Pharmacokinetics and tolerability of single-dose intravenous ertapenem in infants, children, and adolescents.

BACKGROUND: Ertapenem is a carbapenem antibiotic with broad spectrum activity and a pharmacokinetic profile that favors once-daily administration in adults. OBJECTIVES: This investigation was designed to evaluate the dose-exposure profile of ertapenem in children from infancy through adolescence. METHODS: Eighty-four children (3 months-16 years) requiring antibiotic therapy were enrolled in this multicenter trial. Children received a single intravenous dose of ertapenem at 15, 20, or 40 mg/kg followed by repeated blood sampling for 24 hours. Free and total plasma ertapenem concentrations were quantitated by high-performance liquid chromatography, and the pharmacokinetics were determined using a model-independent approach. RESULTS: Ertapenem exposure increased proportionally with increasing dose; however, achievable concentrations were influenced by age. Children older than 12 years attained higher dose-normalized concentrations at the end of the infusion (concentration at the end of the infusion [Ceoi]: 8.7 ± 1.9 mg/L per mg/kg dose) and total body exposure (area under the curve area under the plasma concentration-time curve [AUC]0-∞: 34.7 ± 14.7 mg hr/L per mg/kg dose) as compared with children 2 to 12 years (Ceoi: 6.9 ± 2.4 mg/L per mg/kg dose, AUC0-∞: 18.4 ± 8.0 mg hr/L per mg/kg dose) and children younger than 2 years (Ceoi: 6.1 ± 2.2 mg/L per mg/kg dose, AUC0-∞: 17.0 ± 5.4 mg hr/L per mg/kg dose). These findings were accounted for by age-dependent changes in ertapenem clearance and distribution volume. In 3 children adverse events (nausea, n = 2; injection site reaction, n = 1) were considered related to study drug administration. CONCLUSIONS: Children younger than 12 years require dosing more frequently than once daily to achieve optimal efficacy when treating organisms with a minimum inhibitory concentration near the susceptibility breakpoint.

Comparison of ordinary, weighted, and generalized least-squares straight-line calibrations for LC-MS-MS, GC-MS, HPLC, GC, and enzymatic assay.

The impact of experimental errors in one or both variables on the use of linear least-squares was investigated for method calibrations (response = intercept plus slope times concentration, or equivalently, Y = a(1) + a(2)X ) frequently used in analytical toxicology. In principle, the most reliable calibrations should consider errors from all sources, but consideration of concentration (X) uncertainties has not been common due to complex fitting algorithm requirements. Data were obtained for liquid chromatography-tandem mass spectrometry, gas chromatography-mass spectrometry, high-performance liquid chromatography, gas chromatography, and enzymatic assay. The required experimental uncertainties in response were obtained from replicate measurements. The required experimental uncertainties in concentration were determined from manufacturers' furnished uncertainties in stock solutions coupled with uncertainties imparted by dilution techniques. The mathematical fitting techniques used in the investigation were ordinary least-squares, weighted least-squares (WOLS), and generalized least-squares (GLS). GLS best-fit results, obtained with an efficient iteration algorithm implemented in a spreadsheet format, are used with a modified WOLS-based formula to derive reliable uncertainties in calculated concentrations. It was found that while the values of the intercepts and slopes were not markedly different for the different techniques, the derived uncertainties in parameters were different. Such differences can significantly affect the predicted uncertainties in concentrations derived from the use of the different linear least-squares equations.

Column-switching technique for the sensitive determination of ertapenem in human cerebrospinal fluid using liquid chromatography and ultraviolet absorbance detection.

A sensitive reversed-phase high-performance liquid chromatographic (RP-HPLC) assay with on-line extraction was developed for quantifying ertapenem in human cerebrospinal fluid (CSF). This assay is at least five times more sensitive than previously published ertapenem methods with a lower limit of quantitation at 0.025 microg/ml. In this assay, a CSF sample is extracted on-line using a RP extraction column and an aqueous acidic mobile phase (0.1% formic acid) to wash away polar endogenous materials, while ertapenem is retained on the column. Ertapenem is then back-flushed off the extraction column and directed to a RP analytical column using an acidic mobile phase with an organic modifier (acetonitrile/0.1% formic acid, 15:85 (v/v)) and detected using UV absorbance. The acidic mobile phase provided a sharper chromatographic peak and on-line extraction allowed large injection volumes (> or = 150 microl) of buffered CSF to be injected without compromising column integrity. These assay conditions were necessary to quantify ertapenem at levels expected to be found in human CSF (< 0.05 microg/ml). The method was successfully validated and implemented for a clinical study: intraday precision and accuracy of the CSF assay for calibration standards (0.025-10 microg/ml) and quality control samples (0.1, 0.5, and 2.5 microg/ml) were < 6.2% coefficient of variation and 96.8-104.0% of nominal concentration, respectively.

A semi-automated 96-well protein precipitation method for the determination of montelukast in human plasma using high performance liquid chromatography/fluorescence detection.

A simple, semi-automated, protein precipitation assay for the determination of montelukast (SINGULAIR, MK-0476) in human plasma has been developed. Montelukast is a potent and selective antagonist of the cysteinyl leukotriene receptor used for the treatment of asthma. A Packard MultiPROBE II EX is used to transfer 300 microl of plasma from sample, standard, and QC sample tubes to a microtiter plate (96-well). After addition of the internal standard by a repeating pipettor, a Tomtec QUADRA 96 adds 400 microl of acetonitrile to all plasma sample wells, simultaneously, in the microtiter plate. The Tomtec is also used to transfer the acetonitrile supernatant from the plasma protein precipitation step, batchwise, to another microtiter plate for analysis by HPLC with fluorescence detection. This assay has been validated and implemented for a clinical study of over 1300 plasma samples and is comparable to manual assays in the LLOQ (lower limit of quantitation, 3 ng/ml) and in stability. This is the first semi-automated protein precipitation assay published for the analysis of montelukast in human plasma and it results in significant time savings over the manual methods, both in sample preparation and in HPLC run time.

Assay methodology for the quantitation of unbound ertapenem, a new carbapenem antibiotic, in human plasma.

Ertapenem is a new once-a-day antibiotic with excellent coverage of common community gram negative and gram positive aerobes and anaerobes. It demonstrates nonlinear protein binding in human plasma (about 94% bound). An assay for unbound drug was developed to study the pharmacokinetics of unbound ertapenem in plasma. Unbound drug is separated from plasma samples (1.0 ml) by ultrafiltration using a Centrifree((R)) centrifugal filter device. Ertapenem (vulnerable to hydrolysis of the beta-lactam moiety) is stabilized in the filtrate by adding an equal volume of 0.1 M MES buffer, pH 6.5 and then is analyzed by reversed-phase high-performance liquid chromatography (HPLC) with ultraviolet (UV) absorbance detection (300 nm). Non-specific binding to the Centrifree((R)) device is <3%. A suitable internal standard is not available. The assay is specific and linear over the concentration range of 0.25 to 100 microgram/ml in plasma filtrate. The lower limit of quantitation (LLOQ) is 0.25 microgram/ml. Intra-day precision is C.V.<10% and accuracy ranges from 97 to 101% of nominal concentration. Inter-day precision and accuracy were determined using quality control samples (QCs) prepared in plasma ultrafiltrate at 0.5, 12 and 80 microgram/ml and stored at -70 degrees C with stabilizer. Inter-day assay accuracy and precision ranged from 100 to 111% of nominal concentration and 1.8 to 5.3% C.V. (n=40), respectively. The assay has been used to analyze plasma samples from subjects receiving 500 and 2000 mg i.v. doses of ertapenem (30 min infusion).

Modified high-performance liquid chromatographic method for the determination of ertapenem in human urine: enhanced selectivity and automation.

A column-switching, reversed-phase high-performance liquid chromatographic (HPLC) assay for a new structurally unique carbapenem antibiotic, ertapenem, in urine has been improved for selectivity and automated using a Packard MultiPROBE II EX pipetting station. The method uses column-switching for on-line extraction of the urine sample. The extraction column, analytical column, mobile phase, and timing of the column-switching valve have been changed to enhance selectivity for the analyte over endogenous background material. Sample transfer and dilution prior to direct-injection into the HPLC system have been accomplished using a Packard MultiPROBE II EX robotic liquid handling system.