Probe Electrospray Ionization Tandem Mass Spectrometry for the Detection and Quantification of Benzodiazepines.

BACKGROUND: Legally prescribed benzodiazepines (BZDs) and designer BZDs are widely misused and must be determined in multiple contexts (eg, overdose, drug-facilitated sexual assaults, or driving under the influence of drugs). This study aimed to develop a method for measuring serum BZD levels using probe electrospray ionization (PESI) mass spectrometry and an isotope dilution approach. METHODS: A tandem mass spectrometer equipped with a probe electrospray ionization source in multiple reaction monitoring mode was used. Isotope dilution was applied for quantification using a deuterated internal standard at a fixed concentration for alprazolam, bromazepam, diazepam, nordiazepam, oxazepam, temazepam, zolpidem, and zopiclone. This method included designer BZDs: clonazolam, deschloroetizolam, diclazepam, etizolam, flualprazolam, flubromazepam, flubromazolam, meclonazepam, nifoxipam, and pyrazolam. Sample preparation was done by mixing 10 µL of serum with 500 µL of an ethanol/ammonium formate 0.01 mol/L buffer. Complete validation was performed, and the method was compared with liquid chromatography coupled with mass spectrometry (LC-MS/MS) and immunoassays (IC) by analyzing 40 real samples. RESULTS: The analysis time for identification and quantification of the 18 molecules was 2.5 minutes. This method was fully validated, and the limits of quantification varied from 5 to 50 mcg/L depending on the molecule. In the 40 real samples, 100% of molecules (n = 89) were detected by both LC-MS/MS and PESI-MS/MS, and regression analysis showed excellent agreement between the 2 methods (r2 = 0.98). On IC, bromazepam and zolpidem were not detected in 2 and 1 cases, respectively. CONCLUSIONS: PESI-MS/MS allows serum BZD detection and measurement. Given the isotope dilution approach, a calibration curve was not required, and its performance was similar to that of LC-MS/MS, and its specificity was higher than that of IC.

Early detection of liver injuries by the Serum enhanced binding test sensitive to albumin post-transcriptional modifications.

Early and sensitive biomarkers of liver dysfunction and drug-induced liver injury (DILI) are still needed, both for patient care and drug development. We developed the Serum Enhanced Binding (SEB) test to reveal post-transcriptional modifications (PTMs) of human serum albumin resulting from hepatocyte dysfunctions and further evaluated its performance in an animal model. The SEB test consists in spiking serum ex-vivo with ligands having specific binding sites related to the most relevant albumin PTMs and measuring their unbound fraction. To explore the hypothesis that albumin PTMs occur early during liver injury and can also be detected by the SEB test, we induced hepatotoxicity in male albino Wistar rats by administering high daily doses of ethanol and CCl4 over several days. Blood was collected for characterization and quantification of albumin isoforms by high-resolution mass spectrometry, for classical biochemical analyses as well as to apply the SEB test. In the exposed rats, the appearance of albumin isoforms paralleled the positivity of the SEB test ligands and histological injuries. These were observed as early as D3 in the Ethanol and CCl4 groups, whereas the classical liver tests (ALT, AST, PAL) significantly increased only at D7. The behavior of several ligands was supported by structural and molecular simulation analysis. The SEB test and albumin isoforms revealed hepatocyte damage early, before the current biochemical biomarkers. The SEB test should be easier to implement in the clinics than albumin isoform profiling.

Absolute Quantification of Human Serum Albumin Isoforms by Internal Calibration Based on a Top-Down LC-MS Approach.

Well-characterized biomarkers using reliable quantitative methods are essential for the management of various pathologies such as diabetes, kidney, and liver diseases. Human serum albumin (HSA) isoforms are gaining interest as biomarkers of advanced liver pathologies. In view of the structural alterations observed for HSA, insights into its isoforms are required to establish them as reliable biomarkers. Therefore, a robust absolute quantification method seems necessary. In this study, we developed and validated a far more advanced top-down liquid chromatography-mass spectrometry (LC-MS) method for the absolute quantification of HSA isoforms, using myoglobin (Mb) as an internal standard for quantification and for mass recalibration. Two different quantification approaches were investigated based on peak integration from the deconvoluted spectrum and extracted ion chromatogram (XIC). The protein mixture human serum albumin/myoglobin eluted in well-shaped separated peaks. Mb allowed a systematic mass recalibration for every sample, resulting in extremely low mass deviations compared to conventional deconvolution-based methods. In total, eight HSA isoforms of interest were quantified. Specific-isoform calibration curves showing good linearity were obtained by using the deconvoluted peaks. Noticeably, the HSA ionization behavior appeared to be isoform-dependent, suggesting that the use of an enriched isoform solution as a calibration standard for absolute quantification studies of HSA isoforms is necessary. Good repeatability, reproducibility, and accuracy were observed, with better sensitivity for samples with low albumin concentrations compared to routine biochemical assays. With a relatively simple workflow, the application of this method for absolute quantification shows great potential, especially for HSA isoform studies in a clinical context, where a high-throughput method and sensitivity are needed.

Chlordecone determination in serum by LC-MS/MS and the importance of low limit of detection.

Chlordecone is an organochlorine insecticide that has been used intensively from 1973 to 1993 in the French West Indies banana fields to control root borers. This use has resulted in persistent pollution of soils and waters, and people have been and are still exposed mainly through food. Epidemiological studies showed that this exposure is associated with health disorders, including prostate cancer, prematurity, cognitive or motor development. The measurement of chlordecone in serum is considered as the best surrogate, though no clear and definitive cut-off value has been established. This renders necessary the development of analytical methods with the lowest limit of detection as possible. While most published methods have utilized GC-MS or GC-MS/MS, in the present study we report an LC-MS/MS method based on a simple QuEChERS salts extraction. The whole procedure was validated according to ISO 15189 requirements and reached LOD and LOQ values of 0.007 and 0.02 µg/L, respectively. It was applied to more 10 000 serum samples of French Indies inhabitants. More than a half had a concentration below 0.1 µg/L and more than one third of them exhibiting a concentration below 0.05 µg/L. The capability of this LC-MS/MS method to detect very low concentrations highlights its utility in exploring the health impact of chlordecone.

Semi-synthetic human albumin isoforms: Production, structure, binding capacities and influence on a routine laboratory test.

Human Serum Albumin (HSA) undergoes Post-Translational-Modifications (PTMs) leading to isoforms affecting its oncotic and non-oncotic properties. HSA is comprised of several isoforms whose abundance may vary with pathologies such as diabetes, kidney and liver diseases. Studying their impact separately may help to understand their sources and potential pathogenicity and further their evaluation as biomarkers. The present study examined semi-synthetic HSA isoforms to investigate independently their structure by means of advanced mass spectrometry techniques (LC-TOF-MS and ICP-MS), influence on the HSA binding/antioxidant activities using a binding capacity test, and potential impact on albumin quantification by a routine immunoturbidimetric assay. Applying different chemical reactions to a commercial HSA solution, we obtained different solutions enriched up to 53 % of native HSA, 78 % of acetylated HSA, 71 % of cysteinylated HSA, 94 % of oxidized HSA, 58 % of nitrosylated HSA and 96 % of glycated HSA, respectively. Moreover, the semi-synthetic isoforms showed differently altered binding capacities for a panel of ligands (Cu, Cd, Au, Ds and L-T4). Furthermore, immunoturbidimetry was found to be insensitive to the presence and abundance of the different isoforms. The fully characterized semi synthetic HSA isoforms obtained should be useful to further investigate their pathogenicity and potential roles as biomarkers.

Posttranslational-modifications of human-serum-albumin analysis by a top-down approach validated by a comprehensive bottom-up analysis.

The posttranslational modifications (PTM) of human serum albumin (HSA) can result in the development of isoforms that have been identified as potential biomarkers for advanced hepatic diseases. However, previous approaches using top-down (TD) analysis to identify isoforms based on molecular weight may have resulted in misidentifications. The nature of the identified isoforms has never been confirmed in previous works. Here, we aimed to critically evaluate TD for the characterization and determination of HSA isoforms in patients and make an inventory of HSA-PTM. Serum samples from control subjects and patients with liver dysfunctions were analyzed using both top-down (TD) and bottom-up (BU) approaches. TD analysis involved using a LC-TOF-MS system to obtain a multicharged spectrum of HSA, which was deconvoluted to identify isoforms. Spectra were then used for relative quantitation analysis of albumin isoform abundances based on trapezoidal integration. For BU analysis, serums were reduced +/- alkylated, digested with trypsin and analyzed in the Q-TOF, data-dependent acquisition (DDA) mode to generate a SWATH-MS high-resolution mass spectral library of all HSA peptides. Tryptic digests of another set of serum samples were then analyzed using data-independent acquisition (DIA) mode to confirm the presence of HSA isoforms and their modification sites. TD detected 15 isoforms corresponding to various modifications, including glycation, cysteinylation, nitrosylation, and oxidation (di- and tri-). In BU, the spectral library containing 127 peptides allowed for the characterization of the important isoforms with their modified sites, including some modifications that were only characterized in BU (carbamylation, deamidation, and amino-acid substitution). The method used for determining isoforms offered acceptable reproducibility (intra-/inter-assay CVs < 15%) for all isoforms present at relative abundances higher than 2%. Overall, the study found that several isoforms could be missed or misidentified by TD. However, all HSA isoforms identified by TD and reported to be relevant in liver dysfunctions were confirmed by BU. This critical evaluation of TD approach helped design an adequate and reliable method for the characterization of HSA isoforms in patients and offers the possibility to estimate isoform abundances within 3 min. These findings have significant implications for the diagnosis and treatment of liver dysfunctions.

New perspectives for the therapeutic drug monitoring of tacrolimus: Quantification in volumetric DBS based on an automated extraction and LC-MS/MS analysis.

Volumetric microsampling devices have been developed for home-based capillary blood sampling and are now increasingly proposed for the therapeutic drug monitoring (TDM) of immunosuppressive drugs. Our objective was to validate a LC-MS/MS method for tacrolimus quantification based on both a manual and an automated extraction of dried blood spots (DBS) collected with a volumetric microsampling device. DBS collection was performed by placing a drop of whole blood (WB) pre-spiked with tacrolimus onto a sealing film and placing the hemaPEN® device (Trajan Scientific and Medical, Melbourne, Australia) into the drop according to the device specifications. Tacrolimus was quantified using a fully automatic preparation module connected to a LCMS system (CLAM-3020® and LCMS-8060®, Shimadzu, Marne-la-Vallée, France). The method was validated analytically and clinically in accordance with the EMA and IATDMCT guidelines. The method was linear from 1 to 100 µg/L. Within- and between-run accuracy and precision fulfilled the validation criteria (biases and imprecision <15% or 20% for the lower limit of quantification). No hematocrit effect, matrix effect or carry-over was observed. No selectivity issue was identified and dilution integrity was confirmed. Tacrolimus in DBS was stable for 14 days at room temperature and +4°C, and for 72h at +60°C. There was a good correlation between tacrolimus concentrations measured in WB and in DBS of 20 kidney and liver transplant recipients (r=0.93 and 0.87, for manual and automated extraction respectively). A method for tacrolimus measurement in DBS collected with volumetric micro-sampling device, based on a fully automated process from pre-treatment to LC-MS/MS analysis was developed and validated according to analytical and clinical criteria. This performing sampling and analytical procedure opens the perspective of an easier, faster and more efficient TDM of tacrolimus for patients, clinicians and laboratories.

Mycophenolate Mofetil Dose Adjustment in Pediatric Kidney Transplant Recipients.

BACKGROUND: The Immunosuppressant Bayesian Dose Adjustment web site aids clinicians and pharmacologists involved in the care of transplant recipients; it proposes dose adjustments based on the estimated area under the concentration-time curve (AUCs). Three concentrations (T 20 min , T 1 h , and T 3 h ) are sufficient to estimate mycophenolic acid (MPA) AUC 0-12 h in pediatric kidney transplant recipients. This study investigates mycophenolate mofetil (MMF) doses and MPA AUC values in pediatric kidney transplant recipients, and target exposure attainment when the proposed doses were followed, through a large-scale analysis of the data set collated since the inception of the Immunosuppressant Bayesian Dose Adjustment web site. METHODS: In this study, 4051 MMF dose adjustment requests, corresponding to 1051 patients aged 0-18 years, were retrospectively analyzed. AUC calculations were performed in the back office of the Immunosuppressant Bayesian Dose Adjustment using published Bayesian and population pharmacokinetic models. RESULTS: The first AUC request was posted >12 months posttransplantation for 41% of patients. Overall, only 50% had the first MPA AUC 0-12 h within the recommended 30-60 mg.h/L range. When the proposed dose was not followed, the proportion of patients with an AUC in the therapeutic range for MMF with cyclosporine or tacrolimus at the subsequent request was lower (40% and 45%, respectively) than when it was followed (58% and 60%, respectively): P = 0.08 and 0.006, respectively. Furthermore, 3 months posttransplantation, the dispersion of AUC values was often lower at the second visit when the proposed doses were followed, namely, P = 0.03, 0.003, and 0.07 in the 4 months-1 year, and beyond 1 year with <6-month or >6-month periods between both visits, respectively. CONCLUSIONS: Owing to extreme interindividual variability in MPA exposure, MMF dose adjustment is necessary; it is efficient at reducing such variability when based on MPA AUC.

Can the Area Under the Curve/Trough Level Ratio Be Used to Optimize Tacrolimus Individual Dose Adjustment?

BACKGROUND: The aim of this work was to evaluate, in a large data set of renal transplant recipients, the intraindividual variability of the area under the curve (AUC)/predose concentration (C0) ratio in comparison with that of AUC, C0, AUC/dose, and C0/dose. METHODS: Patients with at least 2 tacrolimus AUC estimation requests were extracted from the Immunosuppressant Bayesian dose Adjustment website, and relative variations between 2 consecutive visits for the different metrics were calculated and compared. RESULTS: Data from 1325 patients on tacrolimus (3827 measured C0 and estimated AUC) showed that the lowest mean relative variation between 2 consecutives visits was for the AUC/C0 ratio (95% confidence interval [CI] relative fold change = -43% to 44% for AUC/C0; 95% CI, -77% to 72% for AUC; 95% CI, -82% to 98% for AUC/dose; 95% CI, -81% to 80% for C0 and 95% CI, -94% to 117% for C0/dose. The correlation between 2 consecutive requests, whether close or far apart, was also best for the AUC/C0 ratio ( r = 0.33 and r = 0.34, respectively) in comparison with C0 ( r = 0.21 and r = 0.22, respectively) and AUC ( r = 0.19 and 0.28, respectively). Regression analysis between AUC0-24 and C0 showed that for some patients, the usual C0 targets translated into some very unusual AUC values. As the AUC/C0 ratio is quite stable during large periods, individualized C0 targets can be derived from the AUC targets, and an algorithm that estimates the individualized C0 was developed for situations in which prior AUC estimates are available or not. CONCLUSIONS: In this study, we confirmed in a large data set that the AUC/C0 ratio yields low intraindividual variability, whereas C0 shows the largest, and we propose to calculate individualized C0 targets based on this ratio.

[Therapeutic drug monitoring of clozapine]. / Suivi thérapeutique pharmacologique de la clozapine.

Clozapine is a prototypical atypical antipsychotic used to treat severe schizophrenia and for which a therapeutic drug monitoring (TDM) is quite commonly proposed. Clozapine is rapidly absorbed (maximum concentration reached within 1 to 4hours), and is extensively metabolized in the liver by CYP1A2 to an active metabolite (and to a lesser extent, to inactive metabolites via other enzymes). Its half-life is 8 to 16h. A therapeutic range has been proposed for clozapine as some studies have reported both a relationship between low plasmatic concentrations and resistance to treatment (threshold level is likely between 250 and 400µg/L), and a relationship between high plasmatic concentrations and an increase in the occurrence of toxicity (alert level=1000µg/L). Given the data obtained in different studies, the TDM was evaluated for this molecule, to recommended.

Metabolic interactions of benzodiazepines with oxycodone ex vivo and toxicity depending on usage patterns in an animal model.

BACKGROUND AND PURPOSE: Opioids and benzodiazepines are frequently combined in medical as well as in non-medical contexts. At high doses, such combinations often result in serious health complications attributed to pharmacodynamics interactions. Here, we investigate the contribution of the metabolic interactions between oxycodone, diazepam and diclazepam (a designer benzodiazepine) in abuse/overdose conditions through ex vivo, in vivo and in silico approaches. EXPERIMENTAL APPROACH: A preparation of pooled human liver microsomes was used to study oxycodone metabolism in the presence or absence of diazepam or diclazepam. In mice, diazepam or diclazepam was concomitantly administered with oxycodone to mimic acute intoxication. Diclazepam was introduced on Day 10 in mice continuously infused with oxycodone for 15 days to mimic chronic intoxication. In silico modelling was used to study the molecular interactions of the three drugs with CYP3A4 and 2D6. KEY RESULTS: In mice, in acute conditions, both diazepam and diclazepam inhibited the metabolism of oxycodone. In chronic conditions and at pharmacologically equivalent doses, diclazepam drastically enhanced the production of oxymorphone. In silico, the affinity of benzodiazepines was higher than oxycodone for CYP3A4, inhibiting oxycodone metabolism through CYP3A4. Oxycodone metabolism is likely to be diverted towards CYP2D6. CONCLUSION AND IMPLICATIONS: Acute doses of diazepam or diclazepam result in the accumulation of oxycodone, whereas chronic administration induces the accumulation of oxymorphone, the toxic metabolite. This suggests that overdoses of opioids in the presence of benzodiazepines are partly due to metabolic interactions, which in turn explain the patterns of toxicity dependent on usage. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Ultrafast Measurement of Metformin in the Clinical Setting Using Probe Electrospray Ionization Mass Spectrometry.

Metformin (MtF) is a treatment used for type 2 diabetes. Lactic acidosis (LA) is a frequent complication that can be either induced by or associated with elevated MtF plasma concentrations. When coupled with a mass spectrometry (MS) system, the probe electrospray ionization (PESI) method allows direct and rapid analysis of different types of matrices without pretreatment. In this study, we developed a PESI-MS method for the determination of MtF in plasma. We used a tandem mass spectrometer equipped with a PESI source in the reaction monitoring mode for the quantitation of MtF. MtF-d6 was chosen as the internal standard (IS), following an isotope dilution (ID) approach. The method was fully validated with six concentration levels (0.5-50 mg/L). The matrix effect was evaluated for each level, and the specificity was tested with a mix of potential co-medications. Using patient samples, the performance was compared with two classical LC-MS-MS and LC-diode array detector (DAD) methods used in external labs. Sample preparation consisted in mixing 10 µL plasma in 1,000 µL ethanol/ammonium formate buffer including MtF-d6 at a fixed concentration of 5 mg/L. The total run time was 0.31 min. ID gave satisfactory results of accuracy and precision (min-max: -12.1 to 15.8% and 1.0-17.1%, respectively). The matrix effect was fully corrected by the internal standard (bias < 1%). The specificity study also reported satisfactory results. Finally, in a representative group of 29 patients (55% with a concentration <5 mg/L, 38% with a concentration >5 mg/L and 7% not detected), we observed almost identical results when comparing LC-DAD and LC-MS-MS to PESI-MS (r2 > 0.99). We propose a specific, sensitive, accurate and ultrafast solution for the measurement of MtF in patient plasma, with no sample preparation or calibration curve building. This could be helpful in a core lab when rapid diagnosis of LA is needed.

Flecainide-induced pneumonitis: a case report.

BACKGROUND: We report a case of acute respiratory distress associated with a histological pattern of acute fibrinous and organizing pneumonia, and discuss the possible responsibility of flecainide therapy. CASE PRESENTATION: A 61-year-old African woman developed a rapidly progressive dyspnea and required admission in the intensive care unit for orotracheal intubation and mechanical ventilation. Chest X-ray examination revealed bilateral infiltrates predominating in the basal part of both lungs. Lung computed tomography disclosed bilateral ground-glass opacities and septal thickening. After exclusion of the most common causes of infectious or immune pneumonia, a toxic origin was investigated and flecainide toxicity was considered. Lung biopsy was consistent with the unusual pattern of acute fibrinous and organizing pneumonia. Clinical and radiological improvement was noted after corticosteroid therapy, but the patient died from septic complications. CONCLUSION: Flecainide-induced lung injury has rarely been reported in the literature and remains a diagnosis of exclusion. The histological pattern of acute fibrinous and organizing pneumonia has been previously observed with amiodarone. There are no firm guidelines for the treatment of acute fibrinous and organizing pneumonia, but some patients may positively respond to corticosteroids.

Impacts of dietary exposure to pesticides on faecal microbiome metabolism in adult twins.

BACKGROUND: Dietary habits have a profound influence on the metabolic activity of gut microorganisms and their influence on health. Concerns have been raised as to whether the consumption of foodstuffs contaminated with pesticides can contribute to the development of chronic disease by affecting the gut microbiome. We performed the first pesticide biomonitoring survey of the British population, and subsequently used the results to perform the first pesticide association study on gut microbiome composition and function from the TwinsUK registry. METHODS: Dietary exposure of 186 common insecticide, herbicide, or fungicide residues and the faecal microbiome in 65 twin pairs in the UK was investigated. We evaluated if dietary habits, geographic location, or the rural/urban environment, are associated with the excretion of pesticide residues. The composition and metabolic activity of faecal microbiota was evaluated using shotgun metagenomics and metabolomics respectively. We performed a targeted urine metabolomics analysis in order to evaluate whether pesticide urinary excretion was also associated with physiological changes. RESULTS: Pyrethroid and/or organophosphorus insecticide residues were found in all urine samples, while the herbicide glyphosate was found in 53% of individuals. Food frequency questionnaires showed that residues from organophosphates were higher with increased consumption of fruit and vegetables. A total of 34 associations between pesticide residue concentrations and faecal metabolite concentrations were detected. Glyphosate excretion was positively associated with an overall increased bacterial species richness, as well as to fatty acid metabolites and phosphate levels. The insecticide metabolite Br2CA, reflecting deltamethrin exposure, was positively associated with the phytoestrogens enterodiol and enterolactone, and negatively associated with some N-methyl amino acids. Urine metabolomics performed on a subset of samples did not reveal associations with the excretion of pesticide residues. CONCLUSIONS: The consumption of conventionally grown fruit and vegetables leads to higher ingestion of pesticides with unknown long-term health consequences. Our results highlight the need for future dietary intervention studies to understand effects of pesticide exposure on the gut microbiome and possible health consequences.

What Is the Therapeutic Reference Range for Levetiracetam? Grand Round/A Case Study.

ABSTRACT: The Therapeutic Drug Monitoring guidelines of Arbeitsgemeinschaft für Neuropsychopharmakologie und Pharmakopsychiatrie had proposed a therapeutic reference range of 10-40 mg/L for levetiracetam in 2011. In the first version of the 2017 update, it was changed to 20-40 mg/L; however, 5 months later, in an erratum version, it was changed back to 10-40 mg/L. In this study, the authors agree with the range to 10-40 mg/L but discuss to what extent a wider interval may be proposed for certain patients.

[Therapeutic drug monitoring of cyamemazine: How to interpret a concentration? A review of literature]. / Suivi thérapeutique pharmacologique de la cyamémazine : comment interpréter une concentration ? Une revue de la littérature.

Cyamemazine is the most prescribed antipsychotic drug in France, often in combination with another antipsychotic, for its sedative and anxiolytic component. Providing to physicians serum concentrations of cyamemazine in different contexts (compliance checking, ineffectiveness, adverse effects, intoxication, modification of pharmacokinetic parameters…) requires to interpret them correctly. This article presents an update on how to interpret a concentration of cyamemazine, wich remains poorly documented. The anxiolysis occurs at steady-state serum trough concentrations of 4 to 5µg/L; the antipsychotic effect from 18-20µg/L. Cyamemazine is not a drug with a narrow therapeutic window and concentrations up to 400µg/L may be sought in cases of partial efficacy; concentrations of 1800µg/L might be fatal; lower concentrations might be fatal if association with high others concentrations of drugs.

Unexplained Metabolic Acidosis: Alcoholic Ketoacidosis or Propylene Glycol Toxicity.

INTRODUCTION: Severe metabolic acidosis with elevated anion and osmol gap is suggestive of toxic alcohol ingestion. The absence of detectable methanol or ethylene glycol in the serum could mean that metabolism is complete or that other hypotheses have to be considered. Ingestion of less common alcohol or alcoholic ketoacidosis should be investigated as illustrated by the present observation. CASE REPORT: A 46-year-old woman was admitted with altered consciousness in the Emergency Department. In the presence of a high anion gap (peak value 39 mEq/L) metabolic acidosis with mildly increased osmol gap (peak value 19 mOsm/kg), there was a high suspicion of toxic alcohol ingestion in an individual with alcohol use disorder (AUD). Serum arterial lactate concentration was particularly high at 27 mmol/L. Urinalysis failed to reveal the presence of ketone bodies or oxalate crystals. The results of the serum determination of ethanol, methanol, ethylene glycol, and isopropanol were obtained within 2 h and were negative. Due to the severity of lactic metabolic acidosis and the persisting suspicion of intoxication by a less common toxic alcohol, antidotal therapy with ethanol was initiated together with hemodialysis. Correction of lactic metabolic acidosis was obtained. Results of urinalysis obtained later revealed the presence not only of propylene glycol and D-lactate but also of significant concentrations of ß-hydroxybutyrate as a marker of alcoholic ketoacidosis. DISCUSSION: The combination of propylene glycol ingestion and alcoholic ketoacidosis may have contributed to the severity of lactic acidosis.

[Therapeutic drug monitoring of antidepressants: Why venlafaxine is the most monitored drug? A review of literature]. / Suivi thérapeutique pharmacologique des antidépresseurs : pourquoi la venlafaxine est le médicament le plus surveillé ? Une revue de la littérature.

Venlafaxine is the third most frequently prescribed antidepressant in France the last decade, with about 400,000 daily doses. Therapeutic drug monitoring (TDM) of this medication, by measuring the active moiety venlafaxine (V) and O-desmethylvenlafaxine (ODV), is recommended (level of recommendation 2). However, this antidepressant seems to be the one for which clinicians most often use TDM, much more frequently than escitalopram, which is more prescribed and for which TDM is also recommended. The main goal of this review is to provide an update on the TDM of venlafaxine: its therapeutic interval, its level of recommendation and the origin of its "success". From the literature does not enable to define a therapeutic interval for the active moiety V+ODV, that is to say a steady-state trough concentration allowing a clinical response without toxicity. Nevertheless, a target concentration from 100 to 400µg/L is certainly relevant for the majority of patients without any pharmacodynamic resistance ; though a greater concentration could result in an earlier response or could be required for a clinical response in a minority of patients. A patient with no clinical response despite a concentration greater than 1000µg/L should be proposed another antidepressant. Measurement of the ODV/V ratio is also a useful tool, values below 0.3 usually reflecting a slow metabolizer phenotype for cytochrome P-450 2D6, which is more at risk of adverse effects. Research for this phenotype probably explains many prescriptions for TDM.

MRP4 is responsible for the efflux transport of mycophenolic acid ß-d glucuronide (MPAG) from hepatocytes to blood.

Mycophenolic acid (MPA) has become a cornerstone of immunosuppressive therapy, in particular for transplant patients. In the gastrointestinal tract, the liver and the kidney, MPA is mainly metabolized into phenyl-ß-d glucuronide (MPAG). Knowledge about the interactions between MPA/MPAG and membrane transporters is still fragmented. The aim of the present study was to explore these interactions with the basolateral hepatic MRP4 transporter. The inhibition of the MRP4-driven transport by various drugs which can be concomitantly prescribed was also evaluated. In vitro experiments using vesicles overexpressing MRP4 showed an ATP-dependent transport of MPAG driven by MRP4 (Michaelis-Menten constant of 233.9 ± 32.8 µM). MPA was not effluxed by MRP4. MRP4-mediated transport of MPAG was inhibited (from -43% to -84%) by ibuprofen, cefazolin, cefotaxime and micafungin. An in silico approach based on molecular docking and molecular dynamics simulations rationalized the mode of binding of MPAG to MRP4. The presence of the glucuronide moiety in MPAG was highlighted as key, being prone to make electrostatic and H-bond interactions with specific residues of the MRP4 protein chamber. This explains why MPAG is a substrate of MRP4 whereas MPA is not.

First-Line Toxicological Screening with Fully Automated Extraction.

In clinical toxicology, laboratories need screening methods allowing unambiguous identification of the compounds in a short turnaround time to either confirm or exclude the hypothesis of drug overdose or poisoning with a toxicant. We developed a fully automated screening procedure designed to identify and quantify in a single run 245 compounds of interest in clinical toxicology. Sample extraction was carried out by a programmable liquid handler directly coupled to a liquid chromatography-tandem mass spectrometry (LC-MS-MS) system. Data acquisition was performed in the positive and negative ionization modes with up to 15 multiple reaction monitoring (MRM) transitions per compound, each with optimized collision energy to enable both qualitative library searching and quantitation. The method was validated according to the ISO 15189 requirements and was applied to real patient samples (n = 127). The 15 MRM transitions per compound provided higher confidence for the identification of all the compounds. The quantitative method was fully validated with satisfactory intra- and inter-assay imprecision and inaccuracy with CV% lower than 20%. For only nine molecules, imprecision and inaccuracy were relatively high but never exceeded 31.7%. Comparison with dedicated quantitative methods using conventional MRM monitoring performed using 127 patient samples (n = 175 pairs of measured concentrations) showed excellent correlation (R2 = 0.96). A robustness study showed that calibration curves prepared for up to 1 month yielded uncertainty < 20%. Retention times ranged from 0.89 min for metformin to 9.72 min for difenacoum. The automated sample preparation required 8 min and was followed by 10 min chromatographic separation. This first-line screening procedure yields high confidence in compound detection and should be useful in core labs facing clinical toxicology situations where rapid and reliable results are needed.