Cannabis profiling of seized samples: An intra-location variability study using gas chromatography-mass spectrometry profiles and multivariate data analysis.

Yearly, cannabis belongs to the most seized drugs worldwide. During judicial investigations, illicit cannabis profiling can be performed to compare seized herbal material. However, comparison is challenging because of the natural heterogeneity of the psychoactive crop. Gas chromatography-mass spectrometry (GC-MS) profiles, consisting of eight cannabinoids, were used to study the intra-location (within) and inter-location (between) variabilities. Decision thresholds were derived from the 95% and 99% confidence limits, applying Pearson correlation coefficients for the intra-location samples. The false negatives and false positives (FPs) determined the discriminative power of different pretreatments applied to obtain the lowest FP error rate possible. Initially, a 97 samples data set was used and with log transformation as pretreatment, a decrease in FPs from 38% and 45% FPs to 17% and 22%, for both confidence limits, respectively, was seen relative to internal standard normalization that was used as reference. An additional intra-plantation variability study with 130 samples verified whether the initial model contained sufficient within-location information, but this was not the case. Hence, a combined data matrix was constructed with all seized samples. Log transformation provided the best FP results for both limits, that is, an improvement from 58% and 64% to 21% and 26%, respectively, was seen. The representativeness of these 'linked' thresholds was demonstrated using both cross-validation and an external set, for which similar FP results as for the calibration set were obtained. By applying data pretreatment, a significant improvement was observed to distinguish seized samples. However, the FP rate is still not at an acceptable level to defend in court.

Applicability of core-shell SiO2 microspheres with a high TiO2 loading as stationary phase for HPLC.

BACKGROUND: Due to its high chemical stability, sufficient rigidity and zwitterionic ion exchange properties, TiO2 can be considered as an alternative stationary phase material to SiO2 for high performance liquid chromatography. TiO2 stationary phase is usually prepared by coating TiO2 onto SiO2 support by sol-gel method. However, in the traditional coating method, in order to overcome the rapid hydrolysis rate of tetrabutyl orthotitanate, only a very low concentration of tetrabutyl orthotitanate can be used, resulting in a low loading of TiO2 on the support. RESULTS: TiO2 core-shell spheres with a good monodispersity were prepared using 0.25 mol L-1 tetrabutyl orthotitanate. The specific surface area, pore volume, pore diameter and TiO2 loading of the TiO2 core-shell spheres were 66 m2 g-1, 0.15 cm3 g-1, 9.8 nm and 57%, respectively. The core-shell spheres were derivatized with n-octadecyltrichlorosilane and then packed into a stainless steel column to test the separation performance for neutral, basic and acidic samples in liquid chromatography. A baseline separation of polyaromatic hydrocarbons was achieved, showing a column efficiency for fluorene of 118075 plates m-1. The prepared stationary phase was also used to separate acidic and basic mixtures, and column efficiencies of 54500 and 25836 plates m-1 were obtained for N,N-dinitroaniline and p-chlorophenol, respectively. The relative standard deviations of the retention times of polyaromatic hydrocarbons for run-to-run, day-to-day and column-to-column repeatability were all below 5.1%. SIGNIFICANCE AND NOVELTY: This work demonstrated that TiO2 can be coated in the pores of the shell of SiO2 core-shell spheres with high TiO2 loading using a high concentration of tetrabutyl orthotitanate as the titania source. The experimental results show that the TiO2 coated core-shell spheres can be a good alternative stationary phase for liquid chromatography.

Modelling the enantiorecognition of structurally diverse pharmaceuticals on O-substituted polysaccharide-based stationary phases.

This study aims to develop models to predict the retention, separation and elution sequence of the enantiomers of structurally diverse pharmaceuticals. More specifically, Quantitative Structure Retention Relationships (QSRR) models are built that describe the relationship between molecular descriptors and retention. Eighteen structurally diverse chiral mixtures, each consisting of a pair of enantiomers, were analyzed on two polysaccharide chiral stationary phases, Chiralcel OD-RH (cellulose tris(3,5-dimethylphenylcarbamate)) and Lux amylose-2 (amylose tris(5-chloro-2-methylphenylcarbamate)), applying either a basic or an acidic mobile phase, and their retention factor and elution sequence were determined. Both achiral and, in-house defined, chiral descriptors were used as descriptive variables to build the models. Linear regression techniques, i.e. stepwise multiple linear regression (sMLR) and partial least squares (PLS) regression, were applied to model the retention or separation as a function of the descriptors. In a first step, models were built with only achiral descriptors to model the global retention of both enantiomers of a chiral molecule. Subsequently, models were built with only chiral descriptors to predict the enantioseparation and elution sequence, and finally, models were considered with both descriptor types to predict the retention, the separation and the elution sequence of the enantiomers. The global retention was predicted well by the sMLR models with only achiral descriptors. The models with only chiral descriptors were not found suitable to predict the enantioseparation and elution sequence. Finally, the models containing both chiral and achiral descriptors allowed predicting the retention well, but their ability to predict the elution sequence and separation of the enantiomers differed widely for the chromatographic systems considered.

Improving the LC-MS/MS analysis of neuromedin U-8 and neuromedin S by minimizing their adsorption behavior and optimizing UHPLC and MS parameters.

Neuromedin U (NmU) and neuromedin S (NmS) are two closely related neuropeptides belonging to the neuromedin family. NmU usually occurs either as a truncated eight amino acid long peptide (NmU-8) or as an 25 amino acid long peptide, although other molecular forms exist depending on the species considered. NmS, on the other hand, is a 36 amino acid long peptide, sharing the same amidated C-terminal heptapeptide with NmU. Nowadays, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the preferred analytical technique for peptide quantification, because of its excellent sensitivity and selectivity. However, reaching the required quantification limits for these compounds in biological samples remains an extremely challenging task, especially because of their nonspecific binding (NSB). This study highlights the difficulties that are faced when quantifying larger neuropeptides (23-36 amino acids) compared to smaller ones (< 15 amino acids). The first part of this work aims to solve the adsorption problem for NmU-8 and NmS, by investigating the different steps involved in the sample preparation, i.e. the different solvents applied and the pipetting protocol. The addition of 0.05% plasma as an adsorption competitor was found to be primordial to avoid peptide loss due to NSB. The second part of this work focusses on further improving the sensitivity of the LC-MS/MS method for NmU-8 and NmS, by evaluating some UHPLC-parameters, including the stationary phase, the column temperature and the trapping conditions. For both peptides of interest, the best results were achieved when combining a C18 trap column with a C18 iKey separation device containing a positively charged surface. Column temperatures of 35 and 45 °C for NmU-8 and NmS respectively, resulted in the highest peak areas and S/N ratios, while applying higher column temperatures substantially decreased sensitivity. Moreover, a gradient starting at 20% organic modifier instead of 5% significantly improved the peak shape of both peptides. Finally, some compound-specific MS parameters, i.e. the capillary and the cone voltages, were evaluated. The peak areas increased with a factor 2 and 7 for NmU-8 and NmS respectively and peptide detection in the low picomolar range is now feasible.

Comparison of supercritical fluid chromatographic methods to predict the skin permeability of pharmaceutical and cosmetic compounds.

Supercritical fluid chromatography (SFC) was explored as an alternative for liquid chromatography to predict the skin permeability of pharmaceutical and cosmetic compounds. Nine dissimilar stationary phases were applied to screen a test set of 58 compounds. The experimental retention factors (log k), in addition to two sets of theoretical molecular descriptors, were applied to model the skin permeability coefficient. Different modelling approaches, i.e. multiple linear regression (MLR) and partial least squares (PLS) regression, were used. In general, the MLR models performed better than the PLS models for a given descriptor set. The results obtained on a cyanopropyl (CN) column provided the best correlation with the skin permeability data. The retention factors obtained on this column were included in a simple MLR model, together with the octanol-water partition coefficient and the number of atoms (r² = 0.81, RMSEC = 0.537 or 20.5% and RMSECV = 0.580 or 22.1%). The overall best MLR model included the chromatographic descriptor from a phenyl column and 18 descriptors (r² = 0.98, RMSEC = 0.167 or 6.2% and RMSECV = 0.238 or 8.9%). This model showed a good fit, on top of very good predictive features. However, stepwise MLR models with a reduced complexity could also be determined, with the best performance parameters obtained with the CN-column based retention and eight descriptors (r² = 0.95, RMSEC = 0.282 or 10.7% and RMSECV = 0.353 or 13.4%). SFC thus provides a suitable alternative to the liquid chromatographic techniques previously applied to model the skin permeability.

Predicting skin permeability of pharmaceutical and cosmetic compounds using retention on octadecyl, cholesterol-bonded and immobilized artificial membrane columns.

In this study, the retention on three types of columns, an immobilized artificial membrane (IAM), a cholesterol-bonded and an octadecyl (C18) column, was applied for the prediction of skin permeability. The first two columns are biomimicking ones, which have certain components of the skin bound to the stationary phase, and were applied in HPLC, while the sub-2 µm C18 column was studied in UHPLC because of its fast features. Fifty-eight compounds were analyzed applying different mobile-phase compositions, with varying percentages of organic modifier on every column, to extrapolate the retention factor to a theoretically purely aqueous mobile phase (log kw). The retention factors, along with two sets of theoretical molecular descriptors, were used to model the skin permeability coefficient (log Kp) using multiple linear regression (MLR) and partial least squares (PLS) regression modelling. Although the retention factors (log k) on the IAM column showed a better correlation with the skin permeability, the overall best model was obtained by applying a stepwise MLR approach on the UHPLC parameters combined with some theoretical descriptors. This model showed a good fit, and on top has potential to accurately predict skin permeability values. Furthermore, the UHPLC method has the advantage of being fast and can thus be classified as a high-throughput approach.

Fabrication of a molecularly imprinted monolithic column via the epitope approach for the selective capillary microextraction of neuropeptides in human plasma.

A novel molecularly imprinted monolithic (MIM) column was designed and fabricated using the epitope approach, and was used for the selective capillary microextraction (CME) of the neuropeptides neurotensin (NT) and neuromedin N (NmN). The MIMs were synthesized in a capillary by thermally initiated polymerization of the functional monomer (methacrylic acid (MAA)), in the presence of a dummy template (Pro-Tyr-Ile-Leu (PYIL)), a crosslinker and porogens. The resulting monoliths were characterized by scanning electron microscopy, pore size distribution measurement, and Fourier transform infrared spectroscopy. Different synthesis conditions of the MIM column were investigated. The parameters affecting the MIM-CME performance, including loading, washing and elution protocols, were optimized as well. The MIMs were used to enrich NT and NmN from human plasma prior to HPLC-UV analysis. The imprinted monolith showed an excellent maximum adsorption capacity of 245-711 mg mL-1 and selectivity (imprinting factor of 5.7-13.4) towards its target peptides. Low detection limits of 0.62 and 1.20 nM, and satisfactory recoveries (82.5-98.8%) were obtained for NT and NmN, respectively. The proposed MIM-CME/HPLC-UV method was found suitable to be used as an effective tool for the highly efficient and specific analysis of NT and NmN in human plasma samples.

Coupling of chiral and achiral stationary phases in supercritical fluid chromatography: Evaluating and improving retention prediction.

Isomers and stereoisomers are always challenging to separate. Column coupling may provide improved chromatographic selectivity, necessary for the separation of the compounds with similar chemical and structural properties. The relatively low viscosity of supercritical fluids, used as mobile phases allows for the coupling of several columns in series in supercritical fluid chromatography (SFC), without exceeding the pressure limits of the system. The aim of this study is to propose reliable prediction of the retention behaviour of analytes on a coupled column system, based on a limited number of initial analyses. The chiral compounds atenolol, ephedrine, propranolol, mianserin, labetalol and nadolol, besides the diastereomers quinine and quinidine, and the structural isomers of aminophenol and aminocresol were used as model analytes. The retention behaviour of the analytes was determined on the individual chiral columns Lux Cellulose-1, Lux Cellulose-2, Lux Cellulose-3, Lux Cellulose-4, Lux Amylose-2 and the achiral columns Luna NH2, Luna Silica, Synergi RP and FluoroSep RP. The mobile phase was composed of CO2 mixed with 20% (v/v) MeOH, which contained 0.1% (v/v) trifluoroacetic acid and 0.1% (v/v) isopropylamine. The retention factors of the analytes on coupled stationary phases were predicted, and subsequently compared to the experimentally obtained ones. Relative deviations of predicted and experimental retention factors were in range from 0.00% to 51.91%. Flow rate and back pressure of the screening conditions were adjusted to improve prediction precision on four column combinations, with varying success rates. The average relative deviations of retention factors were reduced to 2.84% - 6.59% by adjusting flow rate, and to 2.30% - 8.57% by adjusting back pressure. The most successful approach, flow rate adjustment, was then applied to select a column combination providing improved resolution of the structurally similar components of silymarin extract.

Modelling approaches for chiral chromatography on polysaccharide-based and macrocyclic antibiotic chiral selectors: A review.

An overview of molecular modelling approaches, related to chiral separations on polysaccharide-based and macrocyclic antibiotic chiral selectors, is presented. Both atomistic calculations and empirical fitting procedures are discussed. Atomistic calculations, such as docking and molecular dynamics can be used to model the interactions between enantiomers and the chiral stationary phase. This may help obtaining information about the chiral recognition mechanism. Conversely, in empirical fitting procedures, mathematical models for relevant separation parameters are fitted to experimental observations. The latter use theoretical molecular descriptors, calculated from the molecular structure, which are combined into a model to predict a given response, for example, retention. Such relationships, when used in chiral separations, are often called quantitative structure enantioselective retention relationships (QSERR) and an increased interest in them can be observed in the literature. Different regression models are discussed, such as multiple linear regression and partial least squares.

Development of a capillary electrophoresis method for the separation of flavonolignans in silymarin complex.

CE method for the baseline separation of structurally similar flavonolignans silybin A, silybin B, isosilybin A, isosilybin B, silychristin, silydianin, and their precursor taxifolin in silymarin complex has been developed and validated. The optimized background electrolyte was 100 mmol/L boric acid (pH 9.0) containing 5 mmol/L heptakis(2,3,6-tri-O-methyl)-ß-CD and 10% (v/v) of methanol. The separation was carried out in an 80.5/72 cm (50 µm id) fused silica capillary at +25 kV with UV detection at 200 nm. Genistein (10 µg/mL) was used as internal standard. The resolution between the diastereomers of silybin and isosilybin was 1.73 and 2.59, respectively. The method was validated for each analyte in a concentration range of 2.5-50 µg/mL. The calibration curves were rectilinear with correlation coefficients ≥0.9972. The method was applied to determine flavonolignans in two dietary supplements containing Silybum marianum extract. The accuracy was evaluated by comparing the results of the CE analyses of the dietary supplements with those of the reference United States Pharmacopeial HPLC method. The unpaired t-test did not show a statistically significant difference between the results of both the proposed CE and the reference method (p > 0.05, n = 3).

Evaluating micellar liquid chromatographic methods on octadecyl particle-based and monolithic columns to predict the skin permeation of drug and cosmetic molecules.

A micellar liquid chromatographic method was developed to assist in the modeling of the skin permeability of pharmaceutical and cosmetic compounds. The composition of the mobile phase was determined by means of a two-factor central composite design, after which it was tested on both a particle-based and monolithic column. The latter provided the opportunity to increase the flow rate from 1 to 8 mL/min without reaching too high backpressures. The micellar conditions allowed analyzing a large test set of compounds with diverse characteristics with just one mobile-phase composition. The obtained experimental chromatographic descriptors besides two sets of theoretical molecular descriptors were used to model the skin permeability coefficient log Kp, applying multiple linear regression and partial least squares regression approaches. The micellar method on the monolithic column provided useful models with similar or even slightly better performance parameters than the method on the particle-based column. Furthermore, a much faster analysis can be achieved when applying a flow rate of 8 mL/min, making the micellar monolithic method ideal to estimate skin permeability.

Gas Chromatographic Fingerprint Analysis for the Comparison of Seized Cannabis Samples.

Cannabis sativa L. is widely used as recreational illegal drugs. Illicit Cannabis profiling, comparing seized samples, is challenging due to natural Cannabis heterogeneity. The aim of this study was to use GC-FID and GC-MS herbal fingerprints for intra (within)- and inter (between)-location variability evaluation. This study focused on finding an acceptable threshold to link seized samples. Through Pearson correlation-coefficient calculations between intra-location samples, 'linked' thresholds were derived using 95% and 99% confidence limits. False negative (FN) and false positive (FP) error rate calculations, aiming at obtaining the lowest possible FP value, were performed for different data pre-treatments. Fingerprint-alignment parameters were optimized using Automated Correlation-Optimized Warping (ACOW) or Design of Experiments (DoE), which presented similar results. Hence, ACOW data, as reference, showed 54% and 65% FP values (95 and 99% confidence, respectively). An additional fourth root normalization pre-treatment provided the best results for both the GC-FID and GC-MS datasets. For GC-FID, which showed the best improved FP error rate, 54 and 65% FP for the reference data decreased to 24 and 32%, respectively, after fourth root transformation. Cross-validation showed FP values similar as the entire calibration set, indicating the representativeness of the thresholds. A noteworthy improvement in discrimination between seized Cannabis samples could be concluded.

Cytotoxic, Antioxidant, and Antidiabetic Activities versus UPLC-ESI-QTOF-MS Chemical-Profile Analysis of Ipomoea aquatica Fractions.

Ipomoea aquatica is a common green leafy vegetable that has numerous uses in traditional medicine. This study focused on the determination of the cytotoxic, antiradical, and antidiabetic properties of various fractions of the I. aquatica methanolic extract, as well as on the tentative identification of some bioactive compounds in the same fractions. The cytotoxicity was determined by the brine shrimp lethal test. The antioxidant activities of the I. aquatica fractions were investigated through 3 assays. The antidiabetic activity (in vitro) was measured by α-glucosidase and α-amylase inhibition assays. Phytochemical qualitative analyses demonstrated the presence of alkaloids, terpenoids, phenols, and flavonoids in the ethyl acetate-methanol and methanol fractions. The total phenolic and total flavonoid contents were found to be highest in the ethyl acetate-MeOH fractions. The evaluation of the cytotoxicity showed that the hexane-dichloromethane fraction is the most toxic, while the others are moderately toxic. The antioxidant activity assays showed that the ethyl acetate-MeOH fractions are the most potent, while the α-glucosidase and α-amylase assays revealed that the hexane-dichloromethane fraction might contain a potent antidiabetic agent. Some bioactive substances in the MeOH fractions, such as salicylic acid glucoside, 1-O-sinapoyl-ß-D-glucose derivative, and dihydroferulic acid derivative, were tentatively identified. To the best of our knowledge, this is the first report to detect and identify these compounds in this species. Based on the results of this study, it may be concluded that I. aquatica is a potent antioxidant agent and could be a good candidate as a natural antioxidant in food and therapeutics.

Comparison of in-silico modelling and reversed-phase liquid chromatographic retention on an octadecyl silica column to predict skin permeability of pharmaceutical and cosmetic compounds.

This study focuses on the in-silico modelling of the skin permeability using a test set of pharmaceutical and cosmetic compounds. Two sets of theoretical molecular descriptors, obtained from the E-Dragon and Vega ZZ software programs, were used in the models. Different linear regression methods, i.e. Multiple Linear Regression (MLR) and Partial Least Squares (PLS) regression, were applied for modelling and estimating the skin permeability. The best model was obtained using a stepwise MLR approach on the E-Dragon descriptor set. In a second step, the retention of the test set compounds was measured on a C18 column at two pH levels: pH 5.5 and pH 7. Different organic-modifier fractions were applied in the mobile phase to be able to extrapolate the retention factors to a log kw value, with kw the estimated retention factor in an aqueous mobile phase without organic modifier. Thereafter it was examined whether combining this chromatographic descriptor with the theoretical descriptors could improve the modelling of the skin permeability. The chromatographic descriptor often did not show an added value compared to the models containing only theoretical descriptors. Therefore, the in-silico models were preferred, and these models could be useful to predict the skin permeability of pharmaceutical and cosmetic compounds.

A comparative study of UniSpray and electrospray sources for the ionization of neuropeptides in liquid chromatography tandem mass spectrometry.

Despite the extensive use of electrospray ionization (ESI) for the quantification of neuropeptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS), poor ionization and transmission efficiency are described for this ionization interface. A new atmospheric pressure ionization source, named UniSpray, was recently developed and commercialized. In this study, the LC-MS performance of this new ionization interface is evaluated and compared with ESI for the quantification of seven neuropeptides. Besides comparison of signal intensities and charge state distributions, also signal-to-noise (S/N) ratios and accuracy and precision were assessed. Additionally, matrix effects of human precipitated plasma and rat microdialysate were evaluated as well as the effect of three supercharging agents on the ionization of the seven neuropeptides. UniSpray ionization resulted in signal intensities four to eight times higher at the optimal capillary/impactor voltage for all seven neuropeptides. S/N values at the other hand only increased by not more than a twofold when the UniSpray source was used. Moreover, UniSpray ionization resulted in a shift towards lower charge states for some neuropeptides. Evaluation of the matrix effects by a post-column infusion set-up resulted in different infusion profiles between ESI and UniSpray. The charge state distributions of the neuropeptides obtained with UniSpray are highly comparable with ESI. Finally, the effect of the supercharging agents on the ionization of the neuropeptides tends to be peptide-dependent with both ionization sources.

Stationary-phase optimized selectivity in supercritical fluid chromatography using a customized Phase OPtimized Liquid Chromatography kit: comparison of different prediction approaches.

The use of stationary-phase optimized selectivity in liquid chromatography (SOS-LC) was shown to be successful for HPLC to analyze complex mixtures using a Phase OPtimized Liquid Chromatography (POPLC) kit. This commercial kit contains five stationary-phase types of varying lengths, which can be coupled to offer an improved separation of compounds. Recently, stationary-phase optimized selectivity supercritical fluid chromatography (SOS-SFC) has been introduced, transferring the methodology to SFC. In this study, the applicability of a customized POPLC expert kit for isocratic SFC runs was explored. Five stationary-phase chemistries were selected as potentially most suitable for achiral separations of polar compounds: aminopropyl (amino), cyanopropyl (CN), diol, ethylpyridine (EP), and silica. The retention factors (k) on the individual stationary phases were used for the prediction of the best stationary-phase combination, based on the POPLC algorithm (via the included software). As an alternative, the best column combination was predicted using multiple linear regression (MLR) models on the results obtained from a simplex centroid mixture design with only three stationary-phase types (amino, silica, and EP). A third approach applied the isocratic POPLC algorithm on the same three stationary-phase data. The proposed combinations were assembled and tested. The predicted and experimental retention factors were compared. The predictions based on the POPLC algorithm provided a stationary phase showing a complete separation of the mixture. The stationary phase suggested by the MLR models, on the other hand, showed co-elution of two compounds, due to an unexpected experimental retention shift. Overall, the customized POPLC kit showed good potential to be applied in SFC. Graphical abstract.

Rendering A Chiral Screening Step In Supercritical Fluid Chromatography Mass-Spectrometry Compatible.

Nowadays, sensitive chiral methods are required for the determination of chiral impurities and for assays in biological samples. Supercritical fluid chromatography (SFC), one of the main techniques to separate chiral molecules, can be coupled to MS to provide such sensitive methods. Moreover, chiral separation strategies are very useful to reduce the development time and cost of such methods. This study investigates the transfer of an existing non-MS compatible screening step (as part of a separation strategy) into an MS-compatible one. The initial step had a cumulative success rate of 100 % for 57 tested compounds using methanol or 2-propanol as mobile phase modifier on one of four chiral stationary phases. The additives applied in the original mobile phases, i.e. isopropylamine and trifluoroacetic acid, negatively affect the ionization in SFC-MS and thus need to be replaced. Formic acid, acetic acid, water, ammonia, ammonium acetate and ammonium formate were investigated as MS-compatible additives in different combinations and concentrations. Only methanol-based mobile phases were considered in this study because high system pressures were obtained with isopropanol. The other experimental parameters remained the same as in the initial screening step. The effects of the alternative additives on the obtained resolutions as well as on the global success rate were investigated. The best alternative MS-compatible mobile phase contained 0.5 % CH3COOH and 40 mM NH3 as additives. This mobile phase provided the highest number of separations and rather high resolutions. An MS-compatible screening step was defined with this alternative mobile phase. Compared to the original additives, a similar success rate was obtained.

CE-MS metabolic profiling of volume-restricted plasma samples from an acute mouse model for epileptic seizures to discover potentially involved metabolomic features.

Currently, a high variety of analytical techniques to perform metabolomics is available. One of these techniques is capillary electrophoresis coupled to mass spectrometry (CE-MS), which has emerged as a rather strong analytical technique for profiling polar and charged compounds. This work aims to discover with CE-MS potential metabolic consequences of evoked seizures in plasma by using a 6Hz acute corneal seizure mouse model. CE-MS is an appealing technique because of its capability to handle very small sample volumes, such as the 10 µL plasma samples obtained using capillary microsampling in this study. After liquid-liquid extraction, the samples were analyzed with CE-MS using low-pH separation conditions, followed by data analysis and biomarker identification. Both electrically induced seizures showed decreased values of methionine, lysine, glycine, phenylalanine, citrulline, 3-methyladenine and histidine in mice plasma. However, a second provoked seizure, 13 days later, showed a less pronounced decrease of the mean concentrations of these plasma metabolites, demonstrated by higher fold change ratios. Other obtained markers that can be related to seizure activities based on literature data, are isoleucine, serine, proline, tryptophan, alanine, arginine, valine and asparagine. Most amino acids showed relatively stable plasma concentrations between the basal levels (Time point 1) and after the 13-day wash-out period (Time point 3), which suggests its effectiveness. Overall, this work clearly demonstrated the possibility of profiling metabolite consequences related to seizure activities of an intrinsically low amount of body fluid using CE-MS. It would be useful to investigate and validate, in the future, the known and unknown metabolites in different animal models as well as in humans.

Halogen bond in separation science: A critical analysis across experimental and theoretical results.

The halogen bond (XB) is a noncovalent interaction involving a halogen acting as electrophile and a Lewis base. In the last decades XB has found practical application in several fields. Nevertheless, despite the pivotal role of noncovalent interactions in separation science, investigations of XB in this field are still in their infancy, and so far a limited number of studies focusing on solid phase extraction, liquid-liquid microextraction, liquid-phase chromatography, and gas chromatography separation have been published. In addition, in the last few years, our groups have been systematically studying the potentiality of XB for HPLC enantioseparations. On this basis, in the present paper up-to-date results emerging from focused experiments and theoretical analyses performed by our laboratories are integrated with a descriptive presentation of XB features and the few studies published until now in separation science. Then, the aim of this article is to provide a comprehensive and critical discussion of the topic, and account for some still open issues in the application of XB to separate chemical mixtures.