A stability indicating capillary electrophoresis method for analysis of buserelin.

A simple and rapid stability indicating method based on capillary zone electrophoresis has been developed and validated for the analysis of buserelin (BUS). The best separations were achieved by using a bare fused silica capillary (75 µm i.d.; 65.5 cm total, 57.0 cm effective length), phosphate buffer (pH = 3.00; 26.4 mM), at 35 °C. The sample was hydrodynamically injected at 50 mbar for 5 seconds; the applied voltage was 30 kV and detection was carried out by UV-absorbance at 200 nm. Method validation resulted in the following figures of merit : the method was linear in the concentration range between 0.781 and 500 µg/mL (linear regression coefficient 0.9996), accuracy was between 99.3% and 100.9%, intra assay precision was between 0.3% and 1.0% and intermediate precision was between 1.0% and 2.1%. Evaluation of the specificity of the method showed no interference between excipients or products of force degradation and BUS. Under the selected conditions, separation of BUS and its degradation products was completed in less than 10 min, and BUS could be quantified after different stress conditions without any interference. The results enabled the conclusion that under thermal stress upon exposure to 90 °C BUS is degraded by first order kinetics. It was demonstrated that the method can be applied as a rapid and easy to use method for quantification and stability testing of BUS in biopharmaceutical formulations in quality control laboratories.

A critical overview of non-aqueous capillary electrophoresis. Part II: separation efficiency and analysis time.

A survey of the literature on non-aqueous capillary zone electrophoresis leaves one with the impression of a prevailing notion that non-aqueous conditions are principally more favorable than conventional aqueous media. Specifically, the application of organic solvents in capillary zone electrophoresis (CZE) is believed to provide the general advantages of superior separation efficiency, higher applicable electric field strength, and shorter analysis time. These advantages, however, are often claimed without providing any experimental evidence, or based on rather uncritical comparisons of limited sets of arbitrarily selected separation results. Therefore, the performance characteristics of non-aqueous vs. aqueous CZE certainly deserve closer scrutiny. The primary intention of Part II of this review is to give a critical survey of the literature on non-aqueous capillary electrophoresis (NACE) that has emerged over the last five years. Emphasis is mainly placed on those studies that are concerned with the aspects of plate height, plate number, and the crucial mechanisms contributing to zone broadening, both in organic and aqueous conditions. To facilitate a deeper understanding, this treatment covers also the theoretical fundamentals of peak dispersion phenomena arising from wall adsorption; concentration overload (electromigration dispersion); longitudinal diffusion; and thermal gradients. Theoretically achievable plate numbers are discussed, both under limiting (at zero ionic strength) and application-relevant conditions (at finite ionic strength). In addition, the impact of the superimposed electroosmotic flow contributions to overall CZE performance is addressed, both for aqueous and non-aqueous media. It was concluded that for peak dispersion due to wall adsorption and due to concentration overload (electromigration dispersion, leading to peak triangulation) no general conjunction with the solvent can be deduced. This is in contrast to longitudinal diffusion: the plate height (and the plate number) obtainable under limiting conditions (at zero ionic strength) has the same ultimate value for all solvents. However, in background electrolytes with finite ionic strength, the maximum reachable plate number depends on the solvent, and in water it is higher than in the most commonly used organic solvents: methanol and acetonitrile. Thermal peak broadening is also larger in the organic solvents if compared to aqueous solutions under comparable conditions. However, its influence on the plate height is negligible under conditions established with commercial instrumentation. From the laws of electric and thermal conductance, it follows that no general conclusion can be drawn that with organic solvents higher field strength can be applied and shorter analysis time can be reached; the contrary is more evident: under comparable conditions aqueous solutions lead to more favorable results. This comprehensive analysis provides strong evidence that the broadly held notion of non-aqueous CZE being principally superior to aqueous CZE is a myth rather than a fact. However, several studies in which the employment of non-aqueous conditions has been instrumental to solve challenging analytical problems demonstrate that the intelligent use of non-aqueous CE has and will continue having its place in modern separation science.

A critical overview of non-aqueous capillary electrophoresis. Part I: mobility and separation selectivity.

This two-part review critically gives an overview on the theoretical and practical advances in non-aqueous capillary electrophoresis (NACE) achieved over the recent five years. Part I starts out by reviewing the aspects relevant to electromigration in organic solvents and evaluates potential advantages of the latter in comparison to aqueous solvent systems. The crucial role of solubility for the species involved in CE - analytes and back ground electrolyte constituents - is discussed both for ionic and neutral compounds. The impact of organic solvents on the electrophoretic and electroosmotic mobility and on the ionization (pKa values) of weak acids and bases is highlighted. Special emphasis is placed on methanol, acetonitrile and mixtures of these solvents, being the most frequent employed media for NACE applications. In addition, also solvents less commonly used in NACE will be covered, including other alcohols, amides (formamide, N-methylformamide, N,N-dimethylformamide, N,N-dimethylacetamide), propylene carbonate, dimethylsulphoxide, and nitromethane. The discussions address the consequences of dramatic pKa shifts frequently seen for weak acids and bases, and the important contributions of medium-specific electroosmotic flow (EOF) to electromigration in nonaqueous media. Important for NACE, the role of the water content on pKa and mobility is analyzed. Finally, association phenomena rather specific to nonaqueous solvents (ion pairing, homo- and heteroconjugation) will be addressed, along with their potential advantages for the development of NACE separation protocols. It is pointed out that this review is not intended as a listing of all papers that have been published on NACE in the period mentioned above. It rather deals with general aspects of migration and selectivity in organic solvent systems, and discusses - critically - examples from the literature with particular interest to the topic. An analog discussion about the role of the solvent on efficiency will be presented in Part II.

Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas phase electrophoretic mobility molecular analysis: part II.

Human rhinoviruses (HRVs) are valuable tools in the investigation of early viral infection steps due to their far reaching (although still incomplete) characterization. During endocytosis, native virions first loose one of the four capsid proteins (VP4); corresponding particles sediment at 135S and were termed subviral A particles. Subsequently, the viral RNA genome leaves the viral shell giving rise to empty capsids. In continuation of our previous work with HRV serotype 2 (HRV2) intermediate subviral particles, in which we were able to discriminate by CE even between two intermediates (AI and AII) of virus uncoating, we further concentrated on the characterization of AI particles with the electrophoretic mobility of around -17.2 × 10(-9) m(2) /Vs at 20°C. In the course of our present work we related these particles to virions as previously described at the subviral A stage of uncoating (and as such sedimenting at 135S) by determination of their protein and RNA content--in comparison to native virions AI particles did not include VP4, however, still 93% of their initial RNA content. Binding of an mAb specific for subviral particles demonstrated antigenic rearrangements on the capsid surface at the AI stage. Furthermore, we investigated possible factors stabilizing intermediates of virus uncoating. We could exclude the influence of the previously suspected so-called contaminant of virus preparation on HRV2 subviral particle formation. Instead, we regarded other factors being part of the virus preparation system and found a dependence of AI particle formation on the presence of divalent cations.

Characterization of rhinovirus subviral A particles via capillary electrophoresis, electron microscopy and gas-phase electrophoretic mobility molecular analysis: Part I.

During infection, enteroviruses, such as human rhinoviruses (HRVs), convert from the native, infective form with a sedimentation coefficient of 150S to empty subviral particles sedimenting at 80S (B particles). B particles lack viral capsid protein 4 (VP4) and the single-stranded RNA genome. On the way to this end stage, a metastable intermediate particle is observed in the cell early after infection. This subviral A particle still contains the RNA but lacks VP4 and sediments at 135S. Native (150S) HRV serotype 2 (HRV2) as well as its empty (80S) capsid have been well characterized by capillary electrophoresis. In the present paper, we demonstrate separation of at least two forms of subviral A particles on the midway between native virions and empty 80S capsids by CE. For one of these intermediates, we established a reproducible way for its preparation and characterized this particle in terms of its electrophoretic mobility and its appearance in transmission electron microscopy (TEM). Furthermore, the conversion of this intermediate to 80S particles was investigated. Gas-phase electrophoretic mobility molecular analysis (GEMMA) yielded additional insights into sample composition. More data on particle characterization including its protein composition and RNA content (for unambiguous identification of the detected intermediate as subviral A particle) will be presented in the second part of the publication.

Fast analysis of amino acids in wine by capillary electrophoresis with laser-induced fluorescence detection.

A fast analytical method has been developed for the determination of nine amino acids, together with serotonin, in wine samples of different origin and vintage. The method is based on capillary electrophoresis coupled to laser-induced fluorescence detection. Separation was obtained by using a fused-silica capillary (75 µm id, 74.0 cm total length, 60.0 cm length to detector) and a background electrolyte composed of carbonate buffer (20 mM, pH 9.2), applying a 20 kV voltage. Direct hydrodynamic injection of wine samples was made after an original microwave-assisted derivatisation step with 5-(4,6-dichlorotriazinyl)aminofluorescein. Fluorescence was induced by an Ar-Ion laser, exciting at 488 nm. Good linearity (r(2) >0.9990) was obtained for all considered analytes and sensitivity was also good, with limits of detection in the 7-50 ng/mL range. The method was successfully applied for the analysis of commercial Italian wines and thus seems to be suitable for the determination of the relevant amino acids and serotonin, providing good results in terms of accuracy and precision, together with the advantage of a very fast, microwave-assisted derivatisation procedure. Future applications of the method are planned to check for wine adulterations and commercial frauds.

Recent developments in capillary and chip electrophoresis of bioparticles: Viruses, organelles, and cells.

In appropriate aqueous buffer solutions, biological particles usually exhibit a particular electric surface charge due to exposed charged or chargeable functional groups (amino acid residues, acidic carbohydrate moieties, etc.). Consequently, these bioparticles can migrate in solution under the influence of an electric field allowing separation according to their electrophoretic mobilities or their pI values. Based on these properties, electromigration methods are of eminent interest for the characterization, separation, and detection of such particles. The present review discusses the research papers published between 2008 and 2010 dealing with isoelectric focusing and zone electrophoresis of viruses, organelles and microorganisms (bacteria and yeast cells) in the capillary and the chip format.

Liposomal nanocontainers as models for viral infection: monitoring viral genomic RNA transfer through lipid membranes.

After uptake into target cells, many nonenveloped viruses undergo conformational changes in the low-pH environment of the endocytic compartment. This results in exposure of amphipathic viral peptides and/or hydrophobic protein domains that are inserted into and either disrupt or perforate the vesicular membranes. The viral nucleic acids thereby gain access to the cytosol and initiate replication. We here demonstrate the in vitro transfer of the single-stranded positive-sense RNA genome of human rhinovirus 2 into liposomes decorated with recombinant very-low-density lipoprotein receptor fragments. Membrane-attached virions were exposed to pH 5.4, mimicking the in vivo pH environment of late endosomes. This triggered the release of the RNA whose arrival in the liposomal lumen was detected via in situ cDNA synthesis by encapsulated reverse transcriptase. Subsequently, cDNA was PCR amplified. At a low ratio between virions and lipids, RNA transfer was positively correlated with virus concentration. However, membranes became leaky at higher virus concentrations, which resulted in decreased cDNA synthesis. In accordance with earlier in vivo data, the RNA passes through the lipid membrane without causing gross damage to vesicles at physiologically relevant virus concentrations.

Liposomal leakage induced by virus-derived peptides, viral proteins, and entire virions: rapid analysis by chip electrophoresis.

Permeabilization of model lipid membranes by virus-derived peptides, viral proteins, and entire virions of human rhinovirus was assessed by quantifying the release of a fluorescent dye from liposomes via a novel chip electrophoretic assay. Liposomal leakage readily occurred upon incubation with the pH-sensitive synthetic fusogenic peptide GALA and, less efficiently, with a 24mer peptide (P1-N) derived from the N-terminus of the capsid protein VP1 of human rhinovirus 2 (HRV2) at acidic pH. Negative stain transmission electron microscopy showed that liposomes incubated with the rhinovirus-derived peptide remained largely intact. At similar concentrations, the GALA peptide caused gross morphological changes of the liposomes. On a molar basis, the leakage-inducing efficiency of the P1 peptide was by about 2 orders of magnitude inferior to that of recombinant VP1 (from HRV89) and entire HRV2. This underscores the role in membrane destabilization of VP1 domains remote from the N-terminus and the arrangement of the peptide in the context of the icosahedral virion. Our method is rapid, requires tiny amounts of sample, and allows for the parallel determination of released and retained liposomal cargo.

Determination of topiramate in human plasma by capillary electrophoresis with indirect UV detection.

A rapid capillary zone electrophoresis method with indirect UV detection for the determination of topiramate in human plasma was developed and validated. The analyses were carried out with a background electrolyte composed of 10mM sulfamethoxazole as chromophore in phosphate buffer (25 mM, pH 12.0); gabapentin was selected as the internal standard. Application of a voltage of +15 kV led to an analysis time shorter than 5 min; indirect UV detection was operated at 256 nm. Isolation of topiramate from plasma was accomplished by a carefully implemented solid-phase extraction procedure on C18 cartridges. The method provided a linear response over the concentration range of 2-60 microg of topiramate per mL of plasma. The limit of detection (LOD) was 0.8 microg mL(-1) and the limit of quantitation (LOQ) was 2.0 microg mL(-1). Precision, expressed as relative standard deviation, was always lower than 7.3%, extraction yields were always greater than 92%. The results obtained analysing plasma samples from epileptic patients undergoing therapy with topiramate were satisfactory in terms of precision and selectivity.

Chip electrophoretic characterization of liposomes with biological lipid composition: Coming closer to a model for viral infection.

In first attempts at elucidating the transfer of the RNA genome of a human Rhinovirus through lipid membranes in vitro we made use of liposomes decorated with recombinant receptors. This model system was characterized previously by CE but suffered from the requirement for inclusion of polyethylene glycol-modified lipids for reliable separations [Weiss, V. U., Bilek, G., Pickl-Herk, A., Blaas, D., Kenndler, E., Electrophoresis 2009, 30, 2123-2128.]. We here report the analysis of liposomes with a lipid composition much more similar to that of biological lipid bilayers. We found that vesicles containing and lacking this non-physiologic lipid differ significantly in their electrophoretic mobility (by factor 2) although the concentration of charge-bearing lipids in their bilayers is the same. We demonstrate that binding of a human Rhinovirus to the latter liposomes decorated with a cognate receptor can be analysed via electrophoresis on microchips; we support our results with transmission electron microscopy.

Is the general conclusion justified that higher applicable field strength results in shorter analysis time with organic solvents in CE?

In this paper, a widespread opinion in CE with organic solvents for the background electrolyte is critically questioned, namely that in general a shorter analysis time can be achieved due to the higher field strength applicable compared with aqueous electrolyte systems. This view, common in the literature, is based on the supposition that the conductance in organic solvents is lower than in water. Indeed in many organic solvents with higher viscosity than water lower ion mobility is observed, and higher fields can be applied in these cases. However, in this paper the problem is sharper defined and treated two-fold: (i) in all solvents conditions are such that either the same electric power is generated, or (ii) the same temperature increase is taken into account. It was shown that for the same electric power the field strength in the organic solvent can be changed to a less extent than the ionic mobility changes. As a result, the migration velocity of the analytes is lower and the analysis time is longer in most organic solvents compared with water; acetonitrile (MeCN) is an exception (in this solvent the mobilities are higher than in water). The more stringent treatment of the problem takes an equal temperature increase due to Joule heating into account rather than equal electric power. The temperature increase in the capillary depends on the thermal conductivity of the solvent, which is only about one-third of that of water for organic liquids. The consequence is that in none of the organic solvent systems a shorter analysis time can be achieved compared with water (given that the experimental conditions are comparable, e.g. zero EOF). The theoretical predictions were confirmed by measurements with water, methanol, propylenecarbonate, and MeCN as solvents.

Electromigration oscillations occurring in ternary electrolyte systems with complex eigenmobilities, as predicted by theory and ascertained by capillary electrophoresis.

Chemical oscillations are driven by a gradient of chemical potential and can only develop in systems where the substances are far from chemical equilibrium. We have discovered a new analogous type of oscillations in ternary electrolyte mixtures, which we call electromigration oscillations. They appear in liquid solutions of electrolytes and are associated with the electromigration movement of ions when conducting an electric current. These electromigration oscillations are driven by the electric potential gradient, while the system can be close to chemical equilibrium. The unequivocal criterion for the instability of the electrolyte solution and its ability to oscillate is the existence of complex system eigenmobilities. We show how to calculate the system eigenmobilities by utilizing the linear theory of electromigration and how to identify the complex system eigenmobilities to predict electromigration oscillations. To experimentally prove these electromigration oscillations, we employ a commercially available instrument for capillary electrophoresis. The oscillations start a certain period of time after switching on the driving electric current. The axial concentration profiles of the electrolytes in the capillary attain a nearly periodic pattern with a spatial period in the range of 1-4 mm, with almost constant amplitude. This periodic pattern moves in the electric field with mobility that is equal to the real part of the complex eigenmobility pair. We have found several ternary oscillating electrolytes composed of a base and two acids, of which at least one has higher valence than one in absolute value. All the systems have three system eigenmobilities: one is real and close to zero, and the two others form the complex conjugate pair, the real part of which is far from zero.

Mimicking virus attachment to host cells employing liposomes: analysis by chip electrophoresis.

Electrophoresis on a chip increasingly replaces electrophoresis in the capillary format because of its speed and containment of the sample within a disposable cartridge. In this paper we demonstrate its utility in the analysis of the interaction between a virus and a liposome-anchored receptor, mimicking viral attachment to host cells. This became possible because detergents, obligatory constituents of the BGE for capillary electrophoretic separation of the virus, were not necessary in the chip format. Separations were carried out in sodium borate buffer, pH 8.3. Liposomes and virus were both labeled for laser-induced fluorescence detection at lambda(ex)/lambda(em) 630/680 nm. Free virus and virus-receptor complexes were resolved from virus attached to receptor-decorated liposomes in the absence of additives or sieving matrices within about 30 s on commercially available microfluidic chips.

Organic solvents in CE.

In this contribution some fundamental aspects are discussed serving for a critical reflection and elucidation of the role of organic solvents in CE. The implications of the solvent on the parameters governing peak resolution are discussed based on the concepts describing migration and zone broadening in capillary zone electrophoresis. This discussion includes the solvent-dependent influence of the ionic strength on the mobility. The role of the solvent on the plate number in case of the inevitable diffusional peak dispersion is outlined, and its effect on other peak broadening contributions is briefly examined. This paper also deals with the problems of conductance, applicable voltage and analysis time upon application of organic solvents, and tries to clarify some misunderstandings common in the literature.

Determination of duloxetine in human plasma by capillary electrophoresis with laser-induced fluorescence detection.

A method based on capillary electrophoresis has been developed for the analysis of the novel antidepressant drug duloxetine in human plasma. The method makes use of laser-induced fluorescence detection after derivatisation of the analyte with 5-(4,6-dichlorotriazinyl)aminofluorescein at pH 11. A single step liquid/liquid extraction procedure with a mixture of hexane/2-propanol allows the sample clean-up with extraction yields always >or=84% and interference removal. The electrophoretic separation is achieved using uncoated fused silica capillaries (60.0 cm effective length, 75.0 cm total length, 50 microm internal diameter) and a background electrolyte composed of borate buffer (40 mM, pH 10.3), tetrabutylammonium bromide (10 mM), and acetone (10%, v/v). The applied voltage is 20 kV; the samples are injected by pressure (50 mbar x 8 s). The method has been fully validated in terms of linearity range (2.5-150 ng mL(-1)), LOD and LOQ (1.0 and 2.5 ng mL(-1), respectively), precision (R.S.D.<6.7%) and accuracy (recovery >78%). Application to samples obtained from patients under treatment with duloxetine gave good results. The method represents the first application of capillary electrophoresis to the analysis of duloxetine in human plasma.

Formamide as an organic modifier in MEKC with SDS.

The effect of formamide (FA) as a modifier on the retention in MEKC with SDS as the detergent was investigated. The mobility of a series of alkylphenones and of a zwitterionic fluorescent compound as a function of the FA and the SDS concentration was determined for this purpose. Buffering electrolyte was borate, pH 9.23, with total ionic strength of 50 mM. The dependence of the mobility on the FA content - up to 63% w/w - of the BGE (at 10 mM SDS) allows the conclusion that the micelles are destabilized, and the CMC is shifted to higher values. In the system containing 33% FA or more no micelles are present anymore, and the retention factors of all compounds tend to zero. In an MEKC system with 27% v/v FA the CMC of SDS is increased from 2.4 mM in the aqueous BGE with the same buffer composition to 9.7 mM, a behavior that is in contrast to electrolyte-free FA-water systems. The partition constants of free analytes and the formation constants of the adduct between analyte and detergent monomer (assuming 1:1 stoichiometry) were derived from the dependence of the mobility on the SDS concentration. In addition, the involved equilibria were extended by that from the distribution of the analyte-monomer adduct between aqueous and micellar phase, and the according partition constants were derived as well. A selective change in the extent of partitioning was observed for the zwitterionic compound. In general, all binding constants were decreased upon addition of FA, though to a different extent. Although the binding constants of the analyte-monomer associate were only slightly influenced, the most pronounced decrease is found for their partitioning into the micelles.

Virus analysis using electromigration techniques.

We discuss the progress during the last 4 years in the analysis of viruses by electrophoresis in capillaries and microfluidic devices. The paper is the continuation of a review published in this journal in 2005 [Kremser, L., Blaas, D., Kenndler, E., Electrophoresis 2004, 25, 2282-2291]. Eighteen papers on the topic have appeared since; the majority deals with zone electrophoresis and three reports are on IEF. These methods have been applied to human rhinoviruses, poliovirus Semliki Forest virus, norovirus-like particles, and the two bacteriophages MS2 and T5. A main finding was that addition of detergents and salts to the BGEs are essential for the robustness of the CE analysis. Analyte detection was usually via UV absorbance but there are some examples where the viruses were rendered fluorescent via modification of the capsid proteins with reactive dyes and/or by non-covalent attachment of intercalating fluorescent compounds to the nucleic acids making up the viral genome. Interestingly, some viruses are permeable to small molecular mass components; this allows fluorescent dyes to diffuse into the intact virus where they attach to the nucleic acid. Release of a viral genome upon heating was also monitored by using similar methodologies. Interactions of viruses and subviral particles with antibodies, receptors, and receptor-decorated liposomes were investigated with CE methods, all by using a non-equilibrium approach (i.e. co-incubation of the components prior to CE separation). Viruses are multivalent (i.e. possess many identical surface-exposed patches) and most of them are composed of defined numbers of identical subunits. The high resolution of CE has been most remarkably demonstrated by the separation of stoichiometric complexes between virus and a distinct number of soluble recombinant receptors and revealed their concentration-dependent distribution.

An extended description of the effect of detergent monomers on migration in micellar electrokinetic chromatography.

Three equilibria determine the interaction of a neutral analyte with the detergent in micellar electrokinetic chromatography and therefore its migration: (i) that of the free analyte in the aqueous phase with the micelle, (ii) its association with free detergent monomers in the aqueous phase, and (iii) the partition of the associate of analyte and monomer between the aqueous solution and the micelle. For the first equilibrium, non-stoichiometric partitioning between two phases is preferred in the present work over the assumption of complex formation between one molecule of the analyte with one micelle. The second equilibrium is described by the formation of a 1:1 associate of the analyte and monomer. In this paper, thirdly an additional equilibrium is introduced, namely, the distribution of the analyte-monomer associate between the aqueous and the micelle phase; it is expressed by the according partition coefficient. The three equilibrium constants are interrelated. Mobility data for a lipophilic fluorescent compound and a series of n-alkylphenones (differing in chain length) were measured as a function of the SDS concentration below and above the critical micellar concentration. Curve fitting enabled the derivation of the equilibrium constants. It was found that the association constants of the analytes with the detergent monomers are between 2 and 75 M(-1). Interestingly, the partition coefficient of the analyte-monomer associate between the aqueous and micellar phase is by a factor of 5-200 larger than that of the free analyte.

Analysis of the anti-Parkinson drug pramipexole in human urine by capillary electrophoresis with laser-induced fluorescence detection.

A sensitive method based on capillary electrophoresis with laser-induced fluorescence detection has been developed for the analysis of the non-ergoline dopamine agonist pramipexole in human urine. Separation was carried out in uncoated fused silica capillaries (75microm internal diameter, 75.0 and 60.0cm total and effective length, respectively), with a background electrolyte composed of borate buffer (50mM, pH 10.3), tetrabutylammonium bromide (30mM), and acetone (15%, v/v). Applying a 20kV voltage, the electrophoretic run is completed within 12min. A sample pre-treatment procedure based on liquid/liquid extraction with ethyl acetate, followed by derivatisation of pramipexole with fluorescein isothiocyanate at pH 9, allows the complete removal of biological interferences, with extraction yields always higher than 94.5%. Method validation gave good linearity (r(2)=0.9992) in the 25.0-1000ngmL(-1) range; limit of detection and limit of quantitation were 10.0 and 25.0ngmL(-1), respectively; precision was 90.0. The method was applied to the analysis of urine samples from patients undergoing therapy with pramipexole.