A case of fatal idiosyncratic reaction to the designer drug 3,4-methylenedioxypyrovalerone (MDPV) and review of the literature.

The stimulant designer drug 3,4-methylenedioxypyrovalerone (MDPV) was first synthesized by Boehringer Ingelheim in 1969 and introduced on the black market in 2006. Only a small number of fatal intoxication cases have been reported in the literature, all with significant blood MDPV concentrations. In this report, we describe one fatality attributed to an idiosyncratic reaction to MDPV. The victim displayed agitation, violent behavior and delirium followed by cardiac arrest. Hyperthermia was observed at the hospital. The MDPV cardiac and femoral blood concentrations were 6 ng/mL. The presence of excited delirium syndrome and MDPV, a drug with a pharmacology similar to cocaine, leads to the conclusion that the victim suffered a fatal adverse reaction to MDPV. This is the first published case of idiosyncratic reaction to MDPV.

Fiber optic illumination of a poly(dimethylsiloxane) sheath flow cuvette for diode laser induced fluorescence detection in capillary electrophoresis.

A Tee configuration sheath flow cuvette with square cross-section channels has been produced in PDMS for CE detection. The output of a 1.4 W laser diode operating at 450 nm was focused onto the 300 µm core of a 370 µm od fiber optic whose end was inserted into one arm of the Tee for LIF. The optimal configuration had the fiber optic positioned 500 µm downstream from the intersection and the end of the 35 cm 50 µm id 365 µm od capillary just outside the intersection and in the leg of the Tee, resulting in a 90° configuration. Detection limits of 50 and 3 pM and linear calibrations of at least three orders of magnitude were obtained for Lucifer Yellow and fluorescein, respectively.

Dietary nitrate supplementation for preventing and reducing the severity of winter infections, including COVID-19, in care homes (BEET-Winter): a randomised placebo-controlled feasibility trial.

PURPOSE: Infections cause considerable care home morbidity and mortality. Nitric oxide (NO) has broad-spectrum anti-viral, bacterial and yeast activity in vitro. We assessed the feasibility of supplementing dietary nitrate (NO substrate) intake in care home residents. METHODS: We performed a cluster-randomised placebo-controlled trial in UK residential and nursing care home residents and compared nitrate containing (400 mg) versus free (0 mg daily) beetroot juice given for 60 days. Outcomes comprised feasibility of recruitment, adherence, salivary and urinary nitrate, and ordinal infection/clinical events. RESULTS: Of 30 targeted care homes in late 2020, 16 expressed interest and only 6 participated. 49 residents were recruited (median 8 [interquartile range 7-12] per home), mean (standard deviation) age 82 (8) years, with proxy consent 41 (84%), advance directive for hospital non-admission 8 (16%) and ≥ 1 doses of COVID-19 vaccine 37 (82%). Background dietary nitrate was < 30% of acceptable daily intake. 34 (76%) residents received > 50% of juice. Residents randomised to nitrate vs placebo had higher urinary nitrate levels, median 50 [18-175] v 18 [10-50] mg/L, difference 25 [0-90]. Data paucity precluded clinical between-group comparisons; the outcome distribution was as follows: no infection 32 (67%), uncomplicated infection 0, infection requiring healthcare support 11 (23%), all-cause hospitalisation 5 (10%), all-cause mortality 0. Urinary tract infections were most common. CONCLUSIONS: Recruiting UK care homes during the COVID-19 pandemic was partially successful. Supplemented dietary nitrate was tolerated and elevated urinary nitrate. Together, infections, hospitalisations and deaths occurred in 33% of residents over 60 days. A larger trial is now required. TRIAL REGISTRATION: ISRCTN51124684. Application date 7/12/2020; assignment date 13/1/2021.

Increased oxidative modifications of amniotic fluid albumin in pregnancies associated with gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is a state of hyperglycaemia and increased oxidative stress with onset during pregnancy. Human serum albumin (HSA) was extracted from 26 GDM and 26 nonGDM amniotic fluid samples collected at 15 weeks gestation and analyzed by mass spectrometry. The majority of all albumin isoforms were oxidized with the cysteinylated HSA as the base peak in the deconvoluted spectrum. The HSA peak areas, from a control sample, had 36% relative standard deviation (RSD) across the six experimental days but using the relative isoform distribution improved the precision to 3-6%. The results show that the relative contribution of permanently oxidized HSA was greater (P = 0.002) and reversibly oxidized HSA was lower (P = 0.006) for GDM compared to nonGDM in the samples measured. This implies that the path toward GDM has been set prior to 15 weeks gestation and results in increased protein oxidation.

A liquid chromatography to capillary array electrophoresis interface for two-dimensional separations.

A new liquid chromatography-capillary electrophoresis interface has been developed for multidimensional separations. The interface allows two second dimension separation capillaries to sample the chromatography effluent by servo controlled positioning of the capillary inlets in either the effluent or background electrolyte streams. The use of multiple second dimension capillaries allows higher first dimension sampling rates or multiple second dimension separation modes. Fluorescently labelled serum was separated by gel filtration and sub-micellar capillary array electrophoresis producing a multidimensional separation. Alternatively, using different background electrolytes for each capillary, two multidimensional separations with different selectivities were collected simultaneously. An easily fabricated PDMS multichannel sheath-flow cuvette was used for sensitive laser induced fluorescence detection of the labelled serum.

A threshold LC-MS/MS method for 92 analytes in oral fluid collected with the Quantisal® device.

A study of impaired driving rates in the province of Québec is currently planned following the legalization of recreational cannabis in Canada. Oral fluid (OF) samples are to be collected with a Quantisal® device and sent to the laboratory for analysis. In order to prepare for this project, a qualitative decision point analysis method monitoring for the presence of 97 drugs and metabolites in OF was developed and validated. This high throughput method uses incubation with a precipitation solvent (acetone:acetonitrile 30:70 v:v) to boost drug recovery from the collecting device and improve stability of benzodiazepines (e.g., α-hydroxyalprazolam, clonazepam, 7-aminoclonazepam, flunitrazepam, 7-aminoflunitrazepam, N-desmethylflunitrazepam, nitrazepam). The Quantisal® device has polyglycol in its stabilizing buffer, but timed use of the mass spectrometer waste valve proved sufficient to avoid the glycol interferences for nearly all analytes. Interferences from OF matrices and 140 potentially interfering compounds, carryover, ion ratios, stability, recovery, reproducibility, robustness, false positive rate, false negative rate, selectivity, sensitivity and reliability rates were tested in the validation process. Five of the targeted analytes (olanzapine, oxazepam, 7-aminoclonazepam, flunitrazepam and nitrazepam) did not meet the set validation criteria but will be monitored for identification purposes (no comparison to a cut-off level). Blind internal proficiency testing was performed, where six OF samples were tested and analytes were classified as "negative", "likely positive" or "positive" with success. The final validated OF qualitative decision point method covers 92 analytes, and the presence of 5 additional analytes is screened in this high throughput analysis.

The Key Role of Intrinsic Lifetime Dynamics from Upconverting Nanosystems in Multiemission Particle Velocimetry.

Evaluation of particle dynamics at the nano- and microscale poses a challenge to the development of novel velocimetry techniques. Established optical methods implement external or internal calibrations of the emission profiles by varying the particle velocity and are limited to specific experimental conditions. The proposed multiemission particle velocimetry approach aims to introduce a new concept for a luminescent probe, which guarantees accurate velocity measurements at the microscale, independent of the particle concentration or experimental setup, and without need for calibration. The simplicity of these analyses relies on the intrinsic luminescence dynamics of core-shell upconverting nanoparticles. Upon excitation with a focused near-infrared pulsed laser, the nanoparticle emits photons at different wavelengths. The time interval between emissions from different excited states is independent of the local environment or particle velocity. The velocity of the particles is calculated by measuring the distance between the maxima of two different emissions and dividing it by the known difference in luminescence lifetimes. This method is demonstrated using simple digital imaging of nanoparticles flowing in 75-150 µm diameter capillaries. Using this novel approach typically results in a relative standard deviation of the experimental velocities of 5% or lower without any calibration.

Electrospun Upconverting Nanofibrous Hybrids with Smart NIR-Light-Controlled Drug Release for Wound Dressing.

Chronic wounds present a high risk of infection due to delayed and incomplete healing, leading to increased health risks and financial burden to health-care systems. Numerous approaches to promote wound healing have been extensively explored, especially the development of effective wound dressing materials embedded with therapeutic drug molecules. Despite advances made in this area, a remaining challenge to be addressed is the controlled, on-demand release of therapeutic molecules using noncytotoxic stimulus, for example, near-infrared (NIR) excitation. Here, we report a platform that allows for the development of electrospun poly(vinyl alcohol) (PVA) fibrous hybrids embedded with upconverting nanoparticles (UCNPs) and UV-cleavable levofloxacin conjugates for wound dressings. Upon irradiation with NIR light, the excited UCNPs emit UV light around 365 nm, which can cleave the o-nitrobenzyl (ONB) linkage of the levofloxacin conjugates in the PVA fiber, leading to controlled drug release. The release was observed to be triggered only under NIR and UV irradiation, with no effect in the dark. Furthermore, the antibacterial effect against Escherichia coli and Staphylococcus aureus was successfully demonstrated, highlighting the versatility of the electrospun upconverting fiber platform. The development of antibacterial fibrous meshes with on-demand release of encapsulated drugs is imperative for precise treatment of wound infections.

Qualitative threshold method validation and uncertainty evaluation: A theoretical framework and application to a 40 analytes liquid chromatography-tandem mass spectrometry method.

Qualitative methods hold an important place in drug testing, filling central needs in screening and analyses, among others, linked to per se legislation. Nevertheless, the bioanalytical method validation guidelines do not discuss this type of method or describe method validation procedures ill-adapted to qualitative methods. The output of qualitative methods are typically categorical, binary results, such as presence/absence or above cut-off/below cut-off. As the goal of any method validation is to demonstrate fitness for use under production conditions, qualitative validation guidelines should evaluate performance based on discrete, binary results instead of the continuous measurements obtained from the instrument (e.g. area). A tentative validation guideline for threshold qualitative methods was developed by in silico modelling of measurements and derived binary results. This preliminary guideline was applied to a liquid chromatography-tandem mass spectrometry method for 40 analytes, each with a defined threshold concentration. Validation parameters calculated from the analysis of 30 samples spiked above and below the threshold concentration (false negative rate, false positive rate, selectivity rate, sensitivity rate and reliability rate) showed a surprisingly high failure rate. Overall, 13 out of the 40 analytes were not considered validated. A subsequent examination found that this was attributable to an appreciable shift in the standard deviation of the area ratio on a day-to-day basis, a previously undescribed and unaccounted-for behaviour in the qualitative threshold method validation literature. Consequently, the developed guideline was modified and used to validate a qualitative threshold method, based on the binary results for performance evaluation and incorporating measurement uncertainty.

A rapid prototyping technique for valves and filters in centrifugal microfluidic devices.

Using short lengths of fused silica capillary tubing embedded in the disk, a system for valving and filtering samples on centrifugal microfluidic devices has been designed and implemented. Sedimentation of turbid samples and transfer of the clear supernatant was also accomplished. Also demonstrated is the transfer of the liquid through a second capillary valve to the final reservoir. By controlling rotational speed, sedimentation and multiple step valving operations are readily implemented and easily prototyped.

A PDMS sheath flow cuvette for high-sensitivity LIF measurements in CE.

A simple method for producing a sheath flow cuvette in PDMS suitable for post-column detection in CE is described. Two types of cuvette were investigated. In the first, the sheath flow channel had a round cross-section of approximately 635 microm diameter, whereas the second cuvette had a 300x300 microm(2) square channel. Both cuvettes produced laminar flows that ensheathed the separation capillary's effluent allowing sensitive fluorescence measurements. The elasticity of the PDMS allowed the 300x300 microm(2) square sheath flow channel to expand uniformly and accommodate the larger 330-340 microm od round separation capillary, producing a self-aligning cuvette with robust mechanical properties. With this cuvette, linear calibrations of over five orders of magnitude and 15-30 zmol fluorescein detection limits were obtained for 12 and 50 microm id capillaries.

Identification and quantitation of human amniotic fluid components using capillary zone electrophoresis.

A capillary electrophoresis method was used to measure albumin, immunoglobulin G (IgG), transferrin, and uric acid in 230 amniotic fluid (AF) samples collected at 15.15+/-0.06 weeks gestation. Species were quantified by external calibration using thiamine as internal standard. All major components were detected within 10 min. Migration time reproducibility was 3.0% relative standard deviation (RSD) and normalized peak areas were 12% RSD or better at 190 nm from 81 measurements of a pooled AF sample. The separation profile was not affected by 10h of storage at room temperature or by 10 freeze-thaw cycles, suggesting that frozen AF samples are suitable for protein biomarker studies.

Design and optimization of porous polymer enzymatic digestors for proteomics.

Effective protein characterization and identification are demanding and time-consuming operations in proteomics because of long-protein purification/separation procedures, and even longer enzymatic digestions. In this work, polymer-based monolithic enzyme reactors were fabricated in fused-silica capillaries, and performance was characterized through protein digestion and identification by MALDI-MS and ESI-MS. Reactors were prepared by fabricating a porous methacrylate base monolith followed by photografting with glycidyl methacrylate, and immobilization of the enzyme(s) with carbonyldiimidazole. Trypsin and Staphylococcus aureus V-8 protease (Glu-C) were used to produce three types of reactors: trypsin-based, Glu-C-based, and trypsin combined with Glu-C. Protein digestions, performed by perfusing protein solutions through the reactor under pressure, were evaluated based on the peptide map generated when directly coupled to an ESI mass spectrometer. Excellent digestion was observed over flow rates from 0.2 to 1 microL/min, which corresponds to reactor residence times of 0.24-1.4 min. As a proof of principle, chromatographic separation of model proteins followed by the digestion of specific fractions using these proteolytic enzyme reactors and ESI-MS is demonstrated.

A Tool for Automatic Correction of Endogenous Concentrations: Application to BHB Analysis by LC-MS-MS and GC-MS.

Several substances relevant for forensic toxicology purposes have an endogenous presence in biological matrices: beta-hydroxybutyric acid (BHB), gamma-hydroxybutyric acid (GHB), steroids and human insulin, to name only a few. The presence of significant amounts of these endogenous substances in the biological matrix used to prepare calibration standards and quality control samples (QCs) can compromise validation steps and quantitative analyses. Several approaches to overcome this problem have been suggested, including using an analog matrix or analyte, relying entirely on standard addition analyses for these analytes, or simply ignoring the endogenous contribution provided that it is small enough. Although these approaches side-step the issue of endogenous analyte presence in spiked matrix-matched samples, they create serious problems with regards to the accuracy of the analyses or production capacity. We present here a solution that addresses head-on the problem of endogenous concentrations in matrices used for calibration standards and quality control purposes. The endogenous analyte concentration is estimated via a standard-addition type process. This estimated concentration, plus the spiked concentration are then used as the de facto analyte concentration present in the sample. These de facto concentrations are then used in data analysis software (MultiQuant, Mass Hunter, etc.) as the sample's concentration. This yields an accurate quantification of the analyte, free from interference of the endogenous contribution. This de facto correction has been applied in a production setting on two BHB quantification methods (GC-MS and LC-MS-MS), allowing the rectification of BHB biases of up to 30 µg/mL. The additional error introduced by this correction procedure is minimal, although the exact amount will be highly method-dependent. The endogenous concentration correction process has been automated with an R script. The final procedure is therefore highly efficient, only adding four mouse clicks to the data analysis operations.

Tetrahydroquinoxalines induce a lethal evisceration phenotype in Haemonchus contortus in vitro.

In the present study, the anthelmintic activity of a human tyrosine kinase inhibitor, AG-1295, and 14 related tetrahydroquinoxaline analogues against Haemonchus contortus was explored. These compounds were screened against parasitic larvae - exsheathed third-stage (xL3) and fourth-stage (L4) - using a whole-organism screening assay. All compounds were shown to have inhibitory effects on larval motility, development and growth, and induced evisceration through the excretory pore in xL3s. The estimated IC50 values ranged from 3.5 to 52.0 µM for inhibition of larval motility or development. Cytotoxicity IC50 against human MCF10A cells was generally higher than 50 µM. Microscopic studies revealed that this eviscerated (Evi) phenotype occurs rapidly (<20 min) and relates to a protrusion of internal tissues and organs (evisceration) through the excretory pore in xL3s; severe pathological damage in L4s as well as a suppression of larval growth in both stages were also observed. Using a relatively low concentration (12.5 µM) of compound m10, it was established that the inhibitor has to be present for a relatively short time (between 30 h and 42 h) during in vitro development from xL3 to L4, to induce the Evi phenotype. Increasing external osmotic pressure prevented evisceration and moulting, and xL3s remained unaffected by the test compound. These results point to a mode of action involving a dysregulation of morphogenetic processes during a critical time-frame, in agreement with the expected behaviour of a tyrosine kinase inhibitor, and suggest potential for development of this compound class as nematocidal drugs.

Procedure for the Selection and Validation of a Calibration Model II-Theoretical Basis.

In the first part of this paper (I-Description and application), an automated, stepwise and analyst-independent process for the selection and validation of calibration models was put forward and applied to two model analytes. This second part presents the mathematical reasoning and experimental work underlying the selection of the different components of this procedure. Different replicate analysis designs (intra/inter-day and intra/inter-extraction) were tested and their impact on test results was evaluated. For most methods, the use of intra-day/intra-extraction measurement replicates is recommended due to its decreased variability. This process should be repeated three times during the validation process in order to assess the time stability of the underlying model. Strategies for identification of heteroscedasticity and their potential weaknesses were examined and a unilateral F-test using the lower limit of quantification and upper limit of quantification replicates was chosen. Three different options for model selection were examined and tested: ANOVA lack-of-fit (LOF), partial F-test and significance of the second-order term. Examination of mathematical assumptions for each test and LC-MS-MS experimental results lead to selection of the partial F-test as being the most suitable. The advantages and drawbacks of ANOVA-LOF, examination of the standardized residuals graph and residuals normality testing (Kolmogorov-Smirnov or Cramer-Von Mises) for validation of the calibration model were examined with the last option proving the best in light of its robustness and accuracy. Choosing the correct calibration model improves QC accuracy, and simulations have shown that this automated scheme has a much better performance than a more traditional method of fitting with increasingly complex models until QC accuracies pass below a threshold.

Procedure for the Selection and Validation of a Calibration Model I-Description and Application.

Calibration model selection is required for all quantitative methods in toxicology and more broadly in bioanalysis. This typically involves selecting the equation order (quadratic or linear) and weighting factor correctly modelizing the data. A mis-selection of the calibration model will generate lower quality control (QC) accuracy, with an error up to 154%. Unfortunately, simple tools to perform this selection and tests to validate the resulting model are lacking. We present a stepwise, analyst-independent scheme for selection and validation of calibration models. The success rate of this scheme is on average 40% higher than a traditional "fit and check the QCs accuracy" method of selecting the calibration model. Moreover, the process was completely automated through a script (available in Supplemental Data 3) running in RStudio (free, open-source software). The need for weighting was assessed through an F-test using the variances of the upper limit of quantification and lower limit of quantification replicate measurements. When weighting was required, the choice between 1/x and 1/x2 was determined by calculating which option generated the smallest spread of weighted normalized variances. Finally, model order was selected through a partial F-test. The chosen calibration model was validated through Cramer-von Mises or Kolmogorov-Smirnov normality testing of the standardized residuals. Performance of the different tests was assessed using 50 simulated data sets per possible calibration model (e.g., linear-no weight, quadratic-no weight, linear-1/x, etc.). This first of two papers describes the tests, procedures and outcomes of the developed procedure using real LC-MS-MS results for the quantification of cocaine and naltrexone.

Pore size characterization of monolith for electrochromatography via atomic force microscopy studies in air and liquid phase.

This paper investigates the use of scanning electron microscopy (SEM) and atomic force microscopy (AFM) for the characterization of monoliths used in capillary electrochromatography (CEC) while focusing on the nature of the information available from both techniques. SEM imaging revealed a compact structure of non-porous micrometer sized particles homogeneously agglomerated. With a simple AFM methodology, we found by direct observation that the same material exhibits mesopores in the nanometer range while SEM showed non-porous surfaces. These results obtained by AFM clearly showed that micrometer sized particles shrank and micrometer sized pores increased in the monolith when wetted. Thus, AFM was capable of demonstrating the morphological differences between wet and dried monolithic materials that are not possible by other imaging methods at micrometer resolution.

Determination of confidence intervals in non-normal data: application of the bootstrap to cocaine concentration in femoral blood.

Calculating the confidence interval is a common procedure in data analysis and is readily obtained from normally distributed populations with the familiar [Formula: see text] formula. However, when working with non-normally distributed data, determining the confidence interval is not as obvious. For this type of data, there are fewer references in the literature, and they are much less accessible. We describe, in simple language, the percentile and bias-corrected and accelerated variations of the bootstrap method to calculate confidence intervals. This method can be applied to a wide variety of parameters (mean, median, slope of a calibration curve, etc.) and is appropriate for normal and non-normal data sets. As a worked example, the confidence interval around the median concentration of cocaine in femoral blood is calculated using bootstrap techniques. The median of the non-toxic concentrations was 46.7 ng/mL with a 95% confidence interval of 23.9-85.8 ng/mL in the non-normally distributed set of 45 postmortem cases. This method should be used to lead to more statistically sound and accurate confidence intervals for non-normally distributed populations, such as reference values of therapeutic and toxic drug concentration, as well as situations of truncated concentration values near the limit of quantification or cutoff of a method.

High Hydrostatic Pressure Pretreatment of Whey Protein Isolates Improves Their Digestibility and Antioxidant Capacity.

Whey proteins have well-established antioxidant and anti-inflammatory bioactivities. High hydrostatic pressure processing of whey protein isolates increases their in vitro digestibility resulting in enhanced antioxidant and anti-inflammatory effects. This study compared the effects of different digestion protocols on the digestibility of pressurized (pWPI) and native (nWPI) whey protein isolates and the antioxidant and anti-inflammatory properties of the hydrolysates. The pepsin-pancreatin digestion protocol was modified to better simulate human digestion by adjusting temperature and pH conditions, incubation times, enzymes utilized, enzyme-to-substrate ratio and ultrafiltration membrane molecular weight cut-off. pWPI showed a significantly greater proteolysis rate and rate of peptide appearance regardless of digestion protocol. Both digestion methods generated a greater relative abundance of eluting peptides and the appearance of new peptide peaks in association with pWPI digestion in comparison to nWPI hydrolysates. Hydrolysates of pWPI from both digestion conditions showed enhanced ferric-reducing antioxidant power relative to nWPI hydrolysates. Likewise, pWPI hydrolysates from both digestion protocols showed similar enhanced antioxidant and anti-inflammatory effects in a respiratory epithelial cell line as compared to nWPI hydrolysates. These findings indicate that regardless of considerable variations of in vitro digestion protocols, pressurization of WPI leads to more efficient digestion that improves its antioxidant and anti-inflammatory properties.