Determination And Pharmacokinetics Studies Of Two Macrolide Antibiotics In Human Plasma By Ultra Performance Liquid Chromatography-tandem Mass Spectrometry

Objective:To develop and validate rapid,sensitive and specific methods for the determination and pharmacokinetics study of azithromycin and clarithromycin in human plasma by ultra performance liquid chromatography-tandem mass spectrometry.Method:After a liquid-liquid extraction process with ether,azithromycin and roxithromycin (internal standard)were separated on an ACQUITY UPLC^TMBEH C₁₈column with gradient elution at a flow-rate of 0.30 mL/min.The mobile phase was a mixture of 0.05 mol/L ammonium acetate and acetonitrile.The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring(MRM)mode via electrospray ionization(ESI).The quantification was performed using the transitions of m/z 749.7â†'m/z 591.2 for azithromycin and m/z 837.0â†'m/z 679.2 for roxithromycin(internal standard), respectively.Clarithromycin plasma samples were prepared by liquid-liquid extraction,separated on ACQUITY UPLC^TMBEH C₁₈column with gradient elution.The mobile phase was 0.05 mol/L ammonium acetate and acetonitrile.Multiple reaction monitoring(MRM)using the precursorâ†'product ion combination of m/z 748.6â†'m/z 157.9 and m/z 837.0â†'m/z 679.2 was used to quantify clarithromycin and roxithromycin(internal standard),respectively.Result:The linear calibration curves were obtained over the concentration range of 1.00～1.00×10³ ng/mL for azithromycin.The intra- and inter-day precision values(RSD)were not above 9.4%and the accuracy(RE)was -1.3%～5.7%at all three QC levels.The method was applied to study the pharmacokinetics of 20 healthy volunteers after oral administration of 500 mg of azithromycin.Mean peak plasma levels(C_max)of 0.533±.018μg/mL and T_maxof 1.9±0.6 h were observed.The mean t_1/2value of 48±5.2 h was obtained,AUC_0-tand AUC_0-∞ were calculated to be 4.58±0.95μg·h/mL and 5.21±1.1μg·h/mL,respectively.The standard calibration curves for clarithromycin were linear in the concentration range of 1.00～3.00×10³ ng/mL in human plasma.The intra- and inter-day precision values(RSD)were not above 10 %and the accuracy(RE)was -7.6%～6.0%at all three QC levels.The main parameters obtained after single dose of 500 mg clarithromycin to 18 healthy volunteers were as follows: C_maxof 1.22±0.35μg/mL,T_maxof 4.6±1.4 h,the value t_1/2of 6.3±1.8 h,AUC_0-tand AUC_0-∞of 15.8±4.4μg·h/mL and 15.9±4.4μg·h/mL,respectively.After multi dose of 500 mg clarithromycin,C_maxwas found to be 1.16±0.33μg/mL,T_maxobserved was 4.8±1.7 h,the value t_1/2was 6.3±1.9 h,C_avwas 0.595±0.15μg/mL,DF was 0.153±0.035,AUC_sswas 14.3±3.6μg·h/mL.Conclusion:The methods are proved to be of high specificity,speed,sensitivity and have been successfully utilized for the evaluation of the pharmacokinetics of azithromycin and clarithromycin.