Optimisation of a Microwave Synthesis of Silver Nanoparticles by a Quality by Design Approach to Improve SERS Analytical Performances.

A major limitation preventing the use of surface-enhanced Raman scattering (SERS) in routine analyses is the signal variability due to the heterogeneity of metallic nanoparticles used as SERS substrates. This study aimed to robustly optimise a synthesis process of silver nanoparticles to improve the measured SERS signal repeatability and the protocol synthesis repeatability. The process is inspired by a chemical reduction method associated with microwave irradiation to guarantee better controlled and uniform heating. The innovative Quality by Design strategy was implemented to optimise the different parameters of the process. A preliminary investigation design was firstly carried out to evaluate the influence of four parameters selected by means of an Ishikawa diagram. The critical quality attributes were to maximise the intensity of the SERS response and minimise its variance. The reaction time, temperature and stirring speed are critical process parameters. These were optimised using an I-optimal design. A robust operating zone covering the optimal reaction conditions (3.36 min-130 °C-600 rpm) associated with a probability of success was modelled. Validation of this point confirmed the prediction with intra- and inter-batch variabilities of less than 15%. In conclusion, this study successfully optimised silver nanoparticles by a rapid, low cost and simple technique enhancing the quantitative perspectives of SERS.

Quantitative Structure Retention-Relationship Modeling: Towards an Innovative General-Purpose Strategy.

Reversed-Phase Liquid Chromatography (RPLC) is a common liquid chromatographic mode used for the control of pharmaceutical compounds during their drug life cycle. Nevertheless, determining the optimal chromatographic conditions that enable this separation is time consuming and requires a lot of lab work. Quantitative Structure Retention Relationship models (QSRR) are helpful for doing this job with minimal time and cost expenditures by predicting retention times of known compounds without performing experiments. In the current work, several QSRR models were built and compared for their adequacy in predicting the retention times. The regression models were based on a combination of linear and non-linear algorithms such as Multiple Linear Regression, Support Vector Regression, Least Absolute Shrinkage and Selection Operator, Random Forest, and Gradient Boosted Regression. Models were built for five pH conditions, i.e., at pH 2.7, 3.5, 6.5, and 8.0. In the end, the model predictions were combined using stacking and the performances of all models were compared. The k-nearest neighbor-based application domain filter was established to assess the reliability of the prediction for further compound prioritization. Altogether, this study can be insightful for analytical chemists working with RPLC to begin with the computational prediction modeling such as QSRR to predict the separation of small molecules.

Interpretable One-Class Classification of Raman Spectra Using Prediction Bands Estimated by Wavelet Regression.

Previously, we introduced a novel one-class classification (OCC) concept for spectra. It uses as acceptance space for genuine spectra of the target chemical, a prediction band in the wavelengths' space. As a decision rule, test spectra falling substantially outside this band are rejected as noncomplying with the target, and their deviations are documented in the wavelengths' space. This band-based OCC concept was applied to smooth signals like near-infrared (NIR) spectra. A regression model based on a smoothed principal component (PC) representation of the training spectra was used to predict unseen trajectories of future spectra. The boundaries of the most central predicted trajectories were chosen as critical trajectories. We now propose a methodology to construct a similar band-based one-class classifier for Raman spectra, which are sharper and noisier than NIR spectra. The spectra are transformed by a composition of wavelet and principal component (wPC) expansions instead of just a PC expansion in the previous methodology for NIR spectra. Wavelets can capture sharp features of Raman signals and provide a framework to efficiently denoise them. A multinormal prediction model is then used to derive predictions of future wPC scores of unseen spectra. These predicted wPC scores are then backtransformed to obtain predictions of future trajectories of unseen spectra in the wavelengths' space, whose most central region defines the acceptance band or space. This band-based one-class classifier successfully classified the first derivatives of real pharmaceutical Raman spectra, while enjoying the advantage of documenting deviations from the critical trajectories in the wavelengths' space and hence is more interpretable.

A new alternative tool to analyse glycosylation in pharmaceutical proteins based on infrared spectroscopy combined with nonlinear support vector regression.

Almost 60% of commercialized pharmaceutical proteins are glycosylated. Glycosylation is considered a critical quality attribute, as it affects the stability, bioactivity and safety of proteins. Hence, the development of analytical methods to characterise the composition and structure of glycoproteins is crucial. Currently, existing methods are time-consuming, expensive, and require significant sample preparation steps, which can alter the robustness of the analyses. In this work, we suggest the use of a fast, direct, and simple Fourier transform infrared spectroscopy (FT-IR) combined with a chemometric strategy to address this challenge. In this context, a database of FT-IR spectra of glycoproteins was built, and the glycoproteins were characterised by reference methods (MALDI-TOF, LC-ESI-QTOF and LC-FLR-MS) to estimate the mass ratio between carbohydrates and proteins and determine the composition in monosaccharides. The FT-IR spectra were processed first by Partial Least Squares Regression (PLSR), one of the most used regression algorithms in spectroscopy and secondly by Support Vector Regression (SVR). SVR has emerged in recent years and is now considered a powerful alternative to PLSR, thanks to its ability to flexibly model nonlinear relationships. The results provide clear evidence of the efficiency of the combination of FT-IR spectroscopy, and SVR modelling to characterise glycosylation in therapeutic proteins. The SVR models showed better predictive performances than the PLSR models in terms of RMSECV, RMSEP, R2CV, R2Pred and RPD. This tool offers several potential applications, such as comparing the glycosylation of a biosimilar and the original molecule, monitoring batch-to-batch homogeneity, and in-process control.

A New Alternative Tool to Analyse Glycosylation in Monoclonal Antibodies Based on Drop-Coating Deposition Raman imaging: A Proof of Concept.

Glycosylation is considered a critical quality attribute of therapeutic proteins as it affects their stability, bioactivity, and safety. Hence, the development of analytical methods able to characterize the composition and structure of glycoproteins is crucial. Existing methods are time consuming, expensive, and require significant sample preparation, which can alter the robustness of the analyses. In this context, we developed a fast, direct, and simple drop-coating deposition Raman imaging (DCDR) method combined with multivariate curve resolution alternating least square (MCR-ALS) to analyze glycosylation in monoclonal antibodies (mAbs). A database of hyperspectral Raman imaging data of glycoproteins was built, and the glycoproteins were characterized by LC-FLR-MS as a reference method to determine the composition in glycans and monosaccharides. The DCDR method was used and allowed the separation of excipient and protein by forming a "coffee ring". MCR-ALS analysis was performed to visualize the distribution of the compounds in the drop and to extract the pure spectral components. Further, the strategy of SVD-truncation was used to select the number of components to resolve by MCR-ALS. Raman spectra were processed by support vector regression (SVR). SVR models showed good predictive performance in terms of RMSECV, R2CV.

User-Driven Strategy for In Silico Screening of Reversed-Phase Liquid Chromatography Conditions for Known Pharmaceutical-Related Small Molecules.

In the pharmaceutical field, and more precisely in quality control laboratories, robust liquid chromatographic methods are needed to separate and analyze mixtures of compounds. The development of such chromatographic methods for new mixtures can result in a long and tedious process even while using the design of experiments methodology. However, developments could be accelerated with the help of in silico screening. In this work, the usefulness of a strategy combining response surface methodology (RSM) followed by multicriteria decision analysis (MCDA) applied to predictions from a quantitative structure-retention relationship (QSRR) model is demonstrated. The developed strategy shows that selecting equations for the retention time prediction models based on the pKa of the compound allows flexibility in the models. The MCDA developed is shown to help to make decisions on different criteria while being robust to the user's decision on the weights for each criterion. This strategy is proposed for the screening phase of the method lifecycle. The strategy offers the possibility to the user to select chromatographic conditions based on multiple criteria without being too sensitive to the importance given to them. The conditions with the highest desirability are defined as the starting point for further optimization steps.

Comparison of Vibrational Spectroscopic Techniques for Quantification of Water in Natural Deep Eutectic Solvents.

Vibrational spectroscopic techniques, i.e., attenuated total reflectance infrared (ATR-IR), near infrared spectroscopy (NIRS) and Raman spectroscopy (RS), coupled with Partial Least Squares Regression (PLSR), were evaluated as cost-effective label-free and reagent-free tools to monitor water content in Levulinic Acid/L-Proline (LALP) (2:1, mol/mol) Natural Deep Eutectic Solvent (NADES). ATR-IR delivered the best outcome of Root Mean Squared Error (RMSE) of Cross-Validation (CV) = 0.27% added water concentration, RMSE of Prediction (P) = 0.27% added water concentration and mean % relative error = 2.59%. Two NIRS instruments (benchtop and handheld) were also compared during the study, respectively yielding RMSECV = 0.35% added water concentration, RMSEP = 0.56% added water concentration and mean % relative error = 5.13% added water concentration, and RMECV = 0.36% added water concentration, RMSEP = 0.68% added water concentration and mean % relative error = 6.23%. RS analysis performed in quartz cuvettes enabled accurate water quantification with RMECV = 0.43% added water concentration, RMSEP = 0.67% added water concentration and mean % relative error = 6.75%. While the vibrational spectroscopic techniques studied have shown high performance in relation to reliable determination of water concentration, their accuracy is most likely related to their sensitivity to detect the LALP compounds in the NADES. For instance, whereas ATR-IR spectra display strong features from water, Levulinic Acid and L-Proline that contribute to the PLSR predictive models constructed, NIRS and RS spectra are respectively dominated by either water or LALP compounds, representing partial molecular information and moderate accuracy compared to ATR-IR. However, while ATR-IR instruments are common in chemistry and physics laboratories, making the technique readily transferable to water quantification in NADES, Raman spectroscopy offers promising potential for future development for in situ, sample withdrawal-free analysis for high throughput and online monitoring.

Development of a sensitive MEKC-LIF method for synthetic cathinones analysis.

Synthetic cathinones are phenylalkylamine compounds related to natural cathinone from Catha edulis leaves. Due to their sympathomimetic effects comparable to common illicit drugs, these substances are mainly drugs of abuse and constitute the second most frequently seized group of new psychoactive substances. In order to ensure their regulation and to promote public health, reliable analytical tools are required to track these substances. In the present study, we developed a CE hyphenated to laser-induced fluorescence detection method to demonstrate its suitability to perform fast and cost-effective synthetic cathinones analysis. Fourteen compounds including isobaric compounds and position isomers were selected to encompass the large panel of chemical structures. To separate the FITC-labeled analytes (presenting the same negative charge and close mass to charge ratios), MEKC separation mode was selected. Method selectivity was not suitable using common surfactants. In this context, alkyl polyethylene glycol ether surfactants were successfully used as neutral surfactant to overcome this analytical challenge. The effect of surfactant nature on separation performances and migration behaviors of the analytes was also studied. Optimal BGE composition included 75 mM borate buffer at pH 9.3 and 0.4 mM of C12E10 surfactant. Final MEKC separation conditions were proposed to analyze a large panel of synthetic cathinones. This method helped to reach a sensitivity with LOD from 0.1 to 0.4 nM (pg/mL order).

Early Noninvasive Ventilation and Nonroutine Transfusion for Acute Chest Syndrome in Sickle Cell Disease in Children: A Descriptive Study.

OBJECTIVES: To describe the need for transfusion and short- and long-term evolutions of pediatric sickle cell disease patients with acute chest syndrome for whom early continuous noninvasive ventilation represented first-line treatment. DESIGN: Single-center retrospective chart study in PICU. SETTING: A tertiary and quaternary referral PICU. PATIENTS: All sickle cell disease patients 5-20 years old admitted with confirmed acute chest syndrome and not transfused in the previous month were included. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Demographic data, laboratory and radiologic findings, transfusions, invasive ventilation, oxygen and noninvasive ventilation settings, duration of opioid treatment, length of hospital stay, and severe sickle cell disease complications in the ensuing 2 years were extracted from medical charts. Sixty-six acute chest syndrome in 48 patients were included. Continuous early noninvasive ventilation was well tolerated in 65 episodes, with positive expiratory pressure 4 cm H2O and pressure support 10 cm H2O (median) administered continuously, then discontinued during 7 days (median). No patient necessitated invasive ventilation or died. Twenty-three acute chest syndrome (35%) received transfusions; none received blood exchange. Transfused patients had more frequent upper lobe radiologic involvement, more severe anemia, higher reticulocyte counts, and higher C-reactive protein than nontransfused patients. Their evolution was more severe in terms of length of opioid requirement, length of noninvasive ventilation treatment, overall time on noninvasive ventilation, and length of stay. At 2-year follow-up after the acute chest syndrome episode, no difference was observed between the two groups. CONCLUSIONS: Early noninvasive ventilation combined with nonroutine transfusion is well tolerated in acute chest syndrome in children and may spare transfusion in some patients. Early recognition of patients still requiring transfusion is essential and warrants further studies.

Safety and tolerance of cidofovir as a 2% gel for local application in high-grade cervical intraepithelial neoplasia: A phase 1 investigation.

OBJECTIVES: The primary objective was to evaluate the safety and local tolerance of a topical 2% (w/w) cidofovir gel, applied directly to the cervices of women with high-grade cervical intraepithelial neoplasia (CIN 2+). The secondary objective was to evaluate the pharmacokinetics of cidofovir during the treatment. MATERIALS AND METHODS: Nine women with CIN 2+, were treated with a course of 3 g of cidofovir gel, applied locally once per week for 3 weeks in total (9 g). The treatment was administered in a cervical cap, applied to the cervix for 5 or 10 hours (n = 6 and 3 patients, respectively). Follow-up included a structured questionnaire, a gynecological examination, blood analysis for hematology, C-reactive protein (CRP), and renal function assessment plus pharmacokinetic analyses of cidofovir after each treatment and at the end of the full course. RESULTS: No clinically significant hematological/biochemical abnormalities or serious adverse events (SAE) were reported, although 6 mild to moderate adverse events (AE) occurred in relation to the study drug: 1 flu-like syndrome and 5 local AEs. Plasma concentrations of cidofovir were very low (mean Cmax of 103.0 and 99.2 ng/mL after 5 and 10 hours of exposure, respectively). CONCLUSION: Cidofovir, directly applied on CIN 2+, is reasonably well tolerated and the systemic exposure following topical application is much lower than that seen with intravenous administration, at the approved dose.
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"NOTA-PRGD2 and NODAGA-PRGD2 : Bioconjugation, characterization, radiolabelling, and design space".

This work reports on the development of amide bond bioconjugation for the production of -NOTA and -NODAGA PRGD2 using batch strategy and microfluidic reactor technology. The final radiolabelling step was fully optimized using Design of Experiments and Design Space approaches, hence targeting robust labelling yields in routine. Optimal labelling conditions were defined in sodium acetate buffer as 168 µg/mL peptide concentration, 4.9 pH, 47.5°C temperature, and 12.5-minute reaction time. Upon optimization, the Gallium-68 radiolabelling was fully automated. All the work was designed to be compliant to the GMP environment and to support the pharmaceutical scale-up.

Parental perceptions of clown care in paediatric intensive care units.

AIM: The objective of this study was to report family satisfaction with regards to the presence of clowns in the paediatric intensive care unit (PICU). METHODS: This is a single-centre survey-based study, conducted over 4 months in a 12-bed third level PICU in a university hospital. All parents present at the bedside of their child during clowning were considered as potential participants. Eligible parents were approached by one of the two intensivists as investigators and asked to complete a survey within the 48 h following the clowns' intervention. RESULTS: Thirty-three parents consented to complete the survey. Median age of children was 14 months (15 days to 16 years) and median Pediatric Logistic Organ Dysfunction (PELOD) score was 1 (0-22). Twenty-four (72.7%) were considered as clinically stable while the clowns intervened. Twenty-eight parents (84.8%) and 27 (81.8%) considered that clowns had a positive effect on themselves and on their child, respectively. Clown care was considered as necessary in 19 cases (57.6%), optional in 13 (39.4%) and unnecessary in 1 (3.0%). The degree of parental satisfaction was not significantly associated with the child's clinical stability. CONCLUSION: We suggested that medical clowning in the PICU is well accepted by parents, regardless of severity of their child's condition. This study supports the adoption of medical clowning in PICUs as a patient- and family-centred care practice.

Poplar-Root Knot Nematode Interaction: A Model for Perennial Woody Species.

Plant root-knot nematode (RKN) interaction studies are performed on several host plant models. Though RKN interact with trees, no perennial woody model has been explored so far. Here, we show that poplar (Populus tremula × P. alba) grown in vitro is susceptible to Meloidogyne incognita, allowing this nematode to penetrate, to induce feeding sites, and to successfully complete its life cycle. Quantitative reverse transcription-polymerase chain reaction analysis was performed to study changes in poplar gene expression in galls compared with noninfected roots. Three genes (expansin A, histone 3.1, and asparagine synthase), selected as gall development marker genes, followed, during poplar-nematode interaction, a similar expression pattern to what was described for other plant hosts. Downregulation of four genes implicated in the monolignol biosynthesis pathway was evidenced in galls, suggesting a shift in the phenolic profile within galls developed on poplar roots. Raman microspectroscopy demonstrated that cell walls of giant cells were not lignified but mainly composed of pectin and cellulose. The data presented here suggest that RKN exercise conserved strategies to reproduce and to invade perennial plant species and that poplar is a suitable model host to study specific traits of tree-nematode interactions.

High glucose intake and glycaemic level in critically ill neonates with inherited metabolic disorders of intoxication.

UNLABELLED: To investigate glycaemic levels in critically ill neonates with inherited metabolic disorders of intoxication. Thirty-nine neonates with a median age of 7 days (0-24) were retrospectively included (urea cycle disorders (n = 18), maple syrup disease (n = 13), organic acidemias (n = 8)). Twenty-seven neonates were intubated, 21 were haemodialysed and 6 died. During the first 3 days, median total and peak blood glucose (BG) levels were 7.1 mmol/L (0.9-50) and 10 mmol/L (5.1-50), respectively. The median glucose intake rate was 11 mg/kg/min (2.7-15.9). Fifteen and 23 neonates exhibited severe hyperglycaemia (≥2 BG levels >12 mmol/L) and mild hyperglycaemia (≥2 BG levels >7 and ≤12 mmol/L), respectively. Glycaemic levels and number of hyperglycaemic neonates decreased over the first 3 days (p < 0.001) while total glucose intake rate was stable (p = 0.11). Enteral route of glucose intake was associated with a lower number of hyperglycaemic neonates (p = 0.04) and glycaemic level (p = 0.02). CONCLUSION: Hyperglycaemia is common in critically ill neonates receiving high glucose intake with inherited metabolic disorders of intoxication. Physicians should decrease the rate of total glucose intake and begin enteral feeding as quickly as possible in cases of persistent hyperglycaemia. WHAT IS KNOWN: • The risk of hyperglycaemia in the acute phase of critical illness is high. What is New: • Hyperglycaemia is common in the initial management of critically ill neonates with inherited metabolic disorders of intoxication receiving high glucose intake.

Children with Down syndrome: Clinical course and mortality-associated factors in a French medical paediatric intensive care unit.

AIM: To investigate clinical course and mortality-associated factors in children with Down syndrome (DS) managed in a medical paediatric intensive care unit. METHODS: A single-centre, retrospective study conducted between 2001 and 2010 in DS children aged 1 month to 16 years. RESULTS: Sixty-six patients with a median age of 24 months (1-192) and a male/female ratio of 1.5 were analysed. Patients presented with history of congenital heart disease (n = 52, 78.8%), mechanical ventilation (n = 40, 60.6%) and chronic upper airway obstruction (n = 10, 15.1%). The primary reason for admission was respiratory failure (n = 56, 84.8%). Pulmonary arterial hypertension (PAH) (n = 19, 28.8%), acute respiratory distress syndrome (ARDS) (n = 18, 27.2%) and sepsis (n = 14, 21.2%) were observed during their clinical course. Twenty-six patients died (39.4%). Mortality-associated factors included the following: (i) baseline characteristics: history of mechanical ventilation, chronic upper airway obstruction and congenital heart disease; (ii) clinical course during paediatric intensive care unit stay: sepsis, catecholamine support, ARDS, PAH and nosocomial infection. In multivariate logistic analysis, history of mechanical ventilation, ARDS and PAH remained independently associated with death. CONCLUSIONS: The mortality rate in critically ill DS children admitted for medical reasons is high and is predominantly associated with respiratory conditions.

A glial origin for periventricular nodular heterotopia caused by impaired expression of Filamin-A.

Periventricular nodular heterotopia (PH) is a human brain malformation caused by defective neuronal migration that results in ectopic neuronal nodules lining the lateral ventricles beneath a normal appearing cortex. Most affected patients have seizures and their cognitive level varies from normal to severely impaired. Mutations in the Filamin-A (or FLNA) gene are the main cause of PH, but the underlying pathological mechanism remains unknown. Although two FlnA knockout mouse strains have been generated, none of them showed the presence of ectopic nodules. To recapitulate the loss of FlnA function in the developing rat brain, we used an in utero RNA interference-mediated knockdown approach and successfully reproduced a PH phenotype in rats comparable with that observed in human patients. In FlnA-knockdown rats, we report that PH results from a disruption of the polarized radial glial scaffold in the ventricular zone altering progression of neural progenitors through the cell cycle and impairing migration of neurons into the cortical plate. Similar alterations of radial glia are observed in human PH brains of a 35-week fetus and a 3-month-old child, harboring distinct FLNA mutations not previously reported. Finally, juvenile FlnA-knockdown rats are highly susceptible to seizures, confirming the reliability of this novel animal model of PH. Our findings suggest that the disorganization of radial glia is the leading cause of PH pathogenesis associated with FLNA mutations. Rattus norvegicus FlnA mRNA (GenBank accession number FJ416060).

Population pharmacokinetics and haemodynamic effects of norepinephrine in hypotensive critically ill children.

AIM: The aim of the study was to investigate the pharmacokinetics and pharmacodynamics of norepinephrine in hypotensive critically ill children, including associated variability factors. METHODS: This was a prospective study in an 18-bed neonatal and paediatric intensive care unit. All children were aged less than 18 years, weighed more than 1500 g and required norepinephrine for systemic arterial hypotension. The pharmacokinetics and haemodynamic effects were described using the non-linear mixed effect modelling software MONOLIX. RESULTS: Norepinephrine dosing infusions ranging from 0.05 to 2 µg kg(-1) min(-1) were administered to 38 children whose weight ranged from 2 to 85 kg. A one compartment open model with linear elimination adequately described the norepinephrine concentration-time courses. Bodyweight (BW) was the main covariate influencing norepinephrine clearance (CL) and endogenous norepinephrine production rate (q0) via an allometric relationship: CL(BWi) = θCL × (BWi)(3/4) and q0(BWi) = θq0 × (BWi)(3/4) . The increase in mean arterial pressure (MAP) as a function of norepinephrine concentration was well described using an Emax model. The effects of post-conceptional age (PCA) and number of organ dysfunctions were significant on basal MAP level (MAP0i = MAP0 × PCA/9i (0.166) ) and on the maximal increase in MAP (32 mmHg and 12 mmHg for a number of organ dysfunctions ≤3 and ≥4, respectively). CONCLUSION: The pharmacokinetics and haemodynamic effects of norepinephrine in hypotensive critically ill children highlight the between-subject variability which is related to the substantial role of age, BW and severity of illness. Taking into account these individual characteristics may help clinicians in determining an appropriate initial a priori dosing regimen.

Delay and stability of central venous administration of norepinephrine in children: a bench study.

UNLABELLED: In children, because of the dead volume of the central venous catheter (CVC) and the low flow rate of norepinephrine (NE) infusion, the delay between start-up and effective administration can be adversely long. A theoretical calculation enables to estimate the delay and variations of effective administration. However, numerous factors can hinder this theoretical approach. Herein, we measured via bench testing the actual delay and stability of NE administration kinetics. Using an assembly reproducing our currently-implemented catecholamine administration protocol, diluted NE (200 µg ml(-1)) was infused at an initial rate of 2 ml h(-1) (theoretically 6.67 µg min(-1)) for a period of 24 h. An assay measuring the amount of NE (µg) exiting the CVC was conducted by high-pressure liquid chromatography with colorimetric detection. The theoretical calculation of the delay in administered NE, taking into account a CVC dead volume of 0.3 ml, was 9 min. The measured percentage of the administered dose as a function of time in minutes (M) was M0-M3 (0 %), M3-M6 (0 %), M6-M9 (13 %), M9-M12 (28 %), M12-M15 (70 %), and M15-M18 (100 %) The amount of NE (µg) at fixed rate (2 ml h(-1)) was established at 6.9 ± 0.4 µg min(-1) during the 24 h. CONCLUSION: Continuous NE infusion via a CVC at low rate is stable. In children, because of CVC dead volume and low flow rate infusion, the delay in achieving intended dose delivery is significantly longer than that estimated by theoretical calculation. New modalities of initiation of catecholamine infusion adapted to the child are warranted.

The benefits of validation of methods for toxicity testing outweigh its costs

The 4th Annual Forum on Endocrine Disrupters organized by the European Commission brought together the authors of this article around the topic: "From bench to validated test guidelines: (pre)val­idation of test methods". Validation activities are meant to demonstrate the relevance and reliability of methods and approaches used in regulatory safety testing. These activities are essential to facil­itate regulatory use, still they are largely underfunded and unattractive to the scientific community. In the last decade, large amounts of funding have been invested in European research towards the development of approaches that can be used in regulatory decision-making, including for the identification of endocrine disrupters. There is a vast pool of candidate test methods for potential reg­ulatory applications, but most of them will not be used due to the absence of consideration of their relevance and reliability outside the method developer's laboratory. This article explains the reasons why such a gap exists between the outputs of research projects and the uptake in a regulatory context. In parallel, there are also increasing expectations from the regulatory science community that validation becomes more efficient with respect to time and resources. This article shares some of the lessons learned and proposes paths forward for validation of new methods that are not intended as one-to-one replacements of animal studies. This includes submitting only mature methods for validation that were developed following good practices and good documentation, proposing a greater emphasis on well-documented transferability studies, and adopting a cost-sharing model among those who benefit from validated methods.

Validation activities for methods intended to be used to assess chemical safety have a cost but also bring substantial benefits when the validated methods are established as OECD Test Guidelines, which results in mutual acceptance of data generated by the methods across OECD member and adhering countries. The article discusses some of the challenges faced when method validation is underfunded and unattractive for researchers. Proposals are made to improve the current situation, gain efficiency, and make validation a shared responsibility.

Uncertainty management for In Silico screening of reversed-phase liquid chromatography methods for small compounds.

The process of developing new reversed-phase liquid chromatography methods can be both time-consuming and challenging. To meet this challenge, statistics-based strategies have emerged as cost-effective, efficient and flexible solutions. In the present study, we use a Bayesian response surface methodology, which takes advantage of the knowledge of the pKa values of the compounds present in the analyzed sample to model their retention behavior. A multi-criteria decision analysis (MCDA) was then developed to exploit the uncertainty information inherent in the model distributions. This strategic approach is designed to integrate seamlessly with quantitative structure retention relationship (QSRR) models, forming an initial in-silico screening phase. Of the two methods presented for MCDA, one showed promising results. The method development process was carried out with the optimization phase, generating a design space that corroborates the results of the selection phase.