Identification of In Vitro Metabolites of Amoxicillin in Human Liver Microsomes by LC-ESI/MS.

Amoxicillin (AMOX) metabolism in human liver microsomes was studied in vitro using liquid chromatography-mass spectrometry (LC/MS). Amoxicillin was incubated with human liver microsomes along with NADPH, and the reaction mixture was analyzed by LC/MS to obtain the specific metabolic profile of the studied antibiotic drug. Positive electrospray ionization was employed as the ionization source. An ACE C18-column (4.6 mm × 150 mm, 3 µm) was implemented with acetonitrile and water (+0.1 % formic acid) in isocratic mode as the mobile phase at the flow 0.4 mL min-1. The chemical structures of metabolites were proposed on the basis of the accurate mass measurement of the protonated molecule as well as their main product. Six phase I and one phase II metabolites were detected and structurally described. The metabolism of AMOX occurred via oxidation, hydroxylation and oxidative deamination, as well as through combination of these reactions. Compound M7, with glucuronic acid was also observed as phase II metabolite. Neither sulfate nor glutathione conjugates were detected. This study presents novel information about the chemical structure of the potential AMOX metabolites and provides vital data for further pharmacokinetic and in vivo metabolism studies.

Pharmacokinetic study of amoxicillin in human plasma by solid-phase microextraction followed by high-performance liquid chromatography-triple quadrupole mass spectrometry.

Sensitive and selective analytical procedures based on high-performance liquid chromatography with mass spectrometric detection were developed for the determination of amoxicillin in human plasma samples. Samples were prepared by applying in-house manufactured molecularly imprinted solid-phase microextraction probes. The detection of target compounds was performed in multiple reaction monitoring mode. The multiple reaction monitoring detection was operated in the positive electrospray ionization mode using the transitions of m/z 366 ([M + H](+) ) → 349 for amoxicillin and m/z 390 ([M + H](+) ) → 372 for gemifloxacin. The method was validated with precision within 15% relative standard deviation and accuracy within 15% relative error. The method was successfully applied to study of the pharmacokinetics of amoxicillin in human plasma after oral administration of amoxicillin.

Simultaneous determination of selected chemotherapeutics in human whole blood by molecularly imprinted polymers coated solid phase microextraction fibers and liquid chromatography-tandem mass spectrometry.

Development and validation of novel, general liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous determination of amoxicillin (AMOX), cefatoxime (CEF), ciprofloxacin (CIP), daptomycin (DAPTO), fluconazole (FLU), gentamicin (GEN), clindamycin (KLI), linezolid (LIN), metronidazole (MET), moxifloxacin (MOXI) in human whole blood are described. Samples were prepared on solid phase microextraction way with the use of polymeric sorption coatings with molecular imprints and analyzed using a gradient separation over an ACE C18-column (4.6mm×150mm, 3µm) with isocratic elution. Positive electrospray ionization was employed as the ionization source. The mobile phase consisted of acetonitrile-water (0.1% formic acid or 5mM ammonium acetate) at a flow 0.4ml/min. The chromatographic run time was kept less than 9min. The intra- and inter-day relative standard deviation across three validation runs over the entire concentration range was less than 7.3%, while the accuracy was within ±8.4%. The mean recovery of all the analytes ranged from 65.0 to 83.0%. This method was successfully applied to clinical samples from patients with clinically diagnosed bacterial infections process.

A new approach for antibiotic drugs determination in human plasma by liquid chromatography-mass spectrometry.

Sensitive and selective analytical procedures based on high performance liquid chromatography with mass spectrometric detection were developed for the determination of linezolid (LIN) and amoxicillin (AMOX) in human plasma samples. Samples were prepared by applying protein precipitation (PP), solid phase extraction (SPE), and microextraction in packed syringe (MEPS). The analytical separation was carried out using reversed phase liquid chromatography in isocratic mode. All analytes were monitored by mass spectrometry (MS) detection in the product ion mode and the method was validated covering the corresponding therapeutic range of 1-30 µg/mL and 1-50 µg/mL for LIN and AMOX respectively. The assay was linear over AMOX and LIN concentration ranges. The method provided good validation data: accuracy (102.9% (LIN), 100.9% (AMOX)), limit of detection (0.1407 ng/mL (LIN); 0.1341 ng/mL (AMOX); quantification (0.3814 ng/mL (LIN), 0.4249 ng/mL (AMOX)) and acceptable stability within 24h in the auto-sampler. Three different methods were compared as regards precision, accuracy, recovery and matrix effects. The proposed methods offer a fast and simple way to determine selected antibiotic drugs in human plasma that could be applied in pharmacokinetic studies.