Rapid Determination of Hesperidin in Application Brief 142 Peel Using Accelerated Solvent Extraction and UHPLC

Huang Xiongfeng,1 Xu Qun,1 Che Jinshui,1 and Jeffrey Rohrer2 1Thermo Fisher Scientific, Shanghai, People’s Republic of China 2Thermo Fisher Scientific, Sunnyvale, CA, USA

Key Words OH O Pharmacopoeia of the People’s Republic of China (PPRC), Natural Herbal Medicine, , Polyphenolic Bioflavonoid HO O O OH CH 3 O O O OH OH OH Introduction OCH3 Hesperidin, a polyphenolic bioflavonoid, is the OH OH predominant flavonoid in orange peel and other Figure 1. Structure of hesperidin fruits. Figure 1 shows its structure. Hesperidin is an antioxidant that enhances the action of vitamin C to lower cholesterol levels. It is also known to have pharmacological action as an anti-inflammatory, Sample Extraction antihistaminic, and antiviral agent.1,2 The Pharmacopoeia Weigh 1 g of ground orange peel sample and mix it with of the People’s Republic of China (PPRC) 2010 a suitable amount of diatomaceous earth (DE Dispersant recommends its extraction from fruits with a Soxhlet for ASE, P/N 06281). Place the mixture in a separate extraction method using ligarine and methanol.3 10 mL stainless steel extraction cell equipped with two That method is both time- and solvent-consuming, cellulose filters on the bottom. Samples will nearly fill the requiring ≥5 h and >200 mL of ligarine and methanol cells. Extract loaded cells with the Dionex ASE™ 350 for each sample. The PPRC 2010 also recommends the system using the optimized conditions shown in Table 2. determination with a 12 min reversed-phase high- The use of ligarine to extract naphtha ingredients (step 1) performance liquid chromatography (HPLC) method. will eliminate their interference for subsequent hesperidin determination. Discard the extract from this step and Goal extract the sample cell again by methanol (step 2). Transfer the methanol extracts into a separate 25 mL To develop a more efficient and cost-effective method volumetric flask and bring to volume with deionized to determine hesperidin in orange peel and other water. Filter the diluted extracts through a 0.4 μm filter citrus fruits. before injection onto the UHPLC system. Equipment • Thermo Scientific Dionex UltiMate 3000 rapid separation LC (RSLC) system, including: – HPG-3400RS Binary Rapid Separation Pump – WPS-3000RS Rapid Separation Autosampler – TCC-3000RS Thermostatted Column Compartment – DAD-3000RS Rapid Separation Diode Array Detector with Semi-Analytical Flow Cell, SST, 5 µL Volume, 7 mm Path Length • Thermo Scientific Dionex Chromeleon Chromatography Data System software version 6.80, SR9, or above • Thermo Scientific Dionex ASE 350 Accelerated Solvent Extractor Application Brief 142 90 s 5 min Methanol 34 2 60% 6 min  120 °C

 S LPN3066 6 /1 7 Minutes 0 3 min 200 s RSLC 120 C18, RSLC Step 1 Step 2 Ligarine 65) 6289 1190 886) 2 8751 6655 41) 6241) 205 9966 46) 8 473 3380 ™ ( (44) 1276 691722 ( ( ( 1

AB142_E

(2.2 µm, 3 × 30 mm, P/N 071605) 3 × 30 mm, (2.2 µm, standard (2 µg/mL) b. Hesperidin c. Orange peel sample 1 (100-fold dilution) d. Orange peel sample 2 dilution) e. peel sample (50-fold

iwan weden witzerland United Kingdom Ta Singapore S S a c b e d 0 1 2 Conditions Temperature Heat Time Static Time Static Cycles Flush Purge Time Solvent Peak: 1. Hesperidin Mobile Phase: Mobile Flow Rate: acid, 31/65/4 (v/v) Methanol/water/acetic Inj. Volume: Temperature: 0.5 mL/min 1 µL Detection: 40 °C Chromatograms: phase a. Mobile UV, 283 nm Column: Column: Acclaim 40 -10 mAU Figure 2. Chromatograms of hesperidin in orange and lemon peel samples 2. DionexTable ASE 350 instrument conditions 91) 22 2764 2735 81) 6 6885 1213 82) 2 2653 2580 353) 1 644 0064 39) 02 62 51 1267 ( ( ( ( (

1 eland eland aly 35 40 3.1 apan 6.3 Ir It J Korea India Duan, Dionex Dionex

ASE 350 ASE .;

 0 Y

Extraction-

Li,

1 7.4 5.2 200 300 X.W.; Soxhlet

Simultaneous 55) 3731 5140 11 852) 2428 3282 33) 1 39 30 01 1 45) 36 36 90 90 49) 6126 991 0 Assisted

( ( ( ( (

Extraction

Li, ll trademarks are the property of Thermo Fisher Scientific Inc. and its and Inc. Scientific Fisher Thermo of property the are trademarks ll

Hesperidin, and Hesperidin,

H.K.

ubject to change. Not all products are available in all countries. Please consult Please countries. all in available are products all Not change. to ubject s ance ance ermany razil M.F.; B G China Denmark Fr

(2), 203–210. 34 (2), Shin,

Microwave

Xie,

4 by

2011, (5), 640–642. 30 (5), .J.; M.Y.; Y

u, Y Lee,

2011, 61) 3 9757 4486 847) 295 7500 31) 20 683 9768 32) 3 353 42 9 43) 25 51 1 616 3000 RSLC system. The total analysis time— system. 3000 RSLC ( ( ( ( (

W ™

Determination of Hesperidin from Pericarpium Extraction Method

H.K.;

Zhou,

Glycyrrhizin by HPLC-Photodiode Array Detection and Glycyrrhizin by HPLC-Photodiode Arch. Anti-Inflammatory Effect of Pyungwi-san. the Pharmacal Res. Determination of , 826–827. 2010, 1), (Vol. Citri Reticulatae G.L. Herald of High Performance Liquid Chromatography, Medicine Ha, Sample Amount (g) Detected Amount of Hesperidin (%) Solvent Amount (mL) (min) Time RSD Austria Benelux Australia subsidiaries. Specifications, terms and pricing are pricing and terms Specifications, subsidiaries. details. for representative sales local your thermofisher.com reserved. A rights All Inc. Scientific Fisher Thermo ©2016 U.S./Canada RSLC 120 C18 column. 120 C18 RSLC samples separated on the Thermo Scientific Acclaim Thermo Scientific samples separated on the shows the chromatograms of hesperidin in orange peelshows the chromatograms of hesperidin in significant time and solvent with similar results. Figure 2 results. significant time and solvent with similar ASE extractor method; use of the Dionex ASE 350 saves ASE extractor method; use of the Dionex 40 mL. Table 1 compares the Soxhlet approach with the 1 compares the Soxhlet Table 40 mL. separation), and solvent consumed for each extraction was separation), sample was ~40 min (35 min for extraction and 4 min forsample was ~40 min (35 min for extraction including extraction and separation—for an orange peelincluding extraction and separation—for 3. Republic of China Pharmacopoeia of the People’s

1..; J.K.; Lee, H.Y.; Lee, 2. D.Y.; Jung, J.A.; C.S.; Lee, Seo, References to perform quick extractions, was used for the sample to perform quick extractions, This work demonstrates a quick and efficient methodThis work demonstrates uses high temperature and pressureextraction system that UltiMate Conclusion in orange peel and other citrusto determine hesperidin an automated ASE 350 system, The Dionex fruits. separation was performed on a DionexThe extraction. ASE 350 Table 1. Comparison 1. Table of extraction methods using Soxhlet extraction and the Dionex