[Iatrogenic risk in medical practice. About a clinical case report of urinary lithiasis.] / Le cas clinique du mois. Le risque iatrogène dans la pratique médicale. Le point sur le sujet à propos d'un cas de lithiase urinaire.

A case of simple urinary lithiasis resulting in several severe complications is reported to illustrate the problem of iatrogeny in medical practice. It demonstrates the iatrogenic risk related to any medical treatment, including simple procedures. It is critical to identify the kind of iatrogenic events and their severity to reduce their medical, economical or medico-legal impact for the patient, the physicians and the health care system.

Un cas clinique de lithiase urinaire pourtant banal évoluant vers des complications en cascade est décrit pour illustrer l'importance du risque iatrogène dans la pratique médicale courante. Cet exemple, parmi tant d'autres, démontre le risque inhérent à la prise en charge des pathologies et aux actes médicaux, même lorsque ceux-ci semblent anodins. Il est essentiel d'identifier la nature et la sévérité des événements iatrogènes de façon à réduire l'impact clinique, financier ou médico-légal pour le patient, le clinicien et le système de santé publique.

Capillary electrophoresis as a fragment screening tool to cross-validate hits from chromogenic assay: Application to FXIIa.

In this study, a partial-filling affinity capillary electrophoresis (pf-ACE) method was developed for the cross-validation of fragment hits revealed by chromogenic factor XIIa (FXIIa) assay. Chromogenic assay produces false positives, mainly due to spectrophotometric interferences and sample purity issues. pf-ACE was selected as counter-screening technology because of its separative character and the fact that the target does not have to be attached or tagged. The effects of protein plug length, applied voltage and composition of the running buffer were examined and optimized. Detection limit in terms of dissociation constant was estimated at 400 µM. The affinity evaluation was performed close to physiological conditions (pH 7.4, ionic strength 0.13 mol L-1) in a poly (ethylene oxide)-coated capillary of 75 µm internal diameter x 33 cm length with an applied voltage of 3 kV. This method uncovered chromogenic assay's false positives due to zinc contamination. Moreover, pf-ACE supported the evaluation of compounds absorbing at 405 nm.

[Torsade de pointe resulting from a drug interaction between sotalol and ciprofloxacin]. / Torsade de pointe sur interaction médicamenteuse entre sotalol et ciprofloxacine.

Sotalol is a bêta-blocker and class 3 anti-arrhythmic. Ciprofloxacin is a fluoroquinolone antibiotic used against Gram - germs. Both drugs have a common adverse effect : they increase QT interval with a risk of torsade de pointe. The risk increases even more if other risk factors are present such as old age, female gender, renal failure, high blood pressure and ionic disturbances. Because a long QT interval is not associated with symptoms, only an electrocardiogram can establish the diagnosis. However, it's not rare that a torsade de pointe will reveal it. We report a clinical case of a long QT interval due to the association of sotalol and ciprofloxacin, which led to a torsade de pointe. Intravenous magnesium sulphate is the recommended treatment if haemodynamic parameters are good. If not, an external electric shock may be needed.

Le sotalol est un bêta-bloquant utilisé principalement comme anti-arythmique de classe 3. La ciprofloxacine est un antibiotique de la classe des fluoroquinolones, actif sur les germes Gram négatif. Ces deux médicaments présentent, comme effet secondaire commun, le fait d'augmenter l'espace QT avec un risque de torsade de pointe. Si on y ajoute les autres facteurs de risque d'un allongement de QT que sont notamment l'âge, le sexe féminin, l'insuffisance rénale, l'hypertension artérielle et les troubles ioniques, le risque de torsade de pointe est encore majoré. Comme un QT long ne s'accompagne pas de symptômes, seul l'électrocardiogramme permet d'établir le diagnostic. Il n'est néanmoins pas rare qu'une torsade de pointe le révèle. Nous rapportons ici un cas dont le QT long engendré par une association sotalol-ciprofloxacine s'est manifesté par une torsade de pointe chez une patiente âgée avec insuffisance rénale. Le traitement est le sulfate de magnésium par voie intraveineuse si les paramètres hémodynamiques restent bons. S'ils viennent à se dégrader, un choc électrique externe peut s'avérer nécessaire.

Insulin aggregation assessment by capillary gel electrophoresis without sodium dodecyl sulfate: Comparison with size-exclusion chromatography.

Size-exclusion chromatography (SEC) is a method of choice for the analysis of protein aggregates in pharmaceuticals. The United States and European Pharmacopoeias currently use a SEC method with an acidic pH mobile phase to assess the content of aggregates in insulin formulations. In this article, we analyzed aggregated human insulin samples and demonstrated that both methods under neutral conditions, namely neutral pH SEC (nSEC) and capillary gel electrophoresis (CGE), yield to similar aggregate content contrary to SEC under acidic conditions (aSEC). aSEC showed polymeric complexes that were not observed in nSEC and CGE. During method development, the effect on SEC profiles of arginine and acetonitrile were highlighted. In CGE, the effect of SDS on disruption of non-covalent insulin aggregates was confirmed and the benefit of sodium deoxycholate addition in sieving gel was discussed. The three methods were applied to the analysis of an insulin formulation and similar results to those obtained for human insulin as raw material were observed. Finally, the CGE method was used to study the stability of human insulin under different storage conditions. In view of the obtained results one may question the relevance of the current pharmacopoeia method to study insulin aggregates by emphasizing the importance of the mobile phase composition and pH in SEC. The new CGE method developed is an easy method for studying non-covalent aggregates of insulin, which could be applied to other proteins.

Capillary electrophoretic mobility shift displacement assay for the assessment of weak drug-protein interactions.

Only few reports describe the use of capillary electrophoresis in the context of Fragment Based Drug Discovery (FBDD). In this paper, we will present a generic, fully automated, microscale electrophoretic mobility shift displacement assay that can be used in FBDD for primary screening of weak biomolecular interactions between fragments and target protein. The accuracy and reliability of the present method was demonstrated by measuring the IC50 value of two known fragments inhibiting thrombin, namely benzamidine and p-aminobenzamidine and a relatively weak inhibitor, nafamostat. Furthermore, we built a small chemical library to evaluate the performance and the advantage of our newly developed screening-bioassay compared to the direct affinity capillary electrophoresis-binding assay. The results demonstrate the high discriminatory power of the method and above all its ability to screen neutral, negatively or positively charged molecules, as well as molecules that have no or low UV-VIS absorbance, greatly expanding the scope of the assay. Finally, we proved that this approach is able to discriminate between reversible and irreversible binders. Altogether, this work demonstrates that capillary electrophoresis could constitute an important added value in the arsenal used to screen fragments in drug discovery.

Transverse diffusion of laminar flow profiles as a generic capillary electrophoresis method for in-line nanoreactor mixing: Application to the investigation of antithrombotic activity.

Capillary electrophoresis (CE) instrument was used for the generation of a robust and reliable nanoreactor for enzymatic assays in the context of antithrombotic drug screening. The activity of the screened molecules was monitored in a quick and fully automated fashion using only few nanoliters of reactants. To achieve this goal, the targeted enzyme (thrombin) and the chromogenic substrate with or without the screened inhibitor were injected as separate plugs. The mixing of the reactants was then realized using electrophoretically mediated microanalysis (EMMA) or fast transverse diffusion of laminar flow profiles (TDLFP) procedure. The latest provided better mixing performance and was chosen to investigate the inhibitory potency of a fragment library. This very straightforward and fast CE activity assay showed results in good accordance with a previously developed CE affinity assay that confirms the potential of CE at the early stages of drug discovery, providing not only an efficient nanoscale bioreactor but also a selective and integrated separation device.

[Case report : Emphysematous cystitis]. / Le cas clinique du mois : La cystite emphysémateuse.

The emphysematous cystitis is a rare condition characterized by the presence of air in the wall and/or the bladder lumen. The clinical expression of this cystitis is variable. Some patients complain of abdominal pain or urinary symptoms. Other may present only pneumaturia or be totally asymptomatic. This condition is considered as potentially severe since it can lead to an emphysematous pyelonephritis with septicemia and septic shock. Peritonitis may also occur in case of necrosis and perforation of the bladder wall. However, this negative development can be avoided by a diagnosis and an early treatment, and the emphysematous cystitis become therefore of good prognosis. We are here stating the case of a patient with an emphysematous cystitis with symptoms of pneumaturia and lower urinary tract symptoms.

La cystite emphysémateuse est une affection rare caractérisée par la présence d'air dans la paroi et/ou la lumière vésicale. L'expression clinique de cette cystite est variable. Le plus souvent, elle se manifeste par des douleurs abdominales ou des plaintes urinaires, mais les patients peuvent également présenter une pneumaturie ou être symptomatiques. Cette affection est considérée comme potentiellement sévère puisqu'elle peut évoluer vers une pyélonéphrite emphysémateuse avec septicémie et choc septique ou vers une péritonite en cas de nécrose et perforation de la paroi vésicale. Cependant, cette évolution défavorable peut être évitée par un diagnostic et un traitement précoces et la cystite emphysémateuse est, en réalité, de bon pronostic. Nous rapportons ici le cas d'un patient qui a présenté une cystite emphysémateuse s'étant manifestée par une pneumaturie et des mictalgies.

Partial filling affinity capillary electrophoresis as a useful tool for fragment-based drug discovery: A proof of concept on thrombin.

With the emergence of more challenging targets, a relatively new approach, fragment-based drug discovery (FBDD), proved its efficacy and gained increasing importance in the pharmaceutical industry. FBDD identifies low molecular-weight (MW) ligands (fragments) that bind to biologically important macromolecules, then a structure-guided fragment growing or merging approach is performed, contributing to the quality of the lead. However, to select the appropriate fragment to be evolved, sensitive analytical screening methods must be used to measure the affinity in the µM or even mM range. In this particular context, we developed a robust and selective partial filling affinity CE (ACE) method for the direct binding screening of a small fragment library in order to identify new thrombin inhibitors. To demonstrate the accuracy of our assay, the complex dissociation constants of three known thrombin inhibitors, namely benzamidine, p-aminobenzamidine and nafamostat were determined and found to be in good concordance with the previously reported values. Finally, the screening of a small library was performed and demonstrated the high discriminatory power of our method towards weak binders compared to classical spectrophotometric activity assay, proving the interest of our method in the context of FBDD.

Enantioselective capillary electrophoresis-mass spectrometry of amino acids in cerebrospinal fluid using a chiral derivatizing agent and volatile surfactant.

The sensitivity of coupled enantioselective capillary electrophoresis-mass spectrometry (CE-MS) of amino acids (AAs) is often hampered by the chiral selectors in the background electrolyte (BGE). A new method is presented in which the use of a chiral selector is circumvented by employing (+)-1-(9-fluorenyl)ethyl chloroformate (FLEC) as chiral AA derivatizing agent and ammonium perfluorooctanoate (APFO) as a volatile pseudostationary phase for separation of the formed diastereomers. Efficient AA derivatization with FLEC was completed within 10 min. Infusion experiments showed that the APFO concentration hardly affects the MS response of FLEC-AAs and presents significantly less ion suppression than equal concentrations of ammonium acetate. The effect of the pH and APFO concentration of the BGE and the capillary temperature were studied in order to achieve optimized enantioseparation. Optimization of CE-MS parameters, such as sheath-liquid composition and flow rate, ESI and MS settings was performed in order to prevent analyte fragmentation and achieve sensitive detection. Selective detection and quantification of 14 chiral proteinogenic AAs was achieved with chiral resolution between 1.2 and 8.6, and limits of detection ranging from 130 to 630 nM injected concentration. Aspartic acid and glutamic acid were detected, but not enantioseparated. The optimized method was applied to the analysis of chiral AAs in cerebrospinal fluid (CSF). Good linearity (R(2) > 0.99) and acceptable peak area and electrophoretic mobility repeatability (RSDs below 21% and 2.4%, respectively) were achieved for the chiral proteinogenic AAs, with sensitivity and chiral resolution mostly similar to obtained for standard solutions. Next to l-AAs, endogenous levels of d-serine and d-glutamine could be measured in CSF revealing enantiomeric ratios of 4.8%-8.0% and 0.34%-0.74%, respectively, and indicating the method's potential for the analysis of low concentrations of d-AAs in presence of abundant l-AAs.

Impact of injection solvent composition on protein identification in column-switching chip-liquid chromatography/mass spectrometry.

In shotgun proteomics, the gold standard technique is reversed-phase liquid chromatography coupled to mass spectrometry. Many researches have been carried out to study the effects on identification performances of chromatographic parameters such as the stationary phase and column dimensions, mobile phase composition and flow rate, as well as the gradient slope and length. However, little attention is usually paid to the injection solvent composition. In this study, we investigated the effect of the injection solvent on protein identification parameters (number of distinct peptides, amino acid coverage and MS/MS search score) as well as sensitivity. Tryptic peptides from six different proteins, covering a wide range of physicochemical properties, were employed as training set. Design of experiments was employed as a tool to highlight the factors related to the composition of the injection solvent that significantly influenced the obtained results. Optimal results for the training set were applied to analysis of more complex samples. The experiments pointed out optimising the composition of the injection solvent had a strong beneficial effect on all the considered responses. On the basis of these results, an approach to determine optimal conditions was proposed to maximise the protein identification performances and detection sensitivity.

Identification of protein biomarkers associated with cardiac ischemia by a proteomic approach.

Angina is chest pain induced by ischemia of the heart muscle, generally due to obstruction or spasm of the coronary arteries. People that suffer from average to severe cases of angina have an increased percentage of death before the age of 55, usually around 60%. Therefore, prevention of major complications, optimizing diagnosis, prognosis and therapeutics are of primary importance. The main objective of this study was to uncover biomarkers by comparing serum protein profiles of patients suffering from stable or unstable angina and controls. We identified by non-targeted proteomic approach and confirmed by the means of independent techniques, the differential expression of several proteins indicating significantly increased vascular inflammation response, disturbance in the lipid metabolism and in atherogenic plaques stability.

Influence of sample and mobile phase composition on peptide retention behaviour and sensitivity in reversed-phase liquid chromatography/mass spectrometry.

Because the chromatographic behaviour of peptides is totally different from that of small molecules, a good understanding of the mechanisms that occur from injection to detection in reversed-phase LC-MS is strongly recommended to successfully develop not only qualitative but also quantitative methods. In this study, design of experiments was used in order to investigate the influence of the experimental parameters, i.e. sample and mobile phase composition, on a peptide mixture covering a wide range of molecular weights, isoelectric points and hydropathies. First, a screening design was developed to identify the significant factors concerning mobile phase (ion-pairing reagent nature and concentration) and sample composition (organic modifier proportion and ion-pairing reagent nature) on retention and response intensity (sensitivity). Then, after having selected the experimental domain and the significant factors, a full factorial design was used to further investigate the role of the considered factors and their interactions. Interestingly, ion-pairing reagent nature present in the sample had a tremendous effect on retention and response intensity. Optimal conditions leading to good sensitivity and adequate peptide retention without band splitting were selected and could be used as starting point for rapid method development using classical solvents and ion-pairing reagents.

Optimization of the liquid chromatography enantioseparation of chiral acidic compounds using cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector and polar organic mobile phases.

The LC enantioseparation of chiral acidic and zwitterionic drugs selected as model compounds was optimized using chlorine containing cellulose based chiral stationary phases and polar organic mobile phases. The main solvent of the mobile phase was acetonitrile, the temperature was settled at 25°C and a stationary phase with cellulose tris(3-chloro-4-methylphenylcarbamate) as chiral selector (3-Cl-4-Me-PC) was selected. In the screening step, the nature and concentration of both acidic and basic additives were found to have a significant effect on retention, selectivity and resolution. Acetic acid (AcA) was selected as acidic additive for the optimization step since it could lead to the enantioseparation of more acidic compounds than trifluoroacetic acid (TFA) and formic acid (FA), while among the three basic additives tested, diethylamine (DEA) most often gave better results with respect to enantioresolution and selectivity than butylamine (BuA) and triethylamine (TEA). The optimization was performed using a central composite face-centered design with two factors, namely the concentration of acetic acid (0.1-0.3%) and the concentration of DEA (0.01-0.1%) in the mobile phase. On the basis of the results obtained in the screening and optimization steps, a strategy for the rapid development of methods for the enantioseparation of acidic or neutral compounds was proposed.

Development of a nano-liquid chromatography on chip tandem mass spectrometry method for high-sensitivity hepcidin quantitation.

Microfluidic LC systems present undeniable advantages over classical LC in terms of sensitivity. Hepcidin, a peptide marker of clinical disorders linked to iron metabolism, was used as model to demonstrate peptide quantification potentialities of LC-chip coupled to a nanoelectrospray source ion trap mass spectrometer in an aqueous sample. First, stable isotope labelled hepcidin was chosen as internal standard and gradient as well as sample compositions were optimised using design of experiments as development tool. The method was then prevalidated using accuracy profiles in order to select the most appropriate response function and to confirm the ability of the technique to quantify low hepcidin concentration. A reliable and very sensitive quantitation method was finally obtained using this integrated microfluidic technology. Indeed, good results with respect to accuracy, trueness and precision were achieved, as well as a very low limit of quantitation (0.07 ng/ml). Method suitability of nano-LC on chip tandem mass spectrometry for hepcidin quantitation was also demonstrated in complex media such as human plasma.

Development and validation of a LC method for the enantiomeric purity determination of S-ropivacaine in a pharmaceutical formulation using a recently commercialized cellulose-based chiral stationary phase and polar non-aqueous mobile phase.

Ropivacaine is the first enantiomerically pure long-acting local anaesthetic used for surgical anaesthesia and post-operative pain relief. A liquid chromatographic (LC) method using acetonitrile as the main solvent and cellulose tris(4-chloro-3-methylphenylcarbamate) coated on silica as chiral stationary phase was successfully developed and applied for the enantiomeric purity determination of S-ropivacaine in a pharmaceutical formulation (Naropin(®)). The key role played by the acidic additive (trifluoroacetic acid or formic acid) in the enantioseparation of basic drugs in these LC systems was demonstrated by the reversal of ropivacaine enantiomers elution order observed when both acids were compared. In order to elute the enantiomeric impurity (R-ropivacaine) before S-ropivacaine, formic acid (FA) was selected. The temperature and the percentages of acidic additive and hexane in the mobile phase were found to significantly influence the retention and resolution of these enantiomers. The optimized mobile phase consisted of ACN/0.1% DEA/0.2% FA/5% hexane (v/v/v/v). The temperature was set at 35°C to avoid the interference from a peak system related to the presence of water in the sample on ropivacaine enantiomers. The LC method was then fully validated applying the strategy based on total measurement error and accuracy profiles. The accuracy profile obtained by linear regression after square root transformation was selected, the acceptance limits being settled at ±10% for the intended use of this analytical method. The relative bias was lower than 1.5%, while the RSD values for repeatability and intermediate precision were both below 1.0%. The limit of detection (LOD) and the limit of quantification (LOQ) were found to be about 0.2 and 1.0 µg/mL, respectively, corresponding to 0.02 and 0.1% of the enantiomeric impurity in S-ropivacaine.

Development and validation of a sensitive solid phase extraction/hydrophilic interaction liquid chromatography/mass spectrometry method for the accurate determination of glucosamine in dog plasma.

A sensitive and accurate LC/MS method was developed for the monitoring of glucosamine (GLcN) dog plasmatic concentration. In this scope, relatively low plasmatic concentrations of GLcN were expected, ranging from 50 to 1000 ng/mL. Liquid chromatography coupled to simple quadrupole mass spectrometry detection (LC/MS) was selected bringing the selectivity and the sensitivity needed for this application. Additionally, a solid phase extraction (SPE) step was performed to reduce matrix and ion suppression effects. Due to the ionisable character of the compound of interest, a mixed-mode strong cation exchange (Plexa PCX) disposable extraction cartridge (DEC) was selected. The separation was carried out on a Zorbax SB-CN column (5 microm, 4.6mm i.d. x 250 mm), considering hydrophilic interaction liquid chromatography (HILIC). Indeed, the mobile phase was made of methanol and 5mM ammonium hydrogen carbonate buffer at pH 7.5 (95/5, v/v). The detection was led at m/z ratios of 180.0 and 417.0, for GLcN and IS, respectively. Reliability of the results was demonstrated through the validation of the method using an approach based on the accuracy profile allowing managing the risk associated to the use of these methods in routine analysis: it is thus guaranteed that each future result will fall in the +/-30% acceptance limits with a probability of at least 90%. Successful application of the method to a preliminary pharmacokinetic study illustrated the usefulness of the method for pre-clinical studies.

Critical analysis of several analytical method validation strategies in the framework of the fit for purpose concept.

Analytical method validation is a mandatory step at the end of the development in all analytical laboratories. It is a highly regulated step of the life cycle of a quantitative analytical method. However, even if some documents have been published there is a lack of clear guidance for the methodology to follow to adequately decide when a method can be considered as valid. This situation has led to the availability of several methodological approaches and it is therefore the responsibility of the analyst to choose the best one. The classical decision processes encountered during method validation evaluation are compared, namely the descriptive, difference and equivalence approaches. Furthermore a validation approach using accuracy profile computed by means of beta-expectation tolerance interval and total measurement error is also available. In the present paper all of these different validation approaches were applied to the validation of two analytical methods. The evaluation of the producer and consumer risks by Monte Carlo simulations were also made in order to compare the appropriateness of these various approaches. The classical methodologies give rise to inadequate and contradictory conclusions which do not allow them to answer adequately the objective of method validation, i.e. to give enough guarantees that each of the future results that will be generated by the method during routine use will be close enough to the true value. It is found that the validation methodology which gives the most guarantees with regards to the reliability or adequacy of the decision to consider a method as valid is the one based on the use of the accuracy profile.

Nutrient digestibility of Mucuna (Mucuna pruriens var. utilis) bean in guinea fowl (Numida meleagris, L): Effects of heat treatment and levels of incorporation in diets.

1. Mucuna pruriens var. utilis is a legume, the seeds of which are scarcely used in animal diets owing to their high content of 3,4-dihydroxy-L-phenylalanine (L-Dopa). 2. Experiments were conducted on guinea fowl to assess the effects of two types of heat processing (cooking and toasting) on chemical composition and nutrient digestibility of Mucuna seeds offered alone or incorporated at three concentrations (40, 120 or 200 g/kg) in complete diets. 3. Diets containing 200 g/kg seeds had more crude fibre and less ether extract. L-Dopa content increased with the amount of Mucuna inclusion. Cooking reduced markedly L-Dopa content while toasting had no effect. When fed alone, Mucuna seeds dramatically decreased feed intake. 4. Feed intake (FI) and body weight gain (BWG) were not influenced by the complete diets. Cooking significantly increased crude fibre digestibility. 5. It is suggested that cracked and cooked Mucuna bean can be incorporated at a safe level of 120 g/kg in complete diets for guinea fowl production.

[Proteomics studies on arthritis by SELDI-TOF-MS: identification of the S100 proteins family as proteins of interest]. / Protéomique par SELDI-TOF-MS des maladies inflammatoires articulaires: identification des protéines S100 comme protéines d'intérêt.

Clinical proteomics is a technical approach studying the entire proteome expressed by cells, tissues or organs. It describes the dynamics of cell regulation by detecting molecular events related to diseases development. Proteomic techniques focus mainly on identification of new biomarkers or new therapeutic targets. It is a multidisciplinary approach using medical, biological, bioanalytical and bioinformatics knowledges. A strong collaboration between these fields allowed SELDI-TOF-MS proteomics studies to be performed at the CHU and the University of Liège, in GIGA-Research facilities. The aim of these studies was driven along three main axes of research related to the identification of biomarkers specific to a studied pathology, to a common biological pathway and, finally, to a treatment response. This work was presented in the setting of the "Synthèse CHU 2009" meeting.

Validation of a nonaqueous capillary electrophoretic method for the enantiomeric purity determination of R-flurbiprofen using a single-isomer amino cyclodextrin derivative.

Nonaqueous capillary electrophoresis (NACE) was successfully applied to the enantiomeric purity determination of R-flurbiprofen using 6-monodeoxy-6-mono(2-hydroxy)propylamino-beta-cyclodextrin (IPA-beta-CD) as chiral selector. The nonaqueous BGE was made up of 20 mM IPA-beta-CD, 20 mM ammonium camphorsulfonate and 40 mM ammonium acetate in methanol. Flufenamic acid was selected as internal standard. The CE method was carefully optimized in order to prevent the adsorption of the cationic CD onto the capillary wall, and therefore, to avoid loss of peak efficiency and enantioresolution. To achieve this goal, the addition of ammonium camphorsulfonate was found to be necessary. In the selected conditions, the determination of 0.1% of S-flurbiprofen in R-flurbiprofen could be performed using the method of standard additions. The NACE method was then fully validated by applying a novel strategy using accuracy profiles.