Advances in sample preparation and HPLC-MS/MS methods for determining amyloid-ß peptide in biological samples: a review.

Alzheimer's disease (AD), a neurological disorder, is a major public health concern and the most common form of dementia. Its typical symptoms include memory loss, confusion, changes in personality, and cognitive impairment, which result in patients gradually losing independence. Over the last decades, some studies have focused on searching for effective biomarkers as early diagnostic indicators of AD. Amyloid-ß (Aß) peptides have been consolidated as reliable AD biomarkers and have been incorporated into modern diagnostic research criteria. However, quantitative analysis of Aß peptides in biological samples remains a challenge because both the sample and the physical-chemical properties of these peptides are complex. During clinical routine, Aß peptides are measured in the cerebrospinal fluid by immunoassays, but the availability of a specific antibody is critical-in some cases, an antibody may not exist, or its specificity may be inadequate, leading to low sensitivity and false results. HPLC-MS/MS has been reported as a sensitive and selective method for determining different fragments of Aß peptides in biological samples simultaneously. Developments in sample preparation techniques (preconcentration platforms) such as immunoprecipitation, 96-well plate SPME, online SPME, and fiber-in-tube SPME have enabled not only effective enrichment of Aß peptides present at trace levels in biological samples, but also efficient exclusion of interferents from the sample matrix (sample cleanup). This high extraction efficiency has provided MS platforms with higher sensitivity. Recently, methods affording LLOQ values as low as 5 pg mL-1 have been reported. Such low LLOQ values are adequate for quantifying Aß peptides in complex matrixes including cerebrospinal fluid (CSF) and plasma samples. This review summarizes the advances in mass spectrometry (MS)-based methods for quantifying Aß peptides and covers the period 1992-2022. Important considerations regarding the development of the HPLC-MS/MS method such as the sample preparation step, optimization of the HPLC-MS/MS parameters, and matrix effects are described. Clinical applications, difficulties related to analysis of plasma samples, and future trends of these MS/MS-based methods are also discussed.

Direct coupling of fiber-in-tube solid-phase microextraction with tandem mass spectrometry to determine amyloid beta peptides as biomarkers for Alzheimer's disease in cerebrospinal fluid samples.

Current research efforts at neurological diseases have focused on identifying novel biomarkers to aid in diagnosis, to provide accurate prognostic information, and to monitor disease progression. This study presents the direct coupling of fiber-in-tube solid-phase microextraction to tandem mass spectrometry as a reliable method to determine amyloid beta peptides (Aß38, Aß40, and Aß42) as biomarkers for Alzheimer's disease in cerebrospinal fluid (CSF) samples. To obtain the biocompatible fiber-in-tube SPME capillary, a PEEK tube segment was longitudinally packed with fine fibers [nitinol wires coated with a zwitterionic polymeric ionic liquid], to act as selective extraction medium. The fiber-in-tube SPME-MS/MS method integrated analyte extraction/enrichment and sample cleanup (exclusion of interferents) into one step. The method provided lower limits of quantification (LLOQ: 0.2 ng mL-1 for Aß38 and 0.1 ng mL-1 for Aß40 and Aß42), high precision (CV lower than 11.6%), and high accuracy (relative standard deviation lower than 15.1%). This method was successfully applied to determine Aß peptides in CSF samples obtained from AD patients (n = 8) and controls (healthy volunteers, n = 10). Results showed that Aß42 levels in the CSF samples obtained from AD patients were significantly lower compared to healthy controls (p < 0.05). On the basis of the ROC analysis results, the Aß42/Aß40 ratio (AUC = 0.950, p < 0.01; 95%) performed significantly better than Aß42 alone (AUC = 0.913, p < 0.01; 95%) in discriminating between AD patients and healthy controls and presented better diagnostic ability for AD. The novelties of this study are not only related to evaluating Aß peptides as AD biomarkers, but also to demonstrating direct online coupling of fiber-in-tube SPME with MS/MS as a quantitative high-throughput method for bioanalysis.

Magnetic restricted-access carbon nanotubes for SPME to determine cannabinoids in plasma samples by UHPLC-MS/MS.

This manuscript describes the development of magnetic restricted-access carbon nanotubes (M-RACNTs) for use as SPME sorbent to determine cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) in human plasma samples by UHPLC-MS/MS. The adsorptive phase was immobilized on an SPME device by electromagnetic interactions between the M-RACNTs and a cylindrical neodymium magnet (3-mm diameter x 8-mm height) attached to a stainless-steel rod (3-mm diameter x 40-mm height). The M-RACNTs were synthesized by incorporating Fe3O4 magnetic nanoparticles (MNPs) into commercial carbon nanotubes (CNTs); then the surface of the resulting sorbent was further coated with a layer of bovine serum albumin (BSA). Characterization techniques (SEM, FTIR, and Zeta potential) confirmed the presence of both MNPs and BSA layer dispersed through the structure of the CNTs. The M-RACNTs presented adequate sorption capacity, stable physical/chemical characteristics, and appropriate magnetic properties. Protein exclusion capacity (about 98.5%) was attributed to the chemical diffusion barrier created by the BSA network at the outer surface of the sorbent. The SPME parameters (sample pH, equilibrium time, and desorption conditions) were optimized by design of experiments (fraction factorial planning). The method (validated according to the FDA guidelines) presented adequate selectivity and linearity (coefficient of determination higher than 0.99) at concentrations ranging from the lower limit of quantification (LLOQ) (10 ng mL-1) to the upper limit of quantification (ULOQ) (300 ng mL-1) for both CBD and THC. Precision and accuracy varied from 4.47 to 19.84% (LLOQ) and -6.90 to 17.78% (LLOQ), respectively. Carry-over and matrix effect were not significant. The method was successfully applied to determine plasmatic CBD levels in healthy volunteers attending a single session of oral drug administration and THC levels in frequent cannabis smokers.

Current advances and applications of online sample preparation techniques for miniaturized liquid chromatography systems.

Miniaturized liquid chromatography (LC) has been recognized as one of the most important analytical methods in several research fields. Reduced analytical work-scale provides superior chromatographic resolution and decreases sample and organic solvent consumption. However, frequent clogging of tubing connections and use of small sample volumes are significant limitations when high throughput and sensitive analyses are required. Effective sample preparation could help to overcome these limitations. Online coupling of sample preparation techniques (such as column switching and in-tube solid-phase microextraction) with these miniaturized systems may result in more sensitive and reproducible analyses, improving analytical efficiency. This review describes the most common online miniaturized LC configurations, and the main applications of current online sample preparation techniques coupled to miniaturized LC systems in the bioanalytical, omics, and environmental areas. Relevant features, and challenges of these systems, and innovative sorbents, including restricted access materials, monoliths, and immunosorbents is also discussed.

Effects of ayahuasca on the endocannabinoid system of healthy volunteers and in volunteers with social anxiety disorder: Results from two pilot, proof-of-concept, randomized, placebo-controlled trials.

OBJECTIVE: To assess endocannabinoid (anandamide, AEA; 2-arachidonoylglycerol, 2-AG) plasma levels in healthy volunteers and in volunteers with social anxiety disorder (SAD) after a single oral dose of ayahuasca or placebo. METHODS: Post hoc analysis of endocannabinoid plasma levels (baseline, 90 and 240 min after drug intake) from two parallel-group, randomized, placebo-controlled trials. In Study 1, 20 healthy volunteers ingested ayahuasca (average 1.58 mg/ml dimethyltryptamine (DMT)) or placebo, and in Study 2, 17 volunteers with SAD received ayahuasca (average 0.680 mg/ml DMT) or placebo. RESULTS: A significant difference was observed in AEA concentrations in Study 2 after ayahuasca intake (Χ2 (2) = 6.5, p = 0.03, Friedman test), and near significant differences (increases) were observed between baseline and 90 (Z = 0, p = 0.06, Wilcoxon test) and 240 (Z = 10, p = 0.06) minutes after ayahuasca intake. CONCLUSIONS: Although our findings suggest that ayahuasca could modulate AEA levels in SAD patients, the high interindividual variability in both trials and the small samples preclude definitive conclusions. More research with larger samples is needed to better understand the effects of ayahuasca and other hallucinogens in the endocannabinoid system.

Circulating Endocannabinoids in Huntington's Disease: An Exploratory Cross-Sectional Study.

Huntington's disease (HD) is an inherited neurodegenerative disease characterized by motor, cognitive and behavioral deficits. Some evidence suggests that the endocannabinoid system participates in the pathophysiology of HD. We conducted a cross-sectional study comparing plasma levels of anandamide and 2-arachidonoylglycerol in manifest HD gene-expansion carriers (HDGEC) and healthy controls, finding no difference in endocannabinoid levels between the groups. Correlations between endocannabinoid levels and clinical scales (Mini-Mental State Examination, Hospital Anxiety and Depression Scale, Unified Huntington Disease Rating Scale) were non-significant. We found a significant association between body mass index and anandamide levels in healthy controls but not in HDGEC.

Crosslinked zwitterionic polymeric ionic liquid-functionalized nitinol wires for fiber-in-tube solid-phase microextraction and UHPLC-MS/MS as an amyloid beta peptide binding protein assay in biological fluids.

Alzheimer disease (AD) is a neurodegenerative disorder characterized by extracellular accumulation of amyloid-ß peptide (Aß) in the brain interstitium. Human serum albumin (HSA) highly binds to Aß in blood plasma and is thought to inhibit plaque formation in peripheral tissue. Thus, the evaluation of albumin binding to Aß is an important key to understand the dynamics of these molecules in the biological system of patients with AD. In this work, a fiber-in-tube solid-phase microextraction (fiber-in-tube SPME) and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to estimate Aß fraction binding to HSA in cerebrospinal fluid (CSF) and plasma samples. Crosslinked zwitterionic polymeric ionic liquid (zwitterionic PIL)-coated nitinol wires were developed and packed into a polyether ether ketone (PEEK) capillary for a fiber-in-tube SPME and UHPLC-MS/MS method. Zwitterionic PIL sorbent was synthetized from 1-vinyl-3-(butanesulfonate)imidazolium ([VIm+C4SO3-]) and 1,12-di(3-vinylimidazolium)dodecane dibromide ([(VIm)2C12]2[Br]) monomers by in-situ thermally-initiated polymerization. Morphological characterization by scanning electron microscopy (SEM) and atomic force microscopy (AFM) revealed a decrease in the surface roughness of the nitinol wires from ∼17 nm to 1 nm after the in-situ polymerization. The zwitterionic PIL sorbent selectively preconcentrates Aß through a two-pronged interaction mechanism. The fiber-in-tube SPME and UHPLC-MS/MS method presented lower limits of quantification (LLOQ) of 0.4 ng mL-1 for Aß38 and 0.3 ng mL-1 for Aß40 and Aß42, a linear range from LLOQ values to 15 ng mL-1 with coefficients of determination higher than 0.99, precision with coefficient of variation (CV) values ranging from 2.1 to 7.3% and accuracy with relative standard deviation (RSD) values from -0.3 to 7.4. This method was successfully applied to evaluate the binding of HSA to Aß in cerebrospinal fluid (CSF) and plasma samples.

Effects of Ayahuasca on Personality: Results of Two Randomized, Placebo-Controlled Trials in Healthy Volunteers.

Rationale: Previous studies with the serotonergic hallucinogens LSD and psilocybin showed that these drugs induced changes in personality traits, such as increases in Openness. However, results are inconsistent, and the effects of ayahuasca on personality were never investigated in a controlled trial. Objectives: To assess the effects of ayahuasca on personality in two randomized, placebo-controlled trials in healthy volunteers. Methods: Data from two parallel-group, randomized, placebo-controlled trials in healthy volunteers were included. In the first trial, 15 volunteers ingested ayahuasca or placebo, while in the second trial 15 volunteers received placebo+ayahuasca or cannabidiol (CBD)+ayahuasca. Personality was assessed with the NEO-Five Factor Inventory (NEO-FFI) at baseline and 21 days post-treatment. Results: There were significant differences between groups in baseline Openness scores, but not on day 21. A significant increase in Openness scores was observed in the placebo + ayahuasca group in study 2. No other within-group differences were observed for any other domain. Conclusions: Ayahuasca produced inconsistent effects on personality since it induced significant increase in Openness 21 days post-drug intake only in one of the trials. The absence of significant differences in the other ayahuasca groups could be due to small sample sizes and baseline differences among groups. The effects of ayahuasca and other serotonergic hallucinogens on personality should be further investigated in clinical samples.

Innovative extraction materials for fiber-in-tube solid phase microextraction: A review.

Fiber-in-tube solid-phase microextraction (fiber-in-tube SPME) with short capillary longitudinally packed with fine fibers as extraction device allows direct coupling to high performance liquid chromatography (HPLC) systems to determine weakly volatile or thermally labile compounds. This technique associates the advantages of miniaturized and analytical on-line systems. Major achievements include the use of different capillaries (fused-silica, copper, stainless steel, polyetheretherketone (PEEK), or poly(tetrafluoroethylene) (PTFE)) that are packed with neat fibers (Zylon®, silk, or Kevlar 29®) or fibers (stainless steel, basalt, or carbon) functionalized with selective coatings (aerogels, ionic liquids (ILs), polymeric ionic liquids (PILs), molecularly imprinted polymers (MIPs), layered double hydroxides (LDHs), or conducting polymer). This review outlines the fundamental theory and the innovative extraction materials for fiber-in-tube SPME-HPLC systems and highlights their main applications in environmental and bioanalyses.

Novel materials as capillary coatings for in-tube solid-phase microextraction for bioanalysis.

In-tube solid-phase microextraction with a capillary column as extraction device can be directly coupled with high-performance liquid chromatography systems (HPLC). The in-tube solid-phase microextraction technique has been continuously developed since it was introduced in 1997. New couplings have also been evaluated on the basis of state-of-the-art HPLC instruments. Different types of capillaries (wall-coated open tubular, porous layer open tubular, sorbent-packed, porous monolithic rods, or fiber-packed) with selective stationary phases (monoliths, magnetic nanoparticles, conducting polymers, restricted access materials, ionic liquids, carbon, deep eutectic solvents, and hybrid materials) have been developed to boost in-tube solid-phase microextraction performance (sorption capacity and selectivity). This technique has been successfully applied to analyze biological samples (serum, plasma, whole blood, hair, urine, milk, skin, and saliva) for therapeutic drug monitoring, to study biomarkers, to detect illicit drugs, to conduct metabolomics studies, and to assess exposure to drugs. This review describes current advances in in-tube solid-phase microextraction extraction devices and their application in bioanalysis.

Pipette tip micro-solid phase extraction (octyl-functionalized hybrid silica monolith) and ultra-high-performance liquid chromatography-tandem mass spectrometry to determine cannabidiol and tetrahydrocannabinol in plasma samples.

This manuscript describes the development of an innovative method to determine cannabinoids (cannabidiol and tetrahydrocannabinol) in human plasma samples by pipette tip micro-solid phase extraction and liquid chromatography-mass spectrometry/mass spectromtery. An octyl-functionalized hybrid silica monolith, which had been synthesized and characterized, was used as a selective stationary phase. The octyl-functionalized hybrid silica monoliths presented high permeability and adequate mechanical strength. The micro-solid phase extraction variables (sample pH, draw-eject cycles, solvent for phase clean-up, and desorption conditions) were investigated to improve not only the selectivity but also the sorption capacity. The method was linear at concentrations ranging from the lower limit of quantification (10.00 ng/mL) to the upper limit of quantification (150.0 ng/mL). The lack of fit and homoscedasticity tests, as well as the determination coefficients (r2 greater than 0.995), certified that linearity was adequate. The precision assays presented coefficient of variation values lower than 15%, and the accuracy tests provided relative error values ranging from 3.2 to 14%. Neither significant carry-over nor matrix effects were detected. Therefore, the pipette tip micro-solid phase extraction/liquid chromatography-mass spectrometry/mass spectrometry method has demonstrated to be adequate to determine cannabidiol and tetrahydrocannabinol simultaneously in plasma samples for therapeutic drug monitoring of patients undergoing treatment with cannabinoids.

A micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry to determine anandamide and 2-arachidonoylglycerol in rat brain samples.

A simple and reliable method was developed and validated to determine the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples by micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry (SALLLE/UHPLC-MS/MS). The SALLE parameters (brain homogenate volume, salting-out agent, salt concentration, salt solution volume, organic solvent, organic solvent volume, and centrifugation temperature) were optimized to improve sensitivity and selectivity of the method. The SALLE/UHPLC-MS/MS method presented linear ranges from 2.00 to 20.00 ng mL-1 for AEA and from 0.300 to 10.00 µg mL-1 for 2-AG, no significant matrix effect, and inter- and intra-assay precision and accuracy with CV and RSE values lower than 15%, respectively. This innovative method was successfully applied to determine AEA and 2-AG in brain hemispheres from a 6-OHDA animal model of Parkinson's disease (PD).

Endocannabinoid levels in patients with Parkinson's disease with and without levodopa-induced dyskinesias.

Levodopa-induced dyskinesias (LID) in Parkinson's disease (PD) are frequent complications, and the endocannabinoid system has a role on its pathophysiology. To test the hypothesis that the functioning of the endocannabinoid system would be altered in PD and in LID by measuring plasma and CSF levels of α-N-arachidonoylethanolamine (AEA) and 2-arachidonoyl-glycerol (2-AG) in patients with PD with and without LID and in healthy controls. Blood and CSF samples were collected from 20 healthy controls, 23 patients with PD without LID, and 24 patients with PD with LID. The levels of AEA and 2-AG were measured using a highly sensitive column switching ultrahigh-performance liquid chromatography-tandem mass spectrometry method. When pooled together, patients with PD had lower plasma and CSF levels of 2-AG and higher CSF levels of AEA compared to healthy controls (Mann-Whitney statistics = 303.0, p = 0.02). Patients with PD without LID had lower CSF levels of 2-AG (Kruskal-Wallis statistics = 7.76, p = 0.02) and higher CSF levels of AEA levels than healthy controls (Kruskal-Wallis statistics = 8.81, p = 0.01). The findings suggest that the endocannabinoid system participates in the pathophysiology of PD symptoms, but its role in the pathophysiology of LID is still unclear.

Oral Cannabidiol Does Not Convert to Δ8-THC or Δ9-THC in Humans: A Pharmacokinetic Study in Healthy Subjects.

Introduction: Recent studies have suggested that cannabidiol (CBD) could interconvert into Delta-8- and Delta-9- tetrahydrocannabinol. Materials and Methods: Thus, we tested the plasma samples of 120 healthy human subjects (60 male and 60 female), 60 in fasting and the other 60 under normal feeding conditions after acute administration of an oral solution containing CBD 300 mg. To do this, we developed a bioanalytical method to determine CBD and the presence of THC in plasma samples by Ultra-High Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry. Results: The results showed that THC was not detected in plasma after the administration of CBD, and those study participants did not present psychotomimetic effects. Conclusions: The findings presented here are consistent with previous evidence suggesting that the oral administration of CBD in a corn oil formulation is a safe route for the administration of the active substance without bioconversion to THC in humans.

Restricted access carbon nanotube for microextraction by packed sorbent to determine antipsychotics in plasma samples by high-performance liquid chromatography-tandem mass spectrometry.

This manuscript describes the development of the restricted access carbon nanotube (RACNT) as a selective stationary phase for microextraction by packed sorbent (MEPS) to determine antipsychotics (chlorpromazine, clozapine, olanzapine, and quetiapine) in untreated plasma samples from schizophrenic patients by ultra-high liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS). The synthesis was achieved by chemically covering commercial multi-walled carbon nanotubes with bovine serum albumin (BSA) to subsequently pack the material in a polyethylene conical tube (1000 µL). The RACNTs' sorbents were able to exclude about 97% of the plasma proteins, maintaining the same performance for about 100 assays. The MEPS variables (sample pH, draw-eject cycles, desorption and phase cleanup) were evaluated to improve sensibility and selectivity. The MEPS/UHPLC-MS/MS method was linear at concentrations ranging from the lower limit of quantification (10.0 ng mL-1) to the upper limit of quantification (200-700 ng mL-1) with coefficients of determinations higher than 0.99. The precision assays presented relative standard deviation (RSD) values lower than 13%, and the accuracy assays presented relative error (RE) values that ranged from - 8.01 to 11.53%. Neither significant matrix effects nor carryover was observed. The developed method was successfully applied to determine antipsychotics drugs for therapeutic drug monitoring of schizophrenic patients.

In-tube solid-phase microextraction with a dummy molecularly imprinted monolithic capillary coupled to ultra-performance liquid chromatography-tandem mass spectrometry to determine cannabinoids in plasma samples.

A selective and sensitive method that uses automated in-tube solid-phase microextraction coupled to ultra-performance liquid chromatography-tandem mass spectrometry (in-tube SPME/UHPLC-MS/MS) was developed to determine cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) in plasma samples. A new dummy molecularly imprinted monolithic capillary (MIP monolith) for in-tube SPME was prepared by in situ polymerization in a fused silica capillary; hydrogenated cannabidiol was employed as dummy template. Fourier Transform Infrared Spectroscopy (FTIR) confirmed that the synthesis reagents were incorporated into the polymer chain. On the basis of the microscopy images (scanning electron microscopy - SEM and transmission electron microscopy - TEM), the MIP monolithic phase presented larger pores than the non-imprinted monolithic phase (NIP monolith), as well as a skeleton comprising clusters consisting of microspheres. By optimizing the polymerization conditions, the MIP monolith specifically recognized CBD and Δ9-THC. The MIP monolith had CBD and Δ9-THC sorption capacity of 148.05 and 44.49 ng cm-3, respectively. The capillary was reused over fifty times without significant changes in its extraction efficiency. For both CBD and Δ9-THC, in-tube SPME/UHPLC-MS/MS presented linear range from 10 to 300 ng mL-1, precision with coefficient of variation (CV) values ranging from 0.2% to 19.1% (LLOQ), and accuracy with relative standard deviation (RSD) values spanning from -9.3% to 19.6% (LLOQ). The developed method was successfully applied to determine cannabinoid levels in plasma samples from volunteer patients in treatment with CBD.

Determination of anandamide in cerebrospinal fluid samples by disposable pipette extraction and ultra-high performance liquid chromatography tandem mass spectrometry.

This work describes the development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry method that uses disposable pipette extraction (DPX-UHPLC-MS/MS) to determine the endocannabinoid anandamide (AEA) in cerebrospinal fluid samples (CSF). The DPX parameters sorption equilibrium time, sample volume, number of draw-eject cycles, washing solvent volume, and elution solvent volume were optimized by design of experiments (DOE) techniques. The simple DPX protocol proposed herein required a reduced amount of CSF sample and organic solvent. The DPX-UHPLC-MS/MS method presented linear range from 0.10 ng mL-1 (LLOQ) to 3.0 ng mL-1, inter- and intra-assay accuracy with EPR values varying from -8.2% to 9.6%, inter- and intra-assay precision with CV values ranging from 1.3% to 14.8% (except for the LLOQ), and no significant matrix effect. The innovative DPX-UHPLC-MS/MS method was successfully applied to determine AEA in CSF samples from Parkinson's disease (PD) patients and should therefore be used in clinical studies.

Lab-made solid phase microextraction phases for off line extraction and direct mass spectrometry analysis: Evaluating the extraction parameters.

The analyses of drugs and metabolites in complex matrices have been widely studied in recent years. However, due to high levels endogenous compounds and matrix complexity, these analyses require a sample pre-treatment step. To this aim, two lab-made extractive phases were integrated to probe electrospray ionization mass spectrometry (PESI-MS) technique for direct analysis of illicit drugs in biological fluids and phorbol esters in Jatropha curcas extract. The polypyrrole (PPy) phase was electropolymerized onto a platinum wire surface by cyclic voltammetry. The molecularly imprinted polymer (MIP) was synthesized and adhered onto a stainless-steel needle with epoxy resin. The PPy-PESI-MS method showed to be linear in a concentration range from 1 to 500 µg L-1, with accuracy values between -2.1 and 14%, and precision values between 0.8 and 10.8%. The MIP-PESI-MS method showed to be linear in a concentration range from 0.9 to 30 mg L-1, with accuracy values between -1.6 and -15.3%, and precision values between 4.1 and 13.5%.

Tunable Silver-Containing Stationary Phases for Multidimensional Gas Chromatography.

To achieve high separation power of complex samples using multidimensional gas chromatography (MDGC), the selectivity of the employed stationary phases is crucial. The nonpolar × polar column combination remains the most popular column set used in MDGC. However, resolution of mixtures containing light analytes possessing very similar properties remains a formidable challenge. The development of stationary phases that offer unique separation mechanisms have the potential to significantly improve MDGC separations, particularly in resolving coeluting peaks in complex samples. For the first time, a stationary phase containing silver(I) ions was successfully designed and employed as a second-dimension column using comprehensive two-dimensional gas chromatography (GC × GC) for the separation of mixtures containing alkynes, dienes, terpenes, esters, aldehydes, and ketones. Compared with a widely used nonpolar and polar column set, the silver-based column exhibited superior performance by providing better chromatographic resolution of coeluting compounds. A mixture of unsaturated fatty acids was successfully separated using a GC × GC method in which the elution order in the second dimension was highly dependent on the number of double bonds within the analytes.

Butyl Methacrylate-Co-Ethylene Glycol Dimethacrylate Monolith for Online in-Tube SPME-UHPLC-MS/MS to Determine Chlopromazine, Clozapine, Quetiapine, Olanzapine, and Their Metabolites in Plasma Samples.

This manuscript describes a sensitive, selective, and online in-tube solid-phase microextraction coupled with an ultrahigh performance liquid chromatography-tandem mass spectrometry (in-tube SPME-UHPLC-MS/MS) method to determine chlopromazine, clozapine, quetiapine, olanzapine, and their metabolites in plasma samples from schizophrenic patients. Organic poly(butyl methacrylate-co-ethylene glycol dimethacrylate) monolith was synthesized on the internal surface of a fused silica capillary (covalent bonds) for in-tube SPME. Analyte extraction and analysis was conducted by connecting the monolithic capillary to an UHPLC-MS/MS system. The monolith was characterized by scanning electron microscopy (SEM) and Fourier transform infrared spectrometry (FTIR). The developed method presented adequate linearity for all the target antipsychotics: R² was higher than 0.9975, lack-of-fit ranged from 0.115 to 0.955, precision had variation coefficients lower than 14.2%, and accuracy had relative standard error values ranging from -13.5% to 14.6%, with the exception of the lower limit of quantification (LLOQ). The LLOQ values in plasma samples were 10 ng mL-1 for all analytes. The developed method was successfully applied to determine antipsychotics and their metabolites in plasma samples from schizophrenic patients.