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.

A Population Pharmacokinetics and Pharmacodynamic Approach To Optimize Tazobactam Activity in Critically Ill Patients.

The percentage of the time that the free drug concentration remains above a concentration threshold (%fT > concentration threshold) has frequently been identified to be the optimal pharmacokinetic (PK)-pharmacodynamic (PD) target of interest for tazobactam using in vitro infection models. Similar in vitro models suggested that an 85% fT > concentration threshold of 2 µg/ml for tazobactam is required to demonstrate a 2-log10-unit decrease in the number of CFU per milliliter from that at the baseline at 24 h for high-level ß-lactamase-producing Escherichia coli strains. The objective of this study was to characterize the tazobactam concentrations in a cohort of critically ill patients with Gram-negative bacterial infections, determine if traditional dosing regimens achieve a prespecified PK/PD target of an 80% fT > concentration threshold of 2 µg/ml, and propose alternative dosing regimens. Hospitalized critically ill adult patients receiving piperacillin-tazobactam (TZP) for a culture-positive Gram-negative bacterial infection were eligible to consent for study inclusion. Two blood samples were drawn, one during the midpoint of the dosing interval and one at the time of the trough concentration once the patient achieved PK steady state. A population PK model was developed using Phoenix NLME (v8.1) software to characterize the observed concentration-time profile of tazobactam, explore potential covariates to explain the variability in the clearance and volume parameters, and to simulate potential dosing regimens that would achieve the PK/PD target. The PK of tazobactam were adequately described by a one-compartment model with first-order elimination in 18 patients who provided consent. The final model incorporated creatinine clearance as a covariate on clearance. Simulations demonstrated target attainments of less than 50% for tazobactam using traditional dosing regimens (4/0.5 g over 30 min every 6 h). Target attainments of greater than 75% were achieved when using extended infusion times of 4 to 6 h or when administering TZP as a continuous infusion (16/2 g over 24 h). Traditional tazobactam dosing regimens fail to achieve conservative PK/PD targets in critically ill patients. Increases in the tazobactam dose or prolongation of the infusion rate may be warranted to achieve activity against ß-lactamase-producing Gram-negative bacteria.

Ceftolozane-Tazobactam in the Treatment of Experimental Pseudomonas aeruginosa Pneumonia in Persistently Neutropenic Rabbits: Impact on Strains with Genetically Defined Mechanisms of Resistance.

Ceftolozane-tazobactam (C/T) is a novel cephalosporin with in vitro activity against Pseudomonas aeruginosa that is resistant to extended-spectrum penicillins and antipseudomonal cephalosporins. In order to assess the antimicrobial effect of C/T in treatment of Pseudomonas pneumonia, we investigated the pharmacokinetics and efficacy of C/T in persistently neutropenic rabbits. Pseudomonas pneumonia was established by direct endotracheal inoculation. Treatment groups consisted of C/T, ceftazidime (CAZ), piperacillin-tazobactam (TZP), and untreated controls (UC). Rabbits received a dosage of C/T of 80 mg/kg every 4 h (q4h) intravenously (i.v.) (53 mg/kg ceftolozane/26 mg/kg tazobactam) to match the free drug time above the MIC as well as a comparable plasma area under the concentration-time curve (AUC) (humanized doses of ceftolozane-tazobactam of 3 g [2 g/1 g]) q8h, due to the more rapid elimination of ceftolozane in rabbits (0.75 h) than in humans (2.5 h). Four molecularly characterized clinical P. aeruginosa isolates from patients with pneumonia were studied, including one isolate from each classification group: pan-susceptible (PS), outer membrane porin D (OPRD) porin loss (OPRDPL), efflux pump expression (EPE), and AmpC hyperexpression (ACHE). Treatment was continued for 12 days. Treatment with ceftolozane-tazobactam resulted in a ≥105 reduction in residual pulmonary and bronchoalveolar lavage (BAL) fluid bacterial burdens caused by all 4 strains (P ≤ 0.01). This antibacterial activity coincided with reduction of lung weight (an organism-mediated pulmonary injury marker) (P < 0.05). CAZ was less active in ACHE-infected rabbits, and TZP had less activity against EPE, ACHE, and OPRDPL strains. Survival was prolonged in the C/T and CAZ treatment groups in comparison to the TZP and UC groups (P < 0.001). Ceftolozane-tazobactam is highly active in treatment of experimental P. aeruginosa pneumonia in persistently neutropenic rabbits, including infections caused by strains with the most common resistance mechanisms.

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.

Population pharmacokinetics of tazobactam/ceftolozane in Japanese patients with complicated urinary tract infection and complicated intra-abdominal infection.

Tazobactam/ceftolozane is a combination of a ß-lactamase inhibitor and a cephalosporin antibiotic, with recommended dosage for patients with normal renal function of tazobactam 0.5 g/ceftolozane 1 g administered as a 1-h intravenous infusion every 8 h. The doses in patients with moderate and severe renal impairment are recommended to be reduced by half and 1/4th, respectively. The dose in patients undergoing dialysis is a single loading dose of 750 mg followed after 8 h by a 150 mg maintenance dose. In order to evaluate pharmacokinetics (PK) in Japanese patients, individual Bayes PK parameters were derived using the previously developed population PK models. Furthermore, attainment of PK/pharmacodynamic target in Japanese patients was calculated to confirm the recommended dosage. Based on PK data from 200 Japanese patients in the phase 3 studies, including patients with mild and moderate renal impairment, individual tazobactam/ceftolozane PK parameters were derived. No clinically relevant difference was observed in tazobactam/ceftolozane exposures between Japanese and non-Japanese patients. All Japanese patients achieved a target percent of time that free ceftolozane concentrations are above the minimum inhibitory concentration (MIC) of 30% for MICs of up to 8 µg/mL. Also for tazobactam, all Japanese patients achieved a target percent of time that the free tazobactam concentration exceeds a threshold concentration (1 µg/mL) of 20%. The results suggest that the doses will be efficacious in the Japanese population. The results indicate that the recommended dose in patients with normal renal function or renal impairment is appropriate in Japanese patients.

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.

Pharmacokinetics of Ceftolozane-Tazobactam during Prolonged Intermittent Renal Replacement Therapy.

BACKGROUND: Prolonged intermittent renal replacement therapy (PIRRT) eliminates many drugs, and without dosing data, for new antibiotics like ceftolozane/tazobactam, suboptimal concentrations and treatment failure are likely. OBJECTIVES: Herein, we describe the effect of PIRRT on the plasma pharmacokinetics of ceftolozane/tazobactam ad-ministered in a critically ill 55-year-old patient with a polymicrobial sternal wound osteomyelitis, including a multiresistant Pseudomonas aeruginosa. METHOD: Blood samples were taken over 4 days where the patient received a 7.5-h PIRRT treatment. One- and 2-compartment models were tested for ceftolozane and tazobactam separately, and the log-likelihood ratio and goodness-of-fit plots were used to select the final model. RESULTS: Two-compartment models were developed for ceftolozane and tazobactam separately and described significant differences in clearance of ceftolozane and tazobactam with and without PIRRT (8.273 vs. 0.393 and 8.020 vs. 0.767 L/h, respectively). CONCLUSIONS: A ceftolozane/tazobactam dose of 500 mg/250 mg appears to be sufficient to attain pharmacokinetic/pharmacodynamic targets during PIRRT while the manufacturer's recommended dosing of 100 mg/50 mg every 8 h was sufficient during non-PIRRT periods.

Development and validation of a quantitative LC-MS/MS method for the simultaneous determination of ceftolozane and tazobactam in human plasma and urine.

The purpose of this work was to develop and validate a single sensitive, selective and rapid bioanalytical method to determine ceftolozane and tazobactam concentrations in human plasma and urine and to use this method to analyze samples from a human clinical study. Human plasma and urine samples were prepared by protein precipitation using a solution of acetonitrile, water and formic acid. Following protein precipitation, samples were analyzed by liquid chromatography tandem mass spectrometry. Chromatographic resolution was achieved on a Kinetex PFP column using a gradient elution, a flow rate of 0.4 mL/min, and a total run time of 5 min. Positive electrospray ionization was employed and analytes were quantitated using multi-reaction monitoring mode. Method validation was conducted in accordance with Unites States Food and Drug Administration's regulatory guidelines for bioanalytical method validation. Calibration curves were determined to linear over the range of 0.1 to 40 µg/mL for ceftolozane and 0.05 to 20 µg/mL for tazobactam. The method was determined to be accurate (-6.24 to 12.53 percent relative error), precise (less than 13.28 percent standard deviation) and sensitive in both human plasma and urine. Ceftolozane and tazobactam were determined to be stable across a battery of stability studies including autosampler, benchtop, freeze/thaw and long-term stability. This validated method successfully applied to human clinical samples to determine the concentration versus time profiles of the intravenously administered combination of Zerbaxa (ceftolozane-tazobactam) in burn patients.

Plasma pharmacokinetics of ceftolozane/tazobactam in pediatric patients with cystic fibrosis.

BACKGROUND: The antipseudomonal cephalosporin/ß-lactamase inhibitor combination ceftolozane/tazobactam could be a potential treatment option for cystic fibrosis (CF) pulmonary exacerbations. The pharmacokinetics (PK) of ceftolozane/tazobactam in children with CF merits further evaluation. METHODS: This is a retrospective subgroup analysis of a phase 1, noncomparative trial that characterized PK, safety, and tolerability of single intravenous doses of ceftolozane/tazobactam in pediatric patients. This analysis compares ceftolozane and tazobactam plasma PK parameters, estimated from a population PK model, between patients with and without CF enrolled in that trial. Individual attainment of PK/pharmacodynamic (PD) targets of ceftolozane and tazobactam (free ceftolozane concentration >4 µg/mL for >30% and free tazobactam concentration >1 µg/mL for 20% of the dosing interval) in patients with and without CF were evaluated. RESULTS: The study enrolled 18 patients aged greater than or equal to 2 to less than 18 years old, which included 9 with CF. Weight-normalized ceftolozane PK parameters were similar between patients with CF (clearance: 0.16 L/h/kg, half-life: 1.54 hours, volume of distribution: 0.26 L/kg) and without CF (clearance: 0.15 L/h/kg, half-life: 1.62 hours, volume of distribution: 0.26 L/kg), as were most weight-normalized tazobactam PK parameters. Weight-normalized tazobactam clearance was higher in patients with CF (0.73 L/h/kg) than patients without CF (0.42 L/h/kg). All patients achieved the prespecified PK/PD targets for ceftolozane and tazobactam. CONCLUSIONS: This retrospective analysis demonstrated generally similar weight-normalized plasma PK parameters for ceftolozane and tazobactam among children with and without CF; thus, projected doses for treatment of pediatric hospital-acquired/ventilator-associated pneumonia, which are higher than the pediatric complicated urinary tract infection/intra-abdominal infection doses, may be appropriate for treatment of CF pulmonary exacerbation.

Measurement of ceftolozane and tazobactam concentrations in plasma by UHPLC-MS/MS. Clinical application in the management of difficult-to-treat osteoarticular infections.

BACKGROUND: Ceftolozane, in combination with the ß-lactamase inhibitor tazobactam, is a new option in the pipeline against multidrug-resistant Gram-negative bacilli. As for other ß-lactam antibiotics, optimizing the use of ceftolozane-tazobactam is advisable, especially in difficult-to-treat infections. In this regard, therapeutic drug monitoring would be required to guide the treatment of ceftolozane-tazobactam. Thus, we aimed to develop and validate procedures based on UHPLC-MS/MS for measurement of ceftolozane and tazobactam plasma concentrations in clinical practice. MATERIAL AND METHODS: Analyses were conducted using an Acquity® UPLC® integrated system coupled to an Acquity® TQD® tandem-quadrupole mass spectrometer. Ceftolozane, tazobactam and their internal standards (ceftazidime-D5 and sulbactam) were detected by electrospray ionization mass spectrometry in positive and negative ion multiple reaction monitoring modes, using transitions of 667.2 → 199.3/139.0 and 551.9 → 467.9 for ceftolozane and ceftazidime-D5, and 299.0 → 138/254.9 and 232.0 → 140.0 for tazobactam and sulbactam. Measurement procedures developed were used for guiding the treatment and adjusting daily dose of ceftolozane-tazobactam in patients with osteoarticular infections. RESULTS: Coefficients of variation and absolute relative biases were <7.9% and 6.5% in all cases. The lower limit of quantification, linearity, normalized-recoveries, normalized-matrix effects and measurement uncertainties for ceftolozane were: 0.97 mg/L, (0.97-125) mg/L, ≤113.6%, ≤108.7%, and ≤ 18.7%, respectively; and for tazobactam: 1.04 mg/L, (1.04-125) mg/L, ≤103.6%, ≤101.9%, and ≤ 20.0%. No interferences and carry-over were observed. Patients plasma concentrations were higher than the recommended 3-4 times the minimal inhibitory concentrations. CONCLUSIONS: Our measurement procedures are suitable for therapeutic drug monitoring of ceftolozane-tazobactam in patients with osteoarticular infections.

Determination of total and free ceftolozane and tazobactam in human plasma and interstitial fluid by HPLC-UV.

Ceftolozane/tazobactam is a new cephalosporin/beta-lactamase inhibitor combination. An HPLC-UV method is described for the determination of total and free ceftolozane and tazobactam in human plasma and in microdialysate of subcutaneous tissue, respectively. Separation was performed using a reversed-phase column with phosphate buffer/acetonitrile as eluent and photometric detection at 260 nm (ceftolozane) or 220 nm (tazobactam). Linearity has been shown down to ceftolozane/tazobactam 0.1/0.05 mg/L in plasma and 0.03/0.015 mg/L in saline, respectively. The plasma protein binding of both drugs as determined by ultrafiltration was less than 10%. Temperature, pH or relative centrifugation force (up to 3000 x g) had no significant impact on the protein binding. The method was applied to the determination of ceftolozane and tazobactam in plasma and interstitial fluid of healthy volunteers following intravenous infusion of ceftolozane/tazobactam 1.0/0.5 g.