LAAN-A-LC-E120

Application News

High Performance Liquid Chromatography No.L363

High Speed, High Resolution Analysis (Part 13) Analysis of Flavanones in Citrus Juice

Flavanones are a type of flavonoids that are known News introduces examples of high-speed analysis to be widely distributed in plants, either in the of flavanones in citrus juice using the Shimadzu aglycone backbone form itself or as glycosides. In ultra fast LC system, Prominence UFLC, and the citrus fruits especially, they exist in unique forms photodiode array detector SPD-M20A, focusing on depending on the type of fruit, enabling them to be narirutin, naringin, hesperidin and their aglycones used as an indicator of fruit type. This Application naringenin and hesperetin.

Analysis of Flavanone Standard Solution Fig. 1 shows the structures of the flavanones mAU analyzed here. Narirutin and naringin are glycosides 400 Peaks of naringenin, while hesperidin, known as vitamin P, 1. Narirutin 4 is a glycoside of hesperetin. 2. Naringin 350 5 Fig. 2 shows the results of simultaneous analysis of 3. Hesperidin the 5 compounds, and Table 1 shows the analytical 4. Naringenin conditions. A photodiode array detector was used 300 5. Hesperetin for detection. Under these conditions, simultaneous 250 analysis of the glycosides and aglycones was 2 achieved in less than 9 minutes. 3 200 1

OH 150 Naringenin HO O

100

OH O OH 50 HO

O O O OH Naringin OH 0

O OH O OH O 0.0 1.0 2.0 3.0 4.0 5.0 min CH3

OH OH Fig.2 Chromatogram of a Standard Mixture of 5 Flavanones OH (100 mg/L each , 2 µL injected)

OH O CH2 O O O O Narirutin CH3 OH OH Table 1 Analytical Conditions OH OH OH × µ OH O Column : Shim-pack XR-ODS (75 mmL. 3.0 mm I.D., 2.2 m) Mobile Phase : A ; 10 mmol/L Ammonium formate buffer (pH 3.7) OCH3 / Acetonitrile = 9 / 1 (v/v) HO O Hesperetin B ; 10 mmol/L Ammonium formate buffer (pH 3.7) OH / Acetonitrile = 2 / 8 (v/v) B ; 0 % (0.00 min) → 50 % (5.00 min) OH O → 100 % (5.01-6.00 min) → 0 % (6.01-9.00 min) OCH3 Flow Rate : 1.0 mL/min OH O CH2 O O O O Hesperidin Injection Volume : 2 µL CH3 OH OH OH Column Temp. : 35ûC OH OH OH Detection : SPD-M20A (at 285 nm) OH O Flow Cell : Semi-micro Cell

Fig.1 Structures of Flavanones No.L363

■ UV Spectra of Flavanones Fig. 3 shows the UV spectra of the 5 flavanones in qualitative information can be obtained for peaks in the standard mixture acquired with a photodiode an actual sample by conducting spectral array detector. Extremely high absorption is evident comparison with these data, or by calculating the in the vicinity of 285 nm. If these spectra are saved degree of similarity. to a spectrum file or registered in a library,

mAU mAU 600 500 550 450 500 Narirutin 400 450 350 400 Hesperidin 350 300 Naringin 300 250 250 200 200 150 150 100 100

50 50 0 Naringenin 0 Hesperetin -50 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm

Fig.3 UV Spectra of Flavanones

■ Analysis of Citrus Juice Fig. 5 and Fig. 6 show the results of analysis of Sample 3 mL commercial 100% orange juice and 100% grapefruit 60 % Acetonitrile 3 mL juice respectively. The samples were prepared as shown in Fig. 41). Narirutin and hesperidin are Mixing present in the orange juice, while narirutin and naringin are present in the grapefruit juice, all at Centrifugation high concentrations. By comparing these peak Filtration HPLC spectra with those of standard mixture of Fig. 3, (10,000 rpm × 5 min) analysis results with even greater accuracy becomes possible. Fig.4 Preparation of Citrus Juice Sample

mAU mAU 120 225 ■ ■ 110 Peaks Peaks 1. Narirutin 3 200 1. Narirutin 2 100 3. Hesperidin 2. Naringin 3. Hesperidin 90 175

80 150

70 125 60

50 100

40 75 1 1 30 50 20 25 10 3

0 0

0.0 1.0 2.0 3.0 4.0 5.0 min 0.0 1.0 2.0 3.0 4.0 5.0 min

Fig.5 Chromatogram of Orange Juice (2 µL injected) Fig.6 Chromatogram of Grapefruit Juice (2 µL injected)

[References] 1) AOAC Official Method 999.05; Naringin and Neohesperidin in Orange Juice

NOTES: ✽This Application News has been produced and edited using information that was available when the data was acquired for each article. This Application News is subject to revision without prior notice.

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