CoQ10method

Background and Summary of the method  In the present procedure a propanolic plasma extract is directly injected into the HPLC without bringing into dryness. Since plasma, in vivo, contains almost exclusively reduced CoQ10, when a fresh sample is extracted common procedures do not always lead to complete oxidation of ubquinol. As UV methods for assaying CoQ10 usually quantify the oxidised coenzyme at 275 nm, incomplete oxidation during the procedure would lead to underestimation of total CoQ10. Therefore the first phase of our method is chemical oxidation, by p-Benzoquinone, of CoQ10 present in the sample prior to propanol extraction. Plasma obtained from blood anticoagulated with lithium heparin or EDTA, as well as serum, is suitable for the analysis.

Materials/reagents Human plasma or serum CoQ10 standard  Propanol (HPLC grade) Water (HPLC grade) Eppendorf type plastic vials with caps 1,4 benzoquinone   Equipment Vortex stirrer Bench top centrifuge Spectrophotometer  HPLC system with UV detector 
Reference  The proposed method was developed in the laboratory of the International CoQ10 Association, Institute of Biochemistry, Ancona. The method was published in Analytical Biochemistry, 305, 49-54; 2002

Preparation of standards.  CoQ10 stock standard solutions are prepared by dissolving the pure compound in reagent grade ethanol to yield final concentrations close to 25 µM. Approximately 20 mg of pure CoQ10 are weighed and transferred to a 1000 ml flask, which is brought to volume with ethanol: this is the stock solution. Its accurate concentration is determined spectrophotometrically at 275 nm using a molar extinction coefficient of 14,200. Concentration of the standard stock solution (mM) = absorbance of standard/14.2 The stock solution is divided into 0.5 ml aliquots and kept at -80 °C. Each working day, 33 µl of the stock solution is diluted to 2 ml with propanol/water 5:1. The different concentrations of the standards to be used for the calibration curve are obtained by diluting this solution respectively by 1/2, 1/3, 1/4, 1/6, 1/12 with propanol/water 5:1.   Extraction of the Sample 200 µl of plasma are pipetted into an Eppendorf type microtube, supplemented with 50 µl of a 1,4 benzoquinone solution (2mg/ml) and vortexed for 10 seconds. The benzoquinone solution is made freshly every day. After 10 min. 1 ml of n-propanol is added. The test tube is then vortexed for 30 seconds and centrifuged at 10,000 rpm for 2 minutes in order to spin down the protein precipitate. 200 µl of the supernatant are then injected into the HPLC or placed in the autosampler vials (this supernatant, placed in a capped test tube, was found stable for up to three days when kept even at 22°C).   High Performance Liquid Chromatography assembly  The HPLC apparatus consists of a pump and an injector equipped with a 200ml loop. The column is an LC 18 25 cm x 0.46 cm i.d. 5 µ, with a precolumn LC 18S, 2 cm. An in-line filter A-701 is placed between the injector and the precolumn. Mobile phase: ethanol/methanol (65%-35%), flux is 1ml/min. UV detection is performed at 275 nm. 200 µl of different concentrations of pure oxidised CoQ10 are injected as standards. Peak area analysis is performed by a data system. A typical standard curve is presented below. From the area of the sample its concentration is calculated, taking into account that the sample was diluted 1:6 with propanol. Of course the whole calculation can be done by a computer program.

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