LAAN-A-LC-E127

Application News

High Performance Liquid Chromatography No.L366

High Speed, High Resolution Analysis (Part 16) Analysis of Flavanones in Citrus Juice (Part 2)

Previously, Application News No. L363 introduced compound, . Furthermore, the LCMS- the high-speed analysis of 5 flavanones in citrus 2010EV mass spectrometer was added to the juice using the Ultra Fast LC system, Prominence Prominence UFLC / SPD-M20A configuration, and UFLC, and the photodiode array detector SPD- the analysis of minor components were verified M20A. Here we introduce an example of high-speed using UV spectra as well as mass spectra. analysis of the same flavanones plus one additional

■ Analysis of 6 Flavanones Fig. 1 shows the structures of the 6 flavanones mAU(×100) 4.00 5 analyzed here, consisting of the 5 flavanones ■ Peaks described in Application News No. L363 in addition 1.Narirutin 6 2. to neohesperidin. 3.50 Fig. 2 shows the results of simultaneous analysis of 3.Hesperidin 4.Neohesperidin a standard mixture of the 6 flavanones using the 5. SPD-M20A, and Fig. 3 shows the UV spectrum of 3.00 6. neohesperidin. The analytical conditions are shown in Table 1. 2.50 4 R1 2 3 2.00 OR2 1

R3O O 1.50

1.00 OH O

R1 R2 R3 0.50 Narirutin H H Rutinose Naringin H H Neohesperidose 0.00 Hesperidin OH CH3 Rutinose Neohesperidin OH CH3 Neohesperidose 0.0 1.0 2.0 3.0 4.0 5.0 min Naringenin H H H Hesperetin OH CH3 H Fig.2 Chromatogram of a Standard Mixture of 6 Flavanones Fig.1 Structures of the Flavanones (100 mg/L each, 2 μL injected)

mAU Table 1 Analytical Conditions 700 Column : Shim-pack XR-ODS (75 mmL. × 2.0 mmI.D., 2.2 μm) 600 Mobile Phase : A ; 10 mmol/L Ammonium formate buffer (pH 3.7)

500 / Acetonitrile = 9 / 1 (v/v) B ; 10 mmol/L Ammonium formate buffer (pH 3.7) 400 / Acetonitrile = 2 / 8 (v/v) 300 B conc. 0 % (0.00 min)→50 % (5.00 min) →100 % (5.01-6.00 min)→0 % (6.01-9.00 min) 200 Flow Rate : 0.4 mL/min with Semi-micro mixer 100 Injection Volume : 2 μL Column Temp. : 35 ûC 0 Detection : SPD-M20A at 285 nm 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm Flow Cell : Semi-micro cell Fig.3 Spectrum of Neohesperidin No.L366

■ Analysis of Grapefruit Juice Fig. 4 and Fig. 5 show the results of analysis of further supplemented by comparing the UV spectra commercial 100% grapefruit juice using the SPD- and mass spectra. In addition, since the m/z 507 M20A and LCMS-2010EV. Sample preparation was ion in the hesperidin mass spectrum is absent from conducted using the same procedure as that standard solution mass spectrum, it is presumed to described in Application News No. L363, and 2 μL be an unknown compound. Even in this type of was injected. situation, monitoring of the m/z 609 ion can provide From the chromatogram, it is evident that hesperidin very accurate quantitation. Table 2 shows the LCMS- and neohesperidin are included along with narirutin 2010EV analytical conditions. (The other conditions and naringin. However, the qualitative information is are the same as those of Table 1.).

mAU(×100) mAU 500 3000 ■ Peaks 2 450 2750 1.4 1.Narirutin 400 Narirutin 2500 Naringin 2250 2.Naringin 350 Standard 2000 3.Hesperidin 1 300 Grapefruit Juice 1750 1.2 4.Neohesperidin 250 1500

200 1250 1000 150 750 1.0 100 500 50 250

0 0 0.8 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm 200.0 225.0 250.0 275.0 300.0 325.0 350.0 375.0 nm

mAU mAU

70 0.6 60 60

50 Hesperidin Neohesperidin 50

0.4 40 3 40

30 30 4 0.2 20 20

10 10

0.0 0 0

nm 0.0 1.0 2.0 3.0 4.0 5.0 min 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

Fig.4 UV Chromatogram and Spectra of Flavanones in Grapefruit Juice (2 μL injected)

Inten. (×1,000,000)

■ Peaks 2 Inten. (×100,000) Inten. (×100,000) 1.Narirutin [M-H]- 579 [M-H]- 579 2.0 Narirutin Naringin 4.00 2.Naringin 3.Hesperidin 5.0 4.Neohesperidin 1 3 1.5 3.50 4 TIC 1.0 2.5 3.00 0.5 2 271 557 557 0.0 0.0 2.50 300 400 500 600 m/z 300 400 500 600 m/z

1 2.00 Inten. (×10,000) Inten. (×10,000) m/z 579 1.0 609 2.0 Hesperidin507 Neohesperidin - 1.50 [M-H] 4 0.7 1.5 [M-H]- 1.00 609 0.5 3 1.0

0.50 0.5 600 0.2 552 397 433 459 501 0.0 0.0 m/z 609 0.00 300 400 500 600 m/z 300 400 500 600 m/z

0.0 1.0 2.0 3.0 4.0 5.0 min

Fig.5 Mass Chromatograms and Mass Spectra of Flavanones in Grapefruit Juice using Scan Mode (2 μL injected)

Table 2 LC-MS Conditions Probe Voltage : -3.5 kV (ESI-Negative mode) CDL Temp. : 250 ûC Nebulizing Gas Flow : 1.5 L/min Block Heater Temp. : 200 ûC Drying Gas Pressure : 0.1 MPa Scan Range : m/z 100 - 650 CDL, Q-array Voltages : using default values

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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