mL, consecutively.

The drug loading capacity of SNEDDS Rifampicin:

The drug loading capacity of SNEDDS Rifampicin refers to the quantification of the proportion of Rifampicin present in the SNEDDS system. The findings are shown in Table 5.

Table 5: Drug Loading Capacity of Rifampicin

Replication	Rifampicin Concentration (µg/mL)
1	97.477%
2	97.745%
3	97.879%
Mean	2.931
SD	0.002
%RSD	0.070%

CONCLUSION:

The technique devised for Rifampicin SNEDDS was straight forward, uncomplicated, and cost-effective. The validation results indicated that the process meets the linearity, accuracy, and precisioncriteria. UV spectrophotometry is a suitable method for quantifying Rifampicin in SNEDDS dose form. However, to optimize the result, it is advisable to use fresh samples for measurement.

ACKNOWLEDGEMENT:

The author expresses gratitude to the Indonesian Ministry of Education and Culture (BIMA 2023) for providing the funding that enabled the completion of this study.

CONFLICT OF INTEREST:

The writers assert that they have no conflict of interest.

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Received on 10.12.2023 Revised on 08.05.2024

Accepted on 14.08.2024 Published on 24.12.2024

Available online from December 27, 2024

Research J. Pharmacy and Technology. 2024;17(12):6098-6101.

DOI: 10.52711/0974-360X.2024.00924

Concentration (µg/mL)	Day 1 Concentration (µg/mL)	Day 2 Concentration (µg/mL)	Day 3 Concentration (µg/mL)	Mean	S.D	%RSD
20	19,757	17,962	17,328	18,349	1,260	6,866
30	30,370	29,155	29,314	29,613	0,660	2,229
40	39,873	37,181	35,069	37,374	2,408	6,443