Meropenem pharmacokinetics in cerebrospinal fluid: comparing intermittent and continuous infusion strategies in critically ill patients-a prospective cohort study.

Meropenem penetration into the cerebrospinal fluid (CSF) is subject to high interindividual variability resulting in uncertain target attainment in CSF. Recently, several authors recommended administering meropenem as a continuous infusion (CI) to optimize CSF exposure. This study aimed to compare the concentrations and pharmacokinetics of meropenem in CSF after intermittent infusion (II) and CI. This prospective, observational study (NCT04426383) included critically ill patients with external ventricular drains who received either II or CI of meropenem. Meropenem pharmacokinetics in plasma and CSF were characterized using population pharmacokinetic modeling (NONMEM 7.5). The developed model was used to compare the concentration-time profile and probability of target attainment (PTA) between II and CI. A total of 16 patients (8 CI, 8 II; samples: nplasma = 243, nCSF = 263) were recruited, with nine patients (5 CI, 4 II) suffering from cerebral and seven patients from extracerebral infections. A one-compartment model described the plasma concentrations adequately. Meropenem penetration into the CSF (partition coefficient (KP), cCSF/cplasma) was generally low (6.0%), exhibiting substantial between-subject variability (coefficient of variation: 84.0%). There was no correlation between the infusion mode and KP, but interleukin (IL)-6 measured in CSF showed a strong positive correlation with KP (P < 0.001). Dosing simulations revealed no relevant differences in CSF concentrations and PTA in CSF between CI and II. Our study did not demonstrate increased penetration rates or higher concentrations of meropenem in the CSF with CI compared with II. CLINICAL TRIALS: This study is registered with ClinicalTrials.gov as NCT04426383.

Report from the HarmoSter study: different LC-MS/MS androstenedione, DHEAS and testosterone methods compare well; however, unifying calibration is a double-edged sword.

OBJECTIVES: Current liquid chromatography-tandem mass spectrometry (LC-MS/MS) applications for circulating androgen measurements are technically diverse. Previously, variable results have been reported for testosterone. Data are scarce for androstenedione and absent for dehydroepiandrosterone sulfate (DHEAS). We assessed the agreement of androstenedione, DHEAS and testosterone LC-MS/MS measurements among nine European centers and explored benefits of calibration system unification. METHODS: Androgens were measured twice by laboratory-specific procedures in 78 patient samples and in EQA materials. Results were obtained by in-house and external calibration. Intra- and inter-laboratory performances were valued. RESULTS: Intra-laboratory CVs ranged between 4.2-13.2 % for androstenedione, 1.6-10.8 % for DHEAS, and 4.3-8.7 % and 2.6-7.1 % for female and male testosterone, respectively. Bias and trueness in EQA materials were within ±20 %. Median inter-laboratory CV with in-house vs. external calibration were 12.0 vs. 9.6 % for androstenedione (p<0.001), 7.2 vs. 4.9 % for DHEAS (p<0.001), 6.4 vs. 7.6 % for female testosterone (p<0.001) and 6.8 and 7.4 % for male testosterone (p=0.111). Median bias vs. all laboratory median with in-house and external calibration were -13.3 to 20.5 % and -4.9 to 18.7 % for androstenedione, -10.9 to 4.8 % and -3.4 to 3.5 % for DHEAS, -2.7 to 6.5 % and -11.3 to 6.6 % for testosterone in females, and -7.0 to 8.5 % and -7.5 to 11.8 % for testosterone in males, respectively. CONCLUSIONS: Methods showed high intra-laboratory precision but variable bias and trueness. Inter-laboratory agreement was remarkably good. Calibration system unification improved agreement in androstenedione and DHEAS, but not in testosterone measurements. Multiple components, such as commutability of calibrators and EQA materials and internal standard choices, likely contribute to inter-laboratory variability.

Standardization via Post Column Infusion-A Novel and Convenient Quantification Approach for LC-MS/MS.

Mass spectrometry (MS) is a widely used analytical technique including medical diagnostics, forensic toxicology, food and water analysis. The gold standard for quantifying compounds involves using stable isotope-labeled internal standards (SIL-IS). However, when these standards are not commercially available, are prohibitively expensive, or are extremely difficult to synthesize, alternative external quantification techniques are employed. We hereby present a novel, convenient and cheap quantification approach-quantification via post column infusion (PCI). As a proof of concept, we demonstrated PCI quantification for the immunosuppressant tacrolimus in whole blood using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The validation results met the criteria according to the guideline on bioanalytical method validation of the European Medicine Agency (EMA), achieving imprecisions and inaccuracies with coefficient of variation and relative bias below 15%. Anonymized and leftover whole blood samples from immunosuppressed patients receiving tacrolimus were used for method comparison (PCI quantification vs. conventional internal standard (IS) quantification). Both methods showed strong agreement with a Pearson correlation coefficient of r = 0.9532. This novel PCI quantification technique (using the target analyte itself) expands the quantification options available in MS, providing reliable results, particularly when internal standards are unavailable or unaffordable. With the current paper, we aim to demonstrate that our innovative PCI technique has great potential to overcome practical issues in quantification and to provide guidance on how to incorporate PCI in existing or new LC-MS methods. Moreover, this study demonstrated a more convenient method for correcting matrix effects in comparison to alternative PCI techniques.

From research cohorts to the patient - a role for "omics" in diagnostics and laboratory medicine?

Human pathologies are complex and might benefit from a more holistic diagnostic approach than currently practiced. Omics is a concept in biological research that aims to comprehensively characterize and quantify large numbers of biological molecules in complex samples, e.g., proteins (proteomics), low molecular weight molecules (metabolomics), glycans (glycomics) or amphiphilic molecules (lipidomics). Over the past decades, respective unbiased discovery approaches have been intensively applied to investigate functional physiological and pathophysiological relationships in various research study cohorts. In the context of clinical diagnostics, omics approaches seem to have potential in two main areas: (i) biomarker discovery i.e. identification of individual marker analytes for subsequent translation into diagnostics (as classical target analyses with conventional laboratory techniques), and (ii) the readout of complex, higher-dimensional signatures of diagnostic samples, in particular by means of spectrometric techniques in combination with biomathematical approaches of pattern recognition and artificial intelligence for diagnostic classification. Resulting diagnostic methods could potentially represent a disruptive paradigm shift away from current one-dimensional (i.e., single analyte marker based) laboratory diagnostics. The underlying hypothesis of omics approaches for diagnostics is that complex, multigenic pathologies can be more accurately diagnosed via the readout of "omics-type signatures" than with the current one-dimensional single marker diagnostic procedures. While this is indeed promising, one must realize that the clinical translation of high-dimensional analytical procedures into routine diagnostics brings completely new challenges with respect to long-term reproducibility and analytical standardization, data management, and quality assurance. In this article, the conceivable opportunities and challenges of omics-based laboratory diagnostics are discussed.

Report from the HarmoSter study: inter-laboratory comparison of LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) panels that include glucocorticoid-related steroids are increasingly used to characterize and diagnose adrenal cortical diseases. Limited information is currently available about reproducibility of these measurements among laboratories. The aim of the study was to compare LC-MS/MS measurements of corticosterone, 11-deoxycortisol and cortisone at eight European centers and assess the performance after unification of calibration. METHODS: Seventy-eight patient samples and commercial calibrators were measured twice by laboratory-specific procedures. Results were obtained according to in-house and external calibration. We evaluated intra-laboratory and inter-laboratory imprecision, regression and agreement against performance specifications derived from 11-deoxycortisol biological variation. RESULTS: Intra-laboratory CVs ranged between 3.3 and 7.7%, 3.3 and 11.8% and 2.7 and 12.8% for corticosterone, 11-deoxycortisol and cortisone, with 1, 4 and 3 laboratories often exceeding the maximum allowable imprecision (MAI), respectively. Median inter-laboratory CVs were 10.0, 10.7 and 6.2%, with 38.5, 50.7 and 2.6% cases exceeding the MAI for corticosterone, 11-deoxycortisol and cortisone, respectively. Median laboratory bias vs. all laboratory-medians ranged from -5.6 to 12.3% for corticosterone, -14.6 to 12.4% for 11-deoxycortisol and -4.0 to 6.5% for cortisone, with few cases exceeding the total allowable error. Modest deviations were found in regression equations among most laboratories. External calibration did not improve 11-deoxycortisol and worsened corticosterone and cortisone inter-laboratory comparability. CONCLUSIONS: Method imprecision was variable. Inter-laboratory performance was reasonably good. However, cases with imprecision and total error above the acceptable limits were apparent for corticosterone and 11-deoxycortisol. Variability did not depend on calibration but apparently on imprecision, accuracy and specificity of individual methods. Tools for improving selectivity and accuracy are required to improve harmonization.

Rules for mass spectrometry applications in the clinical laboratory.

MS-based analytical methods now play an important role in medical laboratory analysis. Predominantly triple-stage mass spectrometry is used for the quantification of small molecule biomarkers and xenobiotics in blood and urine. The spectrum of applications ranges from completely in-house developed analytical methods, to industrially manufactured kit solutions used on generic equipment, to the first closed MS-based analysis systems. It is to be expected that the weights will shift in the coming years. Thus, operation and evaluation for most applications will remain very challenging and very different from the far more user-friendly and fully automated systems - mainly photometry-based - which are commonly used in clinical laboratories. General regulatory requirements for medical analysis differ significantly between countries globally. General requirements for in-house developed assay methods are valid in some countries, but concrete and methodology-specific rules for operation and quantification when using MS methods in the medical diagnostic laboratory are not applied. This differs significantly from other bio-analytical areas such as food monitoring, pharmaceutical research, or forensics, where legally binding, detailed rules exist in some cases, e.g., for substance identification. Internationally used relevant and helpful general standards with regard to mass spectrometric examination procedures in the clinical laboratory are in particular CLSI 62A and ISO 15189, while the IVDR in the EU primarily regulates the manufacture of diagnostic articles and not their application. In addition, from many years of application experience, some general advice can be recommended as rules that can contribute to robustness and patient safety in the clinical application of MS procedures; with emphasis on: reasonable method description, batch release, competence management, maintenance, and continuity management. This article also proposes some procedural basic requirements for the application of MS procedures in the clinical laboratory.

Non-Invasive Zinc Protoporphyrin Screening Offers Opportunities for Secondary Prevention of Iron Deficiency in Blood Donors.

Background: Frequent blood donors are at high risk of developing iron deficiency. Currently, there is no potent screening during blood donation to detect iron deficient erythropoiesis (IDE) before anemia develops and deferral from donation is inevitable. Study Design and Methods: In addition to capillary and venous hemoglobin, the iron status of 99 frequent blood donors was assessed by various venous blood parameters and zinc protoporphyrin IX (ZnPP). ZnPP was determined by high-performance liquid chromatography (HPLC) and a new prototype fiber-optic device was employed for non-invasive measurements of ZnPP through the blood collection tubing (NI-tubing) and on lip tissue (NI-lip). We aimed to evaluate the feasibility and diagnostic value of the NI-tubing measurement for early detection of severe iron deficiency in blood donors. Results: NI-tubing and HPLC reference measurements of ZnPP showed narrow limits of agreement of 12.2 µmol ZnPP/mol heme and very high correlation (Spearman's Rho = 0.938). Using a cutoff of 65 µmol ZnPP/mol heme, NI-tubing measurements (n = 93) identified 100% of donors with iron deficiency anemia (IDA) and an additional 38% of donors with IDE. Accordingly, NI-tubing measurements would allow detection and selective protection of particularly vulnerable donors. Conclusion: NI-tubing measurements are an accurate and simple method to implement ZnPP determination into the routine blood donation process. ZnPP was able to identify the majority of subjects with IDE and IDA and might therefore be a valuable tool to provide qualified information to donors about dietary measures and adjustments of the donation interval and thereby help to prevent IDA and hemoglobin deferral in the future.

Targeted profiling of 24 sulfated and non-sulfated bile acids in urine using two-dimensional isotope dilution UHPLC-MS/MS.

OBJECTIVES: Bile acids serve as biomarkers for liver function and are indicators for cholestatic and hepatobiliary diseases like hepatitis, cirrhosis, and intrahepatic cholestasis of pregnancy (ICP). Sulfation and renal excretion of bile acids are important elimination steps. The power of ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) allows specific profiling of primary and secondary bile acids as well as their sulfated counterparts. METHODS: Twenty-four sulfated and non-sulfated primary and secondary bile acids were quantified in urine with 15 corresponding stable isotope labeled internal standards by using two-dimensional UHPLC-MS/MS. The sample preparation was based on a simple dilution with a methanolic zinc sulfate solution followed by an automated online solid phase extraction clean up. RESULTS: The validation results of the method fulfilled the criteria of the European Medicine Agency (EMA) "Guideline on bioanalytical method validation". To verify fitness for purpose, 40 urine samples were analyzed which showed an average of 86% sulfation, 9.1% taurine-conjugation, 14% non-conjugation, and 77% glycine-conjugation rates. CONCLUSIONS: Lossless one-pot sample preparation, automated sample purification, and high number of internal standards are major innovations of the presented profiling method, which may allow diagnostic application of BA profiling in the future.

Transport stability profiling - a proposed generic protocol.

OBJECTIVES: Diagnostic samples are exposed to a spectrum of variables during transport to laboratories; therefore, the evaluation of a rather comprehensive stability profile of measurands is warranted. While appropriate testing standards have been established for pharmaceuticals and reagents, this is not the case for diagnostic samples. The aim of our work was to develop and evaluate a protocol applicable to diagnostic samples. METHODS: An isochronous approach with representation of temperature and exposure duration in a two-dimensional matrix was established. The deviations of the measurement results from the baseline associated with the exposure are evaluated with respect to the measurement uncertainty of the analytical measurement procedure applied. Variables of the experiment are documented in a standardized matrix. As a proof-of-concept, we profiled the stability patterns of a number of measurands at four temperature levels over up to 72 h in primary serum sample tubes. RESULTS: The protocol proved to be workable and allowed the description of a comprehensive stability profile of a considerable number of compounds based on 21 small-volume primary samples collected from each volunteer and exposed according to this protocol. CONCLUSIONS: A straightforward and feasible isochronous protocol can be used to investigate in detail the effects of different pre-processing conditions on the stability of measurands in primary samples during transport to diagnostic laboratories. This is of significance as pre-analytical logistics become increasingly important with the centralization of analytical services.

Isotope dilution LC-MS/MS quantification of the cystic fibrosis transmembrane conductance regulator (CFTR) modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their major metabolites in human serum.

OBJECTIVES: Cystic fibrosis (CF) transmembrane conductance regulator (CFTR) modulators have revolutionized the therapeutic landscape in CF treatment. These vital drugs are extensively metabolized via CYP3A, so caution must be exercised in multimodal CF therapy because of the risk of adverse drug interactions. Our goal was to develop a highly sensitive assay for the purpose of therapeutic drug monitoring (TDM) in diagnostic laboratories. METHODS: After protein precipitation, the CFTR modulators ivacaftor, lumacaftor, tezacaftor, elexacaftor, and their metabolites ivacaftor-M1, ivacaftor-M6, and tezacaftor-M1 were separated with a two-dimensional chromatography setup within 5 min, and quantified with stable isotope-labeled internal standards. The method was validated according to the European Medicines Agency (EMA) guideline on bioanalytical method validation and applied to CF patient samples. RESULTS: Inaccuracy was ≤7.0% and the imprecision coefficient of variation (CV) was ≤8.3% for all quality controls (QCs). The method consistently compensated for matrix effects, recovery, and process efficiency were 105-115 and 96.5-103%, respectively. Analysis of CF serum samples provided concentrations comparable to the pharmacokinetic profile data reported in the EMA assessment report for the triple combination therapy Kaftrio. CONCLUSIONS: We hereby present a robust and highly selective isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) assay for the simultaneous quantification of the so far approved CFTR modulators and their metabolites in human serum. The assay is suitable for state-of-the-art pharmacovigilance of CFTR modulator therapy in CF patients, in order to maximize safety and efficacy, and also to establish dose-response relationships in clinical trials.

Report from the HarmoSter study: impact of calibration on comparability of LC-MS/MS measurement of circulating cortisol, 17OH-progesterone and aldosterone.

OBJECTIVES: Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is recommended for measuring circulating steroids. However, assays display technical heterogeneity. So far, reproducibility of corticosteroid LC-MS/MS measurements has received scant attention. The aim of the study was to compare LC-MS/MS measurements of cortisol, 17OH-progesterone and aldosterone from nine European centers and assess performance according to external quality assessment (EQA) materials and calibration. METHODS: Seventy-eight patient samples, EQA materials and two commercial calibration sets were measured twice by laboratory-specific procedures. Results were obtained by in-house (CAL1) and external calibrations (CAL2 and CAL3). We evaluated intra and inter-laboratory imprecision, correlation and agreement in patient samples, and trueness, bias and commutability in EQA materials. RESULTS: Using CAL1, intra-laboratory CVs ranged between 2.8-7.4%, 4.4-18.0% and 5.2-22.2%, for cortisol, 17OH-progesterone and aldosterone, respectively. Trueness and bias in EQA materials were mostly acceptable, however, inappropriate commutability and target value assignment were highlighted in some cases. CAL2 showed suboptimal accuracy. Median inter-laboratory CVs for cortisol, 17OH-progesterone and aldosterone were 4.9, 11.8 and 13.8% with CAL1 and 3.6, 10.3 and 8.6% with CAL3 (all p<0.001), respectively. Using CAL1, median bias vs. all laboratory-medians ranged from -6.6 to 6.9%, -17.2 to 7.8% and -12.0 to 16.8% for cortisol, 17OH-progesterone and aldosterone, respectively. Regression lines significantly deviated from the best fit for most laboratories. Using CAL3 improved cortisol and 17OH-progesterone between-method bias and correlation. CONCLUSIONS: Intra-laboratory imprecision and performance with EQA materials were variable. Inter-laboratory performance was mostly within specifications. Although residual variability persists, adopting common traceable calibrators and RMP-determined EQA materials is beneficial for standardization of LC-MS/MS steroid measurements.

Corticotropin-stimulated steroid profiles to predict shock development and mortality in sepsis: From the HYPRESS study.

RATIONALE: Steroid profiles in combination with a corticotropin stimulation test provide information about steroidogenesis and its functional reserves in critically ill patients. OBJECTIVES: We investigated whether steroid profiles before and after corticotropin stimulation can predict the risk of in-hospital death in sepsis. METHODS: An exploratory data analysis of a double blind, randomized trial in sepsis (HYPRESS [HYdrocortisone for PRevention of Septic Shock]) was performed. The trial included adult patients with sepsis who were not in shock and were randomly assigned to placebo or hydrocortisone treatment. Corticotropin tests were performed in patients prior to randomization and in healthy subjects. Cortisol and precursors of glucocorticoids (17-OH-progesterone, 11-desoxycortisol) and mineralocorticoids (11-desoxycorticosterone, corticosterone) were analyzed using the multi-analyte stable isotope dilution method (LC-MS/MS). Measurement results from healthy subjects were used to determine reference ranges, and those from placebo patients to predict in-hospital mortality. MEASUREMENTS AND MAIN RESULTS: Corticotropin tests from 180 patients and 20 volunteers were included. Compared to healthy subjects, patients with sepsis had elevated levels of 11-desoxycorticosterone and 11-desoxycortisol, consistent with activation of both glucocorticoid and mineralocorticoid pathways. After stimulation with corticotropin, the cortisol response was subnormal in 12% and the corticosterone response in 50% of sepsis patients. In placebo patients (n = 90), a corticotropin-stimulated cortisol-to-corticosterone ratio > 32.2 predicted in-hospital mortality (AUC 0.8 CI 0.70-0.88; sensitivity 83%; and specificity 78%). This ratio also predicted risk of shock development and 90-day mortality. CONCLUSIONS: In this exploratory analysis, we found that in sepsis mineralocorticoid steroidogenesis was more frequently impaired than glucocorticoid steroidogenesis. The corticotropin-stimulated cortisol-to-corticosterone ratio predicts the risk of in-hospital death. Trial registration Clinical trial registered with www. CLINICALTRIALS: gov Identifier: NCT00670254. Registered 1 May 2008, https://clinicaltrials.gov/ct2/show/NCT00670254 .

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.).

Comparing posaconazole and itraconazole for antifungal prophylaxis in critically ill lung transplant recipients: Efficacy and plasma concentrations.

BACKGROUND: Posaconazole and itraconazole are commonly used for systemic antifungal prophylaxis after lung transplantation. The aim of this study on critically ill lung transplant recipients was to assess the rate of adequate plasma concentrations and the frequency of fungal-induced transitions from antifungal prophylaxis to therapy after the administration of either posaconazole or itraconazole for systemic prophylaxis. METHODS: Critically ill lung transplant recipients with postoperative posaconazole or itraconazole prophylaxis and therapeutic drug monitoring from February 2016 to November 2019 were retrospectively included in the study. Positive fungal cultures or Aspergillus antigen tests resulting in a transition from antifungal prophylaxis to therapy were analyzed from the first day of prophylaxis until 7 days after the last sample for each patient. Adequate plasma concentrations were defined as ≥500 µg/L for itraconazole and ≥700 µg/L for posaconazole. RESULTS: Two hundred seventy-five samples from 73 patients were included in the analysis. Overall, 60% of the posaconazole and 55% of the itraconazole concentrations were subtherapeutic. Administration of posaconazole suspension resulted significantly (P < .01) more often in subtherapeutic concentrations than tablets (68% vs 10%). Patients treated with posaconazole showed less positive fungal records resulting in a transition from prophylaxis to therapy than patients treated with itraconazole (10% vs 33%, P-value: .029). The detection of a fungal pathogen was not associated with the measured plasma concentrations or the achievement of the target concentrations. CONCLUSION: Our findings suggest that posaconazole should be used instead of itraconazole for systemic prophylaxis in critically ill lung transplant recipients.

From therapeutic drug monitoring to total drug monitoring and drug-omics.

In view of the role of pharmacotherapy in medicine, on the one hand, and the powerful technical possibilities that are now available on the other hand, therapeutic drug monitoring is a surprisingly neglected area of laboratory medicine. In this viewpoint article, an "omics approach" to pharmacovigilance and drug monitoring is proposed and discussed. A realistic goal for laboratory medicine in the 21st century should indeed be to enable clinicians to check whether the right drug is present in the right patient with an appropriate blood concentration for each compound.

Mass spectrometric sample identification with indicator compounds introduced via labeled sample tubes.

Objectives: The risk of sample confusion continues to be a challenge for the pre-analytical part of the overall testing process. We here describe a novel system to track samples based on a chemical code labeling of test tubes with unique combinations of indicator compounds, which are naturally not present in specimens of human origin. As part of the sample vessel filling, the liquid specimens are permanently labeled with the compound code that can be tracked back to the primary tube. Methods: As a proof of concept we used 10 stable-isotope-labeled derivates of medical drugs as indicator substances to create a combinatory 10-digit binary number ID for individual test tubes, i.e. presence/absence of the respective compound. For this purpose, combinations of indicator compounds were provided in evaporated form in polypropylene tubes prior to filling with anonymized patient whole blood and corresponding plasmas subjected to liquid chromatography tandem-mass spectrometry designed to detect the 10 indicator compounds. Results: In the blind analysis, we correctly identified 307 different whole blood samples by readout of a 10-digit binary number ID based on the detection of indicator compounds with respect to their presence and number. Conclusions: We have demonstrated the feasibility of an internal labeling procedure for diagnostic samples with mass spectrometry-based readout of dissolved indicator compound combinations as a binary number ID. With an increasing number of coding compounds (≫10) a vast number of combinations for sample labeling can be realized beyond the proof of concept setting studied herein.

Isotope dilution-LC-MS/MS method for quantification of the urinary cotinine-to-creatinine ratio.

Background: Appropriate monitoring of tobacco smoking is extremely important in several areas of medicine, e.g. management of chronic obstructive pulmonary disease (COPD), epidemiological surveys, and allocation of heart or lung transplants. The major metabolite of nicotine is cotinine that is increasingly used as a laboratory parameter for assessing tobacco smoke exposure. Methods: Creatinine and cotinine were analyzed simultaneously in urine by ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) in one run within 3 min using a biphenyl column. For quantification, the respective stable-isotope-labeled standards were used. Results: Detuning and measuring a natural isotope of creatinine as precursor and product ion allowed a simultaneous quantification of creatinine and cotinine. The method revealed robust validation results. For both analytes, inaccuracy and imprecision of the quality control and external quality assessment (EQA) samples were ≤-11.1%. Conclusions: One essential novelty of the method presented here is the simultaneous quantification of creatinine and cotinine covered by one analytical method. Despite the very different natural concentrations of creatinine and cotinine, this allows the immediate reporting of the cotinine-to-creatinine ratio without the need for a separate creatinine analysis.

Comparative Routine Therapeutic Drug Monitoring of Mycophenolic Acid in human Plasma with HPLC-UV and Isotope Dilution LC-MS/MS.

BACKGROUND: Therapeutic drug monitoring (TDM) of the immunosuppressant mycophenolic acid (MPA) is especially recommended for the control of personalized immunosuppressive therapy. Various test systems are available for MPA monitoring, including high performance liquid chromatography combined with UV detection (HPLC-UV) and isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS). METHODS: In the present work, commercially available kits for MPA monitoring with HPLC-UV and ID-LC-MS/ MS were subjected to routine use TDM. Following method verification according to the Clinical and Laboratory Standards Institute (CLSI) guidelines, 105 native sample duplicates from patients under therapy with mycophenolate mofetil were assayed with both procedures for comparative testing. RESULTS: Using bi-level quality controls, the estimate of repeatability, within-laboratory imprecision and inaccuracy were ≤ 5.18%, ≤ 5.95% and ≤ 3.86% for all MPA measurements. Weighted Deming regression analysis yielded a slope of 0.93, an intercept of 0.04, and Pearson's correlation coefficient (r) of 0.99, while Bland-Altman analysis showed a combined relative bias of 4.93% (± 1.96 SD: -16.68 - 26.54%). Plasma samples taken from a patient re-peatedly showed the presence of an interferent only in HPLC-UV analysis. CONCLUSIONS: Based on these results, HPLC-UV testing can be considered suitable for routine TDM of MPA in the clinical setting with high precision. Due to the risk of unforeseen analytical interference in ever-increasing multimorbidity and polypharmacy, highly selective ID-LC-MS/MS methodology should be given preference over HPLC-UV analysis whenever feasible.

Ciprofloxacin in critically ill subjects: considering hepatic function, age and sex to choose the optimal dose.

BACKGROUND: Pathophysiological changes often result in altered pharmacokinetics of ciprofloxacin in critically ill patients. Although ciprofloxacin clearance (CLCIP) substantially depends on kidney function in healthy volunteers, its relationship to measured creatinine clearance (CLCRM) is weak in critically ill patients. OBJECTIVES: To assess the need for dose reductions in isolated or combined kidney and liver dysfunction in critically ill patients and to re-evaluate relationships between kidney parameters, demographics and ciprofloxacin pharmacokinetics. METHODS: A population pharmacokinetic model was developed based on 444 ciprofloxacin serum concentrations from 15 critically ill patients with severe infections. CLCIP relationships to parameters reflecting hepatic function, CLCRM, Cockcroft-Gault creatinine clearance (CLCRCG), serum creatinine, sex, weight and age were explored. A simulation study was conducted to integrate knowledge from the new and previously published models. RESULTS: Total bilirubin was identified as a hepatic parameter with a clear relationship to CLCIP. A significant relationship between CLCIP and CLCRCG could be attributed to age and sex only. CLCIP was not associated with CLCRM. The predicted risk of potential overexposure (AUC > 250 mg·h/L) was low even with 1200 mg/day ciprofloxacin daily for patients with reduced CLCRCG (<30 mL/min: risk of 0.7%), while the risk was remarkably higher in elderly female patients with elevated bilirubin (risk of about 20% for 65-year-old women with total bilirubin of 4 mg/dL). CONCLUSIONS: Bilirubin, age and sex should be considered to assess the need for dose reductions. For MICs ≤0.25 mg/L, it might be appropriate to reduce the dose to 400 mg/day for elderly female subjects with high bilirubin.

Investigation on core-fucosylated prostate-specific antigen as a refined biomarker for differentiation of benign prostate hyperplasia and prostate cancer of different aggressiveness.

Prostate cancer represents a major cause of cancer death in men worldwide. Novel non-invasive methods are still required for differentiation of non-aggressive from aggressive tumors. Recently, changes in prostate-specific antigen glycosylation pattern, such as core-fucosylation, have been described in prostate cancer. The objective of this study was to evaluate whether the core-fucosylation determinant of serum prostate-specific antigen may serve as refined marker for differentiation between benign prostate hyperplasia and prostate cancer or identification of aggressive prostate cancer. A previously developed liquid chromatography-mass spectrometry/mass spectrometry-based strategy was used for multiplex analysis of core-fucosylated prostate-specific antigen (fuc-PSA) and total prostate-specific antigen levels in sera from 50 benign prostate hyperplasia and 100 prostate cancer patients of different aggressiveness (Gleason scores, 5-10) covering the critical gray area (2-10 ng/mL). For identification of aggressive prostate cancer, the ratio of fuc-PSA to total prostate-specific antigen (%-fuc-PSA) yielded a 5%-8% increase in the area under the curve (0.60) compared to the currently used total prostate-specific antigen (area under the curve = 0.52) and %-free prostate-specific antigen (area under the curve = 0.55) tests. However, our data showed that aggressive prostate cancer (Gleason score > 6) and non-aggressive prostate cancer (Gleason score ≤ 6) could not significantly (p-value = 0.08) be differentiated by usage of %-fuc-PSA. In addition, both non-standardized fuc-PSA and standardized %-fuc-PSA had no diagnostic value for differentiation of benign prostate hyperplasia from prostate cancer. The %-fuc-PSA serum levels could not improve the differentiation of non-aggressive and aggressive prostate cancer compared to conventional diagnostic prostate cancer markers. Still, it is unclear whether these limitations come from the biomarker, the used patient cohort, or the imprecision of the applied method itself. Therefore, %-fuc-PSA should be further investigated, especially by more precise methods whether it could be clinically used in prostate cancer diagnosis.