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A fast and sensitive LC-MS/MS method for the quantitative analysis of seven steroid hormones in 150 μl of human serum was developed and validated. The following compounds were included: 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, androstenedione, dehydroepiandrosterone, testosterone, pregnenolone, and progesterone. Individual stable isotope-labeled analogues were used as internal standards. Sample preparation was performed by liquid-liquid extraction, followed by oxime derivatization to improve the ionization efficiency of the analytes. In contrast to the common derivatization-based methods, the reaction was incorporated into the sample preparation process and the only additional step due to the derivatization was a short heating of the autosampler vials before the sample injection. Chromatographic separation was achieved on a reversed-phase column using a methanol-water gradient. For the analyte detection, a triple quadrupole instrument with electrospray ionization was used. Total run time was 7.0 min and the lower limits of quantification were in the range of 0.03-0.34 nM (0.01-0.10 ng/ml), depending on the analyte. The method was validated using human serum samples from both sexes and applied for the serum steroid profiling of endometriosis patients., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

This preclinical study investigated the ability of memantine (MEM) to stimulate brain acetylcholine (ACh) release, potentially acting synergistically with donepezil (DON, an acetylcholinesterase inhibitor). Acute systemic administration of either MEM or DON to anesthetized rats caused dose-dependent increases of ACh levels in neocortex and hippocampus, and the combination of MEM (5 mg/kg) and DON (0.5 mg/kg) produced significantly greater increases than either drug alone. To determine whether ACh release correlated with cognitive improvement, rats with partial fimbria-fornix (FF) lesions were treated with acute or chronic MEM or DON. Acute MEM treatment significantly elevated baseline hippocampal ACh release but did not significantly improve task performance on a delayed non-match-to-sample (DNMS) task, whereas chronic MEM treatment significantly improved DNMS performance but only marginally elevated baseline ACh levels. Acute or chronic treatment with DON (in the presence of neostigmine to allow ACh collection) did not significantly improve DNMS performance or alter ACh release. In order to investigate the effect of adding MEM to ongoing DON therapy, lesioned rats pretreated with DON for 3 weeks were given a single intraperitoneal dose of MEM. MEM significantly elevated baseline hippocampal ACh levels, but did not significantly improve DNMS task scores compared to chronic DON-treated animals. These data indicate that MEM, in addition to acting as an NMDA receptor antagonist, can also augment ACh release; however, in this preclinical model, increased ACh levels did not directly correlate with improved cognitive performance., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Although several in vitro models have been reported to predict the ability of drug candidates to cross the blood-brain barrier, their real in vivo relevance has rarely been evaluated. The present study demonstrates the in vivo relevance of simple unidirectional permeability coefficient (P(app)) determined in three in vitro cell models (BBMEC, Caco-2 and MDCKII-MDR1) for nine model drugs (alprenolol, atenolol, metoprolol, pindolol, entacapone, tolcapone, baclofen, midazolam and ondansetron) by using dual probe microdialysis in the rat brain and blood as an in vivo measure. There was a clear correlation between the P(app) and the unbound brain/blood ratios determined by in vivo microdialysis (BBMEC r=0.99, Caco-2 r=0.91 and MDCKII-MDR1 r=0.85). Despite of the substantial differences in the absolute in vitro P(app) values and regardless of the method used (side-by-side vs. filter insert system), the capability of the in vitro models to rank order drugs was similar. By this approach, thus, the additional value offered by the true endothelial cell model (BBMEC) remains obscure. The present results also highlight the need of both in vitro as well as in vivo methods in characterization of blood-brain barrier passage of new drug candidates., (Copyright © 2010 Elsevier B.V. All rights reserved.)

We present a modification of the widely used delayed non-match to sample (DNMS) paradigm for assessment of object recognition memory that can be combined with simultaneous in vivo microdialysis. The present study provides evidence that hippocampal ACh release increases from baseline during active exploration of the test environment and an empty test board, but a specific further increase is seen during the recognition memory task performance. This novel experimental model offers a good tool to study the impact of selective lesions or pharmacological manipulation simultaneously on neurotransmitter levels and memory task performance., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

When a standard atmospheric pressure chemical ionization (APCI) or atmospheric pressure photoionization (APPI) ion source is used without applying the corona discharge or photoirradiation, atmospheric pressure thermospray ionization (APTSI) of various compounds can be achieved. Although largely ignored, this phenomenon has recently gained interest as an alternative ionization technique. In this study, this technique is performed for the first time on a miniaturized scale using a microchip nebulizer. Sample ionization with the presented microchip-APTSI (microAPTSI) is achieved by applying only heat and gas flow to a nebulizer chip, without any other methods to promote gas-phase ionization. To evaluate the performance of the described microAPTSI setup, ionization efficiency for a set of test compounds was monitored as the microchip positioning, temperature, nebulizer gas flow rate, sample solution composition, and solvent flow rate were varied. The microAPTSI mass spectra of the test compounds were also compared to those obtained with ESI and APCI. The microAPTSI produces ESI-like spectra with low background noise, favoring the formation of protonated or deprotonated molecules of compounds that are ionizable in solution. Multiple charging of peptides without in-source fragmentation was also observed. Unlike ESI, however, the microAPTSI source can tolerate the presence of mobile phase additives like trifluoroacetic acid (TFA) without significant ion suppression. The microAPTSI source can be used with standard mass spectrometer ion source hardware, being a unique alternative to the present interfacing techniques., (Copyright 2009 John Wiley & Sons, Ltd.)

Simple and specific hydrophilic interaction liquid chromatography (HILIC) method with ultraviolet (UV) detection was developed for the simultaneous determination of highly water-soluble metformin and its more lipophilic prodrugs in human and rat blood samples. The sample preparation was accomplished by precipitating proteins with acetonitrile, which enabled the direct injection of supernatants to the HPLC. Chromatographic separation was performed on an analytical normal phase silica column using a mixture of 0.01 M ammonium acetate pH 5.0 and acetonitrile (40:60, v/v) as a mobile phase at flow rate of 1 ml/min and at the wavelength of 235 nm. The method was validated in terms of specificity, linearity, accuracy, precision, recovery, and analyte stability. The UV-HILIC method was suitable for detecting both metformin and one of its more lipophilic prodrugs simultaneously in human and rat blood samples.

A rapid and sensitive method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) was developed for the simultaneous determination of acrylamide (AA) and its genotoxic metabolite glycidamide (GA) with a test marker antipyrine (AP) in placental tissue and perfusion medium used in human placental perfusion studies. An internal standard ((13)C-acrylamide) was added to the samples which were then deproteinized with acetonitrile. Chromatographic separation was performed on a reversed phase column with a gradient elution of acetonitrile and 0.01% formic acid at a flow rate of 0.3 mL/min. Detection and quantification of the analytes were carried out with a triple quadrupole mass spectrometer using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The method was validated and linear over a concentration range of 0.5-20 microg/mL for acrylamide and glycidamide and 5-200 microg/mL for antipyrine. The lower limit of quantification for acrylamide and glycidamide was 0.5 microg/mL and for antipyrine 5 microg/mL. The method was selective, and good accuracy, precision, recovery, and stability were obtained for concentrations within the standard curve. The method was successfully used to analyze the placental perfusion medium and tissue samples in a toxicokinetic study for transplacental transfer of acrylamide and glycidamide. This is the first time that acrylamide, glycidamide and antipyrine are measured simultaneously.

Most drugs can penetrate the placenta but there are only a few studies on placental transfer of environmental toxic compounds. In this study, we used dual recirculating human placental perfusion to determine the transfer rate through the placenta of a neurotoxic and carcinogenic compound found in food, acrylamide and its genotoxic metabolite glycidamide. Putative acrylamide metabolism into glycidamide during the 4-h perfusions and acrylamide-derived DNA adducts in placental DNA after perfusions were also analyzed. Placentas were collected immediately after delivery and kept physiologically functional as confirmed by antipyrine kinetics, glucose consumption and leak from fetal to maternal circulation. Acrylamide (5 or 10 microg/ml) or glycidamide (5 microg/ml), both with antipyrine (100 microg/ml), was added to maternal circulation. Acrylamide and glycidamide were analyzed in the perfusion medium by liquid chromatography/mass spectrometry. Acrylamide and glycidamide crossed the placenta from maternal to fetal circulation with similar kinetics to antipyrine, suggesting fetal exposure if the mother is exposed. The concentrations in maternal and fetal circulations equilibrated within 2h for both studied compounds and with both concentrations. Acrylamide metabolism into glycidamide was not detected during the 4-h perfusions. Moreover, DNA adducts were undetectable in the placentas after perfusions. However, fetuses may be exposed to glycidamide after maternal metabolism. Although not found in placental tissue after 4h of perfusion, it is possible that glycidamide adducts are formed in fetal DNA.

The purpose of the study was to evaluate the effect of cyclodextrin (CD) complexation on the chemical and physical stability of a polypeptide hormone glucagon and to study the interactions between glucagon and gamma-cyclodextrin molecules in inclusion complexes. The chemical stability of glucagon at pH 2.0 was studied with HPLC-UV and HPLC-MS/MS. The physical stability of glucagon at pH 2.5 was studied by measuring the turbidity (A(405 nm)) and viscosity (Ostwald capillary viscosimeter) of the samples. The structure of glucagon/gamma-CD complexes at pH 2.5 was studied with 2D-NMR. The presence of various CDs increased the chemical half-life of glucagon at pH 2.0 (37 degrees C, 0.01 M HCl, ionic strength 0.15) and prolonged the lag-time before aggregation at pH 2.5 (0.9% (w/v) NaCl in 3.2 mM HCl). The NMR studies showed that the side chains of all the aromatic amino acid residues (Phe6, Tyr10, Tyr13, Phe22, Trp25) and leucines (Leu14 and Leu26) of glucagon interacted with the cavities of the gamma-CD molecules. The present study shows that glucagon forms inclusion complexes with cyclodextrins in acidic solution, resulting in an improvement in its chemical and physical stability.

Simple HPLC-PAD methods were developed for quantitation of cyclodextrins (CDs) in aqueous matrices from in vitro cell permeation studies. C-18 solid-phase extraction was used for sample pretreatment. Samples were analysed using acetonitrile-water mobile phase with post-column alkalization by 0.5M NaOH. Zorbax SB-Aq (for alpha-CD) and Zorbax SB-Phenyl (for beta-CD and gamma-CD) columns gave excellent peak shape and sufficient resolution of CD to glucose (2.7-3.2). The methods showed good concentration-response relationship (r > or = 0.999), precision (RSD% 0.7-5.1), repeatability (RSD% 3.4-13.7) and accuracy (87-107%). The limits of quantitation were 0.78, 0.46 and 0.52 microg/ml for alpha-CD, beta-CD and gamma-CD (RSD% of 10.6, 8.1 and 16.3, respectively).

A simple, sensitive and selective LC-MS/MS method was developed for the determination of tamsulosin in human aqueous humor and serum to study the recently reported eye-related adverse effects of this alpha(1)-blocker drug. Aqueous humor samples were analyzed by direct injection, after addition of the internal standard, labetalol. Liquid-liquid extraction with ethyl acetate was used for serum sample preparation. The chromatographic separation was performed on a reversed phase column by gradient elution with acetonitrile -0.1% formic acid at a flow-rate of 0.2 ml/min. Detection and quantification of the analytes were carried out with a linear ion trap mass spectrometer, using positive electrospray ionization (ESI) and multiple reaction monitoring (MRM). The limit of quantification was 0.1 ng/ml for both aqueous humor and serum samples and linearity was obtained over the concentration ranges of 0.1-4.7 ng/ml and 0.1-19.3 ng/ml for aqueous humor and serum samples, respectively. Acceptable accuracy and precision were obtained for concentrations within the standard curve ranges. The method has been used for the determination of tamsulosin in aqueous humor and serum samples from patients that were on tamsulosin medication and underwent cataract surgery.

A fast, simple and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed for the determination of acetylcholine in rat brain microdialysis samples. The chromatographic separation was achieved in 3 min on a reversed-phase column with isocratic conditions using a mobile phase containing 2% (v/v) of acetonitrile and 0.05% (v/v) of trifluoroacetic acid (TFA). A stable isotope-labeled internal standard was included in the analysis and detection was carried out with a linear ion trap mass spectrometer using selected reaction monitoring (SRM). Analyte ionization was performed with an atmospheric pressure chemical ionization (APCI) source without applying discharge current (atmospheric pressure spray ionization). This special ionization technique offered significant advantages over electrospray ionization for the analysis of acetylcholine with reversed-phase ion-pairing chromatography. The lower limit of quantification was 0.15 nM (1.5 fmol on-column) and linearity was maintained over the range of 0.15-73 nM, providing a concentration range that is significantly wider than that of the existing LC/MS methods. Good accuracy and precision were obtained for concentrations within the standard curve range. The method was validated and has been used extensively for the determination of acetylcholine in rat brain microdialysis samples., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

Purpose: To study iris-related complications during cataract surgery in patients on tamsulosin medication., Methods: Twenty-one consecutive cataract patients administered tamsulosin and 21 control patients were studied. Characteristics of the iris during surgery were recorded. Pupillary diameters of 16 patients were measured before and after iris dilatation. Tamsulosin concentrations in the aqueous humor and serum were analyzed. In five patients, surgery on the second eye was carried out after a 7- to 28-day pause in tamsulosin medication., Results: Each patient administered tamsulosin had a sluggish hypotonic iris, along with a tendency toward miosis and a tendency for prolapse of the iris into the phaco tunnel or into the side port during cataract surgery. Sluggish irises also often adhered to the phaco tip or to the irrigation-aspiration tip. Despite a pause of 7 to 28 days in the use of tamsulosin, the adverse effects persisted. Tamsulosin concentrations varied between 0.1 and 1.0 ng/mL in the anterior chamber fluid. In three of five cases, tamsulosin remained in detectable amounts the aqueous humor after the 7- to 28-day pause. Preoperative pupillary diameter was smaller in the patients using tamsulosin than in the controls., Conclusions: Tamsulosin has selective alpha1A-adrenoreceptor antagonistic properties and obviously binds for a long period to the postsynaptic nerve endings of the iris dilator muscle, thus affecting iris dilatation and leading to complications in cataract surgery. The iris remained floppy after 7- to 28-day interruption of the tamsulosin regimen.

Novel water-soluble phosphate prodrugs (2b-5b) of buparvaquone-oxime (1a) and buparvaquone-O-methyloxime (1b) were synthesized and evaluated in vitro as potential oral prodrugs against leishmaniasis. Buparvaquone-oxime (1a), and most probably also buparvaquone-O-methyloxime (1b), released the parent buparvaquone via a cytochrome P450-catalysed reaction. The prodrugs 2b-5b showed significantly higher aqueous solubilities (>4 mg/ml) than buparvaquone (< or = 0.03 microg/ml) over a pH range of 3.0-7.4. The prodrugs 2b, 3b and 5b rapidly released (t1/2 = 7 min) the corresponding oximes of buparvaquone (1a and 1b), and prodrug 4b at a moderate rate (t1/2 = 22.5 min) in alkaline phosphatase solution in vitro. Prodrug 3b was the most chemically stable in the aqueous solutions over a pH range of 3.0-7.4 (t1/2 > 8 days). Although buparvaquone-oxime (1a) has been shown to undergo a cytochrome P450-catalysed oxidation in liver microsomes to the parent buparvaquone and behave as a novel bioreversible prodrug, its usefulness is limited in oral drug delivery due to its poor aqueous solubility, like buparvaquone itself. Further phosphorylation of an oxime form of buparvaquone significantly increased water solubility, and this novel approach is therefore useful to improve physicochemical properties of drugs containing a ketone functional group.

Low doses of the intragastrically (i.g.) administered nitric oxide (NO) donors, 1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride (GEA 3162; 0.3 mg/kg) and 3-morpholino-sydnonimine (SIN-1; 1 mg/kg), inhibited gastric ulceration induced by ethanol (94%) in anesthetized rats. In contrast, higher doses of these NO donors administered i.g. exacerbated the damage. When administered intravenously, the NO donors had no effect on ethanol-induced gastric lesions although a clear blood pressure-lowering effect was seen. Neither the inhibition nor the exacerbation of ulceration was correlated with changes in blood pressure or prostaglandin E2 release from the mucosal tissue. The relatively small difference between the gastroprotective and damaging doses suggests that orally administered NO donors, especially in the case of GEA 3162, may have a narrow gastric safety margin.