D printed structure coated with C18 particles in an online flow system coupled to HPLC-DAD for the determination of flavonoids in citrus external peel

Abstract A 3D printed disc coated with C18 resin was developed and used in a flow system coupled to high-performance liquid chromatography (HPLC) with diode array detection (DAD), for the extraction, separation and quantification of six flavonoids: naringin, naringenin, hesperidin, hesperetin, diosmetin and tangeretin in the citrus external peel. The developed method is based on the automatic loading of 16 mL of sample into an extraction tank with a 3D printed C18 disc (8 mm id × 5.5 mm height) with 39.01 mg of immobilized C18. Then, the matrix is cleaned-up in 15 s and the analytes are eluted in 10 s with 0.3 mL of acetonitrile (twice). The extract is directly injected into the HPLC (0.01 mL). A monolithic C18 column is employed to perform the separation using a mixture of acetonitrile and acidified water (pH = 2.5) as a mobile phase in gradient mode. The total procedure time is 28 min. Limits of detection, quantification and relative standard deviations range between 5.86×10-2 - 4.69×10-2 µg/mL, 2.34×10-2 - 1.88×10-1 µg/mL, and 3.29 - 4.49%, respectively. The coupling between the flow-based extraction system and the HPLC provides a fully automated method for the extraction and determination of the studied flavonoids reducing the consumption of time and solvents and improving the analysis sensitivity and throughput. The developed 3D printed device notably eliminates the drawback of the generated backpressure in flow systems using traditional solid-phase extraction columns. Thus, a robust and effective automated solid-phase extraction method has been developed, optimized, and successfully applied for the determination of six flavonoids in 8 citrus external peel samples.