Page 8 - Application Handbook - Liquid Chromatography

Application No.L496
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n Online SFE-SFC Analysis of Reduced Coenzyme Q10
OH
Fig. 2 shows the structure of the reduced coenzyme Q10 O Reduction O
(ubiquinol). It is easily oxidized to form oxidized coenzyme O
Q10 (ubiquinone). In this case, both solvent extraction- O H
SFC and online SFE-SFC were used to analyze the reduced OH 10 Oxidation O H
coenzyme Q10 contained in a supplement capsule. O 10
Pretreatment operations and analytical conditions for Reducd coenzyme Q 10 Coenzyme Q 10
the solvent extraction-SFC analysis are indicated in Fig. 3 (Ubiquinol Q 10) (Ubiquinone Q 10)
and Table 1.
Chromatograms from analyzing the supplement and the Fig. 2 Structural Formulas
oxidized coenzyme Q10 standard sample are shown in
Fig. 4.
Table 1 Analytical Conditions for Solvent Extraction-SFC uV (×100,000)
System : Nexera UC SFC-UV System ■ Peaks
Column : Shim-pack UC-RP (150 mm L. × 4.6 mm I.D., 3 µm) 1.5 1. Coenzyme Q10
Column Temp. : 40 ˚C
Modifier : MeOH 2. Reduced Coenzyme Q10 1
Flowrate : 3 mL/min
Time Program : 5 % (0 min) → 50 % (5 - 8 min)
BPR : 10 MPa 1.0
Detector : UV-VIS (220 nm) Supplement sample
Inj. Vol. : 1 µL
2
0.5
Capsule sample
Standard solution of Coenzyme Q10
Take content in capsule

Ethanol (5 mL) 0.0
Sonoication (5 min)
Supernatant 0.0 2.5 5.0 min

Filtration (0.2 µm) Fig. 4 Chromatograms Obtained by Solvent Extraction-SFC
Inject to SFC (1 µL)
Fig. 3 Pretreatment
uV (×1,000,000)
Analytical conditions for online SFE-SFC are indicated in
Table 2. 2.5 ■ Peaks 2
1. Coenzyme Q10
About 5 µL each of the liquid sealed inside the 2. Reduced Coenzyme Q10
supplement capsule and the standard sample of oxidized 2.0
coenzyme Q10 were dripped onto filter paper. Then a
portion of the filter paper was cut with a punch-out 1.5
device and placed in the extraction vessel for analysis
by online SFE-SFC. Chromatograms from analyzing the 1.0 Supplement sample 1
supplement and the oxidized coenzyme Q10 standard 0.5
sample are shown in Fig. 5.
Standard solution of Coenzyme Q10
0.0
Table 2 Analytical Conditions for Online SFE-SFC
-0.5
System : Nexera UC Online SFE-SFC-UV System
SFE 0.0 2.5 5.0 7.5 10.0 min
Extraction Vessel : 0.2 mL
Static Extraction : Time ; 0 - 2 min,
: B.Conc. ; 5 %
: BPR ; 10 MPa Fig. 5 Chromatograms Obtained by Online SFE-SFC
: Flowrate ; 3 mL/min
Dynamic Extraction : Time ; 2 - 4 min,
: B.Conc. ; 5 % The results show that the coenzyme Q10 was oxidized
: BPR ; 10 MPa during extraction with solvent extraction-SFC, but not
: Flowrate ; 3 mL/min
SFC oxidized and remained as the reduced coenzyme Q10
Column : Shim-pack UC-RP (150 mm L. × 4.6 mm I.D., 3 µm) form throughout extraction, separation, and detection
Column Temp. : 40 ˚C
Mobile Phase : A; CO2 steps with online SFE-SFC. This shows how online SFE-
: B; MeOH SFC is an extremely unique analytical technique that can
Flowrate : 3 mL/min
Time Program : 5 % (4 min) → 50 % (9 - 13 min) be used to analyze unstable compounds without altering
BPR : 10 MPa their original form.
Detector : UV-VIS (220 nm)
First Edition: Oct. 2015

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