LC Technical NoteLT191

Analysis of β-cryptoxanthin in Citrus Unshiu

GL Sciences Inc.

β-Cryptoxanthin is a carotenoid pigment found in the edible part of the fruit Citrus unshiu. In addition to its function asprovitamin A, which is mainly metabolized to the essential nutrient vitamin A, it is also known to have a excellent antioxidant
properties derived from the highly conjugated double bonds in the molecular framework. β-Cryptoxanthin is considered to beeffective in preventing lifestyle-related diseases and in reducing the incidence of osteoporosis. Many foods, supplements, softdrinks, etc. are commercially available that take advantage of these beneficial effects. In this application note, separation andanalysis was made with reference to the food sanitation inspection guidelines, and the JAS amendment. (S.Miura)

Chromatogram with Conditions Described in Japan Agricultural Standards(JAS) Amendment
The Japanese Agricultural Standards (JAS) amendment of 2019 describes HPLC conditions using an Inertsil ODS-3 column.
In this application note we will introduce an example of analysis using Inert Sustain C18 column that has comparable
performance but with greater inactivity. Using this column, it is possible to perform analysis with a separation patternsimilar to ODS-3 column.

Analysis of Standard Solution
1. β-Cryptoxanthin 1 mg/LOH61

Chemical Structure

β-cryptoxanthin

02mAU

4Structures created using Chemistry 4-D Draw, which is provided by
ChemInnovation Software, Inc.

010Time (min)20

Wavelength (nm)Conditions
ColumnColumn Cat. No.
EluentFlow rate
Col. Temp.
Detection
Injection Vol.
SampleUV/Vis absorption spectrum: InertSustain C18(5 μm, 150 x 4.6 mmI.D.): 5020-07345: 0.12 mM Ascorbyl palmitate in (CH3OH / CHCl3 = 24/1, v/v)
: 1.5 mL/min: 40 ℃: VIS 455 nm: 20 μL: Standard

GL Sciences LC Technical Note
Example of Analysis of β-cryptoxanthin
After removing the epicarp part of the Citrus unshiu fruit, it was crushed using a homogenizer, and extraction wasperformed in accordance with the 2019 Japanese Agricultural Standards (JAS) amendment. After saponification, theresidue was dissolved and filtered to obtain a sample solution.

Pretreatment

Recover unsaponifiable agentSample― 20mL of 1% sodium chloride solution
5mL of 2-propanol― Weigh approximately 2.0 g

Add 12mL of ethyl acetate/hexane (9:1) mixtureExtraction― Shake for 5 min― Add 15 mL of pyrogallol solution (30 g / L)

― Centrifuge for 5 min― Sodium sulfate 10 g

― Collect the supernatant (eggplant flask) (②)― Repeat ① for the residue and collect supernatant (③)― Shake extract for 5 min― Centrifuge for 5 min― Repeat operations ③ (④)①― Combine ②③④ and distill under reduced pressure.

― Collect the supernatant (volumetric flask) (②)― Add 15 mL of pyrogallol solution to the residue

Constant volume

repeat ①, and collect the supernatant (③).
― Make up the volume to 5 mL with ethanol― Combine ② and ③ and add pyrogallol solution

Filtrationand make up the volume to 50mL.

― Pass the recovered liquid through a GL
ChromatoDisc 13P (0.45 μm).

Saponification― Take 10mL of constant volume solution
in a centrifuge tube

Sample solution― 1mL of 60% Potassium Hydroxide Solution

HPLC― Heat (70 ℃) for 30 minutes― Cool (to room temperature)

10Analysis of Sample Solutions
1. β-cryptoxanthin※ β-Carotene

24mAU
6810※010Time (min)20GL Sciences LC Technical Note

Chromatogram with Conditions Described in Food Hygiene Inspection Guideline(carotene analysis)

Since β-cryptoxanthin is a type of carotene, it is possible to perform analysis under the same eluent conditions usedfor carotene. In this application note (with reference to the food hygiene inspection guidelines) a relativelyhydrophobic solvent was used as the eluent. In addition, the measurement was performed using an ODS column thatmakes it easy to recognize the difference in structures.

Standard

β-Cryptoxanthin 1 1mg/Lmg/L
1. β-cryptoxanthin

02mAU46110Time (min)0Sample

201mAU24

1. β-cryptoxanthin※ β-Carotene0※Conditions
ColumnColumn Cat. No.
EluentFlow Rate
Col. Temp.
Detection
Injection Vol.
Sample10Time (min): Inertsil ODS-P(5 μm, 150 x 4.6 mmI.D.): 5020-02001: A) CH3CNB) CH3OHC) THF
A/B/C = 55/40/5, v/v/v: 1.5 mL/min: 20 ℃: VIS 455 nm (5430 Diode Array Detector): 20 μL: Standard

20Area0Conc entration(mg/L)

Calibration curve

GL Sciences LC Technical Note
Products used
 Analysis column• InertSustain C18 5 μm 4.6 x 150mmI.D.
Cat.No. 5020-07345• Inertsil ODS-P 5 μm, 4.6 x 150 mmI.D.
Cat.No. 5020-02001
 Vial related products• Polypropylene vial, 1.5mL, 100/pk
Cat.No.1030-51019• Screw Cap with Septa PTFE Red/Silicon White, thickness 1.0mm
Cat.No.1030-51222
 Syringe Filter• GL ChromatoDisc 13P
Cat.No. 5040-28541• Plastic Disposal Syringe, 10mL, Luer-lock type
Cat. No.1030-55110

GL Sciences disclaims any and all responsibility for any injury or damage which may be caused by this data directly or indirectly.
We reserve the right to amend this information or data at any time and without any prior announcement.

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