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.

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.

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.

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.

In-tube solid-phase microextraction directly coupled to tandem mass spectrometry for anandamide and 2-arachidonoylglycerol determination in rat brain samples from an animal model of Parkinson's disease.

To evaluate the endocannabinoid system in an animal model of Parkinson's disease, in-tube solid-phase microextraction (in-tube SPME) was directly coupled to a tandem mass spectrometry (MS/MS) system for determination of the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples. In-tube SPME-which consisted of a microtube of restricted access material (RAM) with a hydrophilic diol external surface and a hydrophobic octyl inner surface-efficiently excluded (up to 95%) macromolecules from the biological samples and selectively pre-concentrated the analytes. In-tube SPME parameters, such as sample volume, mobile phases, flow rate, and pre-concentration time, were evaluated to improve the extraction efficiency and throughput performance. The selectivity of the in-tube SPME and MS/MS (MRM mode) techniques allowed them to be directly coupled online, which dismissed the need for the chromatographic separation step. The in-tube SPME-MS/MS method was validated and shown to be linear from 6.0 to 30.0 ng mL-1 for AEA and from 10.0 to 100.0 ng mL-1 for 2-AG; the intra- and inter-assay accuracy and precision were lower than 15%. Parallelism between the calibration curves constructed in the matrix and aqueous solution confirmed that there was no matrix effect. The method allowed endogenous concentrations of AEA and 2-AG to be determined in rat brain striatum from unilaterally 6-hydroxydopamine-lesioned animals. The concentrations of these endocannabinoids in striatum ipsilateral and contralateral to the lesion differed significantly (p<0.001).

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.

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.

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.

Recent advances in LC-MS/MS methods to determine endocannabinoids in biological samples: Application in neurodegenerative diseases.

Endocannabinoids (ECs) are endogenous lipid-based retrograde neurotransmitters that bind to cannabinoid receptors [cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2)]. Many ECs have been characterized; anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) are still considered the primary ECs signaling mediators. Dysregulation of ECs has been implicated in a wide range of pathologies, including neurodegenerative diseases. Understanding how ECs participate in neurological diseases is important to describe the pathology and to establish new treatments. Considering the physicochemical properties of ECs, liquid chromatography coupled to tandem mass spectrometry has become the reference method to determine these endogenous substances, in trace levels, in different biological samples. This review describes the recent advances in LC-MS/MS methods designed to determine ECs in complex biological matrixes. The advantages, limitations, selectivity, matrix effect, and sensitivity associated with each approach are emphasized. This article comprises three sections: (I) sample preparation techniques (conventional, microextraction, and online systems), (II) chromatographic methods (especially LC-MS/MS), and (III) relationship between ECs levels and neurodegenerative diseases.

Recent development of chromatographic methods to determine parabens in breast milk samples: A review.

Parabens have been widely used as antimicrobial preservatives in food, drugs, and cosmetics for over 60 years. These endocrine disruptors can alter both the wildlife and the human hormone function. Determining these compounds in human milk is important because breast milk plays an important role in infant growth and in neurocognitive development. This article summarizes the current state-of-the-art of chromatographic methods to determine parabens in breast milk samples. Apart from the conventional and modern microextraction sample preparation techniques described herein, the authors discuss the chromatographic systems, primarily LC-MS/MS, and the concentration ranges at which parabens have been detected in milk samples obtained from lactating women over the past few years.

Pipette tip dummy molecularly imprinted solid-phase extraction of Bisphenol A from urine samples and analysis by gas chromatography coupled to mass spectrometry.

Molecularly imprinted polymers (MIPs) were synthesized and used as sorbent for Bisphenol A (BPA) pipette tip solid-phase microextraction from urine samples and BPA analysis by gas chromatography coupled to mass spectrometry (GC-MS). The MIPs were synthesized by the sol-gel methodology. Aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) were used as functional monomer and cross-linking reagent, respectively. BPA and tetrabromobisphenol A (TBBPA) were evaluated as template during MIP synthesis. The BPA-based MIP displayed slightly higher extraction efficiency than the TBBPA-based dummy molecularly imprinted polymer (DMIP), but the TBBPA-based DMIP was selected as sorbent to minimize interference from leaked template. Comparison of the TBBPA-based DMIP, BPA-based MIP, and non-imprinted polymer (NIP) extraction efficiencies attested that the TBBPA-based DMIP was selective. The synthesized polymers were characterized by Scanning Electron Microscopy (SEM) and Fourier Transform Infrared spectroscopy (FTIR). The TBBPA-based DMIP was reused for over 100 times, which confirmed its robustness. The developed method was linear from 50 to 500ngmL-1. Precision values had coefficient of variation (CV) ranging from 4 to 14%. The accuracy relative standard deviation values (RSD) varied from -13.6 to 12.3%.

Bisphenol A release from orthodontic adhesives measured in vitro and in vivo with gas chromatography.

INTRODUCTION: The objectives of this study were to quantify in vitro the Bisphenol A (BPA) release from 5 orthodontic composites and to assess in vivo the BPA level in patients' saliva and urine after bracket bonding with an orthodontic adhesive system. METHODS: For the in-vitro portion of this study, 5 orthodontic composites were evaluated: Eagle Spectrum (American Orthodontics, Sheboygan, Wis), Enlight (Ormco, Orange, Calif), Light Bond (Reliance Orthodontic Products, Itasca, Ill), Mono Lok II (Rocky Mountain Orthodontics, Denver, Colo), and Transbond XT (3M Unitek, Monrovia, Calif). Simulating intraoral conditions, the specimens were immersed in a water/ethanol solution, and the BPA (ng.g-1) liberation was measured after 30 minutes, 24 hours, 1 day, 1 week, and 1 month by the gas chromatography system coupled with mass spectrometry. Twenty patients indicated for fixed orthodontic treatment participated in the in-vivo study. Saliva samples were collected before bracket bonding and then 30 minutes, 24 hours, 1 day, 1 week, and 1 month after bonding the brackets. Urine samples were collected before bonding and then at 1 day, 1 week, and 1 month after bonding. The results were analyzed statistically using analysis of variance and Tukey posttest, with a significance level of 5%. RESULTS: All composites evaluated in vitro released small amounts of BPA. Enlight composite showed the greatest release, at 1 month. Regarding the in-vivo study, the mean BPA level in saliva increased significantly only at 30 minutes after bonding in comparison with measurements recorded before bonding. CONCLUSIONS: All orthodontic composites released BPA in vitro. Enlight and Light Bond had, respectively, the highest and lowest BPA releases in vitro. The in-vivo experiment showed that bracket bonding with the Transbond XT orthodontic adhesive system resulted in increased BPA levels in saliva and urine. The levels were significant but still lower than the reference dose for daily ingestion.

A column switching ultrahigh-performance liquid chromatography-tandem mass spectrometry method to determine anandamide and 2-arachidonoylglycerol in plasma samples.

This study reports a fast, sensitive, and selective column switching ultrahigh-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method to determine the endocannabinoids (eCBs), anandamide (AEA), and 2-arachidonoylglycerol (2-AG) in plasma samples. This bidimensional system used a restricted access media column (RP-8 ADS, 25 mm × 4 mm × 25 µM) in the first dimension and a core-shell Kinetex C18 (100 mm × 2, 1.7 mm × 1 µM) column in the second dimension, followed by detection in a mass spectrometer triple quadrupole (multiple reactions monitoring mode) operating in the positive mode. RP-8 ADS was used for trace enrichment of eCBs (reverse phase partitioning) and macromolecular matrix size exclusion; the core-shell column was used for the chromatographic separation. The column switching UHPLC-MS/MS method presented a linear range spanning from 0.1 ng mL-1 (LOQ) to 6 ng mL-1 for AEA and from 0.04 ng mL-1 (LOQ) to 10 ng mL-1 for 2-AG. Excluding the LLOQ values, the precision assays provided coefficients of variation lower than 8% and accuracy with relative standard error values lower than 14%. Neither carryover nor matrix effects were detected. This high-throughput column switching method compared to conventional methods is time saving as it involves fewer steps, consumes less solvent, and presents lower LLOQ. The column switching UHPLC-MS/MS method was successfully applied to determine AEA and 2-AG in plasma samples obtained from Alzheimer's disease patients. Graphical abstract A column switching ultra high-performance liquid chromatography-tandem mass spectrometry method using RP-8 ADS column and core shell column to determine endocannabinoids in plasma samples.

Evaluation of superficially porous and fully porous columns for analysis of drugs in plasma samples by UHPLC-MS/MS.

This work compares the performance of recently introduced C18 superficially porous columns (four columns) and C18 fully porous columns (two columns), with different particle sizes, during the analysis of drugs in plasma samples by MS/MS detection. The following chromatographic parameters were evaluated for all the columns: reduced plate height (h) vs reduced linear velocity (v), impedance vs v, chromatographic run time vs flow rate (mLmin-1), backpressure vs flow rate (mLmin-1), resolution, peak capacity, asymmetry, and retention factor. By using DAD detection, hydrophobicity, silanol activity, and metal impurities were also assessed for the columns. The columns with charged surface displayed improved chromatographic efficiency for the drugs in their ionized form. The columns with particles smaller than 2µm (Cortecs 1.6µm, Acquity 1.7µm, and Kinetex 1.7µm) presented higher chromatographic efficiency for the drugs, which were in their partially ionized form. The generated mathematical models were able to predict the backpressure and chromatographic run time at different flow rates for all the columns. Considering the efficiency, impedance, resolution, peak capacity, retention factor, and hydrophobicity, Cortecs 1.6µm and Acquity 1.7µm were the columns that given the best performance during analysis of the target drugs in plasma samples.

Determination of Drugs in Plasma Samples by High-Performance Liquid Chromatography-Tandem Mass Spectrometry for Therapeutic Drug Monitoring of Schizophrenic Patients.

This work describes the development of a simple, sensitive and selective method based on high-performance liquid chromatography coupled to tandem mass spectrometry (LC-MS-MS) to determine antipsychotics (olanzapine, quetiapine, clozapine, haloperidol and chlorpromazine) along with antidepressants (mirtazapine, paroxetine, citalopram, sertraline, imipramine, clomipramine and fluoxetine), anticonvulsants (carbamazepine and lamotrigine) and anxiolytics (diazepam and clonazepam) in plasma samples obtained from schizophrenic patients. The samples were prepared by protein precipitation. The target drugs were separated on an XSelect SCH C18 column (100 mm × 2.1 mm × 2.5 µm) within 8.0 min by means of gradient elution. The drugs were then detected on a quadrupole tandem mass spectrometer equipped with an electrospray ionization source, operating in the multiple reactions monitoring mode and in the positive ionization mode. The LC-MS-MS method was linear range from subtherapeutic to toxic concentrations with lower limit of quantification values ranged from 0.2 to 5.0 ng mL(-1), precision with coefficient of variation values lower than 12%, and accuracy ranged from 90 to 108%. The developed method enabled successful analysis of the target drugs in plasma samples obtained from 51 schizophrenic patients. Therapeutic drug monitoring revealed that many of the evaluated schizophrenic patients presented altered plasma concentrations of the analyzed drugs. These altered concentrations resulted from pharmacokinetic interactions among the medications prescribed to treat schizophrenia.

Analysis of drugs in plasma samples from schizophrenic patients by column-switching liquid chromatography-tandem mass spectrometry with organic-inorganic hybrid cyanopropyl monolithic column.

This study reports on the development of a rapid, selective, and sensitive column-switching liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to analyze sixteen drugs (antidepressants, anticonvulsants, anxiolytics, and antipsychotics) in plasma samples from schizophrenic patients. The developed organic-inorganic hybrid monolithic column with cyanopropyl groups was used for the first dimension of the column-switching arrangement. This arrangement enabled online pre-concentration of the drugs (monolithic column) and their subsequent analytical separation on an XSelect SCH C18 column. The drugs were detected on a triple quadrupole tandem mass spectrometer (multiple reactions monitoring mode) with an electrospray ionization source in the positive ion mode. The developed method afforded adequate linearity for the sixteen target drugs; the coefficients of determination (R(2)) lay above 0.9932, the interassay precision had coefficients of variation lower than 6.5%, and the relative standard error values of the accuracy ranged from -14.0 to 11.8%. The lower limits of quantification in plasma samples ranged from 63 to 1250pgmL(-1). The developed method successfully analyzed the target drugs in plasma samples from schizophrenic patients for therapeutic drug monitoring (TDM).