Low Flow Voltage Free Interface for Capillary Electrophoresis and Mass Spectrometry Driven by Vibrating Sharp-Edge Spray Ionization.

Capillary electrophoresis-mass spectrometry is a powerful technique for high-throughput and high efficiency separations combined with structural identification. Electrospray ionization is the primary interface used to couple capillary electrophoresis to mass analyzers; however, improved designs continue to be reported. A new interfacing method based on vibrating sharp-edge spray ionization is presented in this work to overcome the challenges of decoupling applied voltages and to enhance the compatibility with separations performed at near-neutral pH. The versatility and ease of use of this ionization source is demonstrated using ß-blockers, peptides, and proteins. The cationic ß-blocker pindolol was injected electrokinetically, and detected at concentrations ranging from 10 nM to 5 µM, with an estimated detection limit of 2 nM. The vibrating sharp-edge spray ionization functions with flow rates from 70 to 200 nL/min and did not perturb the capillary electrophoresis separation electroosmotic flow as evidenced by the observation that most migration times differed less than 7% (n = 3) across a lab-built system interfaced to mass spectrometry and a commercial system that utilizes absorbance detection. For cationic beta-blockers the theoretical plates achieved in the capillary electrophoresis-mass spectrometry setup were 80%-95% of that observed with a commercial capillary electrophoresis-UV absorbance detection system.

Advantages and Pitfalls of Capillary Electrophoresis of Pharmaceutical Compounds and Their Enantiomers in Complex Samples: Comparison of Hydrodynamically Opened and Closed Systems.

Several research disciplines require fast, reliable and highly automated determination of pharmaceutically active compounds and their enantiomers in complex biological matrices. To address some of the challenges of Capillary Electrophoresis (CE), such as low concentration sensitivity and performance degradation linked to the adsorption and interference of matrix components, CE in a hydrodynamically closed system was evaluated using the model compounds Pindolol and Propranolol. Some established validation parameters such as repeatability of injection efficiency, resolution and sensitivity were used to assess its performance, and it was found to be broadly identical to that of hydrodynamically opened systems. While some reduction in separation efficiency was observed, this was mainly due to dispersion caused by injection and it had no impact on the ability to resolve enantiomers of model compounds even when spiked into complex biological matrix such as blood serum. An approximately 18- to 23-fold increase in concentration sensitivity due to the employment of wide bore capillaries was observed. This brings the sensitivity of CE to a level similar to that of liquid chromatography techniques. In addition to this benefit and unlike in hydrodynamically opened systems, suppression of electroosmotic flow, which is essential for hydrodynamically closed systems practically eliminates the matrix effects that are linked to protein adsorption.

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.

Simultaneous enantiomeric determination of propranolol, metoprolol, pindolol, and atenolol in natural waters by HPLC on new polysaccharide-based stationary phase using a highly selective molecularly imprinted polymer extraction.

A simple high performance liquid chromatography method HPLC-UV for simultaneous enantiomeric determination of propranolol, metoprolol, pindolol, and atenolol in natural water samples was developed and validated, using a molecularly imprinted polymer solid-phase extraction. To achieve this purpose, Lux(®) Cellulose-1/Sepapak-1 (cellulose tris-(3,5-dymethylphenylcarbamate)) (Phenomenex, Madrid, Spain) chiral stationary phase was used in gradient elution and normal phase mode at ambient temperature. The gradient elution program optimized consisted of a progressive change of the mobile phase polarity from n-hex/EtOH/DEA 90/10/0.5 (v/v/v) to 60/40/0.5 (v/v/v) in 13 min, delivered at a flow rate of 1.3 ml/min and a sudden change of flow rate to 2.3 ml/min in 1 min. Critical steps in any molecularly imprinted polymer extraction protocol such as the flow rate to load the water sample in the cartridges and the breakthrough volume were optimized to obtain the higher extraction recoveries for all compounds. In optimal conditions (100 ml breakthrough volume loaded at 2.0 ml/min), extraction recoveries for the four pairs of ß-blockers were near 100%. The MIP-SPE-HPLC-UV method developed demonstrates good linearity (R(2) ≥ 0.99), precision, selectivity, and sensitivity. Method limit detection was 3.0 µg/l for propranolol and pindolol enantiomers and 20.0 and 22.0 µg/l for metoprolol and atenolol enantiomers, respectively. The proposed methodology should be suitable for routine control of these emerging pollutants in natural waters for a better understanding of the environmental impact and fate.

1H and 13C NMR characteristics of ß-blockers.

The ß-blockers are important drugs and decades of clinical experience proved their high medical status. However, to the best of our knowledge, there is no complete assignment of (1)H and (13)C NMR resonances of popular representatives: acebutolol, alpenolol, pindolol, timolol and propranolol and the published NMR data on carvedilol and atenolol are incorrect. Therefore, (1)H and (13)C NMR spectroscopy was applied for the characterization of a series of ß-adrenolytics: carvedilol (1), pindolol (2), alprenolol (3), acebutolol (4), atenolol (5), propranolol (6) and timolol (7). Two-dimensional NMR experiments (COSY, HMQC, HMBC, NOESY) allowed the unequivocal assignment of (1)H and (13)C spectra for solution (DMSO-d(6) ). Salts and bases can be easily distinguished based on (13)C chemical shifts which are within 65.0-65.5 ppm (OC2) and 46.9-47.0 (NC3) for hydrochlorides and larger, ca. 68.4 ppm (OC2) and 50.3-52.6 (NC3) for bases. NMR data of 1-7 should be included in pharmacopoeias.

Cell-based beta2-adrenergic receptor-ligand binding assay using synthesized europium-labeled ligands and time-resolved fluorescence.

High-sensitivity, high-throughput, and user-friendly lanthanide-based assays for receptor-ligand interactions provide an attractive alternative to the traditional radioligand displacement assays. In this study, three small-molecule pindolol ligand derivatives were synthesized and their binding properties were tested in a radioligand displacement assay. The ligand derivatives were further labeled with fluorescent europium(III) chelate for beta(2)-adrenergic receptor-ligand binding assay. The europium-labeled pindolol ligands having no spacer (C0) or a 12-carbon spacer (C12) arm bound to the human beta(2)-adrenergic receptors overexpressed in human embryonic kidney HEK293(i) cells. Europium ligand with a 6-carbon spacer arm (C6) showed no binding. Competitive binding assays were developed with the functional labeled ligands. The IC(50) values for beta(2)-adrenergic antagonist propranolol were 60 and 37 nM, the Z' values were 0.51 and 0.77, and the signal-to-background ratios were 5.5 and 16.0 for C0 and C12, respectively. This study shows that functional time-resolved fluorescent assays can be constructed using fluorescent lanthanide chelates conjugated to small-molecule ligands.

Micelle to solvent stacking of organic cations in capillary zone electrophoresis with electrospray ionization mass spectrometry.

The direction of the effective electrophoretic mobility of small organic cations in micellar electrokinetic chromatography using sodium dodecyl sulphate in a low-pH electrolyte can be reversed in the presence of organic solvent. This effective electrophoretic mobility change is presented here as a new dimension for on-line sample preconcentration of cations in capillary zone electrophoresis (CZE) using a background solution (BGS) modified by an organic solvent. The sample is prepared in a micellar solution without organic solvent. The focusing effect relies on the reversal in the effective electrophoretic mobility at the boundary zone between the micellar matrix and the BGS modified with organic solvent. This on-line sample preconcentration technique, called micelle to solvent stacking (MSS) afforded more than an order of magnitude improvement in concentration sensitivity compared to typical CZE-UV or CZE-electrospray ionization (ESI) MS analysis. The calculated limit of detection (S/N=3) for pindolol and metoprolol analysed by MSS-CZE-ESI-MS was found to be 0.03 and 0.01 microg/mL, respectively.

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.

Determination of pindolol enantiomers in amniotic fluid and breast milk by high-performance liquid chromatography: applications to pharmacokinetics in pregnant and lactating women.

A method for the determination of pindolol enantiomers in amniotic fluid and breast milk was developed, validated, and applied to the investigation of six pregnant women treated with rac-pindolol (10 mg/12 h). Biological samples were extracted with tert-methyl-butyl ether, and the pindolol enantiomers were resolved on a Chiralpak AD column. Amniotic fluid/plasma and milk/plasma concentrations ratios ranged from 0.4 to 4.5 and from 0.6 to 3.7, respectively, for (+)-R-pindolol and from 0.5 to 3.5 and from 1.1 to 2.8, respectively, for (-)-S-pindolol. Preliminary data suggest that amniotic fluid and breast milk are routes of fetal exposure to pindolol enantiomers.

Determination of competitive adsorption isotherm parameters of pindolol enantiomers on alpha1-acid glycoprotein chiral stationary phase.

In this paper, inverse method (IM) was used to determine the binary competitive adsorption isotherm of pindolol enantiomers by a least-square fitting of the proposed model to the experimentally measured elution curves of racemic pindolol. The isotherm parameters were determined by minimizing the least-square error using an adaptation of genetic algorithm, non-dominated sorting genetic algorithm with jumping genes (NSGA-II-JG). An equilibrium dispersive (ED) model combined with bi-Langmuir isotherm was used in predicting the elution profiles. The determined parameters show good agreement with the experimental profiles at various experimental conditions such as sample volume, concentration and flow rates of the racemic mixture. Robustness and validity of the isotherm parameters were also verified by frontal analyses at various step inputs. Results from both the pulse tests and the frontal analysis indicate that adsorption isotherm derived from the inverse method is quite reliable. This method requires relatively less number of experiments to be performed and therefore, lower experimental costs confirming that inverse method is an attractive alternative approach of experimental technique in determining the competitive adsorption isotherm for binary systems.

HPLC and solubility study of the interaction between pindolol and cyclodextrins.

The complexation with beta-cyclodextrin (beta-CD) has been investigated using reversed-phase liquid chromatography. The compounds tested have been pindolol and, for comparison purposes, indole and 4-methoxyindole. The retention behaviour has been analysed on a Kromasil 100 C18 column and the mobile phase used was methanol-pH 6 phosphate buffer (15/85v/v) in which beta-CD was incorporated as a mobile phase additive. The decrease in the retention times with increasing concentrations of beta-CD enables the determination of the apparent stability constants of the complexes. In addition, the low solubility of pindolol, a weak base, in pH 12 aqueous solution has been improved by complexation with different cyclodextrins. The solubility enhancements with 1.4 x 10(-2) M beta-, hydroxypropyl-beta, and gamma-CD have been 1.9, 1.8 and 1.4-fold, respectively, with 2.4 x 10(-2) M methyl-beta-CD it was 2.8-fold whilst no effect was observed with alpha-CD. The stability constants of the complexes at pH 12 have been determined from the solubility isotherms.

Decreased responsiveness to beta-adrenoceptor agonists in arterial strips from spontaneously hypertensive rats is not associated with alterations in beta-adrenoceptors.

Beta-adrenoceptors in femoral and mesenteric arteries from 13-week-old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied using radioligand binding assays and were compared with in vitro measurements of beta-adrenoceptor-mediated relaxation. The relaxant responses to noradrenaline via beta-adrenoceptors were significantly decreased in the SHR femoral artery when compared with the WKY femoral artery. However, under the same conditions, arterial relaxant responses to forskolin, an activator of adenylate cyclase, were not significantly different between SHR and WKY rats. Specific binding of 125I-iodocyanopindolol to membranes prepared from femoral arteries of SHR and WKY rats was saturable and of high affinity. Neither the equilibrium dissociation constant of 125I-iodocyanopindolol, nor the maximum number of binding sites were significantly different between SHR and WKY rats. Similar results were obtained in the case of mesenteric arteries from SHR and WKY rats. These results indicate that the decreased responsiveness to beta-adrenoceptor stimulation in SHR arteries is not associated with alterations in beta-adrenoceptors and further support the hypothesis that a reduced function of the stimulatory guanosine triphosphate-binding protein is responsible for the decreased responsiveness to a variety of receptor agonists whose mechanism of action involves adenylate cyclase activation.

Evaluation of the immobilized artificial membrane phosphatidylcholine. Drug discovery column for high-performance liquid chromatographic screening of drug-membrane interactions.

Chromatographic retention factors (k') of a series of eight beta-adrenoceptor antagonist compounds (beta-adrenolytic drugs) were determined employing an immobilized artificial membrane column (IAM.PC.DD). The influence of mobile phase pH, ionic strength, and organic modifier composition was studied in order to examine column performance. After the IAM.PC.DD columns were exposed to approximately 7000 column volumes of a 0.01 M PBS mobile phase, five out of six columns tested showed significant peak broadening and decreased k' values indicative of premature column failure. The data suggested that the immobilized phospholipids stationary phase was removed by the 0.01 M PBS mobile phase. The beta-adrenolytic drug's log k'IAM values obtained with an IAM.PC.DD column were compared to an esterIAM.PC.MG column for predicting drug membrane interactions. For the linear regression analysis between log k'IAM and the logarithm of the n-octanol-water partition coefficients (rIAM.PC.DD = 0.8710 vs. rIAM.PC.MG = 0.9538), the C18 HPLC retention factors (rIAM.PC.DD = 0.8408 vs. rIAM.PC.MG = 0.9380), the liposome partition coefficients (rIAM.PC.DD = 0.8887 vs. rIAM.PC.MG = 0.9187), and various pharmacokinetic parameters, significantly better correlations were obtained with the esterIAM.PC.MG column than the IAM.PC.DD column.

Determination of pindolol in biological fluids by electron-capture GLC.

An electron-capture GLC method was developed for measuring pindolol in human plasma and urine. The unchanged drug was extracted with benzene from alkalinized plasma or urine using propranolol as the internal standard. Both compounds subsequently were back-extracted into 0.1 M HCl and then into benzene. After evaporation of the organic phase, the compounds were derivatized with trifluoroacetylimidazole to form the trifluoroacetyl ester of pindolol and propranolol. These derivatives then were analyzed by electron-capture GLC. The method allowed the measurement of concentrations as low as 1 ng of pindolol/ml of plasma and was applied successfully to determinations of plasma levels in humans after oral administraton of a single 10-mg dose of pindolol.

High performance liquid chromatographic determination of the enantiomers of beta-adrenoceptor blocking agents in biological fluids. I: Studies with pindolol.

This paper describes a high-performance liquid chromatographic procedure for the analysis of (+)- and (-)-pindolol in biological fluids. Racemic pindolol is extracted from alkalinized plasma or urine into ether, then purified by two steps of back extraction. The final extract is reacted with (S)-(-)-alpha-methylbenzyl isocyanate at room temperature, forming urea diastereoisomers as suggested by mass spectral analysis. Separation of the two diastereoisomers is accomplished by high-performance liquid chromatography with fluorescence detection. The assay is reproducible and precise for both (+)- and (-)-pindolol in human plasma and urine, as judged by a coefficient of variation of less than 10% at most concentrations. The standard curves for (+)- and (-)-pindolol in plasma are linear between 10-100 ng/mL, and between 100-2500 ng/mL in urine. The lower limit of detection is approximately 2 ng/mL for each enantiomer in plasma. This procedure can be readily adapted for the stereospecific assay of other beta-adrenoceptor blocking agents as demonstrated by the base-line separation of atenolol and acebutolol.

Investigation of the pindolol-Fe(III) complex and its use in the spectrophotometric determination of pindolol in bulk drug and tablets.

It was found that pindolol reacts with Fe(III) chloride producing a green water soluble complex (1:1, v/v) with maximum absorbance at 635 nm. By applying the methods of Sommer and Job [Sommer et al., Folia, tomus XI, Chemia 7, 25, 1970] the conditional stability constant of the complex at pH = 1.70 +/- 0.02 was found to be log K' = 4.95 and the molar absorptivity of the complex to be 206 l mol-1 cm-1. Beer's law was obeyed up to a concentration of 220 mumol l-1 of pindolol. The recoveries were 98-101% (n = 7) and the detection limit was 5 micrograms ml-1. The described method was sufficiently simple, selective and sensitive to be suitable for the rapid and accurate determination of pindolol in tablets.

Simultaneous determination of clopamide-pindolol combination in tablets by zero-crossing derivative spectrophotometry.

A first-derivative spectrophotometric method, using a 'zero-crossing' technique of measurement has been used for determining clopamide-pindolol mixture in tablets. In the first-derivative mode the zero-crossing points of clopamide and pindolol occur at 272.6 and 262.4 nm, respectively. The relative ease offered by this technique for the quantification of these drugs with closely overlapping bands was demonstrated. The linearity of the calibration curves was satisfactory (r = 0.9998) and the precision (RSD%) better than 1.89. Detection limits were 0.50 and 0.44 micrograms ml-1 for pindolol and clopamide, respectively. No spectral interferences from tablet excipients were found. Applications are given for the assay of commercial tablets and content uniformity test. The procedures proved to be suitable for rapid and reliable quality control.

Determination of bopindolol in pharmaceuticals by capillary isotachophoresis.

Capillary isotachophoresis (ITP) with conductimetric detection has been used for separating and determining bopindolol (I) in commercial mass-produced pharmaceutical preparations. The optimised operational electrolyte system consisted of 5 mM potassium picolinate and 5 mM picolinic acid (leading electrolyte, LE; pH 5.37) and 10 mM formic acid as the terminating electrolyte (TE). The driving and detection currents were 50 microA (for 350 s) and 10 microA, respectively. The single analysis took about 12 min. Under such conditions the effective mobility of I was determined as 16.73 10(-9)m(2) V(-1) s(-1) (with tetraethylammonium as the mobility standard). The calibration graph relating the ITP zone length to the concentration of I was rectilinear (r=0.99990) in the range 10-100 mg l(-1). The relative standard deviation (R.S.D.) was 0.90% (n=6) when determining 50 mg l(-1) of I in pure test solution. Sample pre-treatment of the tablets involved ice-cooled extraction of I with methanol. The method was suitable for determining I in Sandonorm tablets with R.S.D. value 1.45% (n=6). According to the validation procedure based on the standard addition method the recovery was 97.3%.

Determination of bopindolol using the flow injection technique coupled with solid phase extraction.

In the proposed procedure, the determination of bopindolol using a flow injection analysis (FIA) technique, with spectrophotometric detection at 635 nm, is described. The method is based on the production of a green, water-soluble complex with ferric ions in acid medium. The automated lab-made FIA system was used for the direct determination of bopindolol in tablets. Bopindolol was adsorbed onto the solid phase in a mini-column, which was integrated directly into the flow system. The positive feature of the use of solid phase extraction (SPE) was the pre-concentration of bopindolol (seven times). The sample throughput was 50 samples per hour. Using the SPE method, bopindolol was determined with a linear range from 125 to 1000 microg ml(-1) (Relative standard deviation (R.S.D.)=1.87%), with a detection limit (3sigma) of 70 microg ml(-1). The method was applied to the determination of bopindolol in Sandonorm tablets. The results obtained were compared with a conventional HPLC method, both analytical techniques were in good agreement.

Indirect atomic absorption spectrometric determination of pindolol, propranolol and levamisole hydrochlorides based on formation of ion-associates with ammonium reineckate and sodium cobaltinitrite.

A new simple, accurate, precise and sensitive indirect method for the determination of pindolol HCl (1), propranolol HCl (2) and levamisole HCl (3) using atomic absorption spectrometry has been developed. The method is based on precipitation of the ion-associates formed from the reaction of (1), (2) or (3) with ammonium reineckate and/or sodium cobaltinitrite. The solubility of the solid complexes at the optimum conditions of pH and ionic strength values have been studied. Saturated solutions of each ion-associate were prepared under the optimum conditions and the metal ion content in the supernatant was determined. The method has been used for the determination of 1.14-17.07, 1.18-17.75 and 1.08-16.24 microg/ml of (1), (2) and (3), respectively, using ammonium reineckate, and 1.71-25.60, 1.77-26.62 and 1.62-24.36 microg/ml of (1), (2) and (3), respectively, using sodium cobaltinitrite. The method developed was applied for analysis of bulk drugs and some of their pharmaceutical preparations.