Estimation of linezolid exposure in patients with hepatic impairment using machine learning based on a population pharmacokinetic model.

PURPOSE: To investigate the pharmacokinetic changes of linezolid in patients with hepatic impairment and to explore a method to predict linezolid exposure. METHODS: Patients with hepatic impairment who received linezolid were recruited. A population pharmacokinetic model (PPK) was then built using NONMEM software. And based on the final model, virtual patients with rich concentration values was constructed through Monte Carlo simulations (MCS), which were used to build machine learning (ML) models to predict linezolid exposure levels. Finally, we investigated the risk factors for thrombocytopenia in patients included. RESULTS: A PPK model with population typical values of 3.83 L/h and 34.1 L for clearance and volume of distribution was established, and the severe hepatic impairment was identified as a significant covariate of clearance. Then, we built a series of ML models to predict the area under 0 -24 h concentration-time curve (AUC0-24) of linezolid based on virtual patients from MCS. The results showed that the Xgboost models showed the best predictive performance and were superior to the methods for estimating linezolid AUC0-24 based on though concentration or daily dose. Finally, we found that baseline platelet count, linezolid AUC0-24, and combination with fluoroquinolones were independent risk factors for thrombocytopenia, and based on this, we proposed a method for calculating the toxicity threshold of linezolid. CONCLUSION: In this study, we successfully constructed a PPK model for patients with hepatic impairment and used ML algorithm to estimate linezolid AUC0-24 based on limited data. Finally, we provided a method to determine the toxicity threshold of linezolid.

An eco-friendly and cost-effective HPTLC method for quantification of COVID-19 antiviral drug and co-administered medications in spiked human plasma.

The coronavirus-2 has led to a global pandemic of COVID-19 with an outbreak of severe acute respiratory syndrome leading to worldwide quarantine measures and a rise in death rates. The objective of this study is to propose a green, sensitive, and selective densitometric method to simultaneously quantify remdesivir (REM) in the presence of the co-administered drug linezolid (LNZ) and rivaroxaban (RIV) in spiked human plasma. TLC silica gel aluminum plates 60 F254 were used as the stationary phase, and the mobile phase was composed of dichloromethane (DCM): acetone (8.5:1.5, v/v) with densitometric detection at 254 nm. Well-resolved peaks have been observed with retardation factors (Rf) of 0.23, 0.53, and 0.72 for REM, LNZ, and RIV, respectively. A validation study was conducted according to ICH Q2 (R1) Guidelines. The method was rectilinear over the concentration ranges of 0.2-5.5 µg/band, 0.2-4.5 µg/band and 0.1-3.0 µg/band for REM, LNZ and RIV, respectively. The sensitivities of REM, LIN, and RIV were outstanding, with quantitation limits of 128.8, 50.5, and 55.8 ng/band, respectively. The approach has shown outstanding recoveries ranging from 98.3 to 101.2% when applied to pharmaceutical formulations and spiked human plasma. The method's greenness was assessed using Analytical Eco-scale, GAPI, and AGREE metrics.

UPLC-MS/MS Determination of Linezolid and Heme in Plasma of Infected Patients and Correlation Analysis.

Linezolid can cause serious haematological toxicity, such as thrombocytopenia and aneamia. Heme, composed of iron and porphyrin, is an important component of hemoglobin. In order to investigate the relationship between the concentration of linezolid and heme in the plasma of infected patients, a UPLC-MS/MS method that can determine the concentrations of linezolid and heme simultaneously was developed and validated. A total of 96 healthy subjects and 81 infected patients, who received blood routine blood tests, were included and determined by the UPLC-MS/MS method. The results showed that the concentration of linezolid was 5.08 ± 3.46 µg/mL in infected patients who were treated with linezolid. The heme in healthy subjects was 7.05 ± 8.68 µg/mL, and it was significantly decreased to 0.88 ± 0.79 µg/mL in infected patients (P < 0.01). Spearman correlation analysis showed that linezolid had a high negative correlation with platelet (PLT) (R = -0.309). Heme had a high positive correlation with hemoglobin (Hb) (R = 0.249) in healthy subjects and infected patients. The ROC analysis showed that heme had diagnostic value to distinguish low Hb (110 g/L). In conclusion, there was a positive correlation between heme and Hb, and this correlation was also observed in infected patients. A high concentration of linezolid was inclined to decrease PLT. Monitoring of heme and linezolid helps in the early diagnose of low Hb and PLT.

A sensitive and high-throughput quantitative liquid chromatography high-resolution mass spectrometry method for therapeutic drug monitoring of 10 ß-lactam antibiotics, linezolid and two ß-lactamase inhibitors in human plasma.

An ultra-high pressure liquid chromatography high-resolution mass spectrometric (UHPLC-HRMS) method was developed for the simultaneous and sensitive quantification of 10 ß-lactam antibiotics (cefepime, meropenem, amoxicillin, cefazolin, benzylpenicillin, ceftazidime, piperacillin, flucloxacillin, cefuroxime and aztreonam), linezolid and ß-lactamase inhibitors tazobactam and clavulanic acid in human plasma. Validation according to the EMA guidelines showed excellent within- and between-run accuracy and precision (i.e. between 1.1 and 8.5%) and high sensitivity (i.e. lower limit of quantification between 0.25 and 1 mg/L). The UHPLC-HRMS method enables a short turnaround time and high sensitivity and needs only a small amount of plasma, allowing appropriate routine therapeutic drug monitoring. The short turnaround time is obtained by speeding up the protocol on multiple levels, i.e. fast and workload-efficient sample preparation (i.e. protein precipitation and dilution), short (4 min) instrument run time, simultaneous measurement of all relevant ß-lactam antibiotics used in the intensive care unit and the use of the same instrument, column and mobile phases as for the other routine methods in our laboratory.

Simultaneous quantification of plasma levels of 12 antimicrobial agents including carbapenem, anti-methicillin-resistant Staphylococcus aureus agent, quinolone and azole used in intensive care unit using UHPLC-MS/MS method.

OBJECTIVES: Critically ill patients in intensive care unit (ICU) are susceptible to infectious diseases, thus empirical therapy is recommended. However, the therapeutic effect in ICU patients is difficult to predict due to fluctuation in pharmacokinetics because of various factors. This problem can be solved by developing personalized medicine through therapeutic drug monitoring. However, when different measurement systems are used for various drugs, measurements are complicated and time consuming in clinical practice. In this study, we aimed to develop an assay using ultra-high performance liquid chromatography coupled with tandem mass spectrometry for simultaneous quantification of 12 antimicrobial agents commonly used in ICU: doripenem, meropenem, linezolid, tedizolid, daptomycin, ciprofloxacin, levofloxacin, pazufloxacin, fluconazole, voriconazole, voriconazole N-oxide which is a major metabolite of voriconazole, and posaconazole. DESIGN & METHODS: Plasma protein was precipitated by adding acetonitrile and 50% MeOH containing standard and labeled IS. The analytes were separated with an ACQUITY UHPLC CSH C18 column, under a gradient mobile phase consisting of water and acetonitrile containing 0.1% formic acid and 2 mM ammonium formate. RESULTS: The method fulfilled the criteria of US Food and Drug Administration for assay validation. The recovery rate was more than 84.8%. Matrix effect ranged from 79.1% to 119.3%. All the calibration curves showed good linearity (back calculation of calibrators: relative error ≤ 15%) over wide concentration ranges, which allowed determination of Cmax and Ctrough. Clinical applicability of the novel method was confirmed. CONCLUSIONS: We have developed an assay for simultaneous quantification of 12 antimicrobial agents using a small sample volume of 50 µL with a short assay time of 7 min. Our novel method may contribute to simultaneous calculation of pharmacokinetic and pharmacodynamic parameters.

Analysis of the risk factors of linezolid-related haematological toxicity in Chinese patients.

WHAT IS KNOWN AND OBJECTIVES: Haematological toxicity including thrombocytopenia, anaemia and leucopenia is the main adverse events of linezolid (LZD) therapy. This study aimed to investigate the risk factors for LZD-induced haematological toxicity and define the threshold of plasma trough concentration to minimize the haematological toxicity. METHODS: 145 patients who received LZD for more than 10 days were retrospectively reviewed to determine the incidence of LZD-induced haematological toxicity. Meanwhile, the risk factors of haematological toxicity were confirmed by univariate and multivariate logistic regression analysis. RESULTS AND DISCUSSION: 9 (6.2%) patients developed leucopenia, while 52 (35.9%) and 26 (17.9%) patients developed thrombocytopenia and anaemia, respectively. The estimated glomerular filtration rate (eGFR) <90 ml/min/1.73 m2 (OR, 2.744; 95% CI, 1.117-6.734; p = 0.028) and baseline platelet count <200 × 109 /L (OR, 6.817; 95% CI, 2.870-16.193; p < 0.0001) were found to be significant risk factors for LZD-related thrombocytopenia. Aspartate aminotransferase (AST) >80 U/L (OR, 4.844; 95% CI, 1.207-19.451; p = 0.026) and eGFR <90 ml/min/1.73 m2 (OR, 7.132; 95% CI, 2.088-24.357; p = 0.002) were the risk factors for LZD-related anaemia. However, no significant risk factors were identified for LZD-related leucopenia. Moreover, LZD plasma trough concentration >8 mg/L [OR, 3.047; 95% CI, 1.233-7.539; p = 0.016] could be a predictor for the development of thrombocytopenia and anaemia. WHAT IS NEW AND CONCLUSION: Hepatic and/or renal dysfunction are the risk factors for LZD-related haematological toxicity, while the target plasma trough concentration within 8 mg/L via dose reduction could minimize the haematological toxicity induced by LZD.

Bone penetration of linezolid in osteoarticular tuberculosis patients of China.

BACKGROUND: Linezolid presents strong antimicrobial activity against multidrug-resistant (MDR) pulmonary tuberculosis (TB), but its application in osteoarticular tuberculosis treatment remains understudied. Our objective was to analyze the bone penetration efficiency of linezolid in osteoarticular TB patients. METHODS: Osteoarticular TB patients, treated with 600 mg q 24 h linezolid-containing regimens and undergoing surgery, were prospectively and consecutively enrolled. One dose linezolid was administered before surgery. Blood and bone samples were collected simultaneously during operation, and their linezolid concentrations were then detected using high-performance liquid chromatography-tandem mass spectrometry. Pus samples were subjected to mycobacterial culture and GeneXpert MTB/RIF assay. The minimum inhibition concentrations (MICs) and drug susceptibility testing were performed with the recovered isolates. RESULTS: A total of 36 eligible osteoarticular TB patients were enrolled, including five MDR/rifampicin-resistant cases. All the 12 recovered isolates had MICs ≤0.5 µg/mL for linezolid. Mean concentrations in plasma, collected 100-510 min after the preoperative dosing, were 10.43 ± 4.83 µg/mL (range 3.29-22.26 µg/mL), and median concentrations in bone were 3.93 µg/mL (range 0.61-16.34 µg/mL). The median bone/plasma penetration ratio was 0.42 (range 0.14-0.95 µg/mL). Linezolid concentration in bone had a linear correlation with the drug concentration in plasma (r = 0.7873, p < 0.0001), while plasma concentration could explain 61.98% of the variation of concentration in bone (R2 = 0.6198). Notably, stratification analysis by sampling time demonstrated that samples collected 200-510 min after dosing had very good linear relationships between their bone and plasma concentrations (r = 0.9323). CONCLUSIONS: Linezolid penetrates from blood to bone efficiently, and the penetration further stabilizes ∼3 h after dosing.

Prolonged infusion of linezolid is associated with improved pharmacokinetic/pharmacodynamic (PK/PD) profiles in patients with external ventricular drains.

OBJECTIVE: We previously investigated the pharmacokinetic and pharmacodynamic (PK/PD) parameters of routine linezolid infusions (1 h) in patients with external ventricular drains (EVD). The aim of the study was to determine whether extended linezolid infusions (200 mg/h for 3 h) were more efficacious than short linezolid infusions (600 mg/h for 1 h). METHODS: We collected cerebrospinal fluid (CSF) and plasma samples from 10 patients who received linezolid infusions after cerebral hemorrhage surgery with EVDs. Linezolid concentrations were measured by high-performance liquid chromatography (HPLC). A Monte Carlo simulation was used to measure the probability of target attainments (PTA) and the PK/PD indexes at four minimum inhibitory concentrations (MIC). RESULTS: When the same dose (600 mg) was given as an extended infusion (3 h), linezolid reached its maximum concentrations in the plasma and CSF at 3.00 h and 4.40 h, respectively. The mean penetration of linezolid in CSF was 41.31%. Using the parameter of AUC0-24 h/MIC ≥ 100, the plasma PTA provided good coverage at > 90% when MIC was ≤ 1 µg/mL, while the values were 0 in CSF. Using the parameter %T (time) > MIC ≥ 85%, the PTA in both the plasma and CSF provided good coverage when MIC ≤ 2 µg/mL. Compared with routine infusions, prolonged infusion times (3 h) showed increased PTA of linezolid. CONCLUSIONS: Prolonged infusion times increased the concentration of linezolid in the plasma, leading to improved therapeutic outcomes. However, this improvement did not exist in CSF. Lastly, the PK/PD indicator AUC/MIC ≥ 100 may be used to achieve improved outcomes in patients with critical infections.

Antibiotic therapeutic drug monitoring in intensive care patients treated with different modalities of extracorporeal membrane oxygenation (ECMO) and renal replacement therapy: a prospective, observational single-center study.

BACKGROUND: Effective antimicrobial treatment is key to reduce mortality associated with bacterial sepsis in patients on intensive care units (ICUs). Dose adjustments are often necessary to account for pathophysiological changes or renal replacement therapy. Extracorporeal membrane oxygenation (ECMO) is increasingly being used for the treatment of respiratory and/or cardiac failure. However, it remains unclear whether dose adjustments are necessary to avoid subtherapeutic drug levels in septic patients on ECMO support. Here, we aimed to evaluate and comparatively assess serum concentrations of continuously applied antibiotics in intensive care patients being treated with and without ECMO. METHODS: Between October 2018 and December 2019, we prospectively enrolled patients on a pneumological ICU in southwest Germany who received antibiotic treatment with piperacillin/tazobactam, ceftazidime, meropenem, or linezolid. All antibiotics were applied using continuous infusion, and therapeutic drug monitoring of serum concentrations (expressed as mg/L) was carried out using high-performance liquid chromatography. Target concentrations were defined as fourfold above the minimal inhibitory concentration (MIC) of susceptible bacterial isolates, according to EUCAST breakpoints. RESULTS: The final cohort comprised 105 ICU patients, of whom 30 were treated with ECMO. ECMO patients were significantly younger (mean age: 47.7 vs. 61.2 years; p < 0.001), required renal replacement therapy more frequently (53.3% vs. 32.0%; p = 0.048) and had an elevated ICU mortality (60.0% vs. 22.7%; p < 0.001). Data on antibiotic serum concentrations derived from 112 measurements among ECMO and 186 measurements from non-ECMO patients showed significantly lower median serum concentrations for piperacillin (32.3 vs. 52.9; p = 0.029) and standard-dose meropenem (15.0 vs. 17.8; p = 0.020) in the ECMO group. We found high rates of insufficient antibiotic serum concentrations below the pre-specified MIC target among ECMO patients (piperacillin: 48% vs. 13% in non-ECMO; linezolid: 35% vs. 15% in non-ECMO), whereas no such difference was observed for ceftazidime and meropenem. CONCLUSIONS: ECMO treatment was associated with significantly reduced serum concentrations of specific antibiotics. Future studies are needed to assess the pharmacokinetic characteristics of antibiotics in ICU patients on ECMO support.

Developing an isotope dilution UHPLC-MS/MS method to quantify linezolid in human plasma: application to therapeutic drug monitoring.

Aim: To optimize clinical efficacy and reduce the drug-exposure-related toxicity of linezolid, whose concentrations show wide inter-variabilities, a simple and reliable quantitative assay for therapeutic drug monitoring is necessary. Results: A UHPLC-MS/MS assay has been established for determination of linezolid in human plasma and fully validated according to the US FDA guidelines. After a simple, isotope-dilluted precipitation with methanol, the analytes were separated by a straightforward isocratic mode and the MS/MS was conducted under the ESI+ mode fitted with SRM. The calibration curves proved acceptable linearity in the range of 0.1-30.0 µg/ml. Conclusion: The present assay is currently used in routine clinical practice, being applied to therapeutic drug monitoring and helps to optimize individual dosing regimens and manage adverse effects in ICU patients.

Risk of target non-attainment in obese compared to non-obese patients in calculated linezolid therapy.

OBJECTIVES: The aim was to characterize linezolid population pharmacokinetics in plasma and interstitial space fluid of subcutaneous adipose tissue (target site) of obese compared with non-obese patients and to determine dosing regimens enabling adequate therapy using Monte Carlo simulations. METHODS: In this prospective, parallel group, open-label, controlled, single-centre trial, 30 surgery patients (15 obese, 15 non-obese) received 600 mg of intravenous linezolid. A population pharmacokinetic analysis characterizing plasma and microdialysis-derived target site pharmacokinetics was followed by Monte Carlo simulations using twice/thrice daily 600-1200 mg short-term and extended infusions of linezolid. Adequacy of therapy was assessed by the probability of pharmacokinetic/pharmacodynamic target attainment for time and exposure-related indices. RESULTS: In the model, lean body weight and obesity status largely explained between-patient variability in linezolid PK parameters (12.0-44.9%). Both factors caused lower area under the concentration-time curve in typical obese patients in plasma (-20.4%, 95% CI -22.0% to -15.9%) and at target-site (-37.7%, 95% CI -47.1% to -24.2%) compared with non-obese patients. Probability of target attainment showed improvement with increasing linezolid doses. Depending on lean body weight, adequate therapy was partially attained for 900- and 1200-mg linezolid doses and minimum inhibitory concentrations (MICs) ≤2 mg/L (probability of target attainment 62.5-100%) but could not be reached for MIC = 4 mg/L (probability of target attainment ≤82.3%). Additionally, lower linezolid distribution into the target site in obese patients as described above might compromise the plasma-based probability of target attainment analysis. DISCUSSION: This analysis revealed risks of linezolid underdosing in empirical antibiotic therapy of most resistant bacteria for obese and non-obese patients. Doubling the standard dose is associated with adequate probability of target attainment throughout most body masses for MIC ≤2 mg/L. Further clinical studies with adjusted dosing regimens in for example intensive care patients are needed.

Accumulation of Major Linezolid Metabolites in Patients with Renal Impairment.

In patients with renal impairment (n = 22 of 39), the median serum concentrations of linezolid, PNU-142300, and PNU-142586 were 1.6-, 3.3-, 2.8-fold higher, respectively, than in patients without renal impairment. Metabolite concentrations in paired samples were poorly correlated with linezolid concentrations (r2 = 0.26 for PNU-142300 and 0.06 for PNU-142586). Linezolid and its metabolites share potential toxicophores that deserve characterization to mitigate higher myelosuppression risk in patients with renal impairment.

Development of a sensitive LC-MS/MS method for quantification of linezolid and its primary metabolites in human serum.

Linezolid (LZD) is a widely used antimicrobial that is active against a broad range of disease-causing bacteria. Myelosuppression is major treatment-limiting toxicity of LZD that occurs more frequently in patients with renal insufficiency. Quantification of LZD and its two primary metabolites (PNU-142300 and PNU-142586), which undergo significant renal elimination, may support design of improved dosing strategies to mitigate the risk of myelosuppression. In this study, we established the first liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of LZD and its main metabolites in human serum. Proteins in serum samples were precipitated with acetonitrile containing a deuterated internal standard. Chromatographic separation of analytes was performed with Waters X-bridge column (C18, 150 × 4.6 mm, 3.5 µm) at 25 °C and subjected to mass analysis using positive electro-spray ionization. The mobile phase A was water with 0.1% formic acid, and mobile phase B was acetonitrile with 0.1% formic acid at a flow rate of 0.6 mL/min, within a 15 min run time. Standard curves were linear and correlation coefficients (r2) were ≥0.99 for concentration ranges of 0.1-50 µg/mL for LZD and PNU-142300, and 0.1-25 µg/mL for PNU-142586. The inter- and intra-assay precisions were <15% for all analytes in quality control samples, and the accuracies ranged from 97 to 112%. Extraction recoveries ranged from 78 to 103% for all analytes, and there was no significant matrix effect. Samples from 10 patients (5 with renal impairment) were assayed. Mean (SD) LZD, PNU-142300 and PNU-142586 trough concentrations were 19.4(6.8), 11.6(6.8), 25.7(16.4) µg/mL, respectively, in patients with renal impairment. These values were 2.5-, 5.8-, and 6.8-fold higher for LZD, PNU-142300 and PNU-142586, respectively compared to patients without renal impairment. The method was effectively applied in the determination of LZD and its main metabolites in human serum.

Long-Term Effects on QT Prolongation of Pretomanid Alone and in Combinations in Patients with Tuberculosis.

Concentration-QTc modeling was applied to pretomanid, a new nitroimidazooxazine antituberculosis drug. Data came from eight phase 2 and phase 3 studies. Besides pretomanid alone, various combinations with bedaquiline, linezolid, moxifloxacin, and pyrazinamide were considered; special attention was given to the bedaquiline-pretomanid-linezolid (BPaL) regimen that has demonstrated efficacy in the Nix-TB study in subjects with extensively drug-resistant or treatment-intolerant or nonresponsive multidrug-resistant tuberculosis. Three heart rate corrections to QT were considered: Fridericia's QTcF, Bazett's QTcB, and a population-specific correction, QTcN. QTc increased with the plasma concentrations of pretomanid, bedaquiline's M2 metabolite, and moxifloxacin in a manner described by a linear model in which the three slope coefficients were constant across studies, visits within study, and times postdose within visit but where the intercept varied across those dimensions. The intercepts tended to increase on treatment to a plateau after several weeks, a pattern termed the secular trend. The slope terms were similar for the three QTc corrections, but the secular trends differed, suggesting that at least some of the secular trend was due to the elevated heart rates of tuberculosis patients decreasing to normal levels on treatment. For pretomanid 200 mg once a day (QD) alone, a typical steady-state maximum concentration of drug in plasma (Cmax) resulted in a mean change from baseline of QTcN of 9.1 ms, with an upper 90% confidence interval (CI) limit of 10.2 ms. For the BPaL regimen, due to the additional impact of the bedaquiline M2 metabolite, the corresponding values were 13.6 ms and 15.0 ms. The contribution to these values from the secular trend was 4.0 ms.

Ultrahigh-throughput absolute quantitative analysis of linezolid in human plasma by direct analysis in real time mass spectrometry without chromatographic separation and its application to a pharmacokinetic study.

Therapeutic drug monitoring (TDM) is necessary in the clinical management of linezolid to improve its efficacy and reduce the risk of time- and dose-dependent toxicity. A novel and ultrahigh-throughput analytical method for the determination of linezolid in human plasma was developed based on direct analysis in real-time tandem mass spectrometry (DART-MS/MS) without chromatographic separation. After solid-phase extraction with Waters Oasis HLB, the linezolid and internal standard linezolid-d3 were detected by positive ion electrospray ionization followed by multiple reaction monitoring (MRM) of the transition at m/z 338.1 → 296.2 and 341.2 → 297.3, respectively. The use of DART-MS obviates the need for chromatographic separation and allowed determination of linezolid in a total run time of only 24 s per sample. The method was linear in the concentration range 0.20-25 µg mL-1 with intraday and interday precision <14.5% and accuracy ranging from -3.85% to 12.7%. The method was successfully applied to a pharmacokinetic study of linezolid in healthy male volunteers after oral administration of a 600 mg tablet. DART-MS/MS provides a rapid and sensitive method for the determination of linezolid that does not require chromatographic separation. It is eminently suitable to meet the high-throughput challenge of clinical TDM. Graphical abstract.

Measurement of Linezolid and Its Metabolites PNU-142300 and PNU-142586 in Human Plasma Using Ultra-Performance Liquid Chromatography Method.

An ultra-performance liquid chromatography (UPLC) method was developed and validated for the quantification of linezolid, PNU-142300, and PNU-142586 in human plasma. After protein precipitation using acetonitrile, the protein-free supernatant was separated using reverse-phase chromatography using an ACQUITY UPLC HSS T3 column and monitored at 254 nm. p-Toluic acid was used as the internal standard. No interference peak was observed at the retention times of linezolid, PNU-142300, PNU-142586, and p-toluic acid from blank plasma. The calibration curve of linezolid was linear from 0.2 to 50.0 µg/mL (coefficient of determination (r2) > 0.9999) and those of PNU-142300 and PNU-142586 were linear from 0.2 to 20.0 µg/mL (r2 > 0.9996 and > 0.9998, respectively). The intra- and inter-assay accuracy (%) and precision (relative standard deviation (RSD) %) of the three components were confirmed to meet the criteria of the U.S. Food and Drug Administration guidelines. Tests confirmed the stability of linezolid, PNU-142300, and PNU-142586 in plasma during three freeze-thaw cycles and long-term storage of frozen plasma for up to 30 d; in extracts they were stable in the UPLC autosampler for over 48 h at 4°C. Furthermore, plasma concentrations of linezolid, PNU-142300 and PNU-142586 in patients treated with linezolid could be measured using the UPLC method developed in this study. This assay would be a powerful tool for therapeutic drug monitoring and clinical pharmacokinetic/pharmacodynamic (PK/PD) analyses in the optimization of linezolid treatment.

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.