A new HPLC-MS/MS analytical method for quantification of tazobactam, piperacillin, and meropenem in human plasma.

A simple and fast high-performance liquid chromatography with tandem mass spectrometry method for quantification of tazobactam, piperacillin, and meropenem in human plasma has been developed and validated. Simple sample preparation with a volume of 10 µL was done by protein precipitation with a mixture of methanol-acetonitrile-water (6:2:2, v/v/v). Chromatographic separation was achieved on a Luna column with a precolumn security guard by gradient elution using a mobile phase consisting of water with the addition of 0.1% formic acid (component A) and mixture methanol-acetonitrile (8:2, v/v) with the addition of 0.1% formic acid (component B). The run time was 2.7 min. The lower limits of detection and lower limits of quantification were for piperacillin 0.03 and 0.1 mg/L, for meropenem 0.04 and 0.2 mg/L and for tazobactam 0.16 and 0.5 mg/L. The validated method was used for therapeutic monitoring of tazobactam, piperacillin, and meropenem in samples of patients treated in the intensive care unit.

Meropenem, Cefepime, and Piperacillin Protein Binding in Patient Samples.

BACKGROUND: The mortality rate of patients with a drug-resistant bacterial infection is high, as are the associated treatment costs. To overcome these issues, optimization of the available therapeutic options is required. Beta-lactams are time-dependent antibiotics and their efficacy is determined by the amount of time the free concentration remains above the minimum inhibitory concentration. Therefore, the aim of this study was to assess the extent and variability of protein binding for meropenem, cefepime, and piperacillin. METHODS: Plasma samples for the analysis of meropenem, cefepime, and piperacillin were collected from patients admitted to a tertiary care hospital as part of the standard care. The bound and unbound drug fractions in the samples were separated by ultrafiltration. Validated liquid chromatography-tandem mass spectrometry assays were used to quantify the total and free plasma concentrations, and the protein binding was determined. RESULTS: Samples from 95 patients were analyzed. The median (range) age of patients was 56 years (17-87) and the median (range) body mass index was 25.7 kg/m (14.7-74.2). Approximately 59% of the patients were men. The median (range) unbound fraction (fu) was 62.5% (41.6-99.1) for meropenem, 61.4% (51.6-99.2) for cefepime, and 48.3% (39.4-71.3) for piperacillin. In the bivariate analysis, as the total meropenem concentration increased, the fu increased (r = 0.37, P = 0.045). A decrease in piperacillin fu was observed as the albumin concentration increased (r = -0.56, P = 0.005). CONCLUSIONS: The average fu values were lower than those reported in the literature. There was also a large variability in fu; hence, it should be considered when managing patients administered with these drugs through direct measurements of free drug concentrations.

Comparative LC-MS/MS and HPLC-UV Analyses of Meropenem and Piperacillin in Critically Ill Patients.

BACKGROUND: Therapeutic drug monitoring (TDM) of beta-lactam antibiotics has become a valuable tool to guide dosing in critically ill patients. The main goal of the study was to compare two routinely used techniques for beta-lactam TDM in intensive care unit (ICU) patient samples, namely isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) and high-performance liquid chromatography combined with ultra-violet detection (HPLC-UV). METHODS: A set of 80 sera/plasma samples from ICU patients receiving therapeutic meropenem or piperacillin dosage was investigated. Sample duplicates and quality assessment samples were assayed in parallel with an in-house LC-MS/MS and a commercially available IVD HPLC-UV kit. A pharmacokinetic and pharmacodynamic (PK/PD) target with ≥ 22.5 mg/L for piperacillin and ≥ 8.0 mg/L for meropenem was used for medical assessment of trough sample (n = 40) antibiotic concentrations. RESULTS: There was no difference between serum and Li-heparin plasmas. Concentration deviations were found for 4% of meropenem and 17% of piperacillin samples. Eliminating the influence of the systemic bias of approximately 10% for piperacillin, measurement discrepancies ≥ 25% between LC-MS/MS and HPLC-UV analyses were only observed for ≈ 4 - 6% of all samples. In the same way, identical PK/PD target attainment rates of 50 - 60% could be obtained. CONCLUSIONS: After correction of the analytical bias for piperacillin measurements, both methods showed comparable results, also with respect to clinical decision limits. HPLC-UV analysis is an adequate TDM methodology for testing of beta-lactam antibiotics in centers where no special knowledge in LC-MS/MS based TDM is present. However, potential matrix effects, interferences, and calibration issues for both methods must be taken into account.

Saturable elimination of piperacillin in critically ill patients: implications for continuous infusion.

The study aimed to evaluate saturation of piperacillin elimination in critically ill adult patients. Seventeen critically ill adult patients received continuous and intermittent infusion of piperacillin/tazobactam. Piperacillin plasma concentrations (n = 217) were analysed using population pharmacokinetic (PopPK) modelling. Post-hoc simulations were performed to evaluate the type I error rate associated with the study. Unseen data were used to validate the final model. The mean error (ME) and root mean square error (RMSE) were calculated as a measure of bias and imprecision, respectively. A PopPK model with parallel linear and non-linear elimination best fitted the data. The median and 95% confidence interval (CI) for the model parameters drug clearance (CL), volume of central compartment (V), volume of peripheral compartment (Vp) and intercompartmental clearance (Q) were 9 (7.69-11) L/h, 6.18 (4.93-11.2) L, 11.17 (7.26-12) L and 15.61 (12.66-23.8) L/h, respectively. The Michaelis-Menten constant (Km) and the maximum elimination rate for Michaelis-Menten elimination (Vmax) were estimated without population variability in the model to avoid overfitting and inflation of the type I error rate. The population estimates for Km and Vmax were 37.09 mg/L and 353.57 mg/h, respectively. The bias (ME) was -20.8 (95% CI -26.2 to -15.4) mg/L, whilst imprecision (RMSE) was 49.2 (95% CI 41.2-56) mg/L. In conclusion, piperacillin elimination is (partially) saturable. Moreover, the population estimate for Km lies within the therapeutic window and therefore saturation of elimination should be accounted for when defining optimum dosing regimens for piperacillin in critically ill patients.

Preanalytical Stability of Piperacillin, Tazobactam, Meropenem, and Ceftazidime in Plasma and Whole Blood Using Liquid Chromatography-Tandem Mass Spectrometry.

BACKGROUND: Therapeutic drug monitoring (TDM) is increasingly used to optimize the dosing of beta-lactam antibiotics in critically ill patients. However, beta-lactams are inherently unstable and degrade over time. Hence, patient samples need to be appropriately handled and stored before analysis to generate valid results for TDM. The appropriate handling and storage conditions are not established, with few and conflicting studies on the stability of beta-lactam antibiotics in clinical samples. The aim of this study was to assess the preanalytical stability of piperacillin, tazobactam, meropenem, and ceftazidime in human plasma and whole blood using a liquid chromatography-tandem mass spectrometry method for simultaneous quantification. METHODS: A reverse phase liquid chromatography-tandem mass spectrometry method for the quantification of piperacillin, tazobactam, meropenem, and ceftazidime in plasma after protein precipitation was developed and validated. The preanalytical stability of these beta-lactams was assessed in EDTA- and citrate-anticoagulated plasma at 24, 4, and -20°C. The whole blood stability of the analytes in EDTA-anticoagulated tubes was assessed at 24°C. Stability was determined by nonlinear regression analysis defined by the lower limit of the 95th confidence interval of the time to 15% of degradation. RESULTS: Based on the lower limit of the 95th confidence interval of the time to 15% of degradation, piperacillin, tazobactam, meropenem, and ceftazidime were stable in EDTA-anticoagulated plasma for at least 6 hours at 24°C, 3 days at 4°C, and 4 days at -20°C. Stability in EDTA- and citrate-anticoagulated plasma was similar. Stability in whole blood was similar to plasma at 24°C. CONCLUSIONS: Plasma samples for the TDM of piperacillin, tazobactam, meropenem, and ceftazidime should be processed within 6 hours if kept at room temperature and within 3 days if kept at 4°C. All long-term storage of samples should be at -80°C.

Carboxylesterase catalyzed 18O-labeling of carboxylic acid and its potential application in LC-MS/MS based quantification of drug metabolites.

LC-MS quantification of drug metabolites is sometimes impeded by the availability of internal standards that often requires customized synthesis and/or extensive purification. Although isotopically labeled internal standards are considered ideal for LC-MS/MS based quantification, de novo synthesis using costly isotope-enriched starting materials makes it impractical for early stage of drug discovery. Therefore, quick access to these isotope-enriched compounds without chemical derivatization and purification will greatly facilitate LC-MS/MS based quantification. Herein, we report a novel 18O-labeling technique using metabolizing enzyme carboxylesterase (CES) and its potential application in metabolites quantification study. Substrates of CES typically undergo a two-step oxygen exchange with H218O in the presence of the enzyme, generating singly- and doubly-18O-labeled carboxylic acids; however, unexpected hydrolytic behavior was observed for three of the test compounds - indomethacin, piperacillin and clopidogrel. These unusual observations led to the discovery of several novel hydrolytic mechanisms. Finally, when used as internal standard for LC-MS/MS based quantification, these in situ labeled compounds generated accurate quantitation comparable to the conventional standard curve method. The preliminary results suggest that this method has potential to eliminate laborious chemical synthesis of isotope-labeled internal standards for carboxylic acid-containing compounds, and can be developed to facilitate quantitative analysis in early-stage drug discovery.

UPLC/MS/MS assay for the simultaneous determination of seven antibiotics in human serum-Application to pediatric studies.

A rapid and highly sensitive ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay was developed for quantification of 7 antibiotics in low sample volumes (50 µL): amoxicillin, azithromycin, cefotaxime, ciprofloxacin, meropenem, metronidazole and piperacillin, for both routine monitoring and pharmacokinetic studies. After protein precipitation by acetonitrile, the antibiotics were separated on an Acquity UPLC HSS T3 column (run time, 4 min). The mobile phase consisted of a mixture of (A) ammonium acetate (pH 2.4; 5 mM) and (B) acetonitrile acidified with 0.1% formic acid, delivered at 500 µl/min in a gradient elution mode. Total time run was 2.75 min. Ions were detected in the turbo-ion-spray-positive and multiple-reaction-monitoring modes. The assay was accurate and reproductible for the quantification of the seven antibiotics in serum samples over large concentration ranges.

LC-MS/MS method for simultaneous determination of linezolid, meropenem, piperacillin and teicoplanin in human plasma samples.

Antibiotic therapy is a crucial aspect of the management of hospitalized patients, however, current standard dosing protocols have been shown to often attain inadequate plasmatic concentrations which may impair the clinical outcome and promote the selection of multidrug-resistant bacteria. The aim of this study is to establish and validate a robust and fast liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous analysis of four commonly used antibiotics (Meropenem, Piperacillin, Linezolid and Teicoplanin) in human plasma according to the European Medicines Agency (EMA) guidelines. Samples preparation was performed using a commercially available extraction kit which needs a very small amount of sample (50 µl). Antibiotics were detected, following a 7 min gradient separation, in multiple reactions monitoring (MRM) mode using a Qtrap 5500 triple quadrupole instrument equipped with an electrospray source operating in positive ion mode. The method, covering the antibiotics' clinically relevant concentration ranges, is also able to quantify, individually, the major teicoplanin components. The high reproducibility and the need of a small amount of sample, associated with the use of a commercial kit, together with a short chromatographic time, makes the method particularly suited for high-throughput routine analysis. Monitoring of plasma antibiotic levels, as part of the clinical routine, would result in a quick therapy adjustment leading to a higher probability of eradicating the infection as well as a potential reduction of multidrug-resistance prevalence. The method was successfully applied to monitor the antibiotic concentration of 49 patients under therapy.

Measuring Unbound Versus Total Piperacillin Concentrations in Plasma of Critically Ill Patients: Methodological Issues and Relevance.

BACKGROUND: Piperacillin is considered a moderately protein-bound antibiotic (20%-40%), with albumin being an important binding protein. Although infrequently used in practice, different methods to measure the fraction unbound (fu) are available, but uncertainty remains as to what the most appropriate method is. The main goal of this study was to estimate the impact of the methodology used to measure unbound piperacillin in plasma on the fu of piperacillin; we compared ultrafiltration (UF) at 4°C and 37°C with the reference method, equilibrium dialysis. In addition, we analyzed the impact of other proteins on the fu. METHODS: Anonymized left-over Li-heparin plasma samples (n = 41) from 30 critically ill patients who were treated with piperacillin were used for the analyses. RESULTS: We found that the piperacillin fu, determined by UF, is on average 8% higher at 37°C (91%) than at 4°C (83%). There were no systematic or proportional differences between UF at 4°C and equilibrium dialysis at 4°C. This emphasizes the importance of the temperature during UF, which should therefore be clearly stated in publications that report on the methodology of UF. No significant impact of the albumin-, IgA-, total protein-, or α1-acid glycoprotein concentration on the fu was found. The fu found in this study was higher than the generally assumed fu value of 60%-80%. A possible explanation lies in the studied population or in the temperature used. Based on our results, routine monitoring of unbound piperacillin in intensive care unit patients is not recommended. CONCLUSIONS: Based on the prediction model, we can state that in intensive care patients the fu of piperacillin is 91% (SD 7%), determined with UF at 37°C.

Beta-lactam carryover in arterial and central venous catheters is negligible.

BACKGROUND: Therapeutic drug monitoring is used for aminoglycosides and vancomycin, and has been proposed for ß-lactam antibiotics. Clinical blood samples in the ICU are often obtained via an existing vascular catheter rather than fresh needle phlebotomy. If antibiotics had previously been infused through a vascular catheter then used for blood sampling, carryover of antibiotic from the infusion to the sample might result in misleading assessments of target attainment. To address this concern we conducted a series of in vitro measurements of carryover for three commonly used antibiotics. METHODS: We infused piperacillin-tazobactam, meropenem, and cefepime at pharmacologic concentrations through commonly used vascular catheters at our hospital and flushed the catheters. We then aspirated warmed citrated bovine blood through each catheter and measured antibiotic concentrations in each aspirate. RESULTS: Carryover was below the limits of detection for piperacillin-tazobactam, meropenem, and vancomycin. Cefepime carryover, in contrast, was not negligible and needs to be investigated more fully. CONCLUSION: Carryover from prior infusions does not appear to jeopardize measurements of piperacillin-tazobactam, meropenem, or vancomycin in commonly used vascular catheters at our institution. Caution in interpreting samples obtained for cefepime measurements appears advised until more data is available.

Quantification of Cefepime, Meropenem, Piperacillin, and Tazobactam in Human Plasma Using a Sensitive and Robust Liquid Chromatography-Tandem Mass Spectrometry Method, Part 1: Assay Development and Validation.

The highly variable pharmacokinetics of ß-lactam antibiotics and ß-lactamase inhibitors poses a significant challenge to clinicians in ensuring appropriate antibiotic doses in critically ill patients. Therefore, routine monitoring of plasma concentrations is important for individualization of antimicrobial therapy. Accordingly, a simple and robust analytical method for the simultaneous measurement of multiple ß-lactam antibiotics and ß-lactamase inhibitors is highly desirable to ensure quick decisions on dose adjustments. In this study, a sensitive, simple, and robust method for the simultaneous quantification of cefepime, meropenem, piperacillin, and tazobactam in human plasma was developed and rigorously validated according to FDA guidance. Sample extraction was accomplished by simple protein precipitation. Chromatographic separation of analytes was achieved using stepwise gradient elution. Analytes were monitored using tandem mass spectrometry (MS/MS) with a turbo ion spray source in positive multiple-reaction-monitoring mode. The calibration curve ranged from 0.5 to 150 µg/ml for cefepime, 0.1 to 150 µg/ml for meropenem and piperacillin, and 0.25 to 150 µg/ml for tazobactam. Inter- and intraday precision and accuracy, sensitivity, selectivity, dilution integrity, matrix effect, extraction recovery, and hemolysis effect were investigated for all four analytes, and the results met the acceptance criteria. Compared to other reported methods, our method is more robust because of the combination of the following features: (i) a simple sample extraction procedure, (ii) a short sample run time, (iii) a wide dynamic range, and (iv) the small plasma sample volume needed. Since our method already covers ß-lactams and a ß-lactamase inhibitor with highly heterogeneous physicochemical properties, further antibiotic candidates may easily be incorporated into this multianalyte method.

Piperacillin Population Pharmacokinetics in Critically Ill Adults During Sustained Low-Efficiency Dialysis.

BACKGROUND: Sustained low-efficiency dialysis (SLED), is increasingly being used in intensive care units (ICUs) but studies informing drug dosing for such patients is lacking. OBJECTIVE: To describe the population pharmacokinetics (PKs) of piperacillin/tazobactam in critically ill adults receiving SLED and to provide dosing recommendations. METHODS: This prospective population PK study was conducted in adult ICU patients prescribed piperacillin/tazobactam while receiving SLED; 321 blood samples were obtained from 34 participants during and between approximately 50 SLED treatments for quantification of piperacillin and tazobactam concentrations in plasma. A population PK model was developed. Monte Carlo simulation was used to determine the probability of target attainment and pathogen-specific fractional target attainment at different doses. RESULTS: From a 2-compartment linear model with zero-order input, the mean (SD) clearance of piperacillin on SLED and off SLED were 4.81 (8.48) and 1.42 (1.54) L/h, respectively. Tazobactam concentrations were not sufficient for analysis. For the target of 50% fT>MIC (unbound concentrations of drug are above the minimum inhibitory concentration for >50% of the dosing interval), 3-g of piperacillin infused over 0.5 hours every 8 hours was appropriate for susceptible organisms with MIC ≤16 mg/L. For life-threatening infections where the target of 100% fT>MIC is preferred, a 9-g dose administered as a continuous infusion every 24 hours was appropriate for susceptible organisms with MIC ≤32 mg/L. CONCLUSIONS AND RELEVANCE: In critically ill patients receiving SLED, piperacillin doses need to be guided by the frequency of SLED treatments and susceptibility of the known or suspected pathogen.

A rapid, LC-MS/MS assay for quantification of piperacillin and tazobactam in human plasma and pleural fluid; application to a clinical pharmacokinetic study.

Piperacillin, in combination with tazobactam is a common first-line antibiotic used for the treatment of pleural infection, however its pleural pharmacokinetics and penetration has not previously been reported. The objective of this work was to develop and validate a rapid and sensitive liquid chromatography with tandem mass spectrometry (LC-MS/MS) assay for quantification of piperacillin (PIP) and tazobactam (TAZ). PIP and TAZ were extracted from both human plasma and pleural fluid samples by protein precipitation in methanol containing the internal standards (IS) piperacillin-d5 (PIP-d5) and sulbactam (SUL). Briefly, 5 µL of sample was mixed with 125 µL of methanol containing IS, vortexed and centrifuged. Supernatant (50 µL) was diluted into 500 µL of mobile phase containing 10 mM of ammonium bicarbonate in LCMS grade water and transferred to the autosampler tray. Electrospray ionization in positive mode and multiple reaction monitoring (MRM) were used for PIP and PIP-d5 at the transitions m/z 518.2 → 143.2 and m/z 523.2 → 148.2 respectively, and electrospray ionization in negative mode and MRM were used for TAZ and SUL at the transitions m/z 299.1 → 138.1 and m/z 232.4 → 140.1. The chromatographic separation was achieved using an Acquity BEH C-18 column with gradient elution of mobile phase containing 10 mmol/L ammonium bicarbonate in water and methanol. A linear range was observed over the concentration range of 0.25-352 mg/L and 0.25-50.5 mg/L for PIP and TAZ respectively. Complete method validation was performed according to US FDA guidelines for selectivity, specificity, precision and accuracy, LLOQ, matrix effects, recovery and stability, with all results within acceptable limits. This method was successfully applied to two patients with pleural infection and is suitable for further pharmacokinetic studies and therapeutic drug monitoring.

Pharmacokinetics of piperacillin in critically ill patients with acute kidney injury receiving sustained low-efficiency diafiltration.

Background: Piperacillin is a ß-lactam penicillin antibiotic commonly used for the empirical therapy of sepsis and other hospital-acquired infections. However, knowledge regarding the effect of sustained low-efficiency diafiltration (SLED-f), a technique increasingly being used in ICUs, on piperacillin pharmacokinetics (PK) and dosing in critically ill patients is lacking. Objectives: To describe the PK of piperacillin during SLED-f and compare the results with those reported for other forms of renal replacement therapies. Methods: Serial blood samples were collected at pre- and post-filter ports within the SLED-f circuit during SLED-f in one session and from an arterial catheter during sampling without SLED-f. Piperacillin concentrations were measured using a validated chromatography method. Non-compartmental PK analysis of the data was performed. Results: The median clearance and area under the concentration-time curve during SLED-f were 6 L/h and 532 mg·h/L, respectively. Fifty-eight percent of piperacillin was cleared by a single SLED-f session (6 h) compared with previous reports of 30%-45% clearance by a 3.5 h intermittent haemodialysis session. Clearance, half-life and area under the concentration-time curve during SLED-f obtained from this study were comparable with those reported in the post-dilution mode of continuous veno-venous haemodiafiltration studies. Conclusions: As it can be challenging to accurately predict when SLED-f will be initiated in the critically ill, a maintenance dose of at least 4 g every 12 h with at least a 2 g replacement dose post-SLED-f would be a practical approach to piperacillin dosing in ICU patients with anuria receiving SLED-f with a duration similar to the current study.

Simultaneous quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin in human serum using an isotope-dilution HPLC-MS/MS method.

The aim of the current study was to develop and validate a robust multi-analyte high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS) method for simultaneous quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin, which are the most commonly used antibiotics in intensive care units. Sample clean-up included a protein precipitation protocol, followed by chromatographic separation on a C8 reverse phase HPLC column within 4 min, using a formic acid-ammonium formiate methanol step-elution gradient. All compounds were detected with electrospray ionization (ESI+) mass spectrometry in multiple reaction time monitoring. The method was validated according to the protocol from the European Medicines Agency and was thoroughly evaluated for interferences and quantification linearity. Linear relationships between peak area responses and drug concentrations were obtained in the range of 0.25-200 mg/l for cefepime, 0.25-120 mg/l for meropenem, 0.05-10 mg/l for ciprofloxacin, 0.125-10 mg/l for moxifloxacin, 0.125-50 mg/l for linezolid and 0.5-400 mg/l for piperacillin with an R2 > 0.997. Imprecision and inaccuracy values (both intra- and inter-assay) were ≤ 6.8% and ≤10.9% for all analytes in quality control samples, respectively. The assay proved to be selective for the study antibiotics, and the internal standards consistently compensated for matrix effects. The described simple and reliable HPLC-MS/MS assay is a powerful tool for routine TDM of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid and piperacillin in human serum in clinical laboratories. With a total process time of approximately 30 min, it allows for accurate and selective quantification up to the expected pharmacokinetic peak concentrations.

Considerable variation of trough ß-lactam concentrations in older adults hospitalized with infection-a prospective observational study.

In older adults, few studies confirm that adequate concentrations of antibiotics are achieved using current dosage regimens of intravenous ß-lactam antibiotics. Our objective was to investigate trough concentrations of cefotaxime, meropenem, and piperacillin in older adults hospitalized with infection. We included 102 patients above 70 years of age. Total trough antibiotic concentrations were measured and related to suggested target intervals. Information on antibiotic dose, patient characteristics, and 28-day outcomes were collected from medical records and regression models were fitted. Trough concentrations for all three antibiotics exhibited considerable variation. Mean total trough concentrations for cefotaxime, meropenem, and piperacillin were 6.5 mg/L (range 0-44), 3.4 mg/L (range 0-11), and 30.2 mg/L (range 1.2-131), respectively. When a target range of non-species-related breakpoint - 5× non-species-related breakpoint was applied, only 36% of patients had both values within the target range. Regression models revealed that severe sepsis was associated with varying concentration levels and increasing age and diminishing kidney function with high concentration levels. The study was not powered to demonstrate consequences in clinical outcomes. Conclusively, in older adults treated with cefotaxime, meropenem, or piperacillin-tazobactam, trough antibiotic concentrations varied considerably. Better predictors to guide dosing regimens of ß-lactam antibiotics or increased use of therapeutic drug monitoring are potential ways to address such variations.

Population pharmacokinetics of continuous infusion of piperacillin in critically ill patients.

Dosing recommendations for continuous infusion of piperacillin, a broad-spectrum beta-lactam antibiotic, are mainly guided by outputs from population pharmacokinetic models constructed with intermittent infusion data. However, the probability of target attainment in patients receiving piperacillin by continuous infusion may be overestimated when drug clearance estimates from population pharmacokinetic models based on intermittent infusion data are used, especially when higher doses (e.g. 16 g/24 h or more) are simulated. Therefore, the purpose of this study was to describe the population pharmacokinetics of piperacillin when infused continuously in critically ill patients. For this analysis, 270 plasma samples from 110 critically ill patients receiving piperacillin were available for population pharmacokinetic model building. A one-compartment model with linear clearance best described the concentration-time data. The mean ± standard deviation parameter estimates were 8.38 ± 9.91 L/h for drug clearance and 25.54 ± 3.65 L for volume of distribution. Creatinine clearance improved the model fit and was supported for inclusion as a covariate. In critically ill patients with renal clearance higher than 90 mL/min/1.73 m2, a high-dose continuous infusion of 24 g/24 h is insufficient to achieve adequate exposure (pharmacokinetic/pharmacodynamic target of 100% fT>4 x MIC) against susceptible Pseudomonas aerginosa isolates (MIC ≤16 mg/L). These findings suggest that merely increasing the dose of piperacillin, even with continuous infusion, may not always result in adequate piperacillin exposure. This should be confirmed by evaluating piperacillin target attainment rates in critically ill patients exhibiting high renal clearance.

Development of a Novel Multipenicillin Assay and Assessment of the Impact of Analyte Degradation: Lessons for Scavenged Sampling in Antimicrobial Pharmacokinetic Study Design.

Penicillins are widely used to treat infections in children; however, the evidence is continuing to evolve in defining the optimal dosing. Modern pediatric pharmacokinetic study protocols frequently favor opportunistic, "scavenged" sampling. This study aimed to develop a small-volume single assay for five major penicillins and to assess the influence of sample degradation on inferences made using pharmacokinetic modeling, to investigate the suitability of scavenged sampling strategies. Using a rapid ultrahigh-performance liquid chromatographic-tandem mass spectrometric method, an assay for five penicillins (amoxicillin, ampicillin, benzylpenicillin, piperacillin, and flucloxacillin) in blood plasma was developed and validated. Penicillin stabilities were evaluated under different conditions. Using these data, the impact of drug degradation on inferences made during pharmacokinetic modeling was evaluated. All evaluated penicillins indicated good stability at room temperature (23 ± 2°C) over 1 h, remaining in the range of 98 to 103% of the original concentration. More-rapid analyte degradation had already occurred after 4 h, with stability ranging from 68% to 99%. Stability over longer periods declined: degradation of up to 60% was observed with delayed sample processing of up to 24 h. Modeling showed that analyte degradation can lead to a 30% and 28% bias in clearance and volume of distribution, respectively, and falsely show nonlinearity in clearance. Five common penicillins can now be measured in a single low-volume blood sample. Beta-lactam chemical instability in plasma can cause misleading pharmacokinetic modeling results, which could impact upon model-based dosing recommendations and the forthcoming era of beta-lactam therapeutic drug monitoring.