In vivo investigation of brain and systemic ketobemidone metabolism.

Ketobemidone metabolites have previously been identified in urine and plasma; here we show, for the first time, that norketobemidone and ketobemidone N-oxide are present in in vivo microdialysate from rat brain (striatum) after reverse microdialysis, suggesting striatal metabolism of ketobemidone. Ketobemidone metabolites were also identified in in vivo microdialysate samples from brain and blood, as well as in urine from rats, after subcutaneous administration of ketobemidone. Three Phase I metabolites (norketobemidone, ketobemidone N-oxide and hydroxymethoxyketobemidone) and three Phase II metabolites (glucuronic acid conjugates of ketobemidone, norketobemidone and hydroxymethoxyketobemidone) were identified in the microdialysates after subcutaneous administration. Coupled capillary liquid chromatography tandem mass spectrometry (LC-MS/MS) and SPE (boronate)-MS/MS were utilized for the analysis of the biological samples. The Phase I metabolites were identified by comparing the retention times and tandem mass spectra of the microdialysates with synthetic standards. The Phase II metabolites were identified by determination of exact masses and by comparing the tandem mass spectra of the microdialysates with those of synthetic standards for the aglycones. Hydroxyketobemidone, a catechol-type Phase I metabolite, was selectively isolated by solid-phase boronate-complexation but identified in urine alone. This work demonstrated that the in vivo microdialysis technique in combination with LC-MS/MS can be used to study the local metabolism of a drug in the brain.

The effect of sample salt additives on capillary electrophoresis analysis of intact proteins using surface modified capillaries.

The effect of adding alkali salts to protein samples for capillary electrophoretic (CE) analysis of intact proteins was studied. A high degree of peak stacking, even for large proteins, was found to occur when alkali salts were added to the sample. The addition of salt to the protein sample promotes a strong improvement in the peak efficiency of individual proteins giving up to 2.1x 10(6)apparent plates/m. The concentration of salt required in the sample to reach optimal peak efficiency show dependency on both the molecular weight and molar concentration of the protein. However, adding salt will, at a sufficiently high concentration, cause a mixture of proteins to co-migrate to one very sharp peak. The observed sample stacking effect was obtained with a number of different surface modified silica capillaries indicating a general phenomenon and not surface coating specific.

Coupling of solid-phase microextraction continuous bed (monolithic) capillaries with capillary zone electrophoresis for direct analysis of drugs in biological fluids.

Hyperlink robust biocompatible solid-phase microextraction (SPME) devices were prepared using continuous bed (monolithic) restricted-access media (RAM) as the SPME capillary insert. The RAM-based SPME approach was able to simultaneously separate proteins from a biological sample, while directly extracting the active components of caffeine, paracetamol and acetylsalicylic acid from the drug NeoCitramonum. The devices were interfaced with a CZE system and fully automated analysis for sample preconcentration, desorption, separation and quantification of analytes was evaluated. Comparative study of in-line coupled SPME-CZE using RAM and RP capillary inserts was carried out. Using an SPME (RAM) insert, the calculated caffeine, paracetamol and acetylsalicylic acid LODs in a bovine plasma sample were 0.3, 0.8 and 1.9 ng/mL, respectively.

Restricted-access media development for direct analysis of drugs in biofluids using capillary liquid chromatography.

In analytical sciences the design of novel materials and stationary phases for the sample preparation and separation of analytes from biological fluids is needed. In this work we present different strategies for modification of stationary phases to produce tailored solutions for the analytical problem. In this context a novel shielded polymeric reversed-phase monolithic material was prepared in the presence of different numbers of reactive groups and concentrations of the coating polymer. Chromatographic experiments were performed using benzoic acid propyl ester in order to characterize the hydrophobicity and efficiency of the different restricted-access continuous beds prepared. Inverse size-exclusion chromatography was used for investigation of the pore structure properties of the beds. Capillary columns were applied for nanochromatography of biological fluids containing a mixture of nitrazepamum and medazepamum.

Method development for identification of ketobemidone metabolites in microdialysate samples by coupled-column capillary liquid chromatography-tandem mass spectrometry.

Methodologies for identification of ketobemidone metabolites in microdialysate samples utilizing coupled-column capillary liquid chromatography-electrospray quadrupole time-of-flight tandem mass spectrometry are presented. Two different methods were developed to efficiently analyze the metabolites norketobemidone, ketobemidone N-oxide and hydroxyketobemidone, respectively. Both methods include on-line desalting and trapping of the analytes on micro-solid-phase extraction columns with different retention mechanisms. Norketobemidone and ketobemidone N-oxide were trapped on a C18 column and then eluted by back-flush followed by isocratic separation on a fluorinated reversed-phase type silica gel column (Fluofix). Retention equations are proposed for the chromatographic observations made on the Fluofix column. Hydroxyketobemidone was trapped on a phenylboronic acid column by complex formation at basic pH and then eluted at acidic pH directly into to the mass spectrometer. Oxidation of hydroxyketobemidone to its corresponding quinone was also observed. The methods were successfully used to analyze synthetic ketobemidone metabolites in dilute low-volume microdialysis samples.

Improved properties of the non-covalent coating with N,N-didodecyl-N, N-dimethylammonium bromide for the separation of basic proteins by capillary electrophoresis with acidic buffers in 25 microm capillaries.

Capillaries (25microm I.D.) treated with the double-alkyl-chain cationic surfactant N,N-didodecyl-N, N-dimethylammonium bromide (DDAB) in an improved coating procedure were used for separation of four basic proteins in volatile buffers (ammonium acetate and ammonium hydroxyacetate) as well as in a non-volatile buffer (sodium phosphate) at pH 4. The DDAB coating was stable enough to, without recoating, permit consecutive separations of the proteins up to 9h with good precisions in peak areas (RSD=1.1%) and migration times and with high apparent efficiencies (over 1 million theoretical plates/m) in the presence of a strong anodic electroosmosis. Adsorption of the proteins onto the capillary surface, which in previous studies was found to give a certain contribution to zone broadening, was eliminated with the new modified coating method. Complex formation between the proteins and phosphate buffer was studied and confirmed, and it is proposed that slow protein-buffer component interactions are the main contributions to zone broadening in protein separations by CE.

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers. I. Theoretical studies.

Distortion of the starting zone upon its electrophoretic migration toward the detection window gives rise to both symmetrical zones caused by diffusion, sedimentation in the horizontal section of the capillary and the curvature of the capillary, and asymmetrical zones having their origin in Joule heating, sedimentation in the vertical section of the capillary, pH and conductivity differences between the sample zone and the surrounding buffer, solute adsorption onto the capillary wall, and association-dissociation of complexes between the analyte and a buffer constituent or between analytes. Interestingly and importantly a theoretical study shows that moderate pH and conductivity differences as well as adsorption and all of the above interactions when they are characterized by a fast on/off kinetics do not increase the zone broadening (or only slightly), because the sharpening of one boundary of the zone is about the same as the broadening of the other boundary. In addition the peak symmetry caused by a conductivity difference is in most experiments counteracted by a pH difference. The experimentally determined plate numbers in the absence of electroosmosis exceeded one million per meter in some experiments (Part II). These plate numbers are among the highest reported [Z. Zhao, A. Malik, M.L. Lee, Anal. Chem. 65 (1993) 2747; M. Gilges, K. Kleemiss, G. Schomburg, Anal. Chem. 66 (1994) 2038; H. Wan, M. Ohman, L.G. Blomberg, J. Chromatogr. A 924 (2001) 591 (plate numbers determined in the presence of electroosmosis may be higher, although the width of the zone in the capillary may be larger) [p. 680 in S. Hjertén, Electrophoresis 11 (1990) 665]). Capillary free zone electrophoresis is perhaps the only separation method, which, under optimum conditions, gives a plate number not far from the theoretical limit. A prerequisite for this high performance is that the polyacrylamide-coated capillary is washed with 2 M HCl between the runs and stored in water over night (Part II). The difference between the experimentally determined total variance and the sum of the calculated variances originating from the width of the starting zone, longitudinal diffusion, Joule heating, sedimentation in the vertical section of the capillary, curvature of the capillary (i.e., the sum of all other variances) was in our most successful experiments about 28% of the variance of diffusion. The zone broadening, 2sigma, caused by diffusion was estimated at 0.77 mm. The total zone width (2sigma) calculated from the experimentally determined plate number was as small as 1 mm when the migration distance was 40 cm. Accordingly, the only efficient way to reduce drastically the total zone width is to decrease the analysis time and, thereby, the diffusional broadening. An important finding was that the variance originating from the loops of the capillary is not always negligible in high-performance runs. Therefore, one should employ straight capillaries and avoid CE apparatus with cartridges that require a strong curvature of the capillary, common in most commercial instruments. Mathematical formulas have been derived for the sedimentation of the solute zone, the enrichment factor, and the migration time in experiments where the solute is dissolved in a dilute running buffer. This zone sharpening method gave very narrow starting zones (0.04-0.4 mm). However, upon high dilution of the buffer the enrichment becomes so strong that part of the sample zone probably sediments out of the capillary; the almost inevitable change in pH may decrease the mobility of the proteins and, thus, cause the enrichment factor to become still lower than expected. Diffusion of the protein in the very narrow starting zone (located close to the tip of the capillary) and sometimes the thermal expansion of the buffer in the capillary contributes to additional loss of protein in the enrichment step. In some buffers, the interaction between the protein and the buffer constituents is so slow that the peaks become broad. Therefore, different types of buffers should be tested when high resolution is required. The relation sigma2 (the variance of the interaction between a protein and the buffer constituents) = constant x u (the mobility) seems to be valid for all proteins in the applied sample, at least when they have similar molecular masses. To facilitate the understanding of the progress of a free zone electrophoresis experiment, we have discussed in simple terms how the concentrations of the background electrolytes become rearranged during a run and why the difference between the mobilities of the proteins and the mobilities of the background electrolyte determines whether a peak exhibits fronting or tailing. A theoretical analysis of zone broadening in capillary zone electrophoresis, chromatography, and electrochromatography indicates that electrochromatography in homogeneous gels might be the only chromatographic technique which can compete in performance with free electrophoresis. Using an equation, valid not only for electrophoresis, but also for chromatography and centrifugation, the mobility of a concentration boundary has been calculated for the first time and was, as expected, low. Equations based on the Kohlrausch regulating function do not permit such calculations. Another regulating function (the H function) and some of its characteristics are briefly discussed. The theoretical discussions in this paper and the experimental studies in Part II show that high-performance electrophoresis deserves its prefix when the runs are designed to give minimum zone broadening. Some guidelines are given to facilitate this optimization. The plate numbers are so high that the resolution cannot be increased by more than 30% even if they approach the theoretically maximum values.

Influence of ignored and well-known zone distortions on the separation performance of proteins in capillary free zone electrophoresis with special reference to analysis in polyacrylamide-coated fused silica capillaries in various buffers. II. Experimental studies at acidic pH with on-line enrichment.

The separation of acidic and basic model proteins was studied in capillary free zone electrophoresis in a polyacrylamide-coated, electroosmosis-free capillary at pH below their isoelectric points (pI) using various buffers at pH 2.7-4.8 with UV detection at 200 nm. The separation performance was significantly dependent on the coating quality, which may even differ within the same batch of capillaries. In addition, a washing step with 2 M HCl and the storage of the capillary in distilled water was essential for the performance. For high efficiency and resolution the choice of buffer constituents was extremely important which is discussed in quantitative terms in Part I. The most promising buffers were ammonium acetate and ammonium hydroxyacetate at pH 4 (ionic strengths: 0.12 and 0.15 M, respectively) with plate numbers up to 1,700,000 plates/m, corresponding to a zone width (2sigma) of only 1 mm in a capillary with 40 cm effective length, when the injected samples were dissolved in a 10-fold diluted background electrolyte (BGE), a zone even narrower than those obtained in polyacrylamide gel electrophoresis, the characteristic feature of which is remarkably thin zones. In the experiment giving this plate number, the calculated variance for longitudinal diffusion was larger than all the other calculated variances (those for the width of the starting zone, Joule heating, sedimentation and the curvature of the capillary). Interestingly, the effect of capillary curvature was significant. In addition, the sum of all other imaginable variances (corresponding to various types of slow on/off kinetics and hyper-sharp peaks) was in the most successful experiments only 28-50% of the variance for longitudinal diffusion. One hundred- to two hundred-fold dilution of the BGE improved the detection limits and provided high precision in both migration times and peak areas with ammonium hydroxyacetate and ammonium acetate as background electrolytes. However, that high dilution increased the variance 140-400% for these buffers, respectively, at least partly due to conductivity or pH differences between the sample and buffer zones (hyper-sharp peaks). Sedimentation of the enriched sample, a factor that has not previously been treated theoretically or experimentally, was probably another reason for our finding that peak heights did not increase when the sample was dissolved in a buffer diluted more than 200-fold, although pH changes and in some cases thermal expansion in the capillary also may contribute. Loss of protein may occur at the ionic strength 0.01 and lower due to precipitation. Limits of detection were in the range 4-17 pmol of proteins with ammonium acetate as BGE. No indication of denaturation of proteins at pH 4 was observed. However, the separation performance at pH 3 was not satisfactory and loss of proteins was observed, possibly indicating such problems. The protein mobilities decreased unexpectedly from pH 4 to 3--a further indication of conformation changes.

Microemulsion electrokinetic chromatography of drugs varying in charge and hydrophobicity Part II: Strategies for optimization of separation.

The separation of anionic, cationic, and neutral drugs in microemulsion electrokinetic chromatography (MEEKC) was studied. The concentration of sodium dodecyl sulfate (SDS; surfactant) and 2-propanol (organic solvent) was varied in a three-level full factorial design. 29 different model substances were chosen with different hydrophobicities and charges (neutral, positive, and negative). The models were calculated by means of multiple linear regression (MLR). The compounds were divided into five different subgroups, and different strategies for optimization of the separation within each group were investigated. The optimization was done by maximizing the selectivity using response surface plots in MODDE, by calculation of different chromatographic functions, and by using the software DryLab. For all the different groups, MODDE, almost all chromatographic functions and DryLab gave approximately the same settings of the factors for optimum separation. Attempts were made to fit descriptors of the compounds to the retention data from the three-level full factorial design by means of partial least squares projection to latent structures (PLS). Between 86 and 89% of all predictions of migration times were acceptable (80-120% of the observed value).

Microemulsion electrokinetic chromatography of drugs varying in charge and hydrophobicity: I. Impact of parameters on separation performance evaluated by multiple linear regression models.

The separation of anionic, cationic and neutral drugs in microemulsion electrokinetic chromatography (MEEKC) was studied with a statistical experimental design. The concentration of sodium dodecyl sulfate (SDS, surfactant), 1-butanol (co-surfactant) and borate buffer and the factors Brij 35 (surfactant), 2-propanol (organic solvent) and cassette temperature were varied simultaneously, while the parameters pH (9.2), the concentration of octane (oil, 0.8% w/w), the voltage (10 kV) and the dimension of the fused-silica capillary, were kept constant. Eight different model substances were chosen with different hydrophobicities. Two of the analytes were positively charged, two were negatively charged, and the remaining four were neutral or close to neutral at the pH explored. The importance of each parameter on the separation window, the plate height and the retention factor for each of the analytes was studied by means of multiple linear regression (MLR) models. A new response was evaluated for anions, the quotient between the effective mobility in the microemulsion and the effective mobility in the corresponding buffer. Factors affecting selectivity changes were also explored, and it was found that SDS and 2-propanol had the largest effect on selectivity.

Non-particulate (continuous bed or monolithic) restricted-access reversed-phase media for sample clean-up and separation by capillary-format liquid chromatography.

Restricted-access reversed-phase non-particulate (continuous bed or monolithic) stationary phases of different hydrophobicity synthesized in 100 microm i.d. fused silica capillaries have been evaluated. A specific property of restricted-access media (RAM) is that they interact with small analytes and exclude big molecules, e.g. proteins, from access to the active sites and adsorption on the surface. This dual property facilitates direct injection of biological fluids for drug or drug-metabolite analysis. Different RAM and RAM-precursor capillary columns were tested to assess the influence of chromatographic bed morphology on loadability. Inverse size-exclusion chromatography was used for investigation of pore structural properties of the capillary-format continuous beds. The data obtained were used to discuss the mechanism of separation of the biological samples using capillary columns and to propose a model for the topochemical architecture of the RAM investigated. Different morphology of the non-particulate reversed-phase precursors resulted in two types of RAM material shielded with hydrophilic polymer, classified as homogeneous or heterogeneous topochemistry stationary phases. Capillary columns were applied for chromatography of biological fluids. High resolution was obtained, without the need for column switching, when capillary columns operated in gradient conditions. Extensive evaluation of the chromatographic properties (hydrophobicity, efficiency, separation impedance, and loadability) of the non-particulate reversed-phase materials was performed before and after shielding with hydrophilic polymer to generate restricted-access properties. Minor changes of hydrophobicity, efficiency, or separation impedance were observed after the shielding.

Use of liquid chromatography-diode-array detection and mass spectrometry for rapid product identification in biotechnological synthesis of a hydroxyprogesterone.

In exploratory scale biotechnological process development, the product must be rapidly identified although a reference compound may not always be available. LC-diode-array detection and MS were used for this purpose in a process producing 9alpha-hydroxyprogesterone from progesterone as substrate. The electrospray ionization mass spectrometer was combined with an ion trap mass spectrometer for the second generation MS. The preliminary identification, which could be carried out within the course of a day, confirmed that the product was a hydroxyprogesterone. The final identification step, which was much more material intensive and hence time consuming, involved a two-step preparative separation to yield quantities necessary for definitive product identification based on 1H- and 13C NMR.

Partial filling-micellar electrokinetic chromatography optimization studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry: part II.

We describe the use of partial filling-micellar electrokinetic chromatography-mass spectrometry (PF-MEKC-MS) on the pharmaceutical ingredients ibuprofen and codeine phosphate as well as their degradation products and impurities. The study focuses on the change of the borate buffer to the volatile ammonium acetate and the optimization of critical MS parameters. The sensitivity of the method is also evaluated. The results are compared to an existing MEKC-UV method that is used for quantitative determination of the two main substances as well as for the analysis of the degradation products. It is concluded that the PF-MEKC-MS system is suitable for separation and identification.

A statistical experimental design to study factors affecting enantioseparation of propranolol by capillary electrophoresis with cellobiohydrolase (Cel7A) as chiral selector.

The capillary electrophoretic enantioseparation of rac-propranolol using cellobiohydrolase Tr Cel7A as selector was optimized by an unbiased statistical experimental design. A set of pre-experiments was performed in order to identify critical experimental factors. In the definitive chemometric design pH, ranging from 5 to 7, ionic strength ranging between 0.01 and 0.02 and organic solvent additive in concentration from 1 to 19% v/v were studied. The response surface plot revealed a separation optimum in the pH interval studied. When all parameters were taken into account, a background electrolyte consisting of 0.016 M bistris-acetate buffer with pH 6.5 and 17% v/v acetonitrile gave the optimum separation. The significance of the statistical design was confirmed by the generally good agreement obtained between predicted response and actual experimental data.

Partial filling micellar electrokinetic chromatography optimization studies of ibuprofen, codeine and degradation products, and coupling to mass spectrometry.

Studies have been performed to evaluate whether an on-line partial filling-micellar electrokinetic chromatography (PF-MEKC) system could be applied to a recently developed MEKC method for the separation of ibuprofen, codeine and one of the degradation products. Attempts to couple the PF-MEKC system to MS have also been performed. SDS concentration, micellar zone length and concentration of acetonitrile in the buffer were optimized using factorial design. When a small micelle zone was injected directly after the sample introduction, the results improved markedly. The MS parameters have not been optimized, but the studies show promising results for the use of PF-MEKC-mass spectrometry for identification of the degradation products.