Comparison of two acquisition modes in mass spectrometry for quantification of 12 antibiotics, including beta-lactams, linezolid, fluoroquinolones and clindamycin: Multiple reactions monitoring (MRM) and fullMS/dd-MS2.

The quantification of antibiotics, using mass spectrometry, for monitoring therapeutic drugs is a key benefit in infection management. After an easy work-up of plasma samples, analysis were performed using both two widely used acquisition modes: MRM for the triple quadrupole spectrometer and fullMS/ddMS2 for the HRMS to quantify twelve antibiotics. Comparison between the two acquisition modes were performed. Validation parameters and sample values were used as comparison criteria. The results indicated a good correlation between the two methods, with an advantage for HRMS concerning the matrix effect. Both methods were applied to routine therapeutic drug monitoring.

Comparative Evaluation of Four Commercially Available Immunoassays for Therapeutic Drug Monitoring of Infliximab and Adalimumab.

Therapeutic drug monitoring (TDM) of anti-TNF-α is an important tool in clinical practice for inflammatory diseases. In this study, we have evaluated the performance of several assays for drug and antidrug antibodies (ADA) measurement in the serum. 50 sera from patients treated with infliximab (IFX) and 49 sera from patients treated with adalimumab (ADAL) were monitored with four immunoassays. We have compared Promonitor, i-Track10®, and ez-track1 assays to our gold standard Lisa Tracker® ELISA using Cohen's kappa, Passing-Bablok, and Bland-Altman analysis. The qualitative analysis evaluated by Cohen's kappa values found for IFX measurements an "almost perfect" concordance for Promonitor, "moderate" for i-Track10® and "substantial" for ez-Track1. For ADAL, kappa values were "moderate" for all tested methods. For anti-IFX, kappa values were "almost perfect" for Promonitor, "fair" for i-Track10®, and "substantial" for ez-Track1. For anti-ADAL, kappa values were "almost perfect" for all three assays. For quantitative analysis of drug measurements, Pearson's r values were all above 0.9 and Lin's concordance coefficients of all immunoassays were around 0.80. Performances of the four evaluated immunoassays were acceptable for TDM based on our laboratory experience. Nevertheless, concordance between the four methods for IFX measurement was not perfect and we recommend the use of the same assay for the follow-up of a given patient. The performances of the four immunoassays evaluated were similar and are acceptable for TDM based on our laboratory experience.

Population Pharmacokinetics of Isavuconazole in Critical Care Patients with COVID-19-Associated Pulmonary Aspergillosis and Monte Carlo Simulations of High Off-Label Doses.

Isavuconazole is a triazole antifungal agent recently recommended as first-line therapy for invasive pulmonary aspergillosis. With the COVID-19 pandemic, cases of COVID-19-associated pulmonary aspergillosis (CAPA) have been described with a prevalence ranging from 5 to 30%. We developed and validated a population pharmacokinetic (PKpop) model of isavuconazole plasma concentrations in intensive care unit patients with CAPA. Nonlinear mixed-effect modeling Monolix software were used for PK analysis of 65 plasma trough concentrations from 18 patients. PK parameters were best estimated with a one-compartment model. The mean of ISA plasma concentrations was 1.87 [1.29-2.25] mg/L despite prolonged loading dose (72 h for one-third) and a mean maintenance dose of 300 mg per day. Pharmacokinetics (PK) modeling showed that renal replacement therapy (RRT) was significantly associated with under exposure, explaining a part of clearance variability. The Monte Carlo simulations suggested that the recommended dosing regimen did not achieve the trough target of 2 mg/L in a timely manner (72 h). This is the first isavuconazole PKpop model developed for CAPA critical care patients underlying the need of therapeutic drug monitoring, especially for patients under RRT.

Development and Validation of a Non-Targeted Screening Method for Most Psychoactive, Analgesic, Anaesthetic, Anti-Diabetic, Anti-Coagulant and Anti-Hypertensive Drugs in Human Whole Blood and Plasma Using High-Resolution Mass Spectrometry.

(1) Background: In toxicological laboratories, various screening methods can be used to identify compounds involved in intoxication. High-resolution mass spectrometry has been increasingly used in this context for the last years, because of its sensitivity and reliability. Here, we present the development and validation of a screening method that uses liquid chromatography coupled with a high-resolution mass spectrometer. (2) Methods: This method required only 100 µL of whole blood or plasma sample. Pretreatment consisted of a rapid and simple deproteinisation with methanol/acetonitrile and zinc sulphate. This new assay was validated according to international guidelines. (3) Results: To perform the method validation, 53 compounds were selected. The selection criteria were as follows: various chemical structures and therapeutic families (>15), large m/z distribution, positive or negative ionisation mode, and various elution times. The assays showed high selectivity and specificity, with optimal process efficiency. The identification limits, determined using predefined criteria, were established at sub-therapeutic or therapeutic concentrations. Applicability was evaluated using spiked plasma controls and external quality controls. (4) Conclusions: The new method was then successfully applied to routine clinical and forensic samples.

Quantification of 15 Antibiotics Widely Used in the Critical Care Unit with a LC-MS/MS System: An Easy Method to Perform a Daily Therapeutic Drug Monitoring.

Potential under- or overdose of antibiotics may occur in intensive care units due to high variability in plasma concentrations. The risk is either treatment failure or toxicity. Thus, therapeutic drug monitoring of antibiotics may guide dosing adjustment, maximising antibacterial efficacy and minimising toxicity. The aim of this study was to develop and validate a method for the analysis of 15 antibiotics including beta-lactams, linezolid, fluoroquinolones, daptomycin, and clindamycin to have a complete panel in the management of infections. We proposed to develop a fast, sensitive, and quantitative method for the analysis of 15 antibiotics using ultra-performance liquid chromatography coupled with triple quadrupole mass spectrometer (UPLC-MS/MS) technology. this method required only 100 µL of plasma and consisted of a rapid liquid-liquid deproteinisation using methanol. Calibration curves ranged from 0.078 to 500 mg/L depending on the molecules, and were defined according to a therapeutic range. Inter- and intra-assay precisions values were less than 15%. This work described the development and the full validation of a precise, sensitive and accurate assay using UPLC-MS/MS technology. After validation, this new assay was successfully applied to routine therapeutic drug monitoring.

External Evaluation of Population Pharmacokinetic Models and Bayes-Based Dosing of Infliximab.

Despite the well-demonstrated efficacy of infliximab in inflammatory diseases, treatment failure remains frequent. Dose adjustment using Bayesian methods has shown in silico its interest in achieving target plasma concentrations. However, most of the published models have not been fully validated in accordance with the recommendations. This study aimed to submit these models to an external evaluation and verify their predictive capabilities. Eight models were selected for external evaluation, carried out on an independent database (409 concentrations from 157 patients). Each model was evaluated based on the following parameters: goodness-of-fit (comparison of predictions to observations), residual error model (population weighted residuals (PWRES), individual weighted residuals (IWRES), and normalized prediction distribution errors (NPDE)), and predictive performances (prediction-corrected visual predictive checks (pcVPC) and Bayesian simulations). The performances observed during this external evaluation varied greatly from one model to another. The eight evaluated models showed a significant bias in population predictions (from -7.19 to 7.38 mg/L). Individual predictions showed acceptable bias and precision for six of the eight models (mean error of -0.74 to -0.29 mg/L and mean percent error of -16.6 to -0.4%). Analysis of NPDE and pcVPC confirmed these results and revealed a problem with the inclusion of several covariates (weight, concomitant immunomodulatory treatment, presence of anti-drug antibodies). This external evaluation showed satisfactory results for some models, notably models A and B, and highlighted several prospects for improving the pharmacokinetic models of infliximab for clinical-biological application.

High silver concentrations in biological samples following different exposures: Two case reports.

BACKGROUND: Silver is used in various industrial applications, but also in confectioneries and for therapeutic use due to its antibiotic properties. Its toxicity is not well documented and most often only in the context of professional exposure. AIM: Here we report two cases of high silver concentrations in biological samples in two women: the first patient presented grey marks around her cuticles, probably due to her consumption of silvered sweets and the second patient presented agranulocytosis and thrombocytopenia occurring within 24 h after the topical application of a cream containing sulfadiazine and silver to burns over a large surface area. METHODS: Silver concentrations were determined in blood and urine samples and sweets using inductively coupled plasma- mass spectrometry (ICP-MS). RESULTS: The silver concentrations were elevated compared to population reference values and confirmed the hypotheses for the patients: the significant consumption of sweets coated with silver nanoparticles and the topical application of a cream containing silver to burns over a large area. DISCUSSION-CONCLUSION: After initial questioning by the dermatologist, Patient 1 explained that she consumed more than 30 bags of the sweets per year. She decreased her consumption of the sweets and the control performed one year later showed a plasma silver concentration of 1.5 µg/L. For Patient 2, the absorption of silver through burns over a large area appeared relatively significant, whereas it is very low through undamaged skin. The haematological cells counts returned to normal levels quickly and no other major effects were highlighted. To apply these findings to a larger population, further investigation to determine sulfadiazine and silver concentrations in plasma and urine have been initiated in a cohort of patients with burns over a large area.

Quantification of methadone, buprenorphine, naloxone, opioids, and their derivates in whole blood by liquid chromatography-high-resolution mass spectrometry: Analysis of their involvement in fatal forensic cases.

Opioids represent a broad family of compounds that can be used in several indications: analgesics, antitussives, opioid substitution therapy (e.g. methadone, buprenorphine…). When these products are misused, they are often addictive. Thus, we aimed to develop an analytical method able to rapidly quantify several opiates and opioids (6-monoacetylmorphine, buprenorphine, codeine, dihydrocodeine, 2-ethyl-1,5-dimethyl-3,3-diphenylpyrrolidine, ethylmorphine, heroin, methadone, morphine, nalbuphine, naloxone, norbuprenorphine, norcodeine, norpropoxyphene, oxycodone and propoxyphene) in whole blood by ultra-high performance liquid chromatography combined with high resolution mass spectrometry (UHPLC-HRMS). The validated assay requires only 100 µL of the blood sample. The sample is prepared by a rapid liquid-liquid extraction using 5% zinc sulfate (W/V), methanol and acetonitrile. Calibration curves range from 0.98 to 1000 µg/L, except for buprenorphine (0.39-100 µg/L) and norbuprenorphine (0.20-100 µg/L). Inter- and intra-analytical accuracy was less than 15%. Therefore, we describe the development and full validation of an accurate, sensitive and precise assay using UHPLC-HRMS for the analysis of opioids in whole blood. After validation, this new assay is successfully applied on a routine laboratory application basis.

Real Life Population Pharmacokinetics Modelling of Eight Factors VIII in Patients with Severe Haemophilia A: Is It Always Relevant to Switch to an Extended Half-Life?

We retrospectively analysed the data files of 171 adults and 87 children/adolescents with severe haemophilia, except for 14 patients (moderate; minor) (1), to develop a global population pharmacokinetic (PK) model for eight factors VIII (FVIII) that could estimate individual PK parameters for targeting the desired level of FVIII activity (FVIII:C); and (2) to compare half-life (HL) in patients switching from a standard half-life (SHL) to an extended half-life (EHL) and evaluate the relevance of the switch. One-stage clotting assay for the measurement of FVIII activity (FVIII:C, IU/mL) was used for population PK modelling. The software, Monolix version 2019R1, was used for non-linear mixed-effects modelling. A linear two-compartment model best described FVIII:C. The estimated PK parameters (between-subject variability) were: 2640 mL (23.2%) for volume of central compartment (V1), 339 mL (46.8%) for volume of peripheral compartment (V2), 135 mL/h for Q (fixed random effect), and 204 mL/h (34.9%) for clearance (Cl). Weight, age, and categorical covariate EHL were found to influence Cl and only weight for V1. This model can be used for all of the FVIII cited in the study. Moreover, we demonstrated, in accordance with previous studies, that Elocta had longer half-life (EHL) than SHL (mean ratio: 1.48) as compared to Advate, Factane, Kogenate, Novoeight, and Refacto.

Population Pharmacokinetics Modelling and Simulation of Mitotane in Patients with Adrenocortical Carcinoma: An Individualized Dose Regimen to Target All Patients at Three Months?

Mitotane is the most effective agent in post-operative treatment of adrenocortical carcinoma. In adults, the starting dose is 2-3 g/day and should be slightly increased to reach the therapeutic index of 14-20 mg/L. This study developed a population PK model for mitotane and to simulate recommended/high dosing regimens. We retrospectively analyzed the data files of 38 patients with 503 plasma concentrations for the pharmacokinetic analysis. Monolix version 2019R1 was used for non-linear mixed-effects modelling. Monte Carlo simulations were performed to evaluate the probability of target attainment (PTA ≥ 14 mg/L) at one month and at three months. Mitotane concentration data were best described by a linear one-compartment model. The estimated PK parameters (between-subject variability) were: 8900 L (90.4%) for central volume of distribution (V) and 70 L·h-1 (29.3%) for clearance (Cl). HDL, Triglyceride (Tg) and a latent covariate were found to influence Cl. The PTA at three months for 3, 6, 9, and 12 g per day was 10%, 55%, 76%, and 85%, respectively. For a loading dose of 15 g/day for one month then 5 g/day, the PTA in the first and third months was 57 and 69%, respectively. This is the first PKpop model of mitotane highlighting the effect of HDL and Tg covariates on the clearance as well as a subpopulation of ultrafast metabolizer. The simulations suggest that recommended dose regimens are not enough to target the therapeutic threshold in the third month.

Complementary Role of P2 and Adenosine Receptors in ATP Induced-Anti-Apoptotic Effects Against Hypoxic Injury of HUVECs.

BACKGROUND: Vascular endothelial injury during ischemia generates apoptotic cell death and precedes apoptosis of underlying tissues. We aimed at studying the role of extracellular adenosine triphosphate (ATP) on endothelial cells protection against hypoxia injury. METHODS: In a hypoxic model on endothelial cells, we quantified the extracellular concentration of ATP and adenosine. The expression of mRNA (ectonucleotidases, adenosine, and P2 receptors) was measured. Apoptosis was evaluated by the expression of cleaved caspase 3. The involvement of P2 and adenosine receptors and signaling pathways was investigated using selective inhibitors. RESULTS: Hypoxic stress induced a significant increase in extracellular ATP and adenosine. After a 2-h hypoxic injury, an increase of cleaved caspase 3 was observed. ATP anti-apoptotic effect was prevented by suramin, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), and CGS15943, as well as by selective A2A, A2B, and A3 receptor antagonists. P2 receptor-mediated anti-apoptotic effect of ATP involved phosphoinositide 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), mitoKATP, and nitric oxide synthase (NOS) pathways whereas adenosine receptor-mediated anti-apoptotic effect involved ERK1/2, protein kinase A (PKA), and NOS. CONCLUSIONS: These results suggest a complementary role of P2 and adenosine receptors in ATP-induced protective effects against hypoxia injury of endothelial. This could be considered therapeutic targets to limit the development of ischemic injury of organs such as heart, brain, and kidney.

Population pharmacokinetics of articaine with 1:200,000 epinephrine during third molar surgery and simulation of high-dose regimens.

BACKGROUND AND OBJECTIVES: Articaine is more and more used in third molar surgery under local anesthesia (LA). The objectives of this analysis were to characterize the pharmacokinetics of articaine for this type of surgery and to simulate dosing regimens. METHODS: Non-linear mixed-effects modeling conducted in Monolix 4.4.0 was used to describe articaine plasma concentration-time data from 20 patients. Monte Carlo simulations were then performed to evaluate the probability of cardiotoxic target attainment (PCTA) of various dosage regimens. RESULTS: Articaine concentration data were best described by a linear one-compartment model, with an additional depot compartment for submucosal route with a zero-order transfer to central compartment. Age and gender were found to influence duration transfer (Tk0) and elimination rate constant (Ke), respectively. Simulated maximum recommended dose regimen (7mg/kg) had a PCTA of 0%. Simulated higher doses of 10mg/kg and 15mg/kg had a PCTA of 0% and about 1-4%, respectively. CONCLUSIONS: The model adequately described the articaine pharmacokinetics. This is the first PK model qualified for articaine administered by submucosal route. The simulations suggest that maximum recommended dose regimen is safe concerning the cardiotoxicity in healthy patients.

Population Pharmacokinetic-Pharmacodynamic Modeling of Ropivacaine in Spinal Anesthesia.

BACKGROUND: Ropivacaine is frequently used in spinal anesthesia but the relationship between plasma concentrations and sensory block level remains unknown. OBJECTIVE: The aim of this study was to assess the relationship between plasma ropivacaine concentrations and effects during spinal anesthesia. METHODS: Sixty patients aged between 18 and 82 years were included in this study after providing written informed consent. Patients were randomly assigned to receive intrathecal administration of ropivacaine 15, 20 or 25 mg. Blood samples were drawn to determine ropivacaine concentrations, and sensory blockade was assessed using pinprick testing. Ropivacaine plasma concentrations and sensory block level were analyzed using a nonlinear mixed-effects modeling approach with Monolix 4.2.2. Uncertainty of parameters was estimated by bootstrapping. RESULTS: Overall, 216 plasma ropivacaine values and 407 sensory block-related data were available for pharmacokinetic-pharmacodynamic (PK-PD) model evaluation. A two-compartment open model connected to a spinal compartment was selected to describe the PKs of ropivacaine. Sensory block modeling was performed using a sigmoid E max model assuming an equilibration delay between the amount in the depot or spinal compartment and at the effect site. Using multiple linear regression analysis, we were able to demonstrate the importance of dose, age and weight as major predictors of sensory block-level kinetics. CONCLUSIONS: This first population PK-PD model for ropivacaine in spinal anesthesia confirms the relationship between plasma ropivacaine concentrations and effect. We also clarify the relationship between the spread of sensory block level and dose, age and, for the first time, weight. STUDY REGISTRATION: This study was approved by the Reims University Hospital Ethics Committee (protocol: PHRC-2005; registered at Agence Nationale de Sécurité du Médicament et des Produits de Santé ANSM: D60890). This was an open, prospective, monocentric study conducted in the University Hospital of Reims (France).

Population pharmacokinetics of teicoplanin administered by subcutaneous or intravenous route and simulation of optimal loading dose regimen.

Objectives: To investigate the population pharmacokinetics of teicoplanin in patients treated by the subcutaneous (sc) and/or intravenous (iv) route. Patients and methods: Non-linear mixed-effects modelling described teicoplanin concentrations from 98 patients with infection caused by Gram-positive cocci. Monte Carlo simulations were performed to evaluate the probability of target attainment (PTA) of various dosage regimens. Results: Teicoplanin concentrations were best described by a two-compartment model with clearance predicted by estimated glomerular filtration rate. Estimated absorption rate constant (between-subject variability) was 0.039 h-1 (77%), clearance was 0.305 L/h (28%), central volume was 10.3 L (49%), inter-compartmental clearance was 4.42 L/h (66%) and peripheral volume was 97.4 L (51%). The sc route was associated with lower initial Cmin and AUC (day 3: loading phase) compared with the iv route. This difference appeared to vanish after 14 days, with comparable simulated PTAs based on the Cmin and AUC for all tested dosages (400, 600, 800 and 1000 mg every 12 h). However, a loading dose regimen with five administrations of either 400 or 600 mg was not sufficient to achieve the target Cmin (≥15 mg/L) for both routes. Also, PTAs for higher MIC (≥1.0 mg/L) were poor with all regimens for both routes. Conclusions: This is the first study examining the pharmacokinetic/pharmacodynamic implications of using the sc route for teicoplanin. Subcutaneous administration is associated with lower Cmin and AUC values after the loading phase compared with iv administration. Therefore, iv administration should be preferred in the first few days of therapy. This study also shows that loading doses of teicoplanin higher than currently recommended should be used to improve PTA.

Drowsiness and uncommon fever in a child after cannabis ingestion.

Trivialization of cannabis consumption goes hand in hand with a growing exposure of children and the number of cannabis poisoning cases is steadily increasing. As clinical presentation can be different from what is currently seen in adults, added to the fact that it is not always suspected, diagnosis of cannabis intoxication in children is often delayed or missed. A 16-month-old girl was admitted to the pediatric emergency unit for an important drowsiness combined to moderate fever. After elimination of infectious causes, a toxic origin was considered and biological analyses led to the diagnosis of involuntary acute cannabis intoxication. In conclusion, cannabis intoxication in child has uncommon presentations compared to that seen in adults. In this context, biological analyses have a great importance for a rapid diagnosis and also for the understanding intoxication circumstance. This is of paramount importance because it may lead to consider child protection measures.

Therapeutic drug monitoring of mitotane: Analytical assay and patient follow-up.

Adrenocortical carcinoma (ACC) is an aggressive malignancy of the adrenal gland. Mitotane (o,p'-DDD) is the most effective chemotherapy for ACC. According to the literature, mitotane plasma trough concentrations within 14-20 mg L-1 are correlated with a higher response rate with acceptable toxicity. Therapeutic drug monitoring (TDM) of mitotane is therefore recommended. The aim of this study was to propose a robust and simple method for mitotane quantification in plasma. The validation procedures were based on international guidelines. Sample preparation consisted of a single protein precipitation with methanol using 100 µL of plasma. The supernatant was submitted to liquid chromatography coupled with ultra-violet detection at 230 nm. Mitotane retention time was 7.1 min. The limit of detection was 0.1 mg L-1 and the limit of quantification was 0.78 mg L-1 . The assay demonstrated a linear range of 0.78-25 mg L-1 with correlation coefficients (r2 ) at 0.999. Inter- and intra-assay precision was <4.85%. Evaluation of accuracy showed a deviation <13.69% from target concentration at each quality control level. This method proved easy and rapid to perform mitotane TDM and required a small volume of sample. It was successfully applied to routine TDM in our laboratory.

A Slight Smell of Lemon.

We present here an example of urine substituted with a yellow cleaning product that leads us to develop the main risks to consider in urine toxicology analysis, ie, adulteration and analytical interferences, and how to deal with them. This grand round highlights the importance of the dialog between the clinician and a TDM consultant for optimal care of the patient.