2.7. Validation of the HPLC-ECD Method

Before preparation of calibration curves, the 1000 µM solution of CoQ₁₀ and CoQ₁₀H₂ prepared in hexane was further diluted to 100 µM with 96% ethanol. The CoQ₁₀ stock solution was further diluted 1:1 with 96% ethanol before measurement on a spectrophotometer at 275 nm (ε = 14240). CoQ₁₀H₂ was measured without further dilution at 290 nm (ε = 4010). The spectrophotometric measurements were performed to determine the exact concentrations [28]. Working calibration solutions of CoQ₁₀ and CoQ₁₀H₂ at 10 µM in 96% ethanol were used for preparation of the calibration curves. Eight-point calibration curves from 10 to 1000 nM in 96% ethanol were used for both the HPLC-ECD and LC-MS/MS methods. LLOQ was determined as the concentration giving a signal-to-noise ratio (S/N ratio) of 10:1. The precision was investigated at LLOQ by diluting plasma with PBS buffer and homogenate with extraction solution to the expected LLOQ and analyzing the prepared sample five times. Upper Limit of Quantification (ULOQ) was considered to be equal to the highest concentration in the calibration curve at 1000 nM.

Precision (intra- and inter-assay) and accuracy was estimated in the same set up. 25 µL of canine plasma and around 2 × 135 mg of canine heart tissue were analyzed on three consecutive days (n = 5) as is, and with the addition of 25%, 50%, and 75% of CoQ₁₀ and CoQ₁₀H₂. Precision was acceptable, if the coefficient of variation, CV%, was within 15%, although a CV% of 20% was acceptable at LLOQ. Accuracy was expressed as % recovery. The requirement for accuracy was a CV% of less than 15%.

Stability at 4 °C mimicking storage in a cooled auto sampler was investigated for extracted canine plasma and heart tissue every second hour for 8 h. Furthermore, the effect of BHT on the stability of Q₁₀ in plasma was evaluated. Extracted samples were tested at −20 °C in up to 4 days. Long-term stability at −80 °C of un-extracted plasma and heart tissue was evaluated for 2 months. A deviation of no more than ± 15% of the nominal concentration was accepted.

Concentration ranges of CoQ₁₀H₂ and CoQ₁₀ are expressed as average ± SD. Regarding precision, the variation was stated as CV%, whereas accuracy was expressed as % recovery of measured concentrations from the expected concentration plus the added concentration of calibration standard. Oxidation ratio was calculated as the concentration of oxidized Q₁₀ in relation to the total concentration of Q₁₀. Student’s t-test was applied for comparison of two groups. Single factor ANOVA was used for comparison of more than two groups. Correlation plots, Bland–Altman plots and statistical calculations were performed in GraphPad Prism 6.0 (GraphPad Prism software, La Jolla, CA, USA). Pearson’s r is stated for the correlation plots. Bias ± standard deviation (SD) and 95% limits of agreement are derived from the Bland–Altman plots. A p-value of less than 0.05 was considered to be significant.