ABSTRACT
Critical illness has been shown to affect the pharmacokinetics of antibiotics, which can lead to ineffective antibiotic exposure and the potential emergence of resistant bacteria. The lack of studies describing antibiotic pharmacokinetics in critically ill children has led to significant off-label dosing. This is, in part, due to the ethical and physiological challenges of removing frequent, large-volume samples from children. Capillary microsampling facilitates the collection of small volumes of blood samples to conduct clinical pharmacokinetic studies. A sensitive, rapid, and accurate ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) bioanalytical method to measure cefotaxime and desacetylcefotaxime in 2.8 µL of plasma was developed and validated. Plasma samples were treated with acetonitrile and analytes were separated using a Kinetex C8 (100 × 2.1 mm) column. The chromatographic separation was established using a gradient method, with the mobile phases consisting of acetonitrile and ammonium acetate. An electrospray ionization source interface operated in a positive mode for the multiple reaction monitoring MS/MS analysis of cefotaxime, desacetylcefotaxime, and deuterated cefotaxime (internal standard). The bioanalytical method using microsample volumes met requirements for method validation for both analytes. Cefotaxime had precision within ± 7.3% and accuracy within ± 5% (concentration range of 0.5 to 500 mg/L). Desacetylcefotaxime had precision within ± 9.5% and accuracy within ± 3.5% (concentration range of 0.2 to 10 mg/L). The bioanalytical method was applied for the quantification of cefotaxime and its metabolite to 20 capillary microsamples collected at five time points in one dosing interval from five critically ill children.
Subject(s)
Anti-Bacterial Agents/blood , Cefotaxime/analogs & derivatives , Cefotaxime/blood , Child , Chromatography, High Pressure Liquid/methods , Critical Illness/therapy , Drug Monitoring/methods , Humans , Limit of Detection , Pilot Projects , Reproducibility of Results , Tandem Mass Spectrometry/methodsABSTRACT
An efficient method for the analysis of cefotaxime and its metabolite desacetylcefotaxime residues in eggs was developed based on high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS).The samples were homogenized and extracted with acetonitrile/water (9∶1, V/V) solution.The fat was removed by hexane, and the other impurities were removed with C18 sorbent.The separation of cefotaxime and desacetylcefotaxime was performed on an Agilent Eclipse Plus C18 column (100 mm × 2.1 mm, 3.5 μm) using a mobile phase of 0.2% formic acid(A)-acetonitrile(B) by gradient elution.The analytes were detected by MS/MS in positive electrospray ionization mode (ESI+) and multiple reaction monitoring (MRM), quantitated by matrix-matched extemal standard method.Results showed that the calibration curves had a good linearity in the range of 1.0-143 μg/L (cefotaxime) and 1.0-120 μg/L (desacetylcefotaxime), respectively, with correlation coefficient R.2>0.999.Limits of detection (LOD, S/N=3) for cefotaxime and desacetylcefotaxime were 0.07 and 0.14 μg/kg, and limits of quantitation (LOQ, S/N=10) for cefotaxime and desacetylcefotaxime were 0.23 and 0.99 μg/kg, respectively.At three spiked concentration levels, the recoveries of cefotaxime and desacetylcefotaxime ranged from 83.1% to 103.0% and 88.2% to 101.0%, respectively, both with RSDs (n=6) less than 6.2%.The results demonstrated that the method was simple, quick, sensitive and reliable, and suitable for determination of cefotaxime and desacetylcefotaxime residues in eggs.
