Quantitative Analyses Of Coenzyme Q10 And Hydroxyethyl Starch In Human Plasma And Their Applications

1. Quantitative analysis of coenzyme Q10 in human plasma and its applicationsBackground Coenzyme Q10 (CoQ10) is an endogenous enzyme cofactor that exsits in all living cells in humans. It is an electron carrier in the mitochondrial respiratory chain and act as a lipid-soluble antioxidant.Objective To develop and validate a specific and rapid liquid chromatographic?tandem mass spectrometric method for determination of coenzyme Q10 in human plasma and application to the bioequivalence evaluation.Method Determination of coenzyme Q10 in human plasma by liquid chromatography-tandem mass spectrometry. The plasma sample was extracted twice with n-hexane, then separated on a Capcell Pak C18(35 mm×2.0 mm I.D.,5μm)column using methanol as the mobile phase. A triple quadrupole mass spectrometry equipped with atmospheric pressure chemical ionization (APCI) source was used as the detector and operated in the positive ion mode. Quantitation was performed using multiple reaction monitoring (MRM) of the transitions of m/z 864→m/z 197 and m/z 796→m/z 197 for CoQ10 and the internal standard CoQ9, respectively.Results The linear concentration range of the calibration curve for coenzyme Q10 was 10.0 ? 1000 ng?mL-1, The lower limit of quantification was 10.0 ng?mL-1. The intra-day and inter-day relative standard deviation over the entire concentration range were less than 8.9%. The accuracy was in the range of -0.9% to 3.8% in terms of relative error. Each sample was chromatographed within 4.5 min.Conclusion The validated liquid chromatographic?tandem mass spectrometric method was sensitive, specific, rapid and suitable for the bioequivalence evaluation of coenzyme Q10 in humans.2. Quantitative analysis of hydroxyethyl starch in human plasma and urine and their applicationsBackground Hydroxyethyl starch is a mixture of glucose polymers of varying chain length and various substitution with hydroxyethyl groups. It is necessary to hydrolyze hydroxyethly starch (HES) into glucose or hydroxyethyl glucose monomers when analysis its concentration in samples. Glucose is an endogenous substance. It is fundamental to develop a sensitive and specific method to eliminate the interference of endogenous glucose and quantify the concenteation of glucose monomers after hydrolysis in human plasma and urine samples.Objective To develop and validate a specific, sensitive and rapid analytical method for indirect determination of hydroxyethly starch in human plasma and application to the clinical pharmacokinetic study. Method Indirect determination of hydroxyethly starch in human plasma and urine by hexokinase method. Plasma and urine samples were deproteinated by trichloroacetic acid followed by centrifugation. Supernatant centrifugate was precipitated by acetone, then acidly hydrolyzed with 4M trifluoroaceticacid (1.5 h, 100°C.) to produce monomers. Glucose determination performed using an enzymatic test kit based on hexokinase/glucose-6-phosphatese and hydroxyethly starch concentration was calculated according to the determined concentration.Results The linear concentration range of the calibration curve was 0.25-10.0 mg?mL-1 and the lower limit of quantification was 0.25 mg?mL-1. The intra-day and inter-day relative standard deviation of plasma samples'determination over the entire concentration range were less than 13.2%. The accuracy was in the range of -2.0% to -0.7% in terms of relative error. The intra-day relative standard deviation of urine samples'determination was less than 4.8%. The accuracy was in the range of -3.9% to -1.2%. The mean analysis time was just 8 min for 50 processed samples.Conclusion The validated hexokinase analytical method was sensitive, rapid and suitable for the clinical pharmacokinetic study of hydroxyethly starch in humans.