Ubiquinone analysis

UBIQUINONE DETERMINATION

As coenzyme Q has acquired increasing attention in research concerning diagnosis, therapy and prevention of oxidative damage, several procedures have been developed for the determination of  the components of the coenzyme Q complex.

Extraction

Several extraction procedures may be used as far as they preserve the labile components (reduced forms) against oxidation. One of the best is that reported by Burton that we have successfully used with a slight modification for coenzyme Q and homologues of vitamin E (Leray C et al., J Lipid Res 1998, 39, 2099). 
Another efficient extraction procedure was reported for plasma samples (Menke T et al., Anal Biochem 2000, 282, 209). Briefly, microsamples are mixed with ethanol containing internal standards and antioxidant (di-ter-butyl cresol). Components are extracted by mixing with hexane.

Analysis

Coenzyme Q components, as well as tocopherols and tocotrienols, may be analyzed using a two-isocratic step HPLC combined with an electrochemical detection in the oxidative mode (+ 700mV) following a zinc reduction of all components in a post-column reactor (Leray C et al., J Lipid Res 1998, 39, 2099).  Two eluents were used successively at a flow rate of 1 ml/min: the separation of tocopherols was done for 9 min with eluent A containing 5 mM ZnCl₂ , 2.5 mM Na acetate, and 2.5 mM acetic acid in methanol–water (98:2, v/v) and the separation of coenzyme Q was done for 22 min with eluent B containing 7.5 mM ZnCl₂ , 3.75 mM Na acetate, and 3.75 mM acetic acid in ethanol–methanol–water (49:49:2 v/v).
The high selectivity and sensitivity of the coulometric detection system enabled the use of low oxidation potentials giving little baseline noise, while a fast isolation procedure and quantitative recoveries of all oxidized and reduced forms made it possible to measure a high ubiquinol/ubiquinone ratio in the extracts.
Another method was developed for the simultaneous detection of the lipophilic antioxidants using electrochemical detection (Menke T et al., Anal Biochem 2000, 282, 209). The use of ammonium formate in the HPLC mobile phase improved the reproductibility of the detection. After separation on a reversed-phase C18 PEEK column, the components passed the conditioning cell (-600mV) and the ubiquinones were reduced to the corresponding ubiquinols. By passing the analytical cell (+600 mV) all components were oxidized.
The detection limits for all components were said to be between 1 and 28 fmol. Ten ml plasma samples were used for the simultaneous detection of ubiquinol and ubiquinone 10 and tocopherols.