Target Site Pharmacokinetics of Meropenem: Measurement in Human Explanted Lung Tissue by Bronchoalveolar Lavage, Microdialysis, and Homogenized Lung Tissue.

Pneumonia is one of the most common infections in intensive care patients, and it is often treated with beta-lactam antibiotics. Even if therapeutic drug monitoring in blood is available, it is unclear whether sufficient concentrations are reached at the target site: the lung. The present study was initiated to fill this knowledge gap. Various compartments from 10 patients' explanted lungs were subjected to laboratory analysis. Meropenem was quantified in serum, bronchoalveolar lavage (BAL) fluid, microdialysate, and homogenized lung tissue with isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS). BAL fluid represents diluted epithelial lining fluid (ELF), and microdialysate represents interstitial fluid (IF). Differences between target site and blood concentrations were investigated. The median meropenem concentration in blood, ELF, IF, and tissue were 26.8, 18.0, 12.1, and 9.1 mg/liter, respectively. A total of 37.5% of the target site ELF and IF meropenem concentrations were below the clinical EUCAST breakpoint of 8 mg/liter. The median ELF/serum quotient was 61.8% (interquartile range [IQR], 24.8% to 87.6%), the median IF/serum quotient was 35.4% (IQR, 23.8% to 54.3%), and the median tissue/serum quotient was 34.2% (IQR, 28.3% to 38.2%). We observed a substantial interindividual variability between the blood and the compartments (ELF and IF), whereas the intraindividual variability was relatively low. Target site measurement in different lung compartments was feasible and successfully applied in a clinical setting. A relevant amount of 37.5% of the target site concentrations were below the clinical EUCAST breakpoint, indicating subtherapeutic dosing in high-risk patients receiving perioperative antibiotic prophylaxis in lung transplantation. (This study has been registered at ClinicalTrials.gov under identifier NCT03970265.).

An isotope-dilution LC-MS/MS method for the simultaneous quantification of meropenem and its open-ring metabolite in serum.

BACKGROUND: Therapeutic drug monitoring (TDM) of beta-lactam antibiotics and, among them, especially meropenem gains importance in the field of laboratory medicine. Meropenem is known to be unstable, resulting in a degradation product with an open beta-lactam ring. For a more comprehensive TDM of meropenem, the aim was to develop a LC-MS/MS method for the simultaneous quantification of meropenem and its main degradation product, the open-ring metabolite (ORM). METHODS: The method involves a protein precipitation followed by chromatographic separation using a formic acid-ammonium formate methanol gradient on a pentafluorophenyl column. Multiple reaction monitoring in the positive ion mode and stable isotope labeled internal standards were used for quantification. Validation was performed according to the European Medicines Agency guideline. RESULTS: Validation was successful performed within the linear drug concentration range of 1.0-100.0 mg/l for meropenem and 0.62-62.30 mg/l for the ORM. Investigation of selectivity, accuracy and precision showed good results and potential matrix effects were successfully compensated by the internal standards. The suitability of the method was shown by the comparison of 35 anonymized leftover serum samples from intensive care patients with routine analyses. CONCLUSION: For the first time, we herein describe a liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of meropenem and its ORM in human serum. The ratio of active to inactive compound provides valuable pharmaceutical and pharmacokinetic information, which may contribute to therapeutic efficacy.

Effect of gravimetric correction and type of pipettes used in sample preparation on the precision of LC-MS/MS-based analyses.

BACKGROUND: Currently, manual pipetting of human sample material is still a key process in sample preparation for chromatographic and mass spectrometric analyses in the clinical laboratory. In most cases, however, pipettes used in clinical laboratories are only specified for handling water-like liquids. Actual pipetted liquid volumes can be verified by weighing within the sample preparation process, and the results can be corrected accordingly (gravimetric correction). The aim of our study was to evaluate and compare the effects of gravimetric correction in terms of accuracy and precision for an air cushion and direct replacement pipette. METHODS: Forty-fold serial determination of linezolid in a spiked serum sample by ID-LC-MS/MS was applied as an exemplary measurement procedure. Polypropylene tubes were weighed before the addition of 50 µL serum, after the addition, and after the addition of the internal standard solution. Coefficients of variation (CV) were calculated as an indicator of measurement precision. RESULTS: The use of a direct replacement pipette was associated with improved measurement imprecision than an air cushion pipette (CV 1.70% vs 2.49% for serum, uncorrected results). The results obtained after gravimetric correction showed improved precision with the use of an air cushion pipette compared to the conventional approach (CV 1.51% vs 1.61%). Using a direct replacement pipette, the impact of gravimetric correction on imprecision was negligible. CONCLUSION: Using direct replacement pipettes in sample preparation enables more precise ID-LC-MS/MS analyses than using air cushion pipettes. Gravimetric correction can be a useful tool to improve the precision of LC-MS/MS measurement procedures when complex matrices such as human serum are handled with commonly used air cushion pipettes.

Averaging of results derived from different, simultaneously acquired mass transitions in ID-LC-MS/MS - Potential impact on measurement imprecision.

BACKGROUND: LC-MS/MS allows for many measurands monitoring different mass transitions simultaneously. So far, such alternative mass transitions are usually assessed as "quantifier and qualifier ions" in order to rule out interferences in individual samples. However, quantification can also be based on assessment of alternative mass transitions for both the measurand and its internal standard, with two distinct results for one injection of an individual sample. These paired results can be averaged. The aim of this study was to determine the potential impact of this averaging approach on measurement imprecision. METHODS: We studied the impact of averaging results from different transitions for four exemplary measurands (linezolid (LIN), piperacillin (PIP), voriconazole (VOR), ethylglucuronide (ETG)). Intra-batch studies were performed with 21 injections of single clinical samples in sequence for each analyte (LIN, PIP, VOR), and a between-batch study with evaluation of data from routine QC samples from 20 series over 20 weeks (ETG). CVs and their confidence intervals were assessed comparatively for quantification based on single transitions, and for averaged results from these two transitions, respectively. RESULTS: In all data sets, we observed lower CVs for the averaged results compared to the results obtained from single mass transitions. CVs from averaged results were up to 39.4% lower compared to the CVs observed for results obtained from single transitions for the respective measurands. CONCLUSION: Averaging of quantification results obtained from separate mass transitions acquired simultaneously in ID-LC-MS/MS seems to have the potential to minimize the measurement imprecision for different measurands in short- and long-term settings.

Collision energy-breakdown curves - An additional tool to characterize MS/MS methods.

BACKGROUND: In tandem mass spectrometry, analyte detection is based on collision-induced fragmentation, which is modulated by the collision energy (CE) setting. Variation in CE leads to differential ion yield, and optimization is usually performed empirically as "tuning" during method development. Our aim was to build a method to objectify the impact of collision energy settings on ion yield for individual compounds. METHODS: Collision energy (CE)-breakdown curves were generated based on acquisition files in which a large number of quasi-identical mass transitions were recorded simultaneously, with variation of CE over a defined range within a single injection. Ion yield (normalized to an internal standard recorded with a locked collision energy) was plotted as a curve versus CE settings. Piperacillin and testosterone were studied as exemplary analytes in matrix-free and serum matrix-based liquid chromatography tandem mass spectrometry (LC-MS/MS) measurements. More detailed testosterone CE-breakdown curves were investigated with regard to sample preparation techniques and the isotope labeling pattern of corresponding internal standards. RESULTS: CE-breakdown curves were found characteristically for the piperacillin quantifier transition with respect to CE-related maximum ion yield, as well as curve width and shape. A diverging curve profile was observed for the piperacillin qualifier transition. For testosterone analyses, no impact from different sample preparation techniques or the isotope labeling patterns on the selected CE was shown. CONCLUSION: CE-breakdown curves are a convenient and valuable tool to verify LC-MS/MS methods regarding consistent fragmentation characteristics between sample sources or native analytes and isotope-labeled counterparts.

An isotope dilution LC-MS/MS based candidate reference method for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood.

An isotope dilution LC-MS/MS based candidate reference measurement procedure for the quantification of cyclosporine A, tacrolimus, sirolimus and everolimus in human whole blood is presented to be used for evaluation and standardization of routine assays applied for therapeutic drug monitoring. The assay allows baseline separation of the four immunosuppressive drugs within a total runtime of 9 minutes using a C4 reversed phase column. Sample preparation is based on protein precipitation with zinc sulphate followed by purification with solid phase extraction. Reference materials used in this reference measurement procedure were characterized by qNMR and an absolute content of analytes calculated to guarantee traceability to SI units. As internal standards the corresponding deuterated and 13C-labelled analytes were used. The method allows the measurement of cyclosporine A in the range of 5 ng/mL to 2100 ng/mL; tacrolimus, sirolimus and everolimus were analysed in the range of 0.25 ng/mL to 50 ng/mL. Imprecision for inter-day measurements were found to be ≤3.5% for cyclosporine A and ≤4.4% for tacrolimus, sirolimus and everolimus. Accuracy was found to be within 101% and 108% for cyclosporine A and between 95% and 104% for the macrolide compounds. The uncertainty was evaluated according to the GUM. Expanded measurement uncertainties were found to be ≤7.2% for cyclosporine A, ≤6.8% for tacrolimus, ≤9.0% for sirolimus and ≤8.9% for everolimus (k = 2).

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.

Isotope dilution LC-orbitrap-HRMS with automated sample preparation for the simultaneous quantification of 11 antimycotics in human serum.

INTRODUCTION: The aim of this project was to develop and validate an isotope-dilution liquid chromatography high resolution mass spectrometry (LC-HRMS) method for the quantification of the 11 most widely used systemic antimycotics and to study whether HRMS is a feasible alternative for therapeutic drug monitoring (TDM) when compared to tandem MS (MS/MS) technology. METHODS: After protein precipitation, followed by automated online sample clean-up the analytes were separated within 4 min on a C18 column using an acetonitrile-water gradient. Eleven antimycotics, namely 5-flucytosine, amphotericin B, anidulafungin, fluconazole, isavuconazole, itraconazole, ketoconazole, micafungin, OH-itraconazole, posaconazole and voriconazole were finally quantified in full MS scan mode using positive electrospray ionization (ESI +) with a mass range fromm/z 110-1300 using HRMS. The method was comprehensively validated on the basis of the European Medicines Agengy (EMA) method validation protocol using commercially available IVD kit components. RESULTS: Good linear relationship between peak area responses and drug concentrations (R2 > 0.995) and excellent selectivity were observed for all antimycotics in this study. Inaccuracy and imprecision of all quality controls were consistently below ± 12.6% and ± 8.1%, respectively. Quantification results were in agreement with an IVD LC-MS/MS method. CONCLUSION: HRMS was shown to be suitable for TDM of small molecules when compared to tandem mass spectrometry. The novel HRMS method is quickly installed and may be a robust and reliable tool for routine TDM of antimycotics in clinical laboratories.

Multiplex Therapeutic Drug Monitoring by Isotope-dilution HPLC-MS/MS of Antibiotics in Critical Illnesses.

There is an ever-increasing demand for the therapeutic drug monitoring of antibiotics in many clinical facilities, particularly with regard to the implementation of hospital antibiotic stewardship programs. In the current work, we present a multiplex high-performance liquid chromatography-tandem mass spectrometry (HPCL-MS/MS) protocol for the quantification of cefepime, meropenem, ciprofloxacin, moxifloxacin, linezolid, and piperacillin, commonly used antibiotics in intensive care units. The method was previously comprehensively validated according to the guideline of the European Medicines Agency. After a rapid sample cleanup, the analytes are separated on a C8 reverse-phase HPLC column within 4 minutes and quantified with the corresponding stable isotope-labeled internal standards in electrospray ionization (ESI+) mass spectrometry in multiple reaction time monitoring (MRM). The presented method uses a simple instrumentation setting with uniform chromatographic conditions, allowing for the daily and robust antibiotic therapeutic drug monitoring in clinical laboratories. The calibration curve spans the pharmacokinetic concentration range, thereby including antibiotic amounts close to the minimal inhibitory concentration (MIC) of susceptible bacteria and peak concentrations (Cmax) that are obtained with bolus administration regimens. Without the necessity of the serum dilution before the sample cleanup, the area under the curve for an administered antibiotic can be obtained through multiple measurements.

Chlorinated cobalt alkyne complexes derived from acetylsalicylic acid as new specific antitumor agents.

[(Prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS), an organometallic derivative of the irreversible cyclooxygenase-1/2 (COX-1/2) inhibitor acetylsalicylic acid (ASS), demonstrated high growth-inhibitory potential against various tumor cell lines and inhibition of both COX isoenzymes. With the objective of increasing the selectivity for COX-2, we introduced a chlorine substituent in position 3, 4, 5, or 6 of the ASS moiety, respectively. Increased COX-2 selectivity is desirable as this isoenzyme is predominantly related to the development of cancer and abnormal tissue growth. The new compounds were investigated in comprehensive cellular biological assays to identify the impact of the chlorine substitution at the complex on COX-1/2 inhibition, antiproliferative activity, apoptosis, metabolic activity, cell-based COX inhibition, and cellular uptake. Chlorination distinctly reduced the effects at isolated COX-1 (about 25% inhibition at 10 µM; Co-ASS: 82.7%), while those at COX-2 remained almost unchanged (about 65% inhibition at 10 µM; Co-ASS: 78.5%). In cellular systems, with exception of the 6-Cl derivative, all compounds showed notable antitumor activity in COX-1/2 expressing tumor cells (HT-29 (IC50 = 1.5-2.7 µM), MDA-MB-231 (IC50 = 5.2-8.0 µM)), but were distinctly less active in the COX-1/2-negative MCF-7 breast cancer cell line (IC50 = 15.2-22.9 µM). All complexes possess high selectivity for tumor cells, because they did not influence the growth of the non-tumorigenic, human bone marrow stromal cell line HS-5. These findings clearly demonstrate that the interference with the COX-1/2 cascade contributes to the mode of anticancer action of the cobalt alkyne complexes.