Johannes Zander

Quantification of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in serum using an isotope dilution UHPLC-MS/MS method with semi-automated sample preparation

Abstract Background: Recent studies have demonstrated highly variable blood concentrations of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in critically ill patients with a high incidence of sub-therapeutic levels. Consequently, therapeutic drug monitoring (TDM) of these antibiotics has to be considered, requiring robust and reliable routine analytical methods. The aim of the present work was to develop and validate a multi-analyte ultra high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for the simultaneous quantification of the above mentioned antibiotics. Methods: Sample preparation included a manual protein precipitation step followed by two-dimensional ultra high performance liquid chromatography (2D-UHPLC). Corresponding stable isotope-labeled substances were used as internal standards for all of the analytes, with the exception of tazobactam. The injected sample volume was 7 μL. The run time was 5.0 min. Results: Inaccuracy was ≤8% and imprecision coefficient of variation (CV) was <9% for all analytes. Only minor matrix effects and negligible carry-over was observed. The method was found to be robust during the validation period. Conclusions: We were able to develop a reliable 2D-UHPLC-MS/MS method addressing analytes with highly heterogeneous physico-chemical properties. The novel assay may be an efficient tool for an optimized process workflow in clinical laboratories for important antibiotics in regards to TDM.

Effect of biobanking conditions on short-term stability of biomarkers in human serum and plasma.

Abstract Background: Liquid biobanking is an important tool for laboratory diagnostics in routine settings and clinical studies. However, the current knowledge about adequate storage conditions for different classes of biomarkers is incomplete and, in part, contradictory. Here, we performed a comprehensive study on the effects of different storage conditions on the stability of various biomarkers in human serum and plasma. Methods: Serum and citrated plasma were aliquoted and stored at 4 °C, –20 °C, –80 °C, and <–130 °C for 0, 7, 30, and 90 days, respectively (5–10 pools/condition). Additionally, frozen aliquots were temporarily exposed to higher temperatures during storage to simulate removing individual samples. Stability was tested for 32 biomarkers from 10 different parameter classes (electrolytes, enzymes, metabolites, inert proteins, complement factors, ketone bodies, hormones, cytokines, coagulation factors, and sterols). Results: Biobanking at –80 °C and <–130 °C for up to 90 days did not lead to substantial changes (defined as >3 interassay coefficients of variation and p<0.01) of any biomarker concentration. In contrast, storage at 4 °C and –20 °C induced substantial changes in single biomarker concentrations in most classes. Such substantial changes were increases (<20%) in electrolytes, metabolites, and proteins, and decreases (<96%) in enzymes, ketone bodies, cytokines, and coagulation factors. Biomarker stability was minimally affected by occasional short-term thermal exposure. Conclusions: Based on these results, we provide recommendations for storage conditions of up to 90 days for several biomarkers. Generally, storage at ≤–80 °C for at least 90 days including occasional short-term thermal exposure is an excellent storage condition for most biomarkers.

Quantification of vancomycin in human serum by LC-MS/MS.

Abstract Background: The aim of our work was to develop and validate a reliable LC-MS/MS-based measurement procedure for the quantification of vancomycin in serum, to be applied in the context of efforts to standardize and harmonize therapeutic drug monitoring of this compound using routine assays. Methods: Sample preparation was based on protein precipitation followed by ultrafiltration. In order to minimize differential modulation of ionization by matrix constituents extended chromatographic separation was applied leading to a retention time of 9.8 min for the analyte. Measurement was done by HPLC-ESI-MS/MS. For internal standardization the derivative vancomycin-glycin (ISTD) prepared by chemical synthesis was used, HPLC conditions ensured coelution of ISTD with the analyte. Results: In a bi-center validation total CVs of <4% were observed for quality control material ranging from 5.3 mg/L to 79.4 mg/L; accuracy was ±4%. No relevant ion suppression was observed. Comparative measurement of aliquots from 70 samples at the two validation sites demonstrated close agreement. Conclusions: Employing a closely related homologue molecule for internal standardization and the use of MS/MS following highly efficient sample pre-fractionation by HPLC, the method described here can be considered to offer the highest level of analytical reliability realized so far for the quantification of vancomycin in human serum. Thus, the method is suitable to be used in a comprehensive reference measurement system for vancomycin.

Comparison of five automated hematology analyzers in a university hospital setting: Abbott Cell-Dyn Sapphire, Beckman Coulter DxH 800, Siemens Advia 2120i, Sysmex XE-5000, and Sysmex XN-2000.

Abstract Background: Various types of automated hematology analyzers are used in clinical laboratories. Here, we performed a side-by-side comparison of five current top of the range routine hematology analyzers in the setting of a university hospital central laboratory. Methods: Complete blood counts (CBC), differentials, reticulocyte and nucleated red blood cell (NRBC) counts of 349 patient samples, randomly taken out of routine diagnostics, were analyzed with Cell-Dyn Sapphire (Abbott), DxH 800 (Beckman Coulter), Advia 2120i (Siemens), XE-5000 and XN-2000 (Sysmex). Inter-instrument comparison of CBCs including reticulocyte and NRBC counts and investigation of flagging quality in relation to microscopy were performed with the complete set of samples. Inter-instrument comparison of five-part differential was performed using samples without atypical cells in blood smear (n=292). Automated five-part differentials and NRBCs were additionally compared with microscopy. Results: The five analyzers showed a good concordance for basic blood count parameters. Correlations between instruments were less well for reticulocyte counts, NRBCs, and differentials. The poorest concordance for NRBCs with microscopy was observed for Advia 2120i (Kendall’s τb=0.37). The highest flagging sensitivity for blasts was observed for XN-2000 (97% compared to 65%–76% for other analyzers), whereas overall specificity was comparable between different instruments. Conclusions: To the best of our knowledge, this is the most comprehensive side-by-side comparison of five current top of the range routine hematology analyzers. Variable analyzer quality and parameter specific limitations must be considered in defining laboratory algorithms in clinical practice.

Effects of biobanking conditions on six antibiotic substances in human serum assessed by a novel evaluation protocol

Abstract Background: Although therapeutic drug monitoring (TDM) for antibiotics in critically ill patients is recommended by expert panels, no commercial tests are available for most antibiotics. Therefore, we previously developed a multi-analyte method for the quantification of piperacillin, tazobactam, cefepime, meropenem, ciprofloxacin and linezolid in serum. However, limited stability data were available, and the relevant studies did not address the coefficients of variation of the methods applied, which may be important for verifying the storage dependency of the observed effects. Here we aimed to evaluate the storage effects of antibiotics by applying a novel evaluation protocol. Methods: Serum-based test samples were aliquoted and stored at room temperature, 4 °C, −20 °C or −80 °C for up to 180 days. Using an innovative evaluation protocol (considering the coefficient of variation, p-value, and criterion of monotony of observed changes), we assessed whether relevant changes (defined as ≥15% in comparison with baseline) were storage dependent (defined as substantial changes). Results: Storage at −80 °C for up to 180 days did not lead to substantial changes for any analyte. In contrast, storage at −20 °C induced substantial decreases after ≥7 days for piperacillin, tazobactam, cefepime and meropenem; after 90 days at −20 °C, only ≤23% of the initial concentrations were found for these parameters. No substantial changes were observed for linezolid and ciprofloxacin at any storage condition. All of the observed substantial changes were monotonic decreases. Conclusions: We recommend a storage temperature of −80 °C for β-lactam antibiotics. The applied evaluation protocol yielded conclusive results and may be generally useful for stability studies.

Quantification of linezolid in serum by LC-MS/MS using semi-automated sample preparation and isotope dilution internal standardization

Abstract Background: Linezolid serum concentrations have been shown to be highly variable in critically ill patients with often sub-therapeutic drug levels regarding minimal inhibitory concentrations for relevant pathogens. Consequently, therapeutic drug monitoring of linezolid must be considered, requiring a reliable and convenient analytical method. We therefore developed and validated an LC-MS/MS method applying isotope dilution internal standardization and on-line solid phase extraction for serum linezolid quantification. Methods: Sample preparation was based on protein precipitation and on-line solid phase extraction with two-dimensional liquid chromatography and column switching. Three-fold deuterated linezolid was used as the internal standard. The method was validated involving two separate LC-MS/MS systems covering the concentration range of 0.13–32 mg/L. The run time was 4 min. Results: Validation revealed good analytical performance, with inaccuracy <6% and imprecision of <7.3% (CV) for six quality control samples (0.38–16.0 mg/L). The method was found to be robust during the validation process and during a pharmacokinetic study so far involving 600 samples. Comparative measurements on two LC-MS/MS systems revealed close agreement. Conclusions: This LC-MS/MS assay described herein is a convenient, robust and reliable method for linezolid quantification in serum which can be routinely applied using different LC-MS/MS systems. The method can be used for clinical studies and subsequent TDM of linezolid.

Predictors of Inadequate Linezolid Concentrations after Standard Dosing in Critically Ill Patients

ABSTRACT Adequate linezolid blood concentrations have been shown to be associated with an improved clinical outcome. Our goal was to assess new predictors of inadequate linezolid concentrations often observed in critically ill patients. Fifty-two critically ill patients with severe infections receiving standard dosing of linezolid participated in this prospective observational study. Serum samples (median, 32 per patient) were taken on four consecutive days, and total linezolid concentrations were quantified. Covariates influencing linezolid pharmacokinetics were identified by multivariate analysis and a population pharmacokinetic model. Target attainment (area under the concentration-time curve over 12 h [AUC12]/MIC ratio of >50; MIC = 2 mg/liter) was calculated for both the study patients and a simulated independent patient group (n = 67,000). Target attainment was observed for only 36% of the population on both days 1 and 4. Independent covariates related to significant decreases of linezolid concentrations included higher weight, creatinine clearance rates, and fibrinogen and antithrombin concentrations, lower concentrations of lactate, and the presence of acute respiratory distress syndrome (ARDS). Linezolid clearance was increased in ARDS patients (by 82%) and in patients with elevated fibrinogen or decreased lactate concentrations. In simulated patients, most covariates, including fibrinogen and lactate concentrations and weight, showed quantitatively minor effects on target attainment (difference of ≤9% between the first and fourth quartiles of the respective parameters). In contrast, the presence of ARDS had the strongest influence, with only ≤6% of simulated patients reaching this target. In conclusion, the presence of ARDS was identified as a new and strong predictor of insufficient linezolid concentrations, which might cause treatment failure. Insufficient concentrations might also be a major problem in patients with combined alterations of other covariate parameters. (This study has been registered at ClinicalTrials.gov under registration number NCT01793012.)

Optimization of linezolid therapy in the critically ill: the effect of adjusted infusion regimens

Objectives Insufficient linezolid levels, which are associated with a poorer outcome, are often observed in ICU patients who receive standard dosing. Although strategies to overcome these insufficient levels have been discussed, appropriate alternative dosing regimens remain to be identified. Methods Various infusion regimens (1200-3600 mg/day; q6h, q8h, q12h and continuous) were simulated in 67 000 ICU patients. The probability of attaining pharmacodynamic targets ( T >MIC ≥85%, AUC/MIC ≥100, cumulative fraction of response for Staphylococcus aureus and Enterococcus spp., PTA for an MIC of 0.5-4 mg/L) as well as the avoidance of toxic concentrations and concentrations constantly below the MIC (lack of antibiotic effect) or inside a mutant selection window (resistance development) were evaluated. Results Best target attainment according to T >MIC was observed for continuous infusions, followed by q6h, q8h and q12h. A substantially reduced target attainment was observed in patients with acute respiratory distress syndrome (ARDS). In patients without ARDS, 1200 mg/day was insufficient irrespective of the regimen, while a dose of 1400 mg/day administered q6h or by continuous infusions provided an acceptable target attainment (e.g. cumulative fraction of response with regards to T >MIC  ≥93%). Higher rates of potentially toxic trough concentrations (28% versus 12%) and concentrations constantly inside the mutant selection window (15% versus <0.1%) were observed with continuous infusions compared with q6h infusions (1400 mg/day, patients without ARDS). Conclusions Irrespective of the regimen, 1200 mg/day linezolid might be insufficient for the treatment of ICU patients. Patients without ARDS might particularly benefit from q6h infusions with increased daily doses (e.g. 1400 mg/day).

An LC-MS/MS based candidate reference method for the quantification of total gentamicin in human serum and plasma using NMR characterized calibrator material

BACKGROUND Accurate measurement of gentamicin concentration in serum and plasma is required for therapeutic drug monitoring to ensure appropriate treatment of patients. In this work, we present a validated LC-MS/MS-based candidate reference measurement procedure for total gentamicin quantification to be used for standardization and harmonization of routine assays applied for therapeutic drug monitoring of this compound. Total gentamicin is the sum of the concentrations of five known congeners C1, C1a, C2, C2a and C2b. To our knowledge, there is so far no LC-MS method for quantification of total gentamicin in human serum described in literature. METHODS Sample preparation was based on sample dilution with an aqueous internal standard solution followed by protein precipitation. Stable derivatives of gentamicin-glycine congeners were prepared by chemical synthesis and used as internal standards. The primary calibration material used in this assay was characterized by NMR spectroscopy and the pattern of the gentamicin congeners was determined. The total gentamicin was reported as the sum of the congeners which were quantified individually by LC-MS/MS. RESULTS The method allows the measurement of total gentamicin in human serum and plasma in the concentration range of 0.1 to 12.0μg/ml with an assay imprecision of ≤6% CV and an assay accuracy between 96% and 114%. LOD and LOQ for the total gentamicin were 0.04μg/ml and 0.13μg/ml, respectively. Comparative measurement of 128 native patient samples using this method implemented at two laboratory sites showed an excellent agreement. CONCLUSIONS Validation results proved that this protocol describes a robust and reliable method which is suggested as reference measurement procedure for the standardization and harmonization of routine assays for the quantification of total gentamicin.

Variability of piperacillin concentrations in relation to tazobactam concentrations in critically ill patients

Therapeutic drug monitoring for critically ill patients receiving piperacillin/tazobactam is described as a useful tool. However, the minimum inhibitory concentration of piperacillin depends on a sufficiently high concentration of tazobactam in case of β-lactamase-producing strains. Therefore, the relationship between piperacillin and tazobactam concentrations was assessed in a heterogeneous group of critically ill patients. Sixty patients with severe infections receiving 4.5 g of piperacillin/tazobactam 2-3 times daily by intermittent infusion were included in this prospective observational study (NCT01793012). Over 4 days, multiple serum samples were obtained to determine the total piperacillin and tazobactam concentrations. The target ranges were defined as trough levels >16 mg/L (>22.5 mg/L) and >4 mg/L (>5.7 mg/L) for the calculated unbound concentrations (measured total concentrations) of piperacillin and tazobactam, respectively. Despite a high correlation coefficient (r = 0.93) comparing piperacillin and tazobactam trough levels, the piperacillin/tazobactam quotients varied between ca. 1 and 10. From linear regression analysis of piperacillin versus tazobactam values, it follows that a piperacillin trough level of 22.5 mg/L might be associated with tazobactam trough levels ranging from 1.5 mg/L to 10.1 mg/L. A 70 mg/L threshold for total piperacillin trough levels would be necessary to ensure that tazobactam concentrations are >5.7 mg/L. Because of the observed variability of piperacillin/tazobactam quotients, defining the total piperacillin target range ≥70 mg/L might be useful to ensure that tazobactam concentrations do not fall below 5.7 mg/L. Further studies are necessary to confirm that the used therapeutic ranges are associated with optimal outcomes in critically ill patients.