In-Syringe Electrokinetic Protein Removal from Biological Samples prior to Electrospray Ionization Mass Spectrometry.

Here, an electrokinetic extraction (EkE) syringe is presented allowing for on-line electrokinetic removal of serum proteins before ESI-MS. The proposed concept is demonstrated by the determination of pharmaceuticals from human serum within minutes, with sample preparation limited to a 5× dilution of the sample in the background electrolyte (BGE) and application of voltage, both of which can be performed in-syringe. Signal enhancements of 3.6-32 fold relative to direct infusion of diluted serum and up to 10.8 fold enhancement, were obtained for basic and acidic pharmaceuticals, respectively. Linear correlations for the basic drugs by EkE-ESI-MS/MS were achieved, covering the necessary clinical range with LOQs of 5.3, 7.8, 6.1, and 17.8 ng mL-1 for clomipramine, chlorphenamine, pindolol, and atenolol, respectively. For the acidic drugs, the EkE-ESI-MS LOQs were 3.1 µg mL-1 and 2.9 µg mL-1 for naproxen and paracetamol, respectively. The EkE-ESI-MS and EkE-ESI-MS/MS methods showed good accuracy (%found of 81 % to 120 %), precision (≤20 %), and linearity (r>0.997) for all the studied drugs in spiked serum samples.

No evidence that MDMA-induced enhancement of emotional empathy is related to peripheral oxytocin levels or 5-HT1a receptor activation.

The present study aimed at investigating the effect of MDMA on measures of empathy and social interaction, and the roles of oxytocin and the 5-HT1A receptor in these effects. The design was placebo-controlled within-subject with 4 treatment conditions: MDMA (75 mg), with or without pindolol (20 mg), oxytocin nasal spray (40 IU+16 IU) or placebo. Participants were 20 healthy poly-drug MDMA users, aged between 18-26 years. Cognitive and emotional empathy were assessed by means of the Reading the Mind in the Eyes Test and the Multifaceted Empathy Test. Social interaction, defined as trust and reciprocity, was assessed by means of a Trust Game and a Social Ball Tossing Game. Results showed that MDMA selectively affected emotional empathy and left cognitive empathy, trust and reciprocity unaffected. When combined with pindolol, these effects remained unchanged. Oxytocin did not affect measures of empathy and social interaction. Changes in emotional empathy were not related to oxytocin plasma levels. It was concluded that MDMA (75 mg) selectively enhances emotional empathy in humans. While the underlying neurobiological mechanism is still unknown, it is suggested that peripheral oxytocin does not seem to be the main actor in this; potential candidates are the serotonin 2A and the vasopressin 1A receptors. Trial registration: MDMA & PSB NTR 2636.

Presynaptic selectivity of a ligand for serotonin 1A receptors revealed by in vivo PET assays of rat brain.

A novel investigational antidepressant with high affinity for the serotonin transporter and the serotonin 1A (5-HT(1A)) receptor, called Wf-516 (structural formula: (2S)-1-[4-(3,4-dichlorophenyl)piperidin-1-yl]-3-[2-(5-methyl-1,3,4-oxadiazol-2-yl)benzo[b]furan-4-yloxy]propan-2-ol monohydrochloride), has been found to exert a rapid therapeutic effect, although the mechanistic basis for this potential advantage remains undetermined. We comparatively investigated the pharmacokinetics and pharmacodynamics of Wf-516 and pindolol by positron emission tomographic (PET) and autoradiographic assays of rat brains in order to elucidate their molecular interactions with presynaptic and postsynaptic 5-HT(1A) receptors. In contrast to the full receptor occupancy by pindolol in PET measurements, the binding of Wf-516 to 5-HT(1A) receptors displayed limited capacity, with relatively high receptor occupancy being achieved in regions predominantly containing presynaptic receptors. This selectivity was further proven by PET scans of neurotoxicant-treated rats deficient in presynaptic 5-HT(1A) receptors. In addition, [(35)S]guanosine 5'-O-[γ-thio]triphosphate autoradiography indicated a partial agonistic ability of Wf-516 for 5-HT(1A) receptors. This finding has lent support to reports that diverse partial agonists for 5-HT(1A) receptors exert high sensitivity for presynaptic components. Thus, the present PET data suggest a relatively high capacity of presynaptic binding sites for partial agonists. Since our in vitro and ex vivo autoradiographies failed to illustrate these distinct features of Wf-516, in vivo PET imaging is considered to be, thus far, the sole method capable of pharmacokinetically demonstrating the unique actions of Wf-516 and similar new-generation antidepressants.

Fluorometric determination of bopindolol and celiprolol in pharmaceutical preparations and biological fluids.

Two sensitive fluorometric methods were developed for the determination of both bopindolol malonate (BOP) and celiprolol HCl (CLP) based on measuring their native fluorescence in methanol and acetonitrile, respectively. For BOP, the fluorescence was measured at 316 nm after excitation at 278 nm. The proposed method was successfully applied to the assay of commercial tablets as well as content uniformity testing. For CLP, the fluorescence was enhanced by the addition of carboxymethylcellulose solution and measured at 455 nm after excitation at 339 nm. The method was successfully applied to the analysis of CLP in tablets and biological fluids. In both methods, interference likely to be introduced from co-formulated, co-administered, or chemically related drugs was studied. The results were statistically compared with those obtained by reference methods and were found to be in good agreement.

Preparation and evaluation of propranolol molecularly imprinted solid-phase microextraction fiber for trace analysis of beta-blockers in urine and plasma samples.

An improved multiple co-polymerization technique was developed to prepare a novel molecularly imprinted polymer (MIP)-coated solid-phase microextraction (SPME) fiber with propranolol as template. Investigation was performed for the characteristics and application of the fibers. The MIP coating was highly crosslinked and porous with the average thickness of only 25.0 microm. Consequently, the adsorption and desorption of beta-blockers within the MIP coating could be achieved quickly. The specific selectivity was discovered with the MIP-coated fibers to propranolol and its structural analogues such as atenolol, pindolol, and alprenolol. In contrast, only non-specific adsorption could be shown with the non-imprinted polymer (NIP)-coated fibers, and the extraction efficiencies of propranolol and pindolol with the MIP-coated fibers were higher markedly than that with the commercial SPME fibers. A MIP-coated SPME coupled with high-performance liquid chromatography (HPLC) method for propranolol and pindolol determination was developed under the optimized extraction conditions. Linear ranges for propranolol and pindolol were 20-1000 microg L(-1) and detection limits were 3.8 and 6.9 microg L(-1), respectively. Propranolol and pindolol in the spiked human urine and plasma samples, extracted with organic solvent firstly, could be simultaneous monitored with satisfactory recoveries through this method.

Evaluation of monolithic packed 96-tips and liquid chromatography-tandem mass spectrometry for extraction and quantification of pindolol and metoprolol in human plasma samples.

96-well pipette tips with a chemically bonded monolithic methacrylate sorbent plug were used for solid-phase extraction (SPE) of pindolol and metoprolol in human plasma samples. The sorbent plug was formed by in situ polymerization. Monolithic packed 96-tips are a tool for miniaturized, solid-phase extraction. Using such packed 96-tips, a 96-well plate could be handled in about 2 min. The key aspect of the monolithic phase is that monolithic material can provide both relatively good binding capacity and relatively low backpressure properties. The validation of the methodology showed that the accuracy values of quality-control samples were between 101% and 103% for metoprolol, while between 94% and 114% for pindolol. The precision ranged from 4% to 15%. The standard calibration curves were obtained within the concentration range 5-5000 nM in plasma samples. The coefficients of determination (R2) for plasma samples were >or=0.99. Our prepared polymer based monolithic packed 96-tips were compared with commercial silica based 96-tips and protein precipitation.

Simultaneous determination of beta-blockers in human plasma using liquid chromatography-tandem mass spectrometry.

A detailed procedure for the analysis of four beta-blockers, acebutolol, labetalol, metoprolol and propranolol, in human plasma by high-performance liquid chromatography (LC)-tandem mass spectrometry (MS-MS) using an MSpak GF column, which enables direct injection of crude plasma samples, is presented. Protein and/or macromolecule matrix compounds were eluted first from the column, while the drugs were retained on the polymer stationary phase of the MSpak GF column. The analytes retained on the column were then eluted into an acetonitrile-rich mobile phase using a gradient separation technique. All drugs showed base peak ions due to [M + H]+ ions by LC-MS with positive ion electrospray ionization, and the product ions were produced from each [M + H]+ ion by LC-MS-MS. Quantification was performed by selected reaction monitoring. The recoveries of the four beta-blockers spiked into plasma were 73.5-89.9%. The regression equations for all compounds showed excellent linearity in the range 10-1000 ng/mL of plasma, with the exception of propranolol (10-800 ng/mL). The limits of detection and quantification for each drug were 1-3 and 10 ng/mL, respectively, of plasma. The intra- and inter-day coefficients of variation for all drugs in plasma were not greater than 10.9%.

Room-temperature, phosphorimetric determination of the beta-blocking agent pindolol in pharmaceutical tablets, urine and blood serum.

It is already recognised that heavy-atom-induced, room-temperature phosphorescence can be used to determine pindolol in pharmaceutical samples and biological fluids. We describe here a new, simple, rapid and selective development of this technique. The phosphorescence signals derive from the interaction of pindolol with a relatively high concentration of heavy-atom salts in the presence of sodium sulphite as oxygen scavenger. Phosphorescence was registered in the presence of 1.2 M potassium iodide, 15 mM sodium sulphite and 30% v/v methanol at 450 nm, exciting at 285 nm. The detection limit was 21.1 ng mL(-1). The method has been successfully applied to the determination of pindolol in commercial pharmaceutical tablets, urine and blood serum.

Supercritical fluid chromatography-tandem mass spectrometry for the enantioselective determination of propranolol and pindolol in mouse blood by serial sampling.

Packed-column supercritical fluid chromatography (pSFC) coupled to an atmospheric pressure chemical ionization (APCI) source and a tandem mass spectrometer (MS/MS) with minimum sample pretreatment was explored for the rapid and enantioselective determination of (R,S)-propranolol in mouse blood. Serial bleeding of mice is advantageous for the reduction of animal usage, dosing errors, and animal-to-animal variation. The effects of the eluent flow rate and composition as well as the nebulizer temperatures on the ionization efficiency of racemic propranolol and pindolol as model compounds in the positive ion mode under pSFC conditions were studied. The fundamental parameters on the proposed hyphenated system such as matrix ionization suppression and chromatographic performances were investigated in improving sensitivity and enantiomeric separation for the detection of the analytes. The proposed chiral pSFC-APCI/MS/MS approach requiring approximately 3 min/sample for the determination of (R,S)-propranolol at a low nanogram per milliliter region was partially validated with respect to specificity, linearity, reproducibility, and accuracy and was applied to support a pharmacokinetic study.

Preferential 5-HT1A autoreceptor occupancy by pindolol is attenuated in depressed patients: effect of treatment or an endophenotype of depression?

Using positron emission tomography and the selective 5-HT1A receptor radioligand [11C]WAY100635, we previously demonstrated a preferential occupancy of 5-HT1A autoreceptors, compared to postsynaptic receptors by pindolol in healthy volunteers. We have speculated that preferential occupancy may be clinically important for the purported actions of pindolol in accelerating the antidepressant effects of selective serotonin re-uptake inhibitors (SSRIs). In this study, we have examined the preferential occupancy by pindolol of 5-HT1A autoreceptors, following three different pindolol regimes (10 mg single dose, 2.5 mg t.i.d., and 5 mg t.i.d., in 15 depressed patients on SSRIs. In addition, seven healthy volunteers were examined following a single 10 mg dose of pindolol. We found a preferential occupancy of 22.6+/-7.7% following a single dose of 10 mg of pindolol, in the healthy volunteers, which was attenuated in depressed patients on the same dose of pindolol to 2.9+/-10.8% (Student's t=3.94, df=12, p=0.002). In addition, we found a significant negative correlation between the degree of preferential occupancy and the severity of depression as assessed by the Hamilton depression rating score (HAM-D), Spearman's rho=-0.728, N=14, p=0.003, in the depressed sample. A possible mechanism underlying preferential occupancy and the attenuation of this phenomenon in depressed patients on SSRIs may include changes in the proportion of high affinity 5-HT1A sites in the autoreceptor region of the midbrain raphe. Speculatively, the degree of preferential occupancy may serve as a surrogate marker for depression, or the pharmacological effects of antidepressants.

Direct determination of pindolol enantiomers in human serum by column-switching LC-MS/MS using a phenylcarbamate-beta-cyclodextrin chiral column.

A direct analytical method of pindolol enantiomers in body fluids was developed by means of column-switching semi-microcolumn liquid chromatography/tandem mass spectrometry (LC-MS/MS). A pre-column packed with a silica-based cation-exchanger was used for on-line sample cleanup. Subsequent enantioseparation was conducted with a phenylcarbamate-beta-cyclodextrin (ph-beta-CD) bonded semi-micro chiral column (2.0 mm inner diameter (i.d.)). A 25-microl aliquot of serum/urine samples was directly injected into the system after simple filtration with a membrane filter. Separated enantiomers were monitored with positive electrospray ionization (ESI) and selected reaction monitoring (SRM). R(+)- and S(-)-pindolol in serum and urine were determined separately within 16 min at a resolution factor of 1.9. The detection limits at a signal-to-noise (S/N) ratio of 5 were 0.13 ng ml(-1) for both enantiomers. The linearity of the method was in the range of 0.25-100 ng ml(-1) with regression coefficient greater than 0.997. Recoveries from spiked serum and urine samples, estimated by the external standard method, were between 94.8 and 117.6% with a relative standard deviation (RSD) ranging from 2.1 to 18%.

Is the beneficial antidepressant effect of coadministration of pindolol really due to somatodendritic autoreceptor antagonism?

BACKGROUND: We investigated the combination of selective serotonin reuptake inhibitors (SSRIs) with the beta-adrenoceptor/serotonin 1A (5-HT(1A)) antagonist pindolol, based on the concept that 5-HT(1A) receptor blockade would eliminate the need for desensitization of presynaptic 5-HT(1A) receptors and therefore hasten the onset of action and improve the efficacy of SSRIs. However, since pindolol plasma levels after 2.5 mg three times a day are about 60 nmol/L, and the K(i) for the 5-HT(1A) receptor is 30 nmol/L, it is questionable whether pindolol levels in the brain would be sufficient to antagonize 5-HT(1A) receptors. Using microdialysis in the guinea pig, we correlated brain and plasma levels of pindolol with its capability of augmenting paroxetine-induced increases in brain 5-HT levels. In addition, central beta-receptor antagonism of pindolol was studied by investigating blockade of beta-agonist-induced increases in brain cyclic adenosine monophosphate (cAMP) formation. METHODS: Using microdialysis and jugular vein catheterization, we studied the ability of systemically administered pindolol to antagonize central 5-HT(1A) and beta-adrenoceptors, while simultaneously monitoring pindolol plasma and brain concentrations. RESULTS: Augmentation of paroxetine-induced increases in extracellular 5-HT levels in the ventral hippocampus was only observed at steady state plasma levels exceeding 7000 nmol/L (concurrent brain levels 600 nmol/L). In contrast, antagonism of beta-agonist-induced increases of brain cAMP levels was already observed at pindolol plasma levels of 70 nmol/L (concurrent brain levels < 3 nmol/L). CONCLUSIONS: At plasma levels that are observed in patients after 2.5 mg three times a day ( approximately 60 nmol/L), pindolol produces only a partial blockade of presynaptic 5-HT(1A) autoreceptors and does not augment the SSRI-induced 5-HT increase in the guinea pig brain. It is therefore very unlikely that the favorable effects of combining pindolol with SSRIs, as reported in a number of clinical studies, are due to 5-HT(1A) antagonism. Since pindolol completely blocks central beta-adrenoreceptors at clinically relevant plasma levels, it is possible that beta-adrenoceptor antagonism is involved in mediating pindolol's beneficial effects.

Differential occupancy of somatodendritic and postsynaptic 5HT(1A) receptors by pindolol: a dose-occupancy study with [11C]WAY 100635 and positron emission tomography in humans.

Augmentation of selective serotonin reuptake inhibitors (SSRIs) therapy by the 5-HT(1A) receptor agent pindolol may reduce the delay between initiation of antidepressant treatment and clinical response. This hypothesis is based on the ability of pindolol to block 5-HT(1A) autoreceptors in the dorsal raphe nuclei (DRN) and to potentiate the increase in 5-HT transmission induced by SSRIs. However, placebo-controlled clinical studies of pindolol augmentation of antidepressant therapy have reported inconsistent results. Here, we evaluated the occupancy of 5-HT(1A) receptors during treatment with pindolol controlled release (CR) in nine healthy volunteers with Positron Emission Tomography and [11C]WAY 100635. Subjects were studied four times: at baseline, following one week of pindolol CR 7.5 mg/day (4 and 10 hrs post dose), and following one dose of pindolol CR 30 mg(4 hrs post dose). Occupancy of the DRN was 40 +/- 29% on scan 2, 38 +/- 26% on scan 3, and 64 +/- 15% on scan 4. The average occupancy in all other regions was significantly lower at each doses (18 +/- 5% on scan 2, 12 +/- 3% on scan 3, and 42 +/- 4% on scan 4). These results suggest that the blockade in the DRN reached in clinical studies (7.5 mg/day) might be too low and variable to consistently augment the therapeutic effect of SSRIs. However, these data indicate that pindolol exhibits in vivo selectivity for the DRN 5-HT(1A) autoreceptors. As DRN selectivity is desirable for potentiation of 5-HT function, this observation represents an important proof of concept for the development of 5-HT(1A) agents in this application.

Differential tissue distribution of the enantiomers of racemic pindolol in the rat.

Racemic pindolol, a beta-adrenoceptor and a 5-HT1A/1B receptor antagonist, has been reported to augment and accelerate the clinical efficacy of antidepressants. The (S)-enantiomer is more potent than the (R)-enantiomer both at the beta-adrenergic and at the 5-HT1A/1B receptors. A chiral HPLC column was used for determination of tissue concentrations of the enantiomers of pindolol at 90 min after 8 mg/kg s.c. of the racemate. The (S)/(R) ratio was found to vary between tissues from 1.74 in brain to 0.82 in plasma. The present findings may be important for understanding the pharmacodynamic actions of racemic pindolol.

Determination of pindolol enantiomers in human plasma and urine by simple liquid-liquid extraction and high-performance liquid chromatography.

A simple method for the measurement of pindolol enantiomers by HPLC is presented. Alkalinized serum or urine is extracted with ethyl acetate and the residue remaining after evaporation of the organic layer is then derivatised with (S)-(-)-alpha-methylbenzyl isocyanate. The diastereoisomers of derivatised pindolol and metoprolol (internal standard) are separated by high-performance liquid chromatography (HPLC) using a C18 silica column and detected using fluorescence (excitation gamma: 215 nm, emission gamma: 320 nm). The assay displays reproducible linearity for pindolol enantiomers with a correlation coefficient of r2> or =0.998 over the concentration range 8-100 ng ml(-1) for plasma and 0.1-2.5 microg ml(-1) for urine. The coefficient of variation for accuracy and precision of the quality control samples for both plasma and urine are consistently <10%. Assay parameters are similar to those of previously published assays for pindolol enantiomers, however this assay is significantly easier and cheaper to run. Clinically relevant concentrations of each pindolol enantiomer can readily be measured.

On-line column-switching high-performance liquid chromatography analysis of cardiovascular drugs in serum with automated sample clean-up and zone-cutting technique to perform chiral separation.

A selective and highly reproducible, multi-column HPLC method is described for the analysis of the following cardiovascular drugs: lidocaine, pindolol, metoprolol, oxprenolol, diltiazem and verapamil, in serum. Column-switching devices are employed in combination with advanced separation media technologies for the automated analysis of samples containing complex matrices. The method consists of on-line sample clean-up using a restricted access sorbent, HPLC analysis of the drugs on a microsphere non-porous silica RP-18 column, and front-cutting to perform the chiral separation of pindolol enantiomers on a second HPLC system. Simultaneous control of the two HPLC systems and data analysis is achieved from a single centralized software. The R.S.D. values of the peak areas for spiked serum are less than 1% for metoprolol and oxprenolol, 2-5% for lidocaine, diltiazem and verapamil, and 1.2 and 2.4% for the two pindolol enantiomers. Recoveries, limits of detection and linearities are provided.

Comparison of two extraction methods for determination of propranolol and furosemide in human plasma by mixed-mode chromatography.

An isocratic high performance liquid chromatographic method is described for the determination of the beta-adrenergic blocking drug, propranolol, and the diuretic, furosemide, in human plasma. The two compounds and the internal standard were extracted from plasma using a two-step extraction technique. Propranolol and pindolol (internal standard) were first extracted from alkaline plasma into diethyl ether; this was followed by extraction of furosemide into acidified ether: hexane (65:35). The two extracts were then combined and evaporated under nitrogen, and the reconstituted residues were analysed on a C18/SCX reversed-phase/cation exchange column with a mobile phase of acetonitrile: 0.1 M sodium acetate pH 4 (33:67). The drugs and the internal standard were detected by UV absorption at 230 nm. The drugs were also extracted from plasma by a column-switching technique utilizing a ten-port valve. The drug compounds were retained on a C18 pre-column. A comparison of RSD for within-batch (intra-assay) and between-batch (inter-assay) runs for both methods was carried out, the liquid/liquid extraction method giving better recovery values. The calibration graphs were linear from 25-300 ng ml-1 for furosemide and 50-400 ng ml-1 for propranolol. Recovery values were > 90.0% by liquid/liquid extraction and > 76.0% by column switching.

Selective extraction of beta-blockers from biological fluids by column-switching high-performance liquid chromatography using an internal-surface phenylboronic acid precolumn.

A column-switching HPLC method using an internal-surface phenylboronic acid precolumn for the selective extraction of beta-blockers from biological fluids has been developed. Filtered urine and plasma samples (50 microliters) were injected onto the precolumn equilibrated with methanol-0.05 M disodium hydrogenphosphate (5:95, v/v). After the precolumn had been washed briefly, the selectively retained beta-blockers were eluted with methanol-0.05 M phosphate buffer (pH 2.0) and transferred to a reversed-phase analytical column, on which they were then separated. Even after exposure to at least 160 injections of non-treated urine and plasma samples, the retention efficiency of the precolumn was maintained with no increase in back pressure. Quantitative recoveries and good reproducibility were demonstrated with pindolol.