MINIATURIZED HPLC-UV METHOD FOR ANALYSIS OF COENZYME Q10 IN HUMAN PLASMA

Abstract

Coenzyme Q10 (CoQ10) is a cofactor in the respiratory chain and a potent endogenous antioxidant. Abnormally low levels of CoQ10 in the circulatory system are often involved with pathological states such as heart failure, mitochondrial and muscular diseases, and cancer. The development of simple, rapid, and highly sensitive methods capable of quantifying low CoQ10 levels in plasma is, therefore, increasingly required.

In this work, we developed a miniaturized HPLC-UV system for the determination CoQ10 in human plasma using an XTerra microcolumn (50 mm × 2.1 mm i.d., 3.5 µm particle size), methanol:water (98:2, v/v) as mobile phase, 275 nm, flow rate of 0.3 mL/min, 30 °C column temperature, and 2 µL injection volume. The extraction procedure consists of a plasma precipitation with 1-propanol and evaporation under nitrogen to allow a 1.5-fold enrichment. The chromatographic analysis was accomplished in 7 min. As a result, it was possible to quantitate CoQ10 in small sample volumes down to 0.07 µM and detect as low as 0.02 µM in real plasma, with good accuracy and precision, even in pathological conditions. The proposed method was found to be suitable for routine CoQ10 determination in clinical laboratories.

Keywords:

• coenzyme Q10
• microcolumn
• micro fast HPLC
• mitochondrial disease
• plasma
• UV

Abbreviations:

• CoQ10, coenzyme Q10
• EC, electrochemical detection
• HPLC, high performance liquid chromatography
• LOD, limit of detection
• LOQ, limit of quantitation
• UV, ultraviolet

ACKNOWLEDGMENTS

The authors wish to thank to Dr. Cecilia Dobrecky for assistance.

Notes

^a Calculated according to USP 32 (16).

^b Number of theoretical plates.

^a The mean calibration curves were obtained on three different days with triplicate at five different levels.

^b LOD and LOQ values correspond to a detection and quantitation of 0.02 µM and 0.07 µM of CoQ10 without concentration, respectively.

^c RSD values of normalized areas are the averages from three concentration levels of the calibration curves.

^d Recovery mean values obtained from three individual samples on three different days at three concentration levels. RSD values in parenthesis.

^aStandard deviation.