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Monoclonal antibodies (mAbs) represent a major category of biopharmaceutical products which due to their success as therapeutics have recently experienced the emergence of mAbs originating from different types of trafficking. We report the development of an analytical strategy which enables the structural identification of mAbs in addition to comprehensive characterization and quantification in samples in potentially counterfeit samples. The strategy is based on the concomitant use of capillary zone electrophoresis analysis (CZE-UV), size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and liquid chromatography hyphenated to tandem mass spectrometry (LC–MS/MS). This analytical strategy was applied to the investigation of different samples having unknown origins seized by the authorities, and potentially incorporating an IgG 4 or an IgG 1. The results achieved from the different techniques demonstrated to provide orthogonal and complementary information regarding the nature and the structure of the different mAbs. Therefore, they allowed to conclude unequivocally on the identification of the mAbs in the potentially counterfeit samples. Finally, a LC–MS/MS quantification method was developed which specificity was to incorporate a different mAbs labeled with stable isotopes as internal standard. The LC–MS/MS quantification method was validated and thus demonstrated the possibility to use common peptides with the considered IgG in order to achieve limit of quantification as low as 41.4 nM. The quantification method was used to estimate the concentration in the investigated samples using a single type of internal standard and experimental conditions, even in the case of mAbs with no stable isotope labeled homologues available. Graphical abstract Supplementary Information The online version contains supplementary material available at 10.1007/s00216-022-03913-y.

Background aims Cryopreserved cellular products, as parts of hematopoietic progenitor cell (HPC) transplants, mononuclear cell reinjections for donor lymphocyte infusion or extracorporeal photopheresis, can be washed before being reinjected into the patient or infused directly, depending on local practices. The aim of washing is to reduce the incidence and severity of adverse reactions (ARs) due to the dimethyl sulfoxide (DMSO) used as a cryoprotective agent and other factors, such as dead cell debris. At the authors’ cell therapy laboratory (CTL) in Poitiers, France, as in 76% of Etablissement Francais du Sang (EFS) CTLs, all cryopreserved products undergo thawing in a water bath followed by washing with the COBE 2991. As this device will soon cease to be available, an alternative process needs to be assessed. Methods The authors compared two closed systems: the authors’ semi-automatic system using the traditional centrifugation method (COBE 2991) and an automated device using spinning membrane filtration (Lovo). A total of 72 HPC bags available for research were used. The authors first performed a paired comparison, processing one or two HPC bags washed by each device. A second study was carried out to compare two different washing solutions generally used by EFS CTLs along with variable storage conditions. Finally, the authors studied the efficiency of the Lovo with three or four thawed bags. The main parameters studied were viable CD34+ cell recovery and viability, CD3+ cell recovery, stability up to 6 h after washing, DMSO elimination and center feasibility. Results The Lovo device showed better CD34+ cell recovery compared with the COBE 2991 while maintaining CD34+ viability and stability over 6 h. Moreover, Lovo efficiency seemed to be independent of the number of thawed bags processed and washing solution used in the authors’ study. CD3+ cell recovery met the authors’ internal specifications (cell recovery >50%), with similar results seen when processing with either the COBE 2991 or Lovo. Additionally, on average, 97% of DMSO was removed after washing with Lovo, minimizing the risk of ARs. The storage conditions post-processing indicated preferred storage conditions of 7 ± 3°C. Finally, if processing time seemed shorter using COBE 2991 for one bag washed, the Lovo device required only one staff member regardless of the number of HPC bags processed. Conclusions The Lovo device seems to provide an opportunity to standardize HPC processing, ensuring patient safety, with, on average, 97% of DMSO removed, while improving recovery of cells of interest and maintaining viability over time in case of delayed transplant. The Lovo device consequently seems to be a serious alternative to the COBE 2991.

Resume Une valeur de pH plasmatique inferieure a 7,35 caracterise une acidose respiratoire, metabolique ou mixte. L’exploration des acidoses metaboliques est souvent limitee a la mesure des lactates plasmatiques sans identification ni quantification d’autres acides organiques dont la detection permettrait de preciser l’etiologie des acidoses metaboliques qui peuvent etre d’origine genetiques, pathologiques ou toxicologiques. Les methodes analytiques classiques comme la chromatographie gazeuse ou liquide sont souvent mal adaptees a la separation des acides organiques qui sont des composes de faible poids moleculaire, hydrosolubles et chargees. Nous avons developpe une methode par electrophorese capillaire de zone permettant de separer en moins de 8 minutes 14 acides organiques et 4 anions mineraux. Notre methode permet la quantification des neufs principaux acides organiques retrouves dans des grandes pathologies (diabete, insuffisance renale) ou lors de la consommation de medicaments, de drogues ou de toxiques. Notre methode est lineaire dans le plasma entre 10 et 2 000 mg/L, et dans l’urine entre 25 et 3 000 mg/L pour chacun des 9 acides. Les limites de quantification sont de 10 mg/L et 25 mg/L pour le plasma et les urines, respectivement. Pour la repetabilite et la fidelite intermediaire, les coefficients de variation sont inferieurs a 15 % et les valeurs de biais sont comprises entre ± 10 %. Pour les 2 matrices, les pourcentages de recuperation sont compris entre 80,4 % et 110 %. La specificite de notre methode est assuree par la reproductibilite des temps de migration. Nous n’avons pas mis en evidence d’interference, en particulier avec les anticoagulants comme le citrate, l’EDTA et l’heparine. A travers 15 exemples, nous montrons que cette approche par electrophorese capillaire permet de preciser l’etiologie d’une acidose, quantifier les acides organiques impliques dans differents domaines d’application allant de la clinique au medico-legal, en passant par l’experimentation animale.

An increase of pyroglutamic acid, or 5-oxoproline plasmatic concentration was reported in metabolic acidosis observed after chronic intake of some drugs, as acetaminophen. We developed a simple, fast and reproducible method by capillary zone electrophoresis using a commercial Anion Analysis Kit

Resume La 3,4-methylenedioxymetamphetamine (MDMA) ou ecstasy est connue comme drogue recreative illegale et connait actuellement un regain d’interet dans les soirees festives aupres des jeunes. Elle est consommee pour ses effets empathogenes et entactogenes et les nouvelles tendances de consommations dans ce type de soirees l’associent frequemment au GHB et a l’alcool. Des comprimes tres colores et de formes variees sont retrouves sur les marches pour etre les plus attractifs possible. Et ces dernieres annees, on observe une nette augmentation de leurs teneurs en MDMA. Le depistage rapide de la MDMA par methodes immunochimiques peut etre realise sur des automates de laboratoire ou sur des tests rapides sur les urines ou plus recemment sur des prelevements salivaires. Une methode de confirmation en chromatographie couplee a de la spectrometrie de masse permet d’identifier de facon specifique la MDMA parmi les differentes et nombreuses structures amphetaminiques. Ces recherches et/ou dosages peuvent etre realisees sur de nombreuses matrices biologiques (sang, urine, salive, cheveux etc.) dans des contextes cliniques ou en medico-legal. Les tableaux d’intoxications fatales ou non fatales avec atteinte hepatique sont rapportes dans la litterature avec des concentrations tres variables observees.

Resume L’utilisation des spots de sang seche en biologie remonte aux travaux de Guthrie appliques initialement au depistage neonatal de la phenylcetonurie, puis etendus depuis au depistage d’une dizaine de maladies hereditaires. Initialement qualitative ou semi-quantitative, la methode des spots de sang seche (DBS – Dried Blood Spot) a ete appliquee a la quantification de nombreux analytes, avec une difficulte majeure due au manque de precision du volume de sang preleve. Parmi les facteurs influencant ce volume, l’hematocrite est sans nul doute le plus limitant. Deux nouveaux systemes dits volumetriques, HemaPENTM et MitraTM ont permis de s’affranchir de la valeur de l’hematocrite. Ils permettent de prelever de facon precise des volumes compris entre 2,5 et 30 µL. Comme les DBS, ces nouveaux systemes presentent de nombreux avantages comme la facilite de transport et de stockage. Cependant, les faibles volumes de sang preleves, s’ils sont particulierement adaptes aux etudes en pediatrie ou a l’experimentation animale, necessitent le developpement de methodes analytiques sensibles. Ces methodes sont en general des methodes chromatographiques couplees a de la spectrometrie de masse validees avec une phase de preparation d’echantillon plus longue que pour des prelevements classiques. Malgre leurs couts eleves, les systemes volumetriques ont permis de developper de nombreuses applications quantitatives dans de multiples domaines de la biologie, comme la pharmacocinetique, le suivi therapeutique, la toxicologie clinique et medico-legale.

Resume Nous rapportons le cas d’une intoxication non mortelle d’un homme de 21 ans admis en reanimation apres consommation d’ecstasy et d’alcool dans un contexte de « binge drinking ». Des prelevements de sang, d’urine, de cheveux et un echantillon du produit consomme ont ete adresses au laboratoire pour analyse toxicologique. Un screening par methodes immunochimique, enzymatique, LC-DAD/MS, LC-MS/HR et GC-MS a ete realise. La presence de MDMA et d’une alcoolemie a 2,19 g/L sont rapportees dans le bilan d’admission. Des concentrations elevees de MDMA sont ensuite mesurees a 68 644 ng/mL dans le plasma, 73 250 ng/mL dans l’urine et 112 000 pg/mg dans les cheveux. Les concentrations de son metabolite, la MDA restent plus faibles et respectivement dans les trois matrices de 547 ng/mL, 690 ng/mL et de 1410 pg/mg. Le produit consomme ne contenait pas d’autres substances actives que de la MDMA a un taux de purete estime a 53 %. A notre connaissance, ce cas non mortel est le premier decrit dans la litterature a ces niveaux de concentrations tres elevees dans le plasma, les urines et les cheveux.

Summary Objectives Filters used in continuous renal replacement therapy (CRRT) induce elimination by filtration, dialysis, and adsorption. The worldwide used ST150® filter adsorbs cytokines. However, adsorption is a non-specific process which might alter the pharmacokinetics of drugs. Pharmacodynamic/pharmacokinetic relationship of aminoglycosides evidences the importance of the peak concentration at the first dose. We hypothesize an in vitro study may clarify the routes of elimination of aminoglycosides using the ST150® filter. Methods Prismaflex® and the STX150® filter, Baxter-Gambro were used. The diafiltration mode combined flowrates of dialysis and filtration at 2.5/1.5 L/h, respectively, over 6 h. One ionic solute was used in the different compartments. Pharmacokinetic analyses were performed using the NeckEpur® software. Results Percentages of gentamicin, tobramycin, and amikacin eliminated from the central compartment were 97 ± 1, 95 ± 3, and 94 ± 6, %, respectively. The clearances were 8.4 ± 2.3, 5.4 ± 5, and 4.2 ± 0.4 L/h, respectively. The contributions of dialysis, filtration, and adsorption for gentamicin, tobramycin, and amikacin were 34.3 ± 2.1, 0 ± 0, and 67.7 ± 2.1; 51.1 ± 1.6, 6.3 ± 3.1, and 46.3 ± 2.0, and 37.8 ± 6.3, 46.3 ± 2.0, and 16.0 ± 5.7%, respectively. Among physico-chemical properties, the rate of adsorption linearly and inversely correlated with the polar surface area of aminoglycosides (Y = −0.44 X + 161.7; R2 = 0.9993). Discussion Using the ST150® filter, dialysis, filtration, and adsorption play a role depending on the chemical structure of aminoglycosides. In the diafiltration mode, elimination of gentamicin and tobramycin by filtration is not detected or weak, respectively. Adsorption should be considered as a potential adverse effect of CRRT. Polar surface area of drugs is a physico-chemical parameter which should be considered regarding adsorption of drugs in filters. The risk needs to be systematically assessed.

Introduction: Adsorption of gentamicin in a polyacrylonitrile filter was previously evidenced in a session lasting 6 h using the NeckEpur model. We extended the study over three consecutive days to mimic the 72-h life span of a filter. Methods: Prismaflex® monitor and ST150® filter were used in the continuous diafiltration (CDF) mode at a 2.5 L/h flowrate. The daily session started with a 6-h session of CDF. Thereafter, the 5-L central compartment was changed using a bag free of gentamicin to assess gentamicin release over the following 18 h. Experiments were repeated on Day 2 and stopped at the end of the 6-h session of CDF on Day 3. The experiment was performed in duplicate. Results: At a 2.5 L/h diafiltration flowrate, the mean daily clearances of gentamicin were 5.5, 4.0, and 3.3 L/h, respectively. The mean diafiltration and adsorption ratios in the daily elimination of gentamicin were 32/68%, 58/42%, and 88/12%, respectively. During days 1 and 2, the mean amount of gentamicin released from the ST150® filter were 14 and 34 mg, respectively. Conclusion: The pharmacokinetics of gentamicin over 3 days is strongly altered by adsorption in the same filter with a progressive decrease of elimination by adsorption, suggesting saturation of the filter. One limitation of our study results from the mode of administration using a bolus dose instead of an infusion over 30 min. Adsorption adds a clearance to those of diafiltration. The time-dependency of gentamicin clearance precludes using a constant dosage regimen over the filter’s life span.

Monoclonal antibodies (mAbs) represent a dynamic class of biopharmaceutical products, as evidenced by an increasing number of market authorizations for mAb innovator and biosimilar products. Stability studies are commonly performed during product development, for instance, to exclude unstable molecules, optimize the formulation or determine the storage limit. Such studies are time-consuming, especially for mAbs, because of their structural complexity which requires multiple analytical techniques to achieve a detailed characterization. We report the implementation of a novel methodology based on the accelerated stability assessment program (ASAP) in order to model the long-term stability of mAbs in relation to different structural aspects. Stability studies of innovator infliximab and two different biosimilars were performed using forced degradation conditions alongside in-use administration conditions in order to investigate their similarity regarding stability. Thus, characterization of post-translational modifications was achieved using liquid-chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the formation of aggregates and free chain fragments was characterized using size-exclusion chromatography-multi-angle light scattering (SEC-MALS-UV/RI) analysis. Consequently, ASAP models were investigated with regard to free chain fragmentation of mAbs concomitantly with N57 deamidation, located in the hypervariable region. Comparison of ASAP models and the long-term stability data from samples stored in intravenous bags demonstrated a relevant correlation, indicating the stability of the mAbs. The developed methodology highlighted the particularities of ASAP modeling for mAbs and demonstrated the possibility to independently consider the different types of degradation pathways in order to provide accurate and appropriate prediction of the long-term stability of this type of biomolecule.

Introduction: In continuous renal replacement therapy, conduction and convection are controlled allowing prescribing dosage regimen improving survival. In contrast, adsorption is an uncontrolled property altering drug disposition. Whether adsorption depends on flowrates is unknown. We hypothesized an in vitro model may provide information in conditions mimicking continuous renal replacement therapy in humans. Methods: ST150®-AN69 filter and Prismaflex dialyzer, Baxter-Gambro were used. Simulated blood flowrate was set at 200 mL/min. The flowrates in the filtration (continuous filtration), dialysis (continuous dialysis), and diafiltration (continuous diafiltration) were 1500, 2500, and 4000 mL/h, respectively. Routes of elimination were assessed using NeckEpur® analysis. Results: The percentages of the total amount eliminated by continuous filtration, continuous dialysis, and continuous diafiltration were 82%, 86%, and 94%, respectively. Elimination by effluents and adsorption accounted for 42% ± 7% and 58% ± 5%, 57% ± 7% and 43% ± 6%, and 84% ± 6% and 16% ± 6% of amikacin elimination, respectively. There was a linear regression between flowrates and amikacin clearance: Y = 0.6 X ± 1.7 (R2 = 0.9782). Conversely, there was a linear inverse correlation between the magnitude of amikacin adsorption and flowrate: Y = –16.9 X ± 84.1 (R2 = 0.9976). Conclusion: Low flowrates resulted in predominant elimination by adsorption, accounting for 58% of the elimination of amikacin from the central compartment in the continuous filtration mode at 1500 mL/h of flowrate. Thereafter, the greater the flowrate, the lower the adsorption of amikacin in a linear manner. Flowrate is a major determinant of adsorption of amikacin. There was an about 17% decrease in the rate of adsorption per increase in the flowrate of 1 L/min.

The metabolic consequences in vivo of various balanced solutions are poorly known in critically ill patients. The main objective of this study was to describe the metabolic consequences of Plasmalyte versus Ringer lactate (RL) in critically ill burn patients, with a special focus on the plasma clearance of buffer anions (i.e., gluconate, acetate, and lactate). We conducted a randomized trial between August 2017 and October 2018 in a tertiary teaching hospital in Paris, France. Patients with burn total body surface area >30% were randomized to receive Plasmalyte or RL. The primary end point was the base excess 24 h after inclusion. The secondary end points were acetate, gluconate, and lactate plasma concentration, the strong ion difference (SID). Twenty-eight patients were randomized. Twenty-four hours after inclusion, plasma BE was not significantly different in the Plasmalyte and RL groups {−0.9 [95% confidence interval (95% CI): −1.8–0.9] vs. −2.1 [95% CI: −4.6–0.6] mmol/L, respectively, P = 0.26}. Plasma gluconate concentration was higher in the Plasmalyte group ( P < 0.001), with a maximum level of 1.86 (95% CI: 0.98–4.0) mmol/L versus 0 (95% CI: 0–0.15) mmol/L. Plasma acetate and lactate were not significantly different. Ionized calcium level was lower in the Plasmalyte group ( P = 0.002). Hemodynamics did not differ between groups. To conclude, the alkalinizing effect of Plasmalyte was less important than expected with no difference in base excess compared with RL, in part due to gluconate accumulation. Acetate and lactate did not significantly accumulate. Plasmalyte led to significantly lower ionized calcium levels. NEW & NOTEWORTHY During fluid resuscitation in burns the alkalinizing effect of Plasmalyte was less important than expected, with no difference in base excess compared with Ringer lactate (RL), in part due to gluconate accumulation. Acetate and lactate did not significantly accumulate. Plasmalyte led to significantly lower ionized calcium levels.

Introduction: Continuous renal replacement therapy (CRRT) efficiently eliminates fluconazole. However, the routes of elimination were not clarified. Adsorption of fluconazole by filters is a pending question. We studied the elimination of fluconazole in a model mimicking a session of CRRT in humans using the NeckEpur® model. Two filters were studied. Methods: The AV1000®-polysulfone filter with the Multifiltrate Pro. Fresenius and the ST150®-polyacrylonitrile filter with the Prismaflex. Baxter-Gambro were studied. Continuous filtration used a flowrate of 2.5 L/h in post-dilution only. Session were made in duplicate. Routes of elimination were assessed using the NeckEpur® model. Results: The mean measured initial fluconazole concentration (mean ± SD) for the four sessions in the central compartment (CC) was 14.9 ± 0.2 mg/L. The amount eliminated from the CC at the end of 6 h-session at a 2.5 L/h filtration flowrate for the AV1000®-polysulfone and the ST150®-polyacrylonitrile filters were 90%–93% and 96%–94%, respectively; the clearances from the central compartment (CC) were 2.5–2.6 and 2.4–2.3 L/h, respectively. The means of the instantaneous sieving coefficient were 0.94%–0.91% and 0.99%–0.91%, respectively. The percentages of the amount eliminated from the CC by filtration/adsorption were 100/0%–95/5% and 100/0%–100/0%, respectively. Conclusion: Neither the ST150®-polyacrylonitrile nor the AV1000®-polysulfone filters result in any significant adsorption of fluconazole.

During pharmaceutical development, the stability of the product is assessed during long-term study. If any stability issues are discovered at this point of the process, it will result in re-formulation and important loss of time and cost. Therefore, important efforts are made in order to select the most stable product. Nevertheless, predicting the stability of the developed product at early stage of the development is challenging. Accelerated stability assessment program (ASAP), based on modified Arrhenius equation and isoconversion approach, appears as an interesting tool allowing to evaluate stability and shelf-life of pharmaceutical product in a short period of time. Nevertheless, few studies using these approaches are published in the literature, and the majority concern small drug molecules. Here, this approach was applied on a small drug molecule, ascorbic acid (AA), and on a cyclic hexapeptide named cFEE. AA and cFEE have been exposed to various temperatures for a maximum of 3 weeks, and then analyzed by capillary electrophoresis coupled to UV detection (CZE-UV) for AA or LC–MS for cFEE. The level of major degradation products was used to build ASAP models and predict the stability of both compounds. Comparison between predicted and long-term data were found accurate for both compounds undergoing two different degradation pathways (oxidation and hydrolysis), confirming the real interest of accelerated predicting stability approach for consistent determination of long-term stability shelf-life of pharmaceutical products.

International audience; Background: Tramadol poisoning rarely causes serotonin toxicity, which mechanisms remain poorly understood. We investigated alterations in tramadol pharmacokinetics in a tramadol-poisoned patient who presented with marked and prolonged serotonin toxicity.Case report: A 21-year-old male self-ingested 750 mg-tramadol, 200 mg-sotalol, 400 mg-propranolol and 6 mg-lorazepam. He was a kidney transplant patient treated with mycophenolate, tacrolimus, prednisone, and paroxetine. He developed transitory cardiovascular failure and prolonged serotonin toxicity requiring sedation, muscle paralysis, and cyproheptadine, with a favorable outcome.Methods: We measured plasma concentrations of tramadol, M1, M2, and M5 using liquid-chromatography-tandem mass spectrometry, calculated elimination half-lives and metabolic ratios of the compounds, and genotyped cytochromes involved in tramadol metabolism.Results: Elimination half-lives of tramadol (6.1 h) and M1 (7.1 h) were normal while those of M2 (26.5 h) and M5 (16.7 h) prolonged. M1 metabolic ratio (0.12) was 2-fold reduced, M2 metabolic ratio (197) 1000-fold increased and M5 metabolic ratio (0.12) normal. This metabolic profile in a patient with normal CYP2D6-metabolizer status based on genotyping supports CYP2D6 inhibition by paroxetine and propranolol, two strong mechanism-based inhibitors. Only M2 present in sufficient concentrations up to 48 h could explain the prolonged serotonin toxicity.Conclusion: Marked and prolonged serotonin toxicity was attributed to increased M2 production due to paroxetine- and propranolol-related CYP2D6 inhibition of tramadol metabolism.

Objectifs Developper et valider une methode rapide de dosage de la ricinine par chromatographie liquide couplee a la spectrometrie de masse en haute resolution (LC-HRMS) pour le suivi des intoxications par la ricine. Methode Le dosage plasmatique et urinaire de la ricinine ete developpe en LC-HRMS (Q Exactive Focus™, Thermo Scientific) sur une colonne Luna Omega C18™ (100 × 2,1 mm, 3 μm) (Phenomenex) a 50 °C avec un gradient d’elution eau/acetonitrile (%, v/v), en mode ESI positif couple a une detection en mode full scan-SRM (transition : 165,0659 > 138,0550). Les echantillons biologiques sont prepares en melangeant 100 μL de plasma ou d’urines et 10 ng de ricine-D3 utilisee comme etalon interne. L’extraction est realisee sur micro-SPE Strata-X™ (Phenomenex) sous pression positive (Preston 1000™, Phenomenex) par 50 μL d’un melange eau/methanol (90/10 %, v/v). Apres agitation, 10 μL du melange sont injectes dans le systeme chromatographique. La methode a ete appliquee au suivi d’une intoxication chez une patiente de 47 ans ayant ingere entre 30 et 40 graines de ricin, conjointement a de la cyamemazine et de l’alcool. A son arrivee dans le service de Reanimation, 8 heures apres l’ingestion de graines, la patiente est consciente sans particularite clinique ni biologique. L’evolution, marquee par un episode de convulsions, des diarrhees, et un syndrome extrapyramidal, est favorable et la patiente sort apres une semaine d’hospitalisation. Resultats Dans nos conditions chromatographiques, la ricine est eluee en 1,30 minutes, avec un temps d’analyse total de 4 minutes. Pour les deux matrices biologiques, la methode est lineaire entre 0,25 et 500 ng/mL avec une limite de quantification a 0,25 ng/mL. Les coefficients de variation calcules sur les 3 controles de qualite (0,75, 20 et 400 ng/mL) sont inferieurs a 8 % et 12 % pour la repetabilite et la fidelite intermediaire, respectivement. Aucun effet matrice, et aucun « carryover » n’ont ete detectes. Chez notre patiente, le bilan toxicologique d’admission permet de depister du THC-COOH dans les urines, du paracetamol (21 mg/L) et de la cyamemazine (0,75 mg/L) dans le plasma. Le suivi de la ricinine a ete realise sur 18 echantillons (12 plasmas et 6 urines) recueillis sur 5 jours. Les concentrations plasmatiques de ricinine etaient comprises entre 193 (H + 8 h) et 44 ng/mL (H + 134 h) avec une demi-vie estimee a 70 heures. Les concentrations urinaires etaient comprises entre 535 ng/mL (207 ng/mmol de creatinine) a H + 8 heures et 367 ng/mL (204,5 ng/mmol de creatinine) a H + 100. Conclusion La ricine, puissante toxine naturelle, est retrouvee dans les graines de Ricinus communis, toujours associee a la ricinine, un alcaloide piperidinique. La signature d’un empoisonnement par la ricine est basee sur la detection et le dosage de la ricinine, meme si le dosage ne reflete pas la gravite de l’intoxication. Chez notre patiente, la concentration plasmatique en ricinine est une des plus elevees de celles decrites dans la litterature, comparable a celle rapportee recemment apres ingestion de 50 graines [1] . Nous rapportons une demi-vie plasmatique alors que peu de donnees sur la pharmacocinetique de la ricinine sont decrites. Les dosages realises pour cette patiente, ainsi que pour 4 autres patients ayant ingere des graines de ricin, montrent qu’il n’y a pas de relation entre les concentrations plasmatiques, le nombre de graines supposees ingerees, les signes cliniques et l’evolution de l’intoxication. En conclusion, nous avons developpe une methode specifique et sensible permettant un dosage de la ricinine en moins de 30 minutes, applicable en urgence 24/24 au suivi des patients intoxiques volontairement ou non, mais egalement permettant de surveiller une population dans des situations d’attentats chimiques.

Background Hydrocortisone premedication reduces the risk of antibodies to infliximab [ATIs] formation in patients receiving infliximab [IFX] therapy for inflammatory bowel disease [IBD]. Aim We aimed to determine the safety of hydrocortisone premedication withdrawal in IBD patients with sustained clinical response on maintenance therapy with IFX. Methods We performed an observational prospective pharmacoclinical study in a tertiary referral centre, including all consecutive IBD outpatients with no previous IFX infusion reaction and in clinical remission on maintenance IFX [alone or in combination therapy] for at least 6 months. This cohort was followed for 1 year after discontinuation of hydrocortisone premedication. Results Among the 268 IBD outpatients, 95 patients met the inclusion criteria [mean age 38 years; 64% male; 80% Crohn’s disease; 45% combination therapy]. The median IFX duration was 5 years [0.54–14] with a mean infused dose of 533 mg [200–1000] and a mean interval duration of 7.9 weeks [4–10]. None of the patients developed permanent ATIs or infusion-related reaction at 1 year. Four patients developed transient ATIs without loss of clinical response. There was no significant variation of infliximab serum trough levels [5.5 µg/mL vs 5.9 µg/mL] measured at the time of the three IFX infusions before and after hydrocortisone withdrawal. Loss of response rate to IFX was 18% at 1 year. Conclusions Hydrocortisone discontinuation is safe in IBD patients with sustained clinical remission on maintenance therapy with IFX. Our data suggest that routine premedication with hydrocortisone is unnecessary in patients in prolonged remission under IFX maintenance therapy.

The continuous spread of antimalarial drug resistance is a threat to current chemotherapy efficacy. Therefore, characterizing the genetic diversity of drug resistance markers is needed to follow treatment effectiveness and further update control strategies. Here, we genotyped Plasmodium falciparum resistance gene markers associated with sulfadoxine-pyrimethamine (SP) and artemisinin-based combination therapy (ACT) in isolates from pregnant women in Ghana. The prevalence of the septuple IRN I- A/FG K GS/Tpfdhfr/pfdhps haplotypes, including the pfdhps A581G and A613S/T mutations, was high at delivery among post-SP treatment isolates (18.2%) compared to those of first antenatal care (before initiation of intermittent preventive treatment of malaria in pregnancy with sulfadoxine-pyrimethamine [IPTp-SP]; 6.1%; P = 0.03). Regarding the pfk13 marker gene, two nonsynonymous mutations (N458D and A481C) were detected at positions previously related to artemisinin resistance in isolates from Southeast Asia. These mutations were predicted in silico to alter the stability of the pfk13 propeller-encoding domain. Overall, these findings highlight the need for intensified monitoring and surveillance of additional mutations associated with increased SP resistance as well as emergence of resistance against artemisinin derivatives.

In continuous renal replacement therapy, conduction and convection are controlled allowing prescribing dosage regimen improving survival. In contrast, adsorption is an uncontrolled property altering drug disposition. Whether adsorption depends on flowrates is unknown. We hypothesized an in vitro model may provide information in conditions mimicking continuous renal replacement therapy in humans.ST150The percentages of the total amount eliminated by continuous filtration, continuous dialysis, and continuous diafiltration were 82%, 86%, and 94%, respectively. Elimination by effluents and adsorption accounted for 42% ± 7% and 58% ± 5%, 57% ± 7% and 43% ± 6%, and 84% ± 6% and 16% ± 6% of amikacin elimination, respectively. There was a linear regression between flowrates and amikacin clearance: Y = 0.6 X ± 1.7 (RLow flowrates resulted in predominant elimination by adsorption, accounting for 58% of the elimination of amikacin from the central compartment in the continuous filtration mode at 1500 mL/h of flowrate. Thereafter, the greater the flowrate, the lower the adsorption of amikacin in a linear manner. Flowrate is a major determinant of adsorption of amikacin. There was an about 17% decrease in the rate of adsorption per increase in the flowrate of 1 L/min.

In the last two decades the development of biomaterials has demonstrated an unprecedented progress which has been emphasized by the introduction of a significant number of innovative materials. As such, the advent of biomaterials represents a formidable opportunity for the implementation of therapeutic treatments, able to induce tissue or organ regeneration. Meanwhile, the further understanding of complex biological mechanisms and the improvement of the production of biological (macro)-molecules using state-of-the-art biotechnologies have also experienced some major breakthroughs. These progresses have led to their wide adoption for therapeutic purposes. Indeed, different types of proteins, such as monoclonal antibodies or fusion proteins, are currently approved for therapeutic use and the first biopharmaceutical product based on siRNA has been approved by the FDA on the third semester of 2018. As a consequence, recent research articles describe the incorporation of biomolecules to the structure of biomaterials in order, for instance, to deliver locally biological agents capable of favoring the regeneration of the tissue or to promote the integration of the biomaterial within the host. The incorporation of biomolecules to the material requires a dedicated characterization, based on state-of-the-art analytical techniques, such as mass spectrometry (MS), in order to assess the impact on the structure of the biomolecule and ensure the preservation of the biological activity. From another aspect the implantation of foreign material to a living organism involves some major biological consequences that are not completely understood at the moment. The maturation of different -omics research fields, with genomics and proteomics as flagships, has recently offered the possibility to monitor the faint and complex changes involved by the implantation of biomaterials for tissue regeneration. Thus these advanced analytical workflows should provide crucial information regarding the outcome of biomaterial potentially easing the research and development process. The following chapter focuses on the cutting-edge analytical methodologies which can be implemented to investigate the outcome of biomaterial implantation for tissue from these perspectives. The first part intends to provide an extended description of the proteomics analytical workflow which allows to identify and monitor the expression levels of proteins on a large scale in complex biological samples. The second part aims to present the methodologies based on MS used to perform detailed structural characterization of proteins which can be used to ensure the structural integrity of protein integrated to material consequently to their release.

International audience; Ascorbic acid is a powerful antioxidant compound involved in many biological functions, and a chronic deficiency is at the origin of scurvy disease. A simple, rapid, and cost-effective capillary electrophoresis method was developed for the separation and simultaneous quantification of ascorbic acid and the major degradation products: dehydroascorbic acid, furfural, and furoic acid. Systematic optimization of the conditions was performed that enabled baseline separation of the compounds in less than 10 min. In addition to simultaneous quantification of ascorbic acid alongside to the degradation products, stability studies demonstrated the possibility using capillary electrophoresis to separate and identify the major degradation products. Thus, high-resolution tandem mass spectrometry experiments were conducted in order to identify an unknown degradation product separated by capillary electrophoresis and significantly present in degraded samples. Comparison of mass spectrometry data and capillary electrophoresis electropherograms allowed to identify unambiguously trihydroxy-keto-valeraldehyde. Finally, capillary electrophoresis was successfully applied to evaluate the composition of different pharmaceutical preparation of ascorbic acid. Results showed the excellent performance of the capillary electrophoresis method due to the separation of excipients from the compounds of interest, which demonstrated the relevance of using an electrophoretic separation in order to perform comprehensive stability studies of ascorbic acid.

Clinical toxicology is a subspecialty of toxicology dealing with the bedside management of poisoned patients, including definitive toxicological diagnosis, assessment of immediate severity and long-term prognosis, and selection of treatments including antidotes. Knowledge of potential drug and chemical hazards allows a clinical toxicologist to participate in the preparedness for and prevention of chemical intoxications. Toxicants are a major cause of clinical disease. Therefore clinical toxicology is part of internal medicine, strongly benefiting from other medical specialties. Clinical toxicology requires having a strong basis in pharmacology. However, while pharmacology deals with thousands of substances, a clinical toxicologist may deal with tens of thousands of substances. Therefore clinical toxicology has developed its own subdisciplines, including the newest one, toxicodynetics. Close relationships between clinical toxicologists and toxicological scientists are essential for improving recognition, prevention, and treatment of intoxications. Indeed putative intoxications need clarification of the mechanism of toxicity for a definitive diagnosis. Antidotes are nothing but the reward of knowing precisely the pathophysiology of a poisoning event.

International audience; Protein conjugates such as antibody-drugs conjugates (ADCs) represents the next generation of therapeutic proteins. They allow to combine the biological properties of the protein format with the characteristics of the conjugated ligands. The reac-tion implemented to couple ligands to the peptide backbone represents a crucial aspect of the production of protein conju-gates, influencing the nature and the heterogeneity of the conjugates obtained. Here, we report the concomitant use of MALDI-TOF MS and LC-MS/MS analysis to investigate the chemical functionalization of human serum albumin (HSA) by the intermediate of lysine residues, previously used to generate biopharmaceutical agents for medical imaging. A kinetic was performed by collecting samples after different reaction times and analyzing them using the two techniques. MALDI-TOF MS analyses allowed estimating the number of conjugated ligands in a robust manner and assess the global functionaliza-tion kinetic on the intact protein level. Results demonstrated a maximum of 38 modified residues out of the 59 lysines available showing the limitation of the chemical functionalization. Consequently, LC-MS/MS analysis provided a site-specific characterization of the residues undergoing chemical modification. Data exhibited unique properties due to the presence of the ligands which allowed to identify without ambiguity the residues exhibiting different modification rate and enabled the identification of the unmodified lysine. Results were compared to the structure of HSA described from crystal-lography data. The comparison strongly suggested that accessibility is influencing the residues respective reactivity. The relevant complementarity of the different techniques could be emphasized in order to perform an extensive characterization concerning the evolution of the primary structure of the protein during the chemical reaction, providing an improved insight on the conjugation process and offering the potentiality to tune the reaction.

Resume La cause de complications et de deces la plus frequente chez les transporteurs de drogues intracorporels est la toxicite par rupture des boulettes ou paquets ingeres. La prise en charge du patient peut etre adaptee en fonction de l’etat clinique, des resultats de la radiologie et du bilan toxicologique. Nous rapportons deux cas dans une meme affaire, illustrant l’interet de l’analyse toxicologique. Une etude retrospective entre janvier 2010 et juin 2018 decrit quelques donnees actuelles sur des transporteurs intracorporels necessitant une hospitalisation dans un service de reanimation. Deux jeunes hommes ayant ingere a priori des petits ballots d’aspect identique pour dissimulation pendant leur interpellation par la police, ont necessite des hospitalisations separees. Monsieur LR (20 ans) est pris en charge pour arret cardio-respiratoire par un service de reanimation. Sur les 50 petits ballots estimes ingeres, 30 sont recraches immediatement. Il elimine les autres par voies orale et rectale pendant l’hospitalisation. Des prelevements urinaires et sanguins ainsi que trois ballots sont analyses par le laboratoire. Il decede a J14 des suites d’une encephalopathie post-anoxique. Monsieur NC (13 ans) sans antecedents medicaux est hospitalise dans un service medico-chirurgical pediatrique suite a l’ingestion de 20 ballots similaires. Il est asymptomatique a l’entree et 11 sachets sont visualises et retires par endoscopie gastrique en urgence. Seuls des echantillons urinaires et deux ballots sont analyses. Il est hospitalise pendant 5 jours avant d’etre remis aux autorites de police. Le bilan d’entree toxicologique de Monsieur LR (screenings urinaire immunologique et plasmatique par LC-MS/DAD) est negatif. Pour Monsieur NC, seule la cocaine urinaire est depistee par bandelette (SureStep™ Multi-Drug) jusqu’a J3. L’analyse des ballots expulses permet de confirmer la presence de cocaine dans les deux cas en presence d’un ou plusieurs produits de coupe : phenacetine dans le cas de Monsieur LR et phenacetine, cafeine et levamisole dans le cas de Monsieur NC. Le suivi de l’elimination de la cocaine pour Monsieur LR n’a mis en evidence a aucun moment la presence de son metabolite, la benzoylecgonine (BZE) dans les prelevements biologiques. Ces resultats contribuent a orienter rapidement les cliniciens vers une cause d’arret cardiaque non toxique. Pour Monsieur NC, l’absence de consequences cliniques et la negativation de la presence de BZE dans les urines au bout de 72 h ont permis une sortie du patient apres 5 jours d’hospitalisation. L’analyse toxicologique montrait egalement que dans une meme affaire, des boulettes visuellement identiques n’etaient pas constituees par les memes produits de coupe et etaient donc vraisemblablement d’origine differente. L’etude retrospective decrit 44 patients hospitalises en reanimation dans le cadre d’une prise en charge en tant que transporteurs intracorporels. Environ 20 % etaient des consommateurs habituels (cannabis et/ou cocaine). En somme, 63,6 % avaient ete interpelles dans les aeroports parisiens et l’hospitalisation avait alors dure 3 jours (valeur mediane) avec un suivi toxicologique toutes les 12 h. Environ 46 boulettes (valeur mediane avec un maximum de 129 boulettes) contenant de la cocaine dans 77,3 % des cas, ont ete recuperees chez les porteurs hospitalises. Dans 75 % des cas, la recuperation a ete faite par voie basse. Le bilan toxicologique d’entree montrait que 43,2 % des patients etaient positifs en cocaine et 20,4 % en cannabis.

Objective The efficiency of pralidoxime in the treatment of human organophosphates poisoning is still unclear. In a rat model, we showed that pralidoxime induced a complete but concentration-dependent reversal of paraoxon-induced respiratory toxicity. The aim of this study was to assess the efficiency of pralidoxime in a species other than rats. Methods A dose of diethylparaoxon corresponding to 50% of the median lethal dose was administered subcutaneously to male F1B6D2 mice. Ascending single pralidoxime doses of 10, 50-100 and 150 mg kg-1 were administered intramuscularly 30 min after diethylparaoxon administration. Ventilation at rest was assessed using whole-body plethysmography and mice temperature was assessed using infrared telemetry. Results are expressed as mean±SE. Statistical analysis used non-parametric tests. Results From 30 to 150 min post-injection, diethylparaoxon induced clinical symptoms and a decrease in respiratory frequency, which resulted from an increase in expiratory and inspiratory times associated with an increase in the tidal volume. In the 10-, 50- and 100-mg kg-1 pralidoxime groups, there was a trend towards a non-significant improvement of paraoxon-induced respiratory toxicity. The 150 mg kg-1 dose of pralidoxime induced a significant reversal of all respiratory parameters. Conclusion In the present study, a toxic but non-lethal model of diethylparaoxon in awake, unrestrained mice was observed. By administering an equipotent dose of diethylparaoxon to rats, a 150 mg kg-1 dose of pralidoxime administered alone completely reversed diethylparaoxon-induced respiratory toxicity in mice. The dose dependency of reversal suggests that further studies are needed for assessing plasma concentrations of pralidoxime resulting in reversal of toxicity.