Chuo University, School of Science, 1-13-27, Kasuga Bunkyo 112-0003 Tokyo, Japan
Received date: January 28, 2013; Accepted date: March 15, 2013; Published date: March 20, 2013
Citation:Shintani H (2013) HPLC Analysis of Extracted Coenzyme Q (Coq) Homologues from Animal Tissues. Pharmaceut Anal Acta 4:219. doi: 10.4172/2153-2435.1000219
Copyright: © 2013 Shintani H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Visit for more related articles at Pharmaceutica Analytica Acta
Coenzyme Q (CoQ) plays an important role in ATP synthesis as an electron-carrying component of the mitochondrial respiratory chain. In recent years, increasing attention has focused on the reduced form of CoQ homologues (CoQnH2) as antioxidants.
During lipid peroxidation in disease states it is important to detect the decrease in endogenous antioxidants and to measure the increase in lipid peroxides. Measurement of CoQ homologues in animal tissues by High Performance Liquid Chromatography (HPLC) is described.
1. Animal tissue (approx. 300 mg) is stored in liquid nitrogen.
2. Add ice-cold distilled water (8 vol.).
3. Homogenize tissues by means of a PolytronR under a stream of nitrogen for 20 s at 4°C. A sample of the homogenate is used for protein assay.
4. Place homogenate (1 ml) in individual 10-ml test tubes with a screw cap.
5. Add HPLC-grade ethanol (2 ml) to each tube.
6. Extract each tube with HPLC-grade n-hexane (3 × 5 ml, shaking vigorously for 10 min during each extraction). After each extraction centrifuge at 750 g for 5 min, remove the upper n-hexane layer carefully and pour into 60-ml dark-brown centrifuge tubes with stoppers, previously filled with nitrogen gas.
7. Evaporate the combined n-hexane layers (15 ml) under a stream of nitrogen.
8. Re-dissolve the residue in ice-cold ethanol (0.5-1.0 ml) and pass the solution through a 0.45 μm filter.
9. Analyse by HPLC.
10. HPLC was performed with a JASCO 880-PU pump and an injector. Compounds were separated on a 250 mm × 4.6 mm i.d., 7 μm particle, Chemcosorb ODS-H column. The mobile phase was 0.7% (w/v) NaCIO4.H2O in 700:300;1 HPLC-grade ethanol-HPLC-grade methanol-70% HCIO4 at a flow rate of 1.2 ml/min. The injection volume was 10 μl. A JASCO 870-UV detector (operated at 275 nm) was used for determination of oxidized CoQn and a JASCO 840-EC electrochemical detector (ECD) (+700 mV relative to Ag+ /AgCI) for CoQnH2. The chart speed was 2 em/min.
Preparation of CoQn homologue standards
Standard solutions of CoQ9 and CoQ10: Chromatographically pure CoQ9 and CoQ10 from Eisai (Tokyo, Japan) were dissolved in HPLC-grade ethanol at a concentration of 550 μg/mL and diluted to 5.5 μg/mL with ethanol. When stored in dark-brown vials at –80°C these standard solutions are stable for approximately 1 year.
Standard solutions of CoQ9H2 and CoQ10H2: Standard solutions of CoQ9 and CoQ10 were reduced with NaBH4. CoQn stock solution (200 μL) was vortex-mixed with a mixture of NaBH4 (0.3%, 40 μL) in water and 160 μL HPLC-grade ethanol. CoQnH2 standards should be freshly prepared before use.
Typical UV and ECD detected HPLC chromatograms of CoQ homologues from the liver of a three-week-old rabbit are shown in figure 1.
The ECD is connected to the outlet of the UV detector. ECD is sensitive to electrical noise and the baseline on the ECD chromatogram is not always stable. This seems to be because of dirt in the HPLC system, including the column, and because of mechanical and electrical noise. The working electrode should be cleaned when the sensitivity of the electrochemical detector falls.
1n some animal species, and tissues, an unknown peak co-elutes with the CoQnH2 such as α-tocopherol peak on the chromatogram if the applied potential is +700 mV (Figure 1). If this happens, selectivity is improved by use of a setting near +600 mV. It has previously been reported that electrochemical detection can be performed with an applied potential of +500 mV [1,2].
For UV detection in figure 1, to avoid interference of determination of the interest peaks, solid phase extraction (SPE) is quite recommendable and the automated SPE is commercially available and significant separation results were reported [3-5].