THE DEVELOPMENT AND VALIDATION OF ANALYTICAL METHOD FOR EVALUATING GALLIC ACID IN ETHYL ACETATE FRACTION (EAF) OF SNEDDS FORMULATION: QUANTITATIVE ANALYSIS WITH IN VITRO ASSAY

International Journal of Applied Pharmaceutics Pub Date : 2024-03-07 DOI:10.22159/ijap.2024v16i2.49830

P. Apridamayanti, L. Pratiwi, R. Sari

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Abstract

Objective: This study aimed to develop a simple, accurate, precise, sensitive, robust, and stable analytical method for the evaluation of gallic acid in Self-Nanoemulsifying Drug Delivery System (SNEDDS) incorporating ethyl acetate fraction (EAF) of Melastoma malabathricum leaves in combination with Gentamicin. Methods: Validation process followed ICH guidelines and applied a reverse phase HPLC method with a mobile phase of acetonitrile-phosphoric buffer at pH 3.03 (20:80 v/v). The stationary phase consisted of a VP-ODS shim-pack C-18 column (250x4.6 mm) with a flow rate of 0.2 ml/min and detection at 263 nm using an Ultraviolet detector. Additionally, antioxidant activity was assessed through the DPPH and FRAP methods, and SPF value was determined with a UV/Vis spectrophotometer in the 290-390 nm wavelength range. Results: The results showed that the retention time of quercetin was 16.648 min with a tailing factor of 1.623. The regression equation (y=224689x-989000) had a concentration range of 10-55 µg/ml and a correlation value of 0.9920. Limit of Detection (LOD) and Limit of Quantification (LOQ) were found to be 2.394±0.086 and 7.254±0.260 µg/ml, respectively. Method accuracy, determined by recovery values at concentrations of 50%, 100%, and 150%, ranged from 91.18% to 109.49%. Repeatability inter-day variations were expressed as %RSD values of 1.027-1.963% for AUC and 0.150-0.145 for RT. Moreover, the applied method showed stability within a temperature range of 14 °C–35 °C. Analysis showed gallic acid content of 1.773±0.049 mg/g in SNEDDS EAF formulation. Antioxidant activity measured through the DPPH and FRAP methods yielded IC50 values of 4.167±0.552 µg/ml and 20.253±0.619 µg/ml, respectively, while SPF value at SNEDDS concentration of 150 µg/ml was 36.993±0.183. Conclusion: This study successfully developed a precise, accurate, specific, and stable method for quantifying gallic acid levels in SNEDDS EAF of Melastoma malabathricum leaves in combination with Gentamicin. Therefore, SNEDDS EAF formulation exhibited an effective wound-healing potential, supported by a robust quality control process.

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乙酸乙酯馏分（eaf）中没食子酸分析方法的开发与验证：体外定量分析

研究目的本研究旨在开发一种简便、准确、精确、灵敏、稳健、稳定的分析方法，用于评估含有马拉巴曲叶乙酸乙酯馏分（EAF）的自纳米乳化给药系统（SNEDDS）中没食子酸与庆大霉素的结合情况：验证过程遵循 ICH 指南，采用反相高效液相色谱法，流动相为 pH 值为 3.03 的乙腈-磷酸缓冲液（20:80 v/v）。固定相为 VP-ODS shim-pack C-18 色谱柱（250x4.6 毫米），流速为 0.2 毫升/分钟，紫外检测器的检测波长为 263 纳米。此外，还采用 DPPH 和 FRAP 法评估了抗氧化活性，并用紫外/可见分光光度计在 290-390 nm 波长范围内测定了 SPF 值：结果表明，槲皮素的保留时间为 16.648 分钟，尾数系数为 1.623。回归方程（y=224689x-989000）的浓度范围为 10-55 µg/ml，相关系数为 0.9920。检测限（LOD）和定量限（LOQ）分别为 2.394±0.086 µg/ml 和 7.254±0.260 µg/ml。根据浓度为 50%、100% 和 150% 时的回收率确定，方法的准确度为 91.18% 至 109.49%。AUC和RT的日间重复性差异分别为1.027%-1.963%和0.150%-0.145%。此外，该方法在 14 °C-35 °C 的温度范围内表现出稳定性。分析表明，SNEDDS EAF 配方中的没食子酸含量为 1.773±0.049 mg/g。通过 DPPH 和 FRAP 方法测定的抗氧化活性的 IC50 值分别为 4.167±0.552 µg/ml 和 20.253±0.619 µg/ml，而 SNEDDS 浓度为 150 µg/ml 时的 SPF 值为 36.993±0.183：本研究成功地建立了一种精确、准确、特异、稳定的方法，用于定量分析马缨丹叶SNEDDS EAF与庆大霉素复方制剂中没食子酸的含量。因此，SNEDDS EAF 制剂在健全的质量控制流程的支持下，表现出了有效的伤口愈合潜力。

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International Journal of Applied Pharmaceutics

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