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.

Analysis of endocannabinoids in plasma samples by biocompatible solid-phase microextraction devices coupled to mass spectrometry.

Anandamide (AEA) and 2-arachidonoyl glycerol (2-AG) represent two of the most important endocannabinoids (ECs) investigated in neurobiology as therapeutic targets for several mental disorders. However, the determination of these ECs in biological matrices remains a challenging task because of the low concentrations, low stability and high protein-bound (LogP ∼ 6). This work describes innovative analytical methods based on biocompatible SPME (Bio-SPME), SPME-UHPLC-MS/MS and Bio-SPME-Nano-ESI-MS/MS, to determine AEA and 2-AG in human plasma samples. The direct coupling of Bio-SPME with nano-ESI-MS/MS can be considered an alternative tool for faster analysis. Different Bio-SPME fibers based on silica and polymeric coating (i.e. C18, C30, and HLB) were evaluated. Different desorption solvents based on combinations of methanol, acetonitrile, and isopropanol were also evaluated for efficient elution with minimum carry-over. Given the high protein binding analytes and the fact that SPME extracts the free-concentration of the analytes, the plasma samples were modified with additives such as guanidine hydrochloride (Gu-HCl), trifluoroacetic acid, and acetonitrile. This study was carried out by experimental design to achieve complete protein denaturation and the release of target analytes. The maximum extraction efficiency was obtained under the following conditions: HLB coated fibers (10 mm length, 20 µm coating thickness), matrix modified (300 µL of plasma) with 50 µL of Gu-HCL 1 mol L-1, 75 µL of ACN and 75 µL of water, and desorption with methanol/isopropanol solution (50:50, v/v). Both methods were validated based on current international guidelines and can be applied for monitoring of concentrations of endocannabinoids in plasma samples. SPME-UHPLC-MS/MS method presented lower LOQ values than SPME-nanoESI-MS/MS. The additional separation (chromatographic column) favored the detectability of LC-MS/MS method. However, the SPME-nano-ESI-MS/MS decrease the total analysis time, due to significant reductions in desorption and detection times.

Schizophrenia: recent advances in LC-MS/MS methods to determine antipsychotic drugs in biological samples.

Schizophrenia is one of the most debilitating and costly illnesses worldwide. First-generation antipsychotics such as chlorpromazine and haloperidol succeeded in controlling the positive symptoms of schizophrenia, but had significant extrapyramidal effects that led to the search for new agents and the release of second-generation (or atypical) antipsychotics. These drugs had a lower risk of adverse motor symptoms. Therapeutic drug monitoring has become a useful tool to optimize schizophrenia treatment and HPLC-MS/MS has been considered the primary technique to monitor antipsychotics. This review comprises three sections: schizophrenia pathophysiology and treatment; recent advances in LC-MS/MS methods designed to measure levels of antipsychotics and their metabolites in plasma samples (selectivity, matrix effect and sensitivity); and the importance of therapeutic drug monitoring.

Column switching UHPLC-MS/MS with restricted access material for the determination of CNS drugs in plasma samples.

BACKGROUND: Polypharmacy is a common practice in schizophrenia. Consequently, therapeutic drug monitoring is usually adopted to maintain the concentrations of the drugs in the plasma within a targeted therapeutic range, to maximize therapeutic efficiency and to diminish adverse side effects. METHODOLOGY: This study reports on a column switching UHPLC-MS/MS method to determine psychotropic drugs in plasma samples simultaneously. RESULTS: The method was linear from 0.025 to 1.25 ng ml-1 with R2 above 0.9950 and the lack of fit test (p > 0.05). The precision values presented coefficients of variation lower than 12%, and the relative standard error of the accuracy were lower than 14%. CONCLUSION: The column switching UHPLC-MS/MS method developed herein successfully determined drugs in schizophrenic patients' plasma samples for therapeutic drug monitoring.

Determination of parabens in urine samples by microextraction using packed sorbent and ultra-performance liquid chromatography coupled to tandem mass spectrometry.

A simple, sensitive, and selective method using ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) was developed and validated for simultaneous determination of parabens [methyl paraben (MeP), ethyl paraben (EtP), propyl paraben (PrP), butyl paraben (BuP), and benzyl paraben (BzP)] in human urine samples. After microextraction by packed sorbent (MEPS) using a C18 phase, the parabens were separated on a Kinetex C18 column (100 mm × 2.1 mm × 1.7 µm) within 4.6 min using isocratic elution. These compounds were detected on a triple quadrupole tandem mass spectrometer using the multiple reactions monitoring (MRM) mode via an electrospray ionization source operating in the negative ionization mode. Important factors that influence MEPS performance were evaluated, such as the sample pH, draw-eject sample volume, clean-up step, and desorption conditions. The proposed MEPS/UPLC-MS/MS method presented a linear range from 0.5 ng mL(-1) (limit of quantification - LOQ) to 50 ng mL(-1), and interassay precision with coefficients of variation lower than 15%, and relative standard error values of the accuracy ranged from -8.8% to 15%. The MEPS/UPLC-MS/MS method was applied successfully to determine parabens in urine samples from 30 postpartum volunteers, enabling assessment of human exposure to these compounds.

Simultaneous determination of amino acids and neurotransmitters in plasma samples from schizophrenic patients by hydrophilic interaction liquid chromatography with tandem mass spectrometry.

A sensitive, reproducible, and rapid method was developed for the simultaneous determination of underivatized amino acids (aspartate, serine, glycine, alanine, methionine, leucine, tyrosine, and tryptophan) and neurotransmitters (glutamate and γ-aminobutyric acid) in plasma samples using hydrophilic interaction liquid chromatography coupled to triple quadrupole tandem mass spectrometry. The plasma concentrations of amino acids and neurotransmitters obtained from 35 schizophrenic patients in treatment with clozapine (27 patients) and olanzapine (eight patients) were compared with those obtained from 38 healthy volunteers to monitor the effectiveness of treatment. The chromatographic conditions separated ten target compounds within 3 min. This method presented linear ranges that varied from (lower limit of quantification: 9.7-13.3 nmol/mL) to (upper limit of quantification: 19.4-800 nmol/mL), intra- and interassay precision with coefficients of variation lower than 10%, and relative standard error values of the accuracy ranged from -2.1 to 9.9%. The proposed method appropriately determines amino acids and neurotransmitters in plasma from schizophrenic patients. Compared with the control group (healthy volunteers), the plasma levels of methionine in schizophrenic patients treated with olanzapine are statistically significantly higher. Moreover, schizophrenic patients treated with clozapine tend to have increased plasma levels of glutamate.

In-tube solid-phase microextraction with molecularly imprinted polymer to determine interferon alpha 2a in plasma sample by high performance liquid chromatography.

A molecularly imprinted sol­gel polymer (MIP) based on protein (biopharmaceutical) template with a mild template removal condition using protease was synthetized and evaluated as stationary phase for in-tube solid phase microextraction (in-tube SPME) of the interferon alpha 2a from plasma samples,followed by high performance liquid chromatography analysis with fluorescence detection (HPLC-FD).The developed MIP exhibited high selectivity for the analyte in a complex matrix. The in-tube SPME variables such as draw/eject cycles, draw/eject volume, and desorption conditions were optimized to establish the equilibrium conditions in a short time. The MIP in-tube SPME/HPLC-FD method presented linear response over a dynamic range of 8­300 ng mL−1, with a correlation coefficient of 0.997. The inter-assay precision presented coefficient of variation lower than 9.2%, and accuracy values between 92% and 98%. The developed MIP performed as well as other selective interferon alpha 2a stationary phases (e.g.,immunosorbent and restricted access material), with the advantage that it is robust, easy to handle and cheap to synthesize, in a addition to requiring smaller sample volume (50 L). Based on the analytical validation results, the proposed method (MIP in-tube SPME/HPLC-FD) can be a useful tool to determine interferon alpha 2a in plasma samples from patients receiving therapeutic dosages.

Simultaneous determination of nontricyclic antidepressants in human plasma by solid-phase microextraction and liquid chromatography (SPME-LC).

A sensitive, selective, and reproducible solid-phase microextraction and liquid chromatographic (SPME-LC) method for simultaneous determination of mirtazapine, citalopram, paroxetine, fluoxetine, and sertraline in human plasma was developed, validated, and further applied to analyze plasma samples obtained from patients with depression. Important factors in the optimization of SPME efficiency are discussed, including the fiber coating, extraction time, pH, ionic strength, influence of plasma proteins, and desorption conditions. The limit of quantitation of the nontricyclic antidepressants in plasma varied from 25 to 50 ng/mL with a coefficient of variation lower than 5%. The response of the SPME-LC method for most of the drugs was linear over a dynamic range of 50 to 500 ng/mL, with all of them having correlation coefficients greater than 0.9970. The performance of the SPME-LC method allowed the nontricyclic antidepressants analyses in therapeutic levels.

Reliable HPLC method for therapeutic drug monitoring of frequently prescribed tricyclic and nontricyclic antidepressants.

A new high-performance liquid chromatography method is presented for the determination of 10 frequently prescribed tricyclic and nontricyclic antidepressants: imipramine, amitriptyline, clomipramine, fluoxetine, sertraline, paroxetine, citalopram, mirtazapine, moclobemide and duloxetine. The simple and accurate sample preparation step, consisted of liquid:liquid extraction with recoveries ranging between 72% and 86%, except for moclobemide (59%). Separation was obtained using a reverse phase Select B column under isocratic conditions with UV detection (230 nm). The mobile phase consisted of 35% of a mixture of acetonitrile/methanol (92:8, v/v) and 65% of 0.25 mol L(-1) sodium acetate buffer, pH 4.5. The standard curves were linear over a working range of 2.5-1000 ng mL(-1) for moclobemide, 5-2000 ng mL(-1) for citalopram, duloxetine, fluoxetine, 10-2000 ng mL(-1) for sertraline, imipramine, paroxetine, mirtazapine and clomipramine. The intra-assay and inter-assay precision and accuracy were studied at three concentrations (50, 200, and 500 ng mL(-1)). The intra-assay coefficients of variation (CVs) for all compounds were less than 8.8%, and all inter-CVs were less than 10%. Limits of quantification were 2.5 ng mL(-1) for moclobemide, 5 ng mL(-1) for citalopram, duloxetine and amitriptyline, and 10 ng mL(-1) for mirtazapine, paroxetine, imipramine, fluoxetine, sertraline, and clomipramine. No interference of the drugs normally associated with antidepressants was observed. The method has been successfully applied to the analysis of real samples, for the drug monitoring of ten frequently prescribed tricyclic and non-tricyclic antidepressant drugs.

Optimization of the SPME parameters and its online coupling with HPLC for the analysis of tricyclic antidepressants in plasma samples.

Solid-phase microextraction (SPME)-liquid chromatography (LC) is used to analyze tricyclic antidepressant drugs desipramine, imipramine, nortriptyline, amitriptyline, and clomipramine (internal standard) in plasma samples. Extraction conditions are optimized using a 2(3) factorial design plus a central point to evaluate the influence of the time, temperature, and matrix pH. A Polydimethylsiloxane-divinylbenzene (60-mum film thickness) fiber is selected after the assessment of different types of coating. The chromatographic separation is realized using a C(18) column (150 x 4.6 mm, 5-microm particles), ammonium acetate buffer (0.05 mol/L, pH 5.50)-acetonitrile (55:45 v/v) with 0.1% of triethylamine as mobile phase and UV-vis detection at 214 nm. Among the factorial design conditions evaluated, the best results are obtained at a pH 11.0, temperature of 30 degrees C, and extraction time of 45 min. The proposed method, using a lab-made SPME-LC interface, allowed the determination of tricyclic antidepressants in in plasma at therapeutic concentration levels.

Validation of non-aqueous capillary electrophoresis for simultaneous determination of four tricyclic antidepressants in pharmaceutical formulations and plasma samples.

We present the validation of a method using non-aqueous capillary electrophoresis (NACE) for quantitative analysis of four tricyclic antidepressants (TADs) in pharmaceutical formulations and plasma. The method presented high resolution allowing the separation of the TADs in 4.3 min at optimized conditions: 50 mM ammonium acetate, 1 M acetic acid in acetonitrile, capillary with 48 cm in length, 40 cm to the detector, and voltage of 30 kV. Acceptable precision (relative standard deviation R.S.D.14.1% from plasma samples) and linearity were achieved using the internal standard (IS) method. The limits of quantification determined for plasma, after liquid-liquid extraction (LLE), were between 30 and 50 ng ml-1. These values are beyond the plasmatic therapeutic concentration. Our results were found comparable or better than those described in the literature for high performance liquid chromatography (HPLC)-based methods.