Use of Polyvinyl Alcohol as a Solubility Enhancing Polymer for Poorly Water-Soluble Drug Delivery (Part 2).

The KinetiSol® Dispersing (KSD) technology has enabled the investigation into the use of polyvinyl alcohol (PVAL) as a concentration enhancing polymer for amorphous solid dispersions. Our previous study revealed that the 88% hydrolyzed grade of PVAL was optimal for itraconazole (ITZ) amorphous compositions with regard to solid-state properties, non-sink dissolution performance, and bioavailability enhancement. The current study investigates the influence of molecular weight for the 88% hydrolyzed grades of PVAL on the properties of KSD processed ITZ:PVAL amorphous dispersions. Specifically, molecular weights in the processable range of 4 to 18 mPa · s were evaluated and the 4-88 grade provided the highest AUC dissolution profile. Amorphous dispersions at 10, 20, 30, 40, and 50% ITZ drug loads in PVAL 4-88 were also compared by dissolution performance. Analytical tools of diffusion-ordered spectroscopy and Fourier transform infrared spectroscopy were employed to understand the interaction between drug and polymer. Finally, results from a 30-month stability test of a 30% drug loaded ITZ:PVAL 4-88 composition shows that stable amorphous dispersions can be achieved. Thus, this newly enabled polymer carrier can be considered a viable option for pharmaceutical formulation development for solubility enhancement.

Use of Polyvinyl Alcohol as a Solubility-Enhancing Polymer for Poorly Water Soluble Drug Delivery (Part 1).

Polyvinyl alcohol (PVAL) has not been investigated in a binary formulation as a concentration-enhancing polymer owing to its high melting point/high viscosity and poor organic solubility. Due to the unique attributes of the KinetiSol® dispersing (KSD) technology, PVAL has been enabled for this application and it is the aim of this paper to investigate various grades for improvement of the solubility and bioavailability of poorly water soluble active pharmaceutical ingredients. Solid amorphous dispersions were created with the model drug, itraconazole (ITZ), at a selected drug loading of 20%. Polymer grades were chosen with variation in molecular weight and degree of hydroxylation to determine the effects on performance. Differential scanning calorimetry, powder X-ray diffraction, polarized light microscopy, size exclusion chromatography, and dissolution testing were used to characterize the amorphous dispersions. An in vivo pharmacokinetic study in rats was also conducted to compare the selected formulation to current market formulations of ITZ. The 4-88 grade of PVAL was determined to be effective at enhancing solubility and bioavailability of itraconazole.

Chirally functionalized anion-exchange type silica monolith for enantiomer separation of 2-aryloxypropionic acid herbicides by non-aqueous capillary electrochromatography.

A silica based monolithic capillary column derivatized with O-9-(tert-butylcarbamoyl)quinidine was prepared for CEC enantiomer separation of chiral 2-aryloxypropionic acid herbicides including inter alia dichlorprop, mecoprop and fenoprop. The silica monolith had a relatively low surface coverage with chiral cationic selector moieties. Due to the low selector density retention factors were low as well, yet still enabling enantiomer separations of the target solutes. Both electrophoretically and chromatographically dominated migration and separation modes, respectively, could be established depending on the employed conditions. In the former mode, enantiomers migrated in front of the EOF marker, and faster separations and higher plate numbers could be achieved. In the latter mode, stronger adsorption translated into a typical chromatographic separation in which the enantiomers eluted after the EOF marker whereby separation factors were slightly enhanced compared to the aforementioned separation mode. Reasonable baseline separations of enantiomers were accomplished for all analytes after optimization of relevant mobile phase parameters in the anion-exchange CEC system including sample loadability, and the separations were comparable to such obtained on an optimized high density quinidine-carbamate modified organic polymer monolith column. Overall, it is concluded that monoliths with a high surface density of chiral ion-exchange moieties are favorable because of their enhanced sample loadabilities and improved chromatographic performance with regard to separation factors, plate numbers and peak symmetries. The resultant accompanying longer analysis times may rather be reduced by adjusting effective column length than by reducing selector coverage of the monolith.

Normal-phase automated mass-directed HPLC purification of a pyrrolobenzodiazepine library with vasopressin agonist activity.

A 23-member library of pyrrolobenzodiazepine derivatives with vasopressin agonist activity was purified on a 100-mg per injection scale using normal-phase (NP) automated mass-directed HPLC. Analytical NP APCI-LC/MS on an experimental monolith silica CN column utilizing gradients of methanol in ethoxynonafluorobutane (hexane-like solvent) was used to provide data on chromatographic purity and ionization of the solutes. The analytical data collected were used to program a preparative LC/MS instrument for "smart" fraction collection based on the protonated molecular ion of the component of interest. Preparative HPLC was carried out on a preparative cyano column with gradients of polar organic solvents in heptane containing n-propylamine as a basic additive. Flow rates twice as high as conventional ones were used for purification of library compounds. Small aliquots of the preparative flow were mixed with makeup solvent and introduced into an APCI source of a quadrupole mass spectrometer, which triggered collection of solutes. Two methods with fixed instrument parameters were used for purification. The system utilized commercially available instrumentation and software, which provided excellent recovery and purity of the library components and appeared to be useful as a fast and efficient alternative to traditional purification technologies based on reversed-phase LC/MS.

Profiling of endogenous peptides by multidimensional liquid chromatography: On-line automated sample cleanup for biomarker discovery in human urine.

A simple and flexible system, employing a column switching technique, has been designed to allow the analysis of peptides and proteins smaller than 15 kDa by molecular weight in filtered urine samples by performing a direct on-column injection utilising simultaneous sample clean-up and trace enrichment. The positively charged peptides and small proteins in the sample are attracted to the inner, negatively charged pore structure of the RAM-SCX column while the larger proteins and uncharged or negatively charged compounds are excluded. After preconditioning with the biological sample, large amounts of sample can be injected. Several important and adjustable parameters for the proper use of a RAM-SCX column are described and discussed. The main parameters being: i) the column is sensitive to sample overloading, which may result in drastic changes in the adsorption of peptides; ii) adsorption appears to be flow-rate and concentration dependent, as the sample molecules need time to penetrate into the internal pore structure in order to find complimentary orientated adsorption sites; iii) dilution and pH adjustment of sample during the loading process. The biocompatibility and proof-of-principle of this separation platform was demonstrated using human urine samples. Data are presented on repeatability as well as on the reproducibility of different synthesised batches of restricted access material (RAM).

Deconvolution of electrokinetic and chromatographic contributions to solute migration in stereoselective ion-exchange capillary electrochromatography on monolithic silica capillary columns.

A monolithic silica stationary phase functionalized with an enantioselective strong cation exchanger based on an aminosulfonic acid derivative was used for chiral separations of basic test solutes by nonaqueous CEC and capillary LC. The effects of the applied electric field as well as the ionic strength in the eluent on electrokinetic and chromatographic contributions to the overall separation performance in the electrically driven mode were investigated. Hence, under the utilized experimental conditions, i. e., at an electric field strength in the range of approximately 120-720 V/cm (applied voltages 4-24 kV) and an ionic strength of the counterion between 5 and 25 mM (at constant acid-to-base, i. e., co- to counterion ratio of 2:1), no deviations from the expected linearity of the EOF were observed. This led to the conclusion that an occurrence of the so-called electrokinetic effects of the second kind resulting from electric double layer overlap inside the mesopores of the monolithic stationary phase and concentration polarization phenomena were largely negligible. Additional support to this conclusion was inferred from the observed independence of CEC retention factors on the electric field strength across the investigated ionic strength range of the BGE. As a consequence, a simple framework allowing for calculation of the CEC mobilities from the individual separation contributions, viz. electroosmotic and electrophoretic mobilities as well as retention factors, could be applied to model CEC migration. There was a reasonable agreement between calculated and experimental CEC mobility data with deviations typically below 5%. The deconvolution of the individual contributions to CEC migration and separation is of particular value for the understanding of the separation processes in which electrophoretic migration of ionic sample constituents plays a significant role like in ion-exchange CEC and may aid the optimization procedure of the BGE and other experimental conditions such as the optimization of the surface chemistry of the stationary phase. In combination with the remarkable column performance evident from the low theoretical plate heights observed under CEC conditions for all test solutes (3.5-7.5 microm in the flow rate range of 0.4-1.2 mm/s, corresponding to (130,000-300,000 plates per meter), the presented framework provides an attractive tool as the basis for the assessment of chromatographic selectivities in a miniaturized CEC screening of new selectors and chiral stationary phases (CSPs), respectively, from experimental CEC data and known CE mobilities.

Poly (vinyl alcohol) microneedles: Fabrication, characterization, and application for transdermal drug delivery of doxorubicin.

Poly (vinyl alcohol) microneedles were fabricated, characterized, and applied to enhance in vitro transdermal delivery of doxorubicin. The microneedles were fabricated using the micromolding technique with the drug load in different locations within the needle array. The polymer solution was assessed for rheological properties, drug dissolution, and chemical structurestudies. Microneedles (unloaded) and drug-loaded microneedles were characterized by optical microscopy, fluorescent microscopy, scanning electron microscopy, and drug release kinetics. Successful microporation of dermatomed human cadaver skin was demonstrated by dye binding, pore uniformity, histology, confocal laser microscopy, and skin integrity studies. The microneedles-mediated transdermal delivery of doxorubicin was investigated using vertical Franz diffusion cells. The fabricated microneedles were sharp, strong, and uniform. In vitro permeation studies showed that the microneedle-treated skin (4351.55 ±â€¯560.87 ng/sq.cm) provided a significantly greater drug permeability than the untreated group (0.00 ±â€¯0.00 ng/sq.cm, n = 4, p < 0.01). The drug location within the needle array was found to affect the drug release profile as well as its permeation into and across human skin. Skin microporation achieved by poly (vinyl alcohol) microneedles was found to enhance transdermal delivery of doxorubicin in vitro.

Monolithic silica columns of various format in automated sample clean-up/multidimensional liquid chromatography/mass spectrometry for peptidomics.

The following particulate and monolithic silica columns were implemented in a fully automated and flexible multidimensional LC/MS system with integrated sample clean-up, to perform the analysis of endogeneous peptides from filtered urine and plasma samples: restricted access sulphonic acid strong cation-exchanger (RAM-SCX) for sample clean-up, RP 18 Chromolith guard columns as trap columns and 100 microm I.D. monolithic RP 18 fused silica capillary columns as last LC dimension. The results show sufficient overall system reproducibility and repeatability. Implementation of monolithic silica columns added an additional flexibility with respect to flow rate variation and adjustment due to the low column back pressures. Also, monolithic columns showed a lower clogging rate in long-term usage for biological samples as compared to particulate columns. The applied system set-up was tested to be useful for the routine peptide screening in search of disease biomarkers.

Pore structural characterization of monolithic silica columns by inverse size-exclusion chromatography.

In this work, a parallel pore model (PPM) and a pore network model (PNM) are developed to provide a state-of-art method for the calculation of several characteristic pore structural parameters from inverse size-exclusion chromatography (ISEC) experiments. The proposed PPM and PNM could be applicable to both monoliths and columns packed with porous particles. The PPM and PNM proposed in this work are able to predict the existence of the second inflection point in the experimental exclusion curve that has been observed for monolithic materials by accounting for volume partitioning of the polymer standards in the macropores of the column. The appearance and prominence of the second inflection point in the exclusion curve is determined to depend strongly on the void fraction of the macropores (flow-through pores), (b) the nominal diameter of the macropores, and (c) the radius of gyration of the largest polymer standard employed in the determination of the experimental ISEC exclusion curve. The conditions that dictate the appearance and prominence of the second inflection point in the exclusion curve are presented. The proposed models are applied to experimentally measured ISEC exclusion curves of six silica monoliths having different macropore and mesopore diameters. The PPM and PNM proposed in this work are able to determine the void fractions of the macropores and silica skeleton, the pore connectivity of the mesopores, as well as the pore number distribution (PND) and pore volume distribution (PVD) of the mesopores. The results indicate that the mesoporous structure of all materials studied is well connected as evidenced by the similarities between the PVDs calculated with the PPM and the PNM, and by the high pore connectivity values obtained from the PNM. Due to the fact that the proposed models can predict the existence of the second inflection point in the exclusion curves, the proposed models could be more applicable than other models for ISEC characterization of chromatographic columns with small diameter macropores (interstitial pores) and/or large macropore (interstitial pore) void fractions. It should be noted that the PNM can always be applied without the use of the PPM, since the PPM is an idealization that considers an infinitely connected porous medium and for materials having a low (<6) pore connectivity the PPM would force the PVD to a lower average diameter and larger distribution width as opposed to properly accounting for the network effects present in the real porous medium.

Impact of pore structural parameters on column performance and resolution of reversed-phase monolithic silica columns for peptides and proteins.

In this work, monolithic silica columns with the C4, C8, and C18 chemistry and having various macropore diameters and two different mesopore diameters are studied to access the differences in the column efficiency under isocratic elution conditions and the resolution of selected peptide pairs under reversed-phase gradient elution conditions for the separation of peptides and proteins. The columns with the pore structural characteristics that provided the most efficient separations are then employed to optimize the conditions of a gradient separation of a model mixture of peptides and proteins based on surface chemistry, gradient time, volumetric flow rate, and acetonitrile concentration. Both the mesopore and macropore diameters of the monolithic column are decisive for the column efficiency. As the diameter of the through-pores decreases, the column efficiency increases. The large set of mesopores studied with a nominal diameter of approximately 25 nm provided the most efficient column performance. The efficiency of the monolithic silica columns increase with decreasing n-alkyl chain length in the sequence of C18

Mesoporous silica-based dosage forms improve bioavailability of poorly soluble drugs in pigs: case example fenofibrate.

OBJECTIVES: Mesoporous silicas (SLC) have demonstrated considerable potential to improve bioavailability of poorly soluble drugs by facilitating rapid dissolution and generating supersaturation. The addition of certain polymers can further enhance the dissolution of these formulations by preventing drug precipitation. This study uses fenofibrate as a model drug to investigate the performance of an SLC-based formulation, delivered with hydroxypropyl methylcellulose acetate succinate (HPMCAS) as a precipitation inhibitor, in pigs. The ability of biorelevant dissolution testing to predict the in vivo performance was also assessed. KEY FINDINGS: Fenofibrate-loaded mesoporous silica (FF-SLC), together with HPMCAS, displayed significant improvements in biorelevant dissolution tests relative to a reference formulation consisting of a physical mixture of crystalline fenofibrate with HPMCAS. In vivo assessment in fasted pigs demonstrated bioavailabilities of 86.69 ± 35.37% with combination of FF-SLC and HPMCAS in capsule form and 75.47 ± 14.58% as a suspension, compared to 19.92 ± 9.89% with the reference formulation. A positive correlation was identified between bioavailability and dissolution efficiency. CONCLUSIONS: The substantial improvements in bioavailability of fenofibrate from the SLC-based formulations confirm the ability of this formulation strategy to overcome the dissolution and solubility limitations, further raising the prospects of a future commercially available SLC-based formulation.

Monolithic silica-based capillary column with strong chiral cation-exchange type surface modification for enantioselective non-aqueous capillary electrochromatography.

A silica-based monolithic stationary phase prepared by the sol-gel process in a 100 microm I.D. fused-silica (FS) capillary has been modified chemically with 3-mercaptopropyl trimethoxysilane followed by immobilization of a strong cation-exchange (SCX) type chiral selector, (S)-N-(4-allyloxy-3,5-dichlorobenzoyl)-2-amino-3,3-dimethylbutane phosphonic acid, by radical addition reaction onto the reactive sulfhydryl surface. After a fine-tuning of the mobile phase composition, the enantioselective capillary column was evaluated for the separation of various chiral basic drugs by enantioselective non-aqueous capillary electrochromatography (CEC), in comparison to capillary column analogs packed with 3.5 microm silica particles having attached the same selector. The performance of the monolithic silica column was further compared to corresponding polymethacrylate-based organic polymer monoliths. The study indicated that strong counter-ions such as 2-aminobutanol or N,N,N',N'-tetramethylethylenediamine are needed, although they reduce the electroosmotic flow velocity and separation factors in comparison to less efficient counter-ions, in order to allow the elution of the oppositely charged solutes in the ion-exchange retention mode within reasonable run time and as sharp zones. In contrast, weak counter-ions such as N,N-diisopropylethylamine (Huenig base) provided stronger electroosmotic flow and much better separation factors, but relatively poor peak efficiencies. Overall, with the chemically functionalized monolithic silica column the high quality separations of packed column analogs could be approximated, with regards to both separation factors and peak performances. On the other hand, the monolithic capillary column certainly outperformed the packed column in terms of system robustness under capillary electrochromatography conditions and showed excellent column longevity. The enantioselective strong cation-exchange-type monolithic silica column performed also well in comparison to the organic polymer monolith.

Fast, high-efficiency peptide separations on a 50-microm reversed-phase silica monolith in a nanoLC-MS set-up.

Proteomic studies have stimulated the development of novel stationary phases in miniaturised chromatographic columns that permit high linear flow velocities and exhibit high resolving power. In this work, a 50-microm reversed-phase silica-based monolith was chromatographically characterised for its use in proteomics applications using a nanoLC-MS set-up. It showed high efficiency for the separation of tryptic peptides under isocratic elution conditions (HETP(min)=5-10 microm at 2.4 mm/s). Flow rates up to 1.95 microL/min (18.4 mm/s) and gradient slopes up to an unusually fast 9% could be used. This resulted in rapid separations of peptide mixtures, with peak widths at half height of between 5 and 10 s. The 50-microm monolithic column was used to analyse depleted serum from a cervical cancer patient at a throughput of one sample per 30 min.

Mesoporous silica-based dosage forms improve release characteristics of poorly soluble drugs: case example fenofibrate.

OBJECTIVES: Mesoporous silica-based dosage forms offer the potential for improving the absorption of poorly soluble drugs after oral administration. In this investigation, fenofibrate was used as a model drug to study the ability of monomodal ('PSP A') and bimodal ('PSP B') porous silica to improve release by a 'spring' effect in in vitro biorelevant dissolution tests. Also investigated was the addition of various polymers to provide a 'parachute' effect, that is, to keep the drug in solution after its release. KEY FINDINGS: Loading fenofibrate onto PSP A or PSP B porous silica substantially improved the dissolution profile of fenofibrate under fasted state conditions compared with both pure drug and the marketed product, TriCor® 145 mg. Adding a polymer such as hydroxypropyl methylcellulose acetate succinate, polyvinylpyrrolidone or copovidon (HPMCAS, PVP or PVPVA) sustains the higher release of fenofibrate from the PSP A silica, resulting in a combination 'spring and parachute' effect - loading the drug onto the silica causes a 'spring' effect while the polymer enhances the spring effect (HPMCAS, PVP) and adds a sustaining 'parachute'. Interestingly, a silica to polymer ratio of 4:1 w/w appears to have an optimal effect for fenofibrate (HPMCAS, PVP). Dissolution results under conditions simulating the fasted state in the small intestine with the PSP A or the PSP B silica with HPMCAS added in a 4:1 w/w ratio show very substantial improvement over the marketed, nanosized product (TriCor® 145 mg). CONCLUSIONS: Further experiments to determine whether the highly positive effects on fenofibrate release observed with the silica prototypes investigated to date can be translated to further poorly soluble drugs and to what extent they translate into improved in-vivo performance are warranted.

Enabling thermal processing of ritonavir-polyvinyl alcohol amorphous solid dispersions by KinetiSol® Dispersing.

Polyvinyl alcohol has received little attention as a matrix polymer in amorphous solid dispersions (ASDs) due to its thermal and rheological limitations in extrusion processing and limited organic solubility in spray drying applications. Additionally, in extrusion processing, the high temperatures required to process often exclude thermally labile APIs. The purpose of this study was to evaluate the feasibility of processing polyvinyl alcohol amorphous solid dispersions utilizing the model compound ritonavir with KinetiSol® Dispersing (KSD) technology. The effects of KSD rotor speed and ejection temperature on the physicochemical properties of the processed material were evaluated. Powder X-ray diffraction and modulated differential scanning calorimetry were used to confirm amorphous conversion. Liquid chromatography-mass spectroscopy was used to characterize and identify degradation pathways of ritonavir during KSD processing and (13)C nuclear magnetic resonance spectroscopy was used to investigate polymer stability. An optimal range of processing conditions was found that resulted in amorphous product and minimal to no drug and polymer degradation. Drug release of the ASD produced from the optimal processing conditions was evaluated using a non-sink, pH-shift dissolution test. The ability to process amorphous solid dispersions with polyvinyl alcohol as a matrix polymer will enable further investigations of the polymer's performance in amorphous systems for poorly water-soluble compounds.

Combining restricted access material (RAM) and turbulent flow for the rapid on-line extraction of the cyclooxygenase-2 inhibitor rofecoxib in plasma samples.

Restricted access material (RAM) has been used in the packing of a solid-phase extraction (SPE) column for on-line extractions under turbulent flow conditions. The bio-compatible RAM material works by the principle of size exclusion in addition to conventional reversed-phase chromatography, thereby allowing the extraction and preconcentration of small analyte molecules from biological samples such as plasma. Using small column dimensions (0.76 mm x 50 mm) and a consequently high linear velocity, turbulent flow was achieved during online sample extractions. The improved mass-transfer rate characteristic of turbulent flow allows fast sample cleanup without decreased extraction efficiency. The novel use of the RAM column, connected upstream to a C18 monolithic column, allowed the direct injection, extraction, separation, and MS/MS detection of plasma samples spiked with rofecoxib in a span of 5 min. Calibration curves obtained using this RAM turbulent flow coupled column method showed good linearity (R2 > 0.99) and reproducibility (%RSD < or = 7%). The lower limit of quantitation of rofecoxib in plasma samples was found to be 40 ng/ml. The extraction method showed good recovery of rofecoxib from a plasma matrix with minimal signal loss and robustness after more than 200 plasma injections.

Stir bar sorptive extraction based on restricted access material for the direct extraction of caffeine and metabolites in biological fluids.

A biocompatible stir bar sorptive extraction (SBSE) device was prepared using an alkyl-diol-silica (ADS) restricted access material (RAM) as the SBSE coating. The RAM-SBSE bar was able to simultaneously fractionate the protein component from a biological sample, while directly extracting caffeine and its metabolites, overcoming the present disadvantages of direct sampling in biological matrices by SBSE, such as fouling of the extraction coating by proteins. Desorption of the analytes was performed by stirring the bar in a water/ACN mixture (3/1, v/v) and subsequently reconcentrating the sample solution in water to enable HPLC-UV analysis to be performed. The limit of detection, based on a signal to noise ratio of 3, for caffeine was 25 ng/mL in plasma. The method was confirmed to be linear over the range of 0.5-100 microg/mL of caffeine with an average linear coefficient (R2) value of 0.9981. The injection repeatability and intra-assay precision of the method were evaluated over ten injections, resulting in a %RSD of approximately 8%. The RAM-SBSE device was robust (>50 extraction in plasma without significant signal loss) and simple to use, providing many direct extractions and subsequent determination of caffeine and its metabolites in biological fluids. In contrast to existing sample preparation methods for the analysis of caffeine and selected metabolites in biological fluids, this feasibility study using a biocompatible SBSE approach was advantageous in terms of simplifying the sample preparation procedures.

Comprehensive pore structure characterization of silica monoliths with controlled mesopore size and macropore size by nitrogen sorption, mercury porosimetry, transmission electron microscopy and inverse size exclusion chromatography.

The porosity of monolithic silica columns is measured by using different analytical methods. Two sets of monoliths were prepared with a given mesopore diameter of 10 and 25 nm, respectively and with gradated macropore diameters between 1.8 and 7.5 microm. After preparing the two sets of monolithic silica columns with different macro- and mesopores the internal, external and total porosity of these columns are determined by inverse size-exclusion chromatography (ISEC) using polystyrene samples of narrow molecular size distribution and known average molecular weight. The ISEC data from the 4.6 mm analytical monolithic silica columns are used to determine the structural properties of monolithic silica capillaries (100 microm I.D.) prepared as a third set of samples. The ISEC results illustrate a multimodal mesopore structure (mesopores are pores with stagnant zones) of the monoliths. It is found by ISEC that the ratio of the different types of pores is dependent on the change in diameter of the macropores (serve as flow-through pores). The porosity data achieved from the mercury penetration measurement and nitrogen adsorption as well of scanning electron microscopy (SEM) and transmission electron microscopy (TEM) pictures are correlated with the results we calculated from the ISEC measurements. The ISEC results, namely the multimodal pore structure of the monoliths, reported in several publications, are not confirmed analyzing the pore structures of the different silica monoliths using all other analytical methods.

Approaches for coupling solid-phase microextraction to nanospray.

Biocompatible solid-phase microextraction (SPME) devices were prepared using two restricted access materials (RAM) as the SPME coating. The restricted access materials were immobilized on steel and platinum wires. The selective coating eliminated most of the matrix interference, which allowed the coupling to mass spectrometry without further purification. The SPME devices were interfaced to mass spectrometry by electronanospray. Several experimental set-ups are described and discussed herein. For the in situ extraction of peptides from the tryptic digests, trypsin was immobilized both on steel wires and on the inside wall of a vial. The devices were incubated together with the RAM-SPME devices and a protein (casein) solution. After the protein digestion, the resulting peptides were analyzed by SPME/nanospray. The vial approach provided the best results; up to eight peptides could be identified which corresponds to a sequence coverage of 58%. The limit of detection of SPME/nanospray for the extraction of peptides from an aqueous solution was about 50 fmol/mL. The results demonstrate that the direct coupling of SPME to nanospray can reduce analysis time and is an attractive alternative to conventional approaches like Zip-Tip purification.

Monolithic silica columns with chemically bonded tert-butylcarbamoylquinine chiral anion-exchanger selector as a stationary phase for enantiomer separations.

An enantioselective silica rod type chiral stationary phase (CSP) is presented as a novel combination of the well-known enantiomer separation properties of immobilized tert-butyl-carbamoylquinine chiral anion-exchanger selector with the unique properties of monolithic silica material. The chromatographic behavior of the tert-butyl-carbamoylquinine silica rod was studied and compared with a similar prepared particulate material. Good selectivities were achieved for a spectrum of chiral test components like N-derivatized amino acids (DNB- Ac-, DNZ-, Bz-, Z-amino acids) and for Suprofen. The influence of mobile phase parameters, as well as the effect of serially coupling up to six 10 cm monolithic silica columns was studied and put in context to conventional columns of particulate 5 microm type CSP. Using that 60 cm long monolithic column it was possible to improve the enantiomer separation of Suprofen and achieve a baseline separation in less than 10 min of total separation time.