Determination of naringin content by HPLC - Master's thesis - Dissertation

Zhuanggu Capsule is a traditional herbal preparation made from eight key ingredients, including Sclerotium, Epimedium, and Myrrh. It is known for its ability to strengthen the kidneys, reinforce bones, and relieve pain. Among these components, Sclerotherapy is one of the essential herbs, and its active compound, naringin, serves as an important marker for quality assessment. This article presents a high-performance liquid chromatography (HPLC) method for determining naringin content in Zhuanggu Capsule, based on the methodology described in the Chinese Pharmacopoeia [5]. The method was validated through various experiments, including linearity, precision, stability, reproducibility, and recovery rate, confirming its reliability and accuracy for quality control purposes. **Instruments and Reagents** - **Instrument**: Shimadzu HPLC system with SPD-20AVP UV detector, LC-20ATVP pump, and Zhejiang University N200O chromatography workstation. - **Balance**: FA2004 electronic balance (Shanghai Precision Science Instrument Co., Ltd.) - **Reference Substance**: Naringin standard (batch no. 110722-200309), provided by the National Institutes for Food and Drug Control. - **Reagents**: Analytical grade reagents, double-distilled water, and HPLC-grade methanol. **Methods and Results** **Chromatographic Conditions** - Column: Phenomenex ODS (250 mm × 4.6 mm, 5 μm) - Mobile Phase: Methanol-acetic acid-water (37:2:61) - Detection Wavelength: 283 nm - Flow Rate: 1.2 mL/min - Column Temperature: Room temperature - Theoretical Plates: ≥ 3,000 **Preparation of Reference Solution** Accurately weigh a suitable amount of naringin reference substance dried at 110°C and dissolve it in methanol to prepare a solution of 0.08 mg/mL. **Preparation of Test Solution** Weigh 1.0 g of capsule contents, grind into fine powder, and extract with 30 mL methanol using ultrasonic bath for 30 minutes. After cooling, filter and evaporate the solvent. Dissolve the residue in methanol and dilute to 10 mL for analysis. **Method Validation** **Linearity Study** Naringin solutions at concentrations of 0.184–0.920 g/L were injected. A linear relationship was observed with regression equation Y = 17215X + 43318, R² = 0.9995. **Precision Test** Six injections of 10 μL of the reference solution showed an RSD of 0.9%, indicating good repeatability. **Stability Test** Sample solutions remained stable for up to 8 hours, with RSD of 1.2%. **Reproducibility Test** Six independently prepared samples showed an RSD of 1.6%, confirming the method's consistency. **Recovery Test** Average recovery was 99.1% with an RSD of 1.9%, demonstrating the method’s accuracy. **Sample Analysis** Six batches of samples were analyzed, and the naringin content ranged from 0.32 to 0.41 mg/g. **Discussion** Various extraction methods, including cold maceration, reflux, and ultrasonic extraction, were compared. Methanol proved to be the most effective solvent, and 30 minutes of sonication was sufficient for complete extraction. This method ensures efficient and accurate determination of naringin in Zhuanggu Capsules, making it suitable for routine quality control.

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