Quantitative Analyses Of Four Antibiotics In Plasma By Liquid Chromatography-tandem Mass Spectrometry

Nowadays liquid chromatography-tandem mass spectrometry (LC/MS/MS), due to its higher sensitivity and selectivity, has become a valuable technique in the determination of biological samples and in the pharmacokinetic studies. Two rapid, sensitive and specific methods for quantitative analyses of two tetracycline antibiotics and two macrolide antibiotics in plasma were developed and validated by liquid chromatography-tandem mass spectrometry in this thesis. The methods have been successfully applied to pharmacokinetic studies.1. Determination of methacycline and doxycycline in plasma by LC/MS/MSA sensitive and selective liquid chromatography-tandem mass spectrometric (LC/MS/MS) method for the determination of methacycline in human plasma was developed. This method was applied to doxycycline. After a simple solid-phase extraction, the post-treatment samples were analyzed on a Diamonsil-C₁₈ column interfaced with a triple quadrupole tandem mass spectrometer. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of methanol-water-formic acid (80: 20: 1, v/v/v). Selected reaction monitoring (SRM) using the precursor→product ion combination of m/z 443→m/z 426, m/z 445→m/z 428 and m/z 461→m/z 426 was used to quantify methacycline, doxycycline and oxytetrarycline, respectively. The linear calibration curves were obtained in the concentration range of 5.0-4000 ng/mL for methacycline and 5.0-8000 ng/mL for doxycycline. The method has a lower limit of quantification (LLOQ) of 5.0 ng/mL for both methacycline and doxycycline. The method was applicated to study the pharmacokinetics of 18 healthy volunteers after oral administration of 300 mg of methacycline or 200 mg doxycycline. Mean peak plasma levels (C_max) of 2693±686 ng/mL and 3647±939 ng/mL and T_max of 3.11±0.76 h and 2.5±1.0h were observed The mean t_1/2 value of 14.7±2.2 h and 21.5±3.2 h were obtained, AUC_0-t were calculated to be 38.95±11.69μg·h/mL and 70.78±19.81μg·h/mL. The method was shown sensitive in the determination of methacycline and doxycycline levels in plasma samples, and was successfully used in pharmacokinetics investigation of methacycline and doxycycline. 2. Determination of azithromycin and roxithromycin in plasma by LC/MS/MSA rapid, sensitive and highly selective liquid chromatography-tandem mass spectrometry method was developed and validated for determination of azithromycin in human plasma. This method was applied to roxithromycin. The analytes were extracted from plasma samples by liquid-liquid extraction, then separated on a Zorbax XDB C₈ column. The mobile phase consisted of methanol-water-formic acid (80: 20: 0.5, v/v/v). A Finnigan TSQ tandem mass spectrometer equipped with ESI source was used as detector and was operated in the positive ion mode. Selected reaction monitoring (SRM) using the precursor→product ion combinations of m/z 749→m/z 591, m/z 837→m/z 158 and m/z 748→m/z 158 was used to quantify azithromycin, roxithromycin and internal standard clarithromycin, respectively. The method has a lower limit of quantification (LLOQ) of 1.0 ng/mL and 20 ng/mL for azithromycin and roxithromycin. The linear calibration curves were obtained in the concentration range of 1.0-1000 ng/mL for azithromycin and 20-8000 ng/mL for roxithromycin. The method was applicated to study the pharmacokinetics of 20 healthy volunteers after oral administration of 500 mg of azithromycin or 150 mg roxithromycin. Mean peak plasma levels (C_max) of 320±123 ng/mL and 4.14±2.58μg/mL and T_max of 1.9±0.7 h and 3.48±1.96 h were observed. The mean t_1／2 value of 25.9±5.0 h and 15.11±5.70 h were obtained, AUC_0-t were calculated to be 3088±1095 ng.h/mL and 57.91±31.12μg·h/mL. The method is simple, sensitive and selective, and has been successfully utilized for oral azithromycin and roxithromycin clinical studies.