Analytical Method Development for Coq10 Determination in Human Plasma Using HPLC-UV and HPLC/MS/MS

Background: CoQ10 is a very important compound which is found in every tissue of our organism. It participates in the processes of cellular respiration and ATP production. Also, it acts as a strong antioxidant. In an organism, it is represented in two forms: oxidized (ubiquinone) and reduced (ubiquinol). Its low blood level may be a signal for a list of diseases. Materials and Methods: This study developed and compared two methods of CoQ10 determination in order to find the fastest and the most convenient one. The first one involved HPLC-UV with the wavelength of ubiquinone determination equivalent to 290 nm and 275 nm for ubiquinol, respectively. The second one was carried out on an HPLC/MS/MS system utilizing Electrospray Ionization (ESI) and triple quadrupole mass analyzer for quantification in MRM positive mode. Results: Two methods of ubiquinol and ubiquinone determination were developed and validated. HPLC-UV included sample preparation based on liquid-liquid extraction. The LLOQ was 0.50 μg/ml. HPLC-MS/MS method sample preparation was based on protein precipitation. The LLOQ was 0.10 μg/ml. Conclusion: During the investigation, a conclusion was drawn that the HPLC-UV method is too insensitive for simultaneous determination of ubiquinol and ubiquinone. Furthermore, ubiquinol is very unstable and during exogenous factors’ exposure, it rapidly turns into ubiquinone. While, the HPLCMS/ MS method turned out to be sensitive, selective, rapid as it provides an accurate determination of both forms of CoQ10 in spiked human plasma.