Determination of Reduced and Oxidized Coenzyme Q10 in Canine Plasma and Heart Tissue by HPLC-ECD: Comparison with LC-MS/MS Quantification

Abstract

Coenzyme Q10 (Q10) plays an important role in mammals for energy production in the mitochondria, and as a potent antioxidant. Oxidation ratio (% oxidized in relation to total Q10) has been proposed as an important biomarker. A sensitive and reproducible HPLC-ECD method was developed for determination of reduced and oxidized Q10 in canine plasma and heart tissue. Chromatographic separation was achieved in 10 min using a Waters Nova-pak C18 column and a mobile phase with lithium perchlorate in ethanol/methanol/2-propanol. The validation showed satisfying results. Excellent linear correlation was found (r2 > 0.9997), intra- and inter-day precisions were below 6.5% (n = 5) and recoveries were between 89 and 109% (n = 5). Sensitivity stated as Lower Limit of Quantification (LLOQ) was 10 nM. Acceptable stability of both extracted and un-extracted samples was observed. The plasma concentration range of total Q10 was found to be between 0.64 and 1.24 µg/mL. Comparison with a developed LC-MS/MS method showed a correlation of r = 0.85 for reduced Q10 and r = 0.60 for oxidized Q10 (N = 17). However, average results were around 30% lower for ubiquinol using the LC-MS/MS method as compared with the HPLC-ECD analysis. The two methods are therefore not considered to be interchangeable.

Pharmacokinetics of Repeated Oral Dosing with Coenzyme Q10 in Cavalier King Charles Spaniels with Myxomatous Mitral Valve Disease

“…A minimum concentration of total circulating Q10 concentration of 2.0 µg/mL was proposed as a relevant target concentration in human heart failure patients [14]. This is above normal endogenous circulating concentrations reported in both healthy adult humans (means of serum Q10 concentration range: 0.6-1.0 µg/mL) [15] and in dogs (0.4-1.5 µg/mL) [16,17]. In a recent clinical trial done in human heart failure patients (Q-SYMBIO), the 2.0 µg/mL concentration of total circulating Q10 was used as a target with the administration of 300 mg ubiquinone daily and showed a positive effect of Q10 on clinical endpoints [18].…”

“…Heparinized plasma samples obtained at the end of each treatment period in all dogs and from six dogs on five consecutive days following Q10 treatment were analyzed for Q10 concentrations (total, reduced, and oxidized Q10) using high-performance liquid chromatography (HPLC) with electrochemical detection, as previously described [17].…”

Ascorbate maintains a low plasma oxygen level

“…Ascorbate concentration was quantified by high-performance liquid chromatography with coulometric detection, as described previously 18 . Likewise, using high-performance liquid chromatography with coulometric detection, α-and γ-tocopherol were analyzed as described by Sattler et al 19 , and ubiquinone and ubiquinol as described elsewhere 20 .…”

Ascorbate maintains a low plasma low oxygen level

“…The adaptation of circulating leucocytes to plasma low oxygen level has not previously 17 been investigated. In other cell-types, it has been reported that under hypoxic 18 conditions, mitochondrial abundance and oxygen consumption is reduced [11][12][13] . In 19 addition, mitochondria play a key function in leucocyte metabolism and function, 20 beyond metabolism regulation 14 .…”