Validated reversed phase-high performance liquid chromatography-diode array detector method for the quantitation of Rutin, a natural immunostimulant for improving survival in aquaculture practice, in toonea sinensis folium

Background: Rutin is a biofl avonoid of strong immunostimulating activity from the Toonea Sinensis Folium, which has shown a signifi cant ability to increase the survival rate of white shrimp with bacterial infection. However, no method for the quantitation of this active ingredient in the herb has been reported to date. Materials and Methods: A reversed phase-high performance liquid chromatography-diode array detector (RP-HPLC-DAD) method was developed to quantify Rutin in the Toonea Sinensis Folium, with the HPLC conditions optimized, followed by validation for linearity, accuracy, precision, limit of detection (LOD), repeatability, and stability. Then, the established method was used to determine the content of Rutin in two samples. Results: The separation was performed on a Waters XBridge Shield RP18 column (150 mm × 4.6 mm, 5 μm) kept at 25°C, and acetonitrile and water containing 0.1% acetate acid (18:82, v / v)-composed mobile phase was constantly driven at 1.0 mL / minute during the analysis. Twenty microliters of sample solution or standard solution were injected into the HPLC system and 254 nm was selected to monitor the separation. A strong linear relationship between the peak area and concentration of Rutin was observed within the range of 0.01044 – 0.2610 mg / mL (r2 = 1.0000). The LOD was 0.03915 μg / mL, and recovery of Rutin was from 97.6 to 99.6%. In addition, the method was also validated to be repeatable, stable, precise, and accurate. Conclusions: An effi cient and reliable RP-HPLC-DAD method was established, which could be used for routine analysis of Rutin in Toonea Sinensis Folium and to assist in the quality control of this herb.