FeS2 nanosheets-luminol-O2 chemiluminescence method for determination of venlafaxine hydrochloride, imipramine hydrochloride, and cefazolin sodium.

This study introduces a novel chemiluminescence (CL) approach utilizing FeS2 nanosheets (NSs) catalyzed luminol-O2 CL reaction for the measurement of three pharmaceuticals, namely venlafaxine hydrochloride (VFX), imipramine hydrochloride (IPM), and cefazolin sodium (CEF). The CL method involved the phenomenon of quenching induced by the pharmaceuticals in the CL reaction. To achieve the most quenching efficacy of the pharmaceuticals in the CL reaction, the concentrations of reactants comprising luminol, NaOH, and FeS2 NSs were optimized accordingly. The calibration curves demonstrated exceptional linearity within the concentration range spanning from 4.00 × 10-7 to 1.00 × 10-3 mol L-1, 1.00 × 10-7 to 1.00 × 10-4 mol L-1, and 4.00 × 10-6 to 2.00 × 10-4 mol L-1 with detection limits (3σ) of 3.54 × 10-7, 1.08 × 10-8, and 2.63 × 10-6 mol L-1 for VFX, IPM, and CEF, respectively. This study synthesized FeS2 NSs using a facile hydrothermal approach, and then the synthesized FeS2 NSs were subjected to a comprehensive characterization using a range of spectroscopic methods. The proposed CL method was effective in measuring the aforementioned pharmaceuticals in pharmaceutical formulations as well as different water samples. The mechanism of the CL system has been elucidated.

On-Chip Electromembrane Surrounded Solid Phase Microextraction for Determination of Tricyclic Antidepressants from Biological Fluids Using Poly(3,4-ethylenedioxythiophene)-Graphene Oxide Nanocomposite as a Fiber Coating.

In the present study, on-chip electromembrane surrounded solid phase microextraction (EM-SPME) was employed in the determination of tricyclic antidepressants (TCAs), including amitriptyline, nortriptyline, imipramine, desipramine, maprotiline, and sertraline, from various biological fluids. In this regard, poly(3,4-ethylenedioxythiophene)-graphene oxide (PEDOT-GO) was electrodeposited on an SPME fiber as a conductive coating, then the fiber played the acceptor-electrode role during the extraction. Thus, the immigration of the analytes under the influence of an electric field and their absorption onto the fiber coating were accomplished simultaneously. Under the optimized conditions, the limits of detection for the target analytes were acquired in the range of 0.005-0.025 µg L-1 using gas chromatography-mass spectrometry. The linearity of the method was 0.010-500 µg L-1 for the imipramine and sertraline, 0.025-500 µg L-1 for the amitriptyline, nortriptyline, and desipramine, and 1.000-250 µg L-1 for the maprotiline (R2 ≥ 0.9984). Moreover, this method provided suitable precision and fiber-to-fiber reproducibility, with RSDs ≤ 8.4%. The applicability of the proposed setup was eventually investigated for extraction of the drugs from human bone marrow aspirate, urine, plasma, and well water samples, in which satisfactory relative recoveries, from 93-105%, were obtained.

Evaluation of molecularly imprinted polymers for chlorpromazine and bromopromazine prepared by multi-step swelling and polymerization method-The application for the determination of chlorpromazine and its metabolites in rat plasma by column-switching LC.

Monodisperse molecularly imprinted polymers (MIPs) for chlorpromazine (CPZ) and bromopromazine (BPZ), MIPCPZ and MIPBPZ, were prepared using methacrylic acid as a functional monomer and ethylene glycol dimethacrylate as a crosslinker by multi-step swelling and polymerization. The retention and molecular-recognition properties of MIPCPZ and MIPBPZ were evaluated using a mixture of potassium phosphate buffer and acetonitrile or a mixture of water and acetonitrile including ammonium formate as a mobile phase in reversed-phase LC. On MIPBPZ, CPZ, BPZ and imipramine (IMP) gave the maximal retention factors at a mobile-phase pH 8, while the maximal imprinting factors were obtained at a mobile-phase pH 7. Each MIP recognized a template molecule the most, while CPZ metabolites, desmethyl CPZ (DM-CPZ), CPZ sulfoxide (CPZ-SO) and 7-hydroxy CPZ (7-OH-CPZ), were moderately recognized on MIPCPZ and MIPBPZ. Furthermore, both MIPs gave the similar retention and molecular-recognition for CPZ and its metabolites. For avoiding the template-leakage problems, MIPBPZ was used as the pretreatment column for the determination of CPZ and its metabolites in rat plasma in column-switching LC with UV detection. In addition to DM-CPZ and CPZ-SO, didesmethyl CPZ (DDM-CPZ) and CPZ N-oxide (CPZ-NO) were speculated as the metabolite in rat plasma after administration of CPZ using LC-ESI-TOF-MS, while 7-OH-CPZ was not detected. The column-switching LC method was validated and applied for the determination of CPZ and its metabolites, DM-CPZ, DDM-CPZ, CPZ-SO and CPZ-NO, in rat plasma after intravenous and oral administration of CPZ using IMP as an internal standard.

Selective drug metabolite trace analysis by very high-volume injections and heartcut two-dimensional (2D)-ultrahigh performance liquid chromatography (UHPLC).

The application of two-dimensional liquid chromatography (2D-LC) is gradually growing also in the area of metabolite profiling and identification. The current contribution describes a heartcut 2D-UHPLC configuration that is applied in support of drug metabolism studies in development. The setup applies four LC columns: two analytical UHPLC columns to perform the first and second dimension separations, which are both preceded by a short HPLC column operated as trapping column. The first HPLC column allows a significant online preconcentration by large volume injection. The second short HPLC column is placed between the first and second dimension columns and enables the selection of orthogonal conditions in the second dimension independent of the first dimension making the heartcutting 2D approach more generic. The value of the setup was demonstrated with selective ultraviolet chromatograms obtained for the two major hydroxylated metabolites of atorvastatin separating them from a very high biological background, originating from an injection of 4 mL feces extract, by heartcut 2D-LC. In a second application, the main metabolite of imipramine was baseline separated from some minor metabolites that were co-eluting in the first dimension, allowing accurate and sensitive quantification. A quantification limit in the attogram/mL range was achieved thanks to the injection of 200 mL diluted urine, corresponding to 100 mL urine on column.

Simple and green synthesis of carbon dots (CDs) from valerian root and application of modified mesoporous boehmite (AlOOH) with CDs as a fluorescence probe for determination of imipramine.

A novel, sensitive, rapid, and simple fluorescent probe has been developed based on green-synthesized carbon dots (CDs). In this work, CDs have been synthesized from valerian root by hydrothermal method. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) results confirm the formation of CDs with sizes of less than 10 nm. Fluorescence quenching of CDs was due to the aggregation of the negative charges of CDs with the positive charge of imipramine (IMI) and was then used as the signal for determination of IMI. In addition, the cytotoxicity of CDs was determined using the MTT assay. The probe responses under optimum conditions were linear in the range of 1.0-200.0 ng mL-1 with a limit of detection of 0.6 ng mL-1. Afterwards, mesoporous boehmite (MB) was modified with synthesized CDs (CDs/MB). TEM images confirmed MB modification with CDs. In this case, the variations in the fluorescence signal for different concentrations of IMI increased leading to the higher sensitivity for IMI detection. The limit of detection and linear range for determination of IMI with CDs/MB were obtained as 0.2 and 0.5-200.0 ng mL-1, respectively. To evaluate the fluorescent probe, IMI was measured in real samples. Graphical abstract.

Toward Higher Sensitivity in Quantitative MALDI Imaging Mass Spectrometry of CNS Drugs Using a Nonpolar Matrix.

Tissue-specific ion suppression is an unavoidable matrix effect in MALDI mass spectrometry imaging (MALDI-MSI), the negative impact of which on precision and accuracy in quantitative MALDI-MSI can be reduced to some extent by applying isotope internal standards for normalization and matrix-matched calibration routines. The detection sensitivity still suffers, however, often resulting in significant loss of signal for the investigated analytes. An MSI application considerably affected by this phenomenon is the quantitative spatial analysis of central nervous system (CNS) drugs. Most of these drugs are low molecular weight, lipophilic compounds, which exhibit inefficient desorption and ionization during MALDI using conventional polar acidic matrices (CHCA, DHB). Here, we present the application of the (2-[(2 E)-3-(4- tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile) matrix for high sensitivity imaging of CNS drugs in mouse brain sections. Since DCTB is usually described as an electron-transfer matrix, we provide a rationale (i.e., computational calculations of gas-phase proton affinity and ionization energy) for an additional proton-transfer ionization mechanism with this matrix. Furthermore, we compare the extent of signal suppression for five different CNS drugs when employing DCTB versus CHCA matrices. The results showed that the signal suppression was not only several times lower with DCTB than with CHCA but also depended on the specific tissue investigated. Finally, we present the application of DCTB and ultrahigh resolution Fourier transform ion cyclotron resonance mass spectrometry to quantitative MALDI imaging of the anesthetic drug xylazine in mouse brain sections based on a linear matrix-matched calibration curve. DCTB afforded up to 100-fold signal intensity improvement over CHCA when comparing representative single MSI pixels and >440-fold improvement for the averaged mass spectrum of the adjacent tissue sections.

Combination of electromembrane extraction and electro-assisted liquid-liquid microextraction: A tandem sample preparation method.

As a well-known extraction procedure, electromembrane extraction (EME) was combined with electro-assisted liquid-liquid microextraction (EA-LLME) in the present work, which resulted in a promising method. This hyphenated sample preparation method, named EME-EA-LLME, was followed by GC for the determination of two model analytes (clomipramine and imipramine). The effective parameters of both EME and EA-LLME (such as organic solvent, pH of acceptor and sample solutions, voltage and extraction time) were optimized. The proposed EME-EA-LLME procedure demonstrated good linearity with coefficients of determination, R2 ≥ 0.998 over the concentration range of 0.5-750 ng/mL. Limit of detection for both analytes was 0.15 ng/mL. The corresponding repeatability ranged from 6.9 to 12.2% (n = 3). The high enrichment factors were obtained as 770.3 and 561.4 for imipramine and clomipramine, respectively. The advantages of this tandem sample preparation method were low detection limits, simplicity, low cost, and short analysis time (<10 min). Finally, the optimized method was used to extract and determine the analytes in urine and wastewater samples. Overall, the results revealed that the developed EME-EA-LLME procedure had better extraction efficiency in comparison with EME and EA-LLME alone.

Liquid-Phase Microextraction and Gas Chromatographic-Mass Spectrometric Analysis of Antidepressants in Vitreous Humor: Study of Matrix Effect of Human and Bovine Vitreous and Saline Solution.

In forensic bioanalytical methods, there is a general agreement that calibrators should be prepared by fortifying analytes in matrix-based blank samples (matrix-based). However, in the case of vitreous humor (VH), the collection of blank samples for the validation and for routine analysis would require the availability of many cadavers. Besides the difficulty of obtaining enough blank VH, this procedure could also represent an ethical issue. Here, a study of matrix effect was performed taking into consideration human and bovine vitreous and saline solution (SS) (NaCl 0.9%). Tricyclic antidepressants [amitriptyline (AMI), nortriptyline (NTR), imipramine (IMI) and desipramine (DES)] were used as model analytes and were extracted from samples by means of liquid-phase microextraction and detected by gas chromatography-mass spectrometry. Samples of human and bovine VH and SS were prepared in six different concentrations of antidepressants (5, 40, 80, 120, 160 and 200 ng/mL) and were analyzed. Relative matrix effect was evaluated by applying a two-tailed homoscedastic Student's t-test, comparing the results obtained with the set of data obtained with human VH and bovine VH and SS. No significant matrix effect was found for AMI and NTR in the three evaluated matrices. However, a great variability was observed for IMI and DES for all matrices. Once compatibilities among the matrices were demonstrated, the method was fully validated for AMI and NTR in SS. The method was applied to six VH samples deriving from real cases whose femoral whole blood (FWB) was analyzed by a previously published method. An average ratio (VH/FWB) of ∼ 0.1 was found for both compounds.

Synthesis of palladium nanoparticles on organically modified silica: application to design of a solid-state electrochemiluminescence sensor for highly sensitive determination of imipramine.

Organically modified silica substrate containing amine and vinyl functional groups were used for reduction and stabilization of palladium nanoparticles. Uniform spherical nanoparticles of palladium with average diameter of 10 nm were formed on silica substrate by direct contact of the substrate with an aqueous solution of palladium precursor, without the addition of any chemical reducer. Moreover, a sensitive and selective solid state electrochemiluminescence sensor was fabricated for the determination of imipramine, based on Ru(bpy)3(2+)-palladium nanoparticles doped carbon ionic liquid electrode. In this process, imipramine acts as a co-reactant for Ru(bpy)3(2+). It is believed that the enhancement of the electrochemiluminescence signal in the presence of palladium nanoparticles in the composite is due to palladium catalytic effect on electrochemical and also chemical process involved in formation of Ru(byp)3(2+)*. In addition, the results confirmed that, the rigid composite electrode shows the characteristic of microelectrode arrays. The proposed method was applied to the determination of imipramine in tablets and urine samples. The electrochemiluminescence intensity showed good linearity with the imipramine concentration from 1-100 pM, with a detection limit of 0.1 pM.

Adsorptive stripping voltammetric determination of imipramine, trimipramine and desipramine employing titanium dioxide nanoparticles and an Amberlite XAD-2 modified glassy carbon paste electrode.

An Amberlite XAD-2 (XAD2) and titanium dioxide nanoparticles (TNPs) modified glassy carbon paste electrode (XAD2-TNP-GCPE) was developed for the determination of imipramine (IMI), trimipramine (TRI) and desipramine (DES). The electrochemical behavior of these molecules was investigated employing cyclic voltammetry (CV), chronocoulometry (CC), electrochemical impedance spectroscopy (EIS) and adsorptive stripping differential pulse voltammetry (AdSDPV). After optimization of analytical conditions using a XAD2-TNP-GCPE electrode at pH 6.0 phosphate buffer (0.1 M), the peak currents were found to vary linearly with its concentration in the range of 1.30 × 10(-9) to 6.23 × 10(-6) M for IMI, 1.16 × 10(-9) to 6.87 × 10(-6) M for TRI and 1.43 × 10(-9) to 5.68 × 10(-6) M for DES. The detection limits (S/N = 3) of 3.93 × 10(-10), 3.51 × 10(-10) and 4.35 × 10(-10) M were obtained for IMI, TRI and DES respectively using AdSDPV. The prepared modified electrode showed several advantages such as a simple preparation method, high sensitivity, very low detection limits and excellent reproducibility. The proposed method was employed for the determination of IMI, TRI and DES in pharmaceutical formulations, blood serum and urine samples.

Comparación de los efectos del D-004, imipramina y sertralina en el modelo de nado forzado en ratones / Comparative effects of D-004, Imipramine and Sertraline in the forced swimming test in mice

Introducción: La finasterida, inhibidor de la 5 a-reductasa, se emplea en el tratamiento de la hiperplasia prostática benigna, y ocasiona entre sus efectos adversos un aumento de cuadros de depresión. El D-004 es un extracto lipídico extraído a partir del fruto de la palma real (Roystonea regia), inhibe la 5 a-reductasa y previene la hiperplasia prostática benigna, y muestra un efecto antidepresivo moderado en el ensayo de nado forzado y suspensión por la cola. Objetivo: comparar el efecto del D-004 con la imipramina y la sertralina sobre la duración de las conductas de inmovilidad, nado y escalado en el ensayo de nado forzado. Métodos: se distribuyeron los ratones en ocho grupos: control (vehículo), tres tratados con D-004 (100, 250 y 500 mg/kg), dos con sertralina y dos con imipramina (30 y 50 mg/kg) respectivamente. Estos se colocaron en un cilindro de cristal que contenía agua a una altura de 6 cm y se cuantificaron las conductas. Resultados: la administración oral de D-004 (100, 250 y 500 mg/kg) durante 14 días redujo significativamente el tiempo de inmovilidad con respecto al grupo control (17, 22 y 25 porciento) y aumentó significativamente la conducta de nado en 1,58, 1,68 y 1,74 veces. Este efecto resulta moderado (25 porciento) comparado con las reducciones alcanzadas por la sertralina y la imipramina (³ 60 porciento). Las dosis mayores (250 y 500 mg/kg) ocasionaron incrementos de la conducta de escalado, 2,79 y 3,55 veces superiores a la del grupo control, lo que mostró semejanza con la imipramina, aunque con una menor eficacia. Conclusiones: el D-004 ejerce un moderado efecto antidepresivo, lo que pudiera contribuir al manejo de los pacientes con hiperplasia prostática benigna en los cuales se informa coincidencia de cuadros depresivos

Introduction: Finasteride is a 5 a-reductase inhibitor to treat benign prostatic hyperplasia (BPH) and one of the adverse effects is the increase of depressive symptoms. D-004 is a lipid extract from the real palm fruit (Roystonea regia) that is effective to prevent prostatic hyperplasia by inhibiting 5 a-reductase and shows moderate antidepressant effects in the forced swimming test (FST) and tail suspension test. Objective: to compare the effects of D-004, Imipramine and Sertraline on the duration of behaviours under conditions of immobility, swimming and climbing in the forced swimming test. Methods: mice were randomly distributed in 8 groups: control (vehicle), 3 treated with D-004 (100, 250 and 500 mg/kg), 2 with Sertraline and 2 with Imipramine (30 and 50 mg/kg) respectively. Mice were placed in a glass cylinder containing 6 cm high column of water and their behaviours were quantified. Results: oral administration of D-004 (100, 250 and 500 mg/kg) during 14 days reduced the length of time of immobility with respect to the control group (17, 22 and 25 percent), and significantly increased the behaviours at swimming by 1.58, 1.68 and 1.74 times. This is a moderate effect (25 percento) if compared with Sertraline and Imipramine (³ 60 percent) The doses of 250 and 500 mg/kg showed that climbing behaviours were 2.79 and 3.55 times higher than the control group. The results were similar to those of Imipramine but less effective. Conclusions: D-004 showed moderate antidepressant effect. This fact could help in the treatment of patients with benign prostatic hyperplasia, who reported similar depressive status

Comparación de los efectos del D-004, imipramina y sertralina en el modelo de nado forzado en ratones / Comparative effects of D-004, Imipramine and Sertraline in the forced swimming test in mice

Introducción: La finasterida, inhibidor de la 5 a-reductasa, se emplea en el tratamiento de la hiperplasia prostática benigna, y ocasiona entre sus efectos adversos un aumento de cuadros de depresión. El D-004 es un extracto lipídico extraído a partir del fruto de la palma real (Roystonea regia), inhibe la 5 a-reductasa y previene la hiperplasia prostática benigna, y muestra un efecto antidepresivo moderado en el ensayo de nado forzado y suspensión por la cola. Objetivo: comparar el efecto del D-004 con la imipramina y la sertralina sobre la duración de las conductas de inmovilidad, nado y escalado en el ensayo de nado forzado. Métodos: se distribuyeron los ratones en ocho grupos: control (vehículo), tres tratados con D-004 (100, 250 y 500 mg/kg), dos con sertralina y dos con imipramina (30 y 50 mg/kg) respectivamente. Estos se colocaron en un cilindro de cristal que contenía agua a una altura de 6 cm y se cuantificaron las conductas. Resultados: la administración oral de D-004 (100, 250 y 500 mg/kg) durante 14 días redujo significativamente el tiempo de inmovilidad con respecto al grupo control (17, 22 y 25 porciento) y aumentó significativamente la conducta de nado en 1,58, 1,68 y 1,74 veces. Este efecto resulta moderado (25 porciento) comparado con las reducciones alcanzadas por la sertralina y la imipramina (³ 60 porciento). Las dosis mayores (250 y 500 mg/kg) ocasionaron incrementos de la conducta de escalado, 2,79 y 3,55 veces superiores a la del grupo control, lo que mostró semejanza con la imipramina, aunque con una menor eficacia. Conclusiones: el D-004 ejerce un moderado efecto antidepresivo, lo que pudiera contribuir al manejo de los pacientes con hiperplasia prostática benigna en los cuales se informa coincidencia de cuadros depresivos(AU)

Introduction: Finasteride is a 5 a-reductase inhibitor to treat benign prostatic hyperplasia (BPH) and one of the adverse effects is the increase of depressive symptoms. D-004 is a lipid extract from the real palm fruit (Roystonea regia) that is effective to prevent prostatic hyperplasia by inhibiting 5 a-reductase and shows moderate antidepressant effects in the forced swimming test (FST) and tail suspension test. Objective: to compare the effects of D-004, Imipramine and Sertraline on the duration of behaviours under conditions of immobility, swimming and climbing in the forced swimming test. Methods: mice were randomly distributed in 8 groups: control (vehicle), 3 treated with D-004 (100, 250 and 500 mg/kg), 2 with Sertraline and 2 with Imipramine (30 and 50 mg/kg) respectively. Mice were placed in a glass cylinder containing 6 cm high column of water and their behaviours were quantified. Results: oral administration of D-004 (100, 250 and 500 mg/kg) during 14 days reduced the length of time of immobility with respect to the control group (17, 22 and 25 percent), and significantly increased the behaviours at swimming by 1.58, 1.68 and 1.74 times. This is a moderate effect (25 percento) if compared with Sertraline and Imipramine (³ 60 percent) The doses of 250 and 500 mg/kg showed that climbing behaviours were 2.79 and 3.55 times higher than the control group. The results were similar to those of Imipramine but less effective. Conclusions: D-004 showed moderate antidepressant effect. This fact could help in the treatment of patients with benign prostatic hyperplasia, who reported similar depressive status(AU)

Controlled-pH tissue cleanup protocol for signal enhancement of small molecule drugs analyzed by MALDI-MS imaging.

The limit of detection of low-molecular weight compounds in tissue sections, analyzed by matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI), was significantly improved by employing sample washing using a pH-controlled buffer solution. The pH of the washing solutions were set at values whereby the target analytes would have low solubility. Washing the tissue sections in the buffered solution resulted in removal of endogenous soluble ionization-suppressing compounds and salts, while the target compound remained in situ with minor or no delocalization during the buffered washing procedure. Two pharmaceutical compounds (cimetidine and imipramine) and one new protease inhibitor compound were successfully used to evaluate the feasibility of the pH-controlled tissue washing protocol for MALDI-MSI. Enhancement in signal-to-noise ratio was achieved by a factor of up to 10.

Evaluation of the potential of surface enhancement Raman spectroscopy for detection of tricyclic psychotropic drugs. Case studies on imipramine and its metabolite.

The potential use of surface Raman enhanced spectroscopy (SERS) for confirmatory identification and the semi-quantitative analysis of selected tricyclic antidepressants (TCAs) is examined utilizing a conventional silver colloid. Raman and SERS spectra of aqueous solutions of imipramine (Imi) and its metabolite, desipramine (Des), were recorded as the function of concentration using NIR excitation. A good linear correlation is observed for the dependence of the SERS signal at 684 cm(-1) (R(2) = 0.9997) on Imi concentration over the range of 0.75-7.5 µM. The limit of detection of imipramine in the silver colloidal solution is 0.98 µM. SERS spectra of Imi and Des were also recorded for blood plasma samples without prior purification as well as after the use of standard solid phase extraction. All spectra show the characteristic spectral profile of the molecules and moreover, stronger signal enhancement is observed for Imi in the "raw" samples as opposed to Imi extracted from a biological matrix.

Liquid chromatography-tandem mass spectrometry method for measurement of nicotine N-glucuronide: a marker for human UGT2B10 inhibition.

Nicotine is considered to be a specific substrate for UGT2B10, an isoform of human uridine diphosphate glucuronosyltransferase (UGT). In the present study, a sensitive and selective liquid chromatography/tandem mass spectrometry (LC-MS-MS) method for quantification of nicotine N-glucuronide in pooled human liver microsomal incubates was developed and validated. Proteins in a 200µL aliquot of incubation solution were precipitated by adding 40µL 35% perchloric acid. The overall extraction efficiency was greater than 98%. Nicotine N-glucuronide and internal standard were recorded using selected reaction monitoring in positive ion electrospray with ion transitions of m/z 339-163 and m/z 342-166, respectively. The linear calibration curve was obtained over the concentration range of 10-1000nM, with a lower limit of quantification of 10nM. The intra-day and inter-day precision (% CV) and accuracy (% bias) of the method were within 15% at all quality control levels. Nicotine glucuronide in processed samples was stable for 24h at room temperature and 48h at 4°C based on the stability experiments performed in this study. This established method was employed to evaluate the inhibitory effects of five target compounds including amitriptyline, hecogenin, imipramine, lamotrigine, and trifluoperazine on enzymatic activity of UGT2B10. IC(50) values for inhibition of nicotine N-glucuronidation by amitriptyline, imipramine, lamotrigine, and trifluoperazine were calculated. Trifluoperazine was found to be a non-substrate inhibitor for human UGT2B10.

Determination of imipramine and trimipramine by capillary electrophoresis with electrochemiluminescence detection.

The tricyclic antidepressants (TCA) imipramine (Imi) and trimipramine (Tri) were successfully analyzed by capillary electrophoresis (CE) coupling with Tris(2,2-bipyridyl) ruthenium(II)-based (Ru(bpy)(3)(2+)) end-column electrochemiluminescence (ECL) detection. The addition of ß-CD to the running buffer was found to enable baseline separation of the two analytes and the addition of acetonitrile (ACN) as an organic additive to improve the repeatability and sensitivity of the CE method. Under the optimized separation and detection conditions (50mM PBS (pH=7.0) and 2mM Ru(bpy)(3)(2+) in the ECL detection cell, 20 mM Tris (pH=2.0), 0.2 mM ß-CD and 20% ACN (v/v) as running buffer), wide linear ranges of 0.1-5 µM and 0.1-5 µM were achieved, with the correlation coefficients of 0.9990 (n=8) and 0.9980 (n=8) for Imi and Tri, respectively. Detection limits 5 nM and 1 nM (S/N=3) were obtained for Imi and Tri, respectively. The method was also successfully applied for the determination of Imi in pharmaceutical dosage form.

Kinetic studies of drug-protein interactions by using peak profiling and high-performance affinity chromatography: examination of multi-site interactions of drugs with human serum albumin columns.

Carbamazepine and imipramine are drugs that have significant binding to human serum albumin (HSA), the most abundant serum protein in blood and a common transport protein for many drugs in the body. Information on the kinetics of these drug interactions with HSA would be valuable in understanding the pharmacokinetic behavior of these drugs and could provide data that might lead to the creation of improved assays for these analytes in biological samples. In this report, an approach based on peak profiling was used with high-performance affinity chromatography to measure the dissociation rate constants for carbamazepine and imipramine with HSA. This approach compared the elution profiles for each drug and a non-retained species on an HSA column and control column over a board range of flow rates. Various approaches for the corrections of non-specific binding between these drugs and the support were considered and compared in this process. Dissociation rate constants of 1.7 (±0.2) s(-1) and 0.67 (±0.04) s(-1) at pH 7.4 and 37°C were estimated by this approach for HSA in its interactions with carbamazepine and imipramine, respectively. These results gave good agreement with rate constants that have determined by other methods or for similar solute interactions with HSA. The approach described in this report for kinetic studies is not limited to these particular drugs or HSA but can also be extended to other drugs and proteins.

Functional expression of milligram quantities of the synthetic human serotonin transporter gene in a tetracycline-inducible HEK293 cell line.

The serotonin transporter (SERT), a member of the solute carrier 6 family, is responsible for reuptake of the monoamine neurotransmitter serotonin (5-hydroxytryptamine) from the synaptic cleft on the neural cells, and a vital target for several antidepressants. To investigate biophysical studies of this pharmacologically relevant transporter, we developed a mammalian expression system with tetracycline-inducible HEK293 cells using synthetic human SERT genes produced by PCR-based self-assembly method. Codon-optimization of this de novo constructed genes and construction of stable cell lines improved expression 3.5-fold and single-step immunoaffinity purification with FLAG-epitope tag yielded around one milligram functional SERT per liter culture medium assessed by [(3)H] imipramine ligand binding. Some characterizations including electrospray ionization MS/MS analysis, subcellular localization and cellular-uptake assay demonstrated that expressed human SERT was properly expressed, folded and fully functional. The long cytosolic N-terminal of SERT was predicted as containing 'intrinsically disordered region (IDR)' (∼85 residues) by DISOPRED2 program. We engineered this salient region by step-wise truncation and ligand binding assay determined that dissociation constant for a series of de novo designed truncation constructs was close to the one for full-length wild type SERT. Our expression platform using synthetic codon-optimized gene and mammalian stable cell lines is feasible to produce milligram-scale functional membrane transporter for further biophysical and biochemical studies.

Simultaneous determination of imipramine hydrochloride and chlordiazepoxide in pharmaceutical preparations by spectrophotometric, RP-HPLC, and HPTLC methods.

A binary mixture of imipramine HCl and chlordiazepoxide was determined by three different methods. The first involved determination of imipramine HCl and chlordiazepoxide using the first derivative spectrophotometric technique at 219 and 231.5 nm over the concentration ranges of 1-20 and 2-24 microg/mL with mean accuracies of 99.47 +/- 0.78 and 101.43 +/- 1.20%, respectively. The second method utilized RP-HPLC with methanol-acetonitrile-0.065 M ammonium acetate buffer (45 + 25 + 30, v/v/v, pH adjusted to 5.6 +/- 0.02 with phosphoric acid) as the mobile phase pumped at a flow rate of 1.0 mL/min. Quantification was achieved using UV detection at 240 nm over concentration ranges of 0.25-4.0 and 0.1-1.6 microg/mL, with mean accuracies of 101.17 +/- 0.56 and 100.67 +/- 0.40% for imipramine HCl and chlordiazepoxide, respectively. The third method was HPTLC with carbon tetrachloride-acetone-triethylamine (pH 8.3; 6 + 3 + 0.3, v/v/v) as the mobile phase. Quantification was achieved with UV detection at 240 nm over concentration ranges of 50-600 and 20-240 ng/spot with mean accuracies of 99.51 +/- 0.59 and 100.59 +/- 0.84% for imipramine HCl and chlordiazepoxide, respectively. The suggested procedures were checked using prepared mixtures, and were successfully applied for the analysis of pharmaceutical preparations. The accuracy and precision of the methods were confirmed when the standard addition technique was applied. The results obtained by applying the proposed methods were statistically analyzed.

Quantitative determination of fluoxetine in human serum by high performance thin layer chromatography.

A high performance thin layer chromatographic method was developed and validated for the quantification of fluoxetine in human serum. Fluoxetine was extracted by liquid-liquid extraction method with diethyl ether as extraction solvent. Imipramine was used as internal standard. The chromatographic separation was achieved on precoated silica gel F 254 high performance thin layer chromatographic plates using a mixture of toluene/acetic acid glacial (4:5 v/v) as mobile phase. 4-Dimethylamino-azobenzene-4-sulphonyl chloride was used as derivatization reagent. Densitometric detection was done at 272 nm. The method was linear between 12.5 and 87.5 ng/spot, corresponding to 0.05 and 0.35 ng/microL of fluoxetine in human serum after extraction process and applying 25 microL to the chromatographic plates. The method correlation coefficient was 0.999. The intra-assay and inter-assay precisions, expressed as the RSD, were in the range of 0.70-2.01% (n=3) and 0.81-3.90% (n=9), respectively. The LOD was 0.23 ng, and the LOQ was 0.70 ng. The method proved be accurate, with a recovery between 94.75 and 98.95%, with a RSD not higher than 3.61% and was selective for the active principle tested. This method was successfully applied to quantify fluoxetine in patient serum samples. In conclusion, the method is useful for quantitative determination of fluoxetine in human serum.