Textural characterization of native and n-alky-bonded silica monoliths by mercury intrusion/extrusion, inverse size exclusion chromatography and nitrogen adsorption.

Native and n-alkyl-bonded (n-octadecyl) monolithic silica rods with mesopores in the range between 10 and 25 nm and macropores in the range between 1.8 and 6.0 microm were examined by mercury intrusion/extrusion, inverse size exclusion chromatography (ISEC) and nitrogen sorption. Our results reveal very good agreement for the mesopore size distribution obtained from nitrogen adsorption (in combination with an advanced NLDFT analysis) and ISEC. Our studies highlight the importance of mercury porosimetry for the assessment of the macropore size distribution and show that mercury porosimetry is the only method which allows obtaining a combined and comprehensive structural characterization of macroporous/mesoporous silica monoliths. Our data clearly confirm that mercury porosimetry hysteresis and entrapment have different origin, and indicate the intrinsic nature of mercury porosimetry hysteresis in these silica monoliths. Within this context some silica monoliths show the remarkable result of no entrapment of mercury after extrusion from the mesopore system (i.e. for the first intrusion/extrusion cycle). The results of a systematic study of the mercury intrusion/extrusion behavior into native silica monoliths and monoliths with bonded n-alkyl groups reveals that the macro (through) pore structure, which controls the mass transfer to and from the mesopores, here mainly controls the entrapment behavior. Our data suggest that mercury intrusion/extrusion porosimetry does not only allow to obtain a comprehensive pore structure analysis, but can also serve as a tool to estimate the mass transport properties of silica monoliths to be employed in liquid-phase separation processes.

Sulphonic acid strong cation-exchange restricted access columns in sample cleanup for profiling of endogenous peptides in multidimensional liquid chromatography. Structure and function of strong cation-exchange restricted access materials.

In this work, the pore structural parameters and size exclusion properties of LiChrospher strong cation-exchange and reverse phase restricted access materials (RAM) are analysed. The molecular weight size exclusion limit for polystyrenes was found to be about 17.7 kDa, while for standard proteins, the molecular weight size exclusion limit was higher, at approximately 25 kDa. The average pore diameter on a volume basis calculated from the pore network model changes from 8.5 nm (native LiChrospher) to 8.6 nm (diol derivative) to 8.2 nm (sulphonic acid derivative) to 6.9 nm (n-octadecyl derivative). Additional characterisations were performed on restricted access materials with nitrogen sorption at 77 K, water adsorption at 25 degrees C, intrusion-extrusion of water (in order to evaluate the hydrophobic properties of the pores of the hydrophobic RAM), and zeta potential measurements by microelectrophoresis. For peptide analysis out of the biofluids, the strong cation-exchange functionality seems to be particularly suitable mainly because of the high loadability of the strong cation-exchange restricted access material (SCX-RAM) and the fact that one can work under non-denaturing conditions to perform effective chromatographic separations. For bacitracin, the dynamic capacity of the SCX-RAM columns does not reach its maximum value in the analysed range. For lysozyme, the dynamic capacity reaches a value of 0.08 mg/ml of column volume before column is overloaded. Additionally, the proper column operating conditions that lead to the total effective working time of the RAM column to be equal to approximately 500 injections (depending on the type of sample), is comprehensively described. The SCX-RAM column was used in the same system analysing urine samples for the period of 1 month (approximately 150 injections) with run-to-run reproducibility below 5% RSD and below 10% RSD for the relative fractions.

Optimization of a trypsin-bioreactor coupled with high-performance liquid chromatography-electrospray ionization tandem mass spectrometry for quality control of biotechnological drugs.

The optimization of a silica-based trypsin bioreactor and its use in the quality control of biotechnological drugs like peptides and proteins is described. Five bioreactors based on monolithic material have been prepared, with different amount of bound trypsin. The performances of these bioreactors were compared to the proteolytic activity of a bioreactor based on silica material. The trypsin-based chromatographic columns were coupled on-line with an LC/ESI/MS/MS system for digestion and identification of proteins. First, human serum albumin has been used as test protein to compare the ability of the bioreactors to hydrolyse high-molecular-weight proteins. The best chromatographic material (epoxy monolithic silica) and the optimum amount of enzyme bound (7.13 mg) have been identified to obtain the highest protein recovery and an analytical reproducibility of the whole digestion, separation and identification process. The optimized enzyme-reactor has been used for the on-line digestion of some biotechnological drugs such as somatotropin. Somatotropin for parentheral use has been analyzed, without sample pre-treatment, with both an on-line procedure and the traditional off-line procedure described in the European Pharmacopoeia. It was found that the cleavage efficiency (aminoacidic recovery, %AA) achieved within minutes by the developed protocol is at least comparable or even better than the conventional 4h consuming method.

Dynamic electric field assisted multi-dimensional liquid chromatography of biological samples.

Complex biological samples require very high resolution separation strategies. The platform introduced here capitalises on the hyphenation of liquid chromatographic (LC) and electric potential gradient electrochromatographic multi-dimensional separation genres. First-dimension selectivity is provided by simultaneous size exclusion (SEC) and strong cation exchange (SCX) chromatography modes, while the second dimension comprises reversed phase (RP) characteristics in a dynamic (time-variant) electric field. The time-variant potential gradient with reversal of polarity is applied across the second dimension monolithic capillary throughout the duration of the solvent strength gradient elution. Hence, the platform offers comprehensive on-line sample clean-up (matrix depletion, analyte enrichment), fractionation (first dimention LC), and separation (second dimension LC) with the prospect of altering selectivity via polarity reversal dynamic electric field tuning.

Integrated on-line sample clean-up using cation exchange restricted access sorbent for the LC determination of atropine in human plasma coupled to UV detection.

A new, simple and fully automated liquid chromatographic (LC) method with UV detection has been developed for the direct determination of atropine in plasma. Sample clean-up was based on the use of cation exchange restricted access material (RAM) in a pre-column, coupled to LC by means of a column switching system. After direct injection of a 200 microl-volume of plasma sample, the biological matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate adjusted to pH 3.0 and methanol (97:3; v/v). By rotation of the switching valve, atropine was then eluted in the back-flush mode for 2 min and transferred to the analytical column packed with octadecyl silica by the LC mobile phase constituted of a mixture of acetonitrile and potassium phosphate buffer (pH 3.0; 50 mM) containing 2 mM sodium heptanesulfonate (16:84; v/v). The UV detection was performed at 220 nm. The method was validated according to a new approach based on accuracy profile over a concentration range from 25 ng/ml, corresponding to the limit of quantitation, to 1000 ng/ml. The method was then applied for the determination of atropine in plasma after intravenous administration to hospitalised patients.

Development and validation of a fully automated LC method for the determination of cloxacillin in human plasma using anion exchange restricted access material for sample clean-up.

In the framework of a preliminary investigation on the plasma profile of cloxacillin after oral administration, a simple and rapid LC method was developed for the direct determination of this compound in human plasma. The on-line sample clean-up was carried out using a weak anion exchanger (diethylaminoethyl groups) as restricted access material (RAM). The effects of the washing liquid pH, the ionic strength and the addition of organic modifier to the washing liquid were studied in order to obtain an efficient sample clean-up and a high recovery of cloxacillin. The separation was achieved on octadecylsilica stationary phase using a mobile phase consisting in a mixture of phosphate buffer (pH 4.0; 25 mM) and acetonitrile (72:28, v/v). The UV detection was performed at 215 nm. The most appropriate regression model of the response function as well as the limit of quantitation (LOQ) were first selected during the pre-validation step. These criteria were then assessed during the formal validation step. The LOQ was 50 ng/ml. The method was also validated with respect to analyte recovery, precision, trueness, accuracy and linearity. Finally, it was successfully applied for the analysis of the first plasma samples obtained from patients having taken an oral dose of 500 mg cloxacillin.

Development of a bioreactor based on trypsin immobilized on monolithic support for the on-line digestion and identification of proteins.

The preparation and characterization of a new trypsin-based bioreactor is here described for on-line protein digestion and peptide analysis. Trypsin was immobilized on an epoxy-modified silica monolithic support with a single reaction step and the amount of immobilized enzyme was found to be 66.07 mg (+/-11.75 S.D.)/column (n = 6). The bioreactor was coupled through a switching valve to an analytical column for the on-line digestion, peptide separation and identification of test proteins by ESI-MS-MS. The influence of various parameters (flow rate, temperature, buffer pH and molarity, etc.) on enzymatic activity was investigated by an experimental design and the mostly significant factor was found to be the flow rate. The efficacy of the reported on-line bioreactor for tryptic mapping is reported for somatostatin and myoglobin, selected as model compounds. Tryptic peptide maps obtained by on-line digestion of myoglobin were compared to those obtained by traditional off-line digestion. Sequence coverage obtained with the on-line protocol (21 peptides, 75.16% coverage of myoglobin sequence) was found to be comparable to the one obtained with the off-line protocol (18 peptides, 76.47% coverage). Sensitivity for myoglobin digestion and identification was 0.1 mg/ml. The reproducibily of the peptide maps in terms of retention time was from 1.53 to 4.31%, R.S.D.

Development of a chromatographic bioreactor based on immobilized beta-glucuronidase on monolithic support for the determination of dextromethorphan and dextrorphan in human urine.

We here reported the development and application of an immobilized enzyme reactor (IMER) based on beta-glucuronidase to the on-line determination of urinary molar ratios of dextromethorphan (DOMe)/dextrorphan (DOH) for the assessment of the metabolic activity of CYP2D6, a genetically variable isoform of cytochrome P-450 (CYP). beta-Glucuronidase was immobilized on an HPLC monolithic aminopropyl silica support. Catalytic activity and stability of the chromatographic reactor were evaluated using 8-hydroxyquinoline glucuronide (8-HQG) as substrate. The IMER was coupled through a switching valve to a reversed-phase column (C8) for the simultaneous determination of dextromethorphan and its main metabolite dextrorphan. On purpose a selective reversed-phase ion pair HPLC method coupled with fluorescence detection was developed. Urine samples were first centrifuged to remove insoluble materials and then aliquots of the supernatants were injected into the coupled-column analyser. Linearity, precision and accuracy of the method were established. The method reliability was verified by comparing our data with previous data of a phenotyping study carried out by the Poison Control Centre of Pavia-Clinical Toxicology Division.

Evaluation of a monolithic epoxy silica support for penicillin G acylase immobilization.

In this work, a new type of penicillin G acylase (PGA)-based monolithic silica support was developed and evaluated for the chiral separation in HPLC. The preparation procedure consisted of two steps: preparation of an epoxy derivatized monolithic silica column and chemical modification of the epoxide groups with the enzyme chiral selector. The epoxy Silica-Rod column for the immobilization of PGA was prepared with the in situ modification process by using epoxy-silanes and the identification of the species bound to the surface was achieved by solid-state nuclear magnetic resonance. The enzyme was covalently immobilized to the surface of the derivatized monolithic column. The enantioselectivity and the performance of the developed column are discussed and compared to the corresponding experimental data obtained with a PGA-based microparticulate (5 microm) silica column.

Evaluation of a novel anion-exchange restricted-access sorbent for on-line sample clean-up prior to the determination of acidic compounds in plasma by liquid chromatography.

A new kind of silica-based restricted-access material (RAM) with anionic properties has been tested in pre-columns for on-line solid-phase extraction of acidic compounds from directly injected plasma samples prior to their determination by reversed-phase liquid chromatography (LC), using the column-switching technique. The outer surface of the porous RAM particles contains hydrophilic diol groups while diethylaminoethyl (DEAE) groups are bound to the internal surface which gives the sorbent the properties of a weak anion exchanger towards low-molecular-mass compounds. Due to an appropriate pore diameter (about 6 nm), macromolecules, such as proteins, are physically excluded from the pores and flushed directly out during the sample clean-up process, while small compounds have access to the inner surface and can be retained mainly by electrostatic interactions. The retention capability of this novel packing material has been tested for some hydrophilic acidic compounds such as aspartic acid, glutamic acid, ascorbic acid and acetylcysteine as well as for some more hydrophobic drugs such as naproxen, ibuprofen and diclofenac, used as model compounds. The influence of the composition of the washing liquid on the retention of the analytes in the pre-column has been investigated. The efficiency of the sorbent to clean-up complex matrices was also tested using human plasma and urine samples. A generic washing liquid composition was then selected in order to obtain efficient and selective sample clean-up as well as a high recovery of the acidic analytes.

Monolithic silica columns with chemically bonded beta-cyclodextrin as a stationary phase for enantiomer separations of chiral pharmaceuticals.

An enantioselective silica rod type chiral stationary phase (CSP) is presented; a novel combination of the well known enantiomer separation properties of beta-cyclodextrin and the unique properties concerning the flow behavior of silica monoliths. Two different synthesis routes are described, and it was found that the in situ modification of a plain silica rod column turned out to be the best. The chromatographic behaviour of the beta-cyclodextrin silica rod was studied and compared with a very similar commercially available beta-cyclodextrin bonded particulate material (ChiraDex). Even if the amount of beta-cyclodextrin bound to the silica rod was only about half of the amount of beta-cyclodextrin bound to ChiraDex) particles, good resolutions were achieved for a set of chiral test components like Chromakalin, Prominal, Oxazepam, Methadone and some other drugs. By taking advantage of the unique features of the silica rods relating to their flat H/u (Van Deemter) curves, fast enantiomer separations could be demonstrated.

Fully automated LC method for the determination of sotalol in human plasma using restricted access material with cation exchange properties for sample clean-up.

A simple and rapid fully automated bio-analytical method for the liquid chromatographic (LC) determination of sotalol in human plasma has been described. The method is based on the use of a new kind of porous silica restricted access material (RAM) with cation exchange properties for sample clean-up. 100 microl of plasma samples were directly injected into the precolumn coupled on-line to a reversed-phase column (RP-Select B) by means of column switching system. The plasma matrix was washed out for 10 min using a washing liquid composed of 2 mM lithium perchlorate and methanol (97:3; v/v). By rotation of the switching valve, the analytes were then eluted in back-flush mode for 2 min and transferred to the analytical column by the LC mobile phase constituted of a mixture of methanol and 50 mM potassium phosphate buffer (pH 7.0) containing 1 mM 1-octanesulphonic acid sodium salt (20:80; v/v). The flow-rate was 1.0 ml/min and sotalol was detected using fluorescence detection at 235 and 300 nm as excitation and emission wavelengths, respectively. The method was then validated using a new approach based on accuracy profile over a concentration range from 5 to 500 ng/ml. The limit of quantitation (LOQ) was 5 ng/ml and the total analysis time was 19 min.

Use of a novel cation-exchange restricted-access material for automated sample clean-up prior to the determination of basic drugs in plasma by liquid chromatography.

A new kind of silica-based restricted-access material (RAM) has been tested in pre-columns for the on-line solid-phase extraction (SPE) of basic drugs from directly injected plasma samples before their quantitative analysis by reversed-phase liquid chromatography (LC), using the column switching technique. The outer surface of the porous RAM particlescontains hydrophilic diol groups while sulphonic acid groups are bound to the internal surface, which gives the sorbent the properties of a strong cation exchanger towards low molecular mass compounds. Macromolecules such as proteins have no access to the internal surface of the pre-column due to their exclusion from the pores and are then flushed directly out. The retention capability of this novel packing material has been tested for some hydrophilic basic drugs, such as atropine, fenoterol, ipratropium, procaine, sotalol and terbutaline, used as model compounds. The influence of the composition of the washing liquid on the retention of the analytes in the pre-column has been investigated. The elution profiles of the different compounds and the plasma matrix as well as the time needed for the transfer of the analytes from the pre-column to the analytical column were determined in order to deduce the most suitable conditions for the clean-up step and develop on-line methods for the LC determination of these compounds in plasma. The cationic exchange sorbent was also compared to another RAM, namely RP-18 ADS (alkyl diol silica) sorbent with respect to retention capability towards basic analytes.

Alkyl diol silica: restricted access pre-column packings for fast liquid chromatography-integrated sample preparation of biological fluids.

A silica-based pre-column packed with a 'restricted access material' was used for fully automated solid-phase extraction of drugs and metabolites from untreated biological fluids via a simple valve-switching method. The bifunctional sorbent employed, alkyl diol silica, is characterised by a different chemical modification of the outer surface (diol groups) and the pore surface of the particles (alkyl chains: C4, C8 or C18). This allows for a fast and efficient clean-up of complex biological samples, such as blood, serum, urine or milk, with exclusion of the macromolecular matrix (proteins, nucleic acids) in less than 1.5 min.

Analysis of neuropeptide Y and its metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry and integrated sample clean-up with a novel restricted-access sulphonic acid cation exchanger.

A novel restricted access cation exchanger with sulphonic acid groups at the internal surface was proven to be highly suitable in the sample clean up of peptides on-line coupled to HPLC-electrospray ionization (ESI)-MS. Neuropeptide Y (NPY) and several of its fragments in plasma were subjected to the sample clean-up procedure. The peptides were eluted by a step gradient from the restricted access column, applying 10 mM phosphate buffer pH 3.5 from 5 to 20% (v/v) of acetonitrile with 1 M NaCl and transferred to a Micra ODS II column (33x4.6 mm). The separation of the peptides and their fragments was performed by a linear gradient from 20 to 60% (v/v) acetonitrile in water with 0.1% formic acid and 0.01% trifluoroacetic acid in 4 min at a flow-rate of 0.75 ml/min. An integrated and completely automated system composed of sample clean up-HPLC-ESI-MS was used to analyze real life samples. The sample volumes ranged between 20 and 100 microl. Peaks due to the fragments NPY 1-36, 3-36 and 13-36 in porcine plasma were identified by ESI-MS. The limit of detection was in the 5 nmol/ml range. The total analysis required 21 min and allowed the direct injection of plasma.

Shape selectivity of C30 phases for RP-HPLC separation of tocopherol isomers and correlation with MAS NMR data from suspended stationary phases.

Vitamin E (tocopherol) acts in various organisms as the main free radical scavenger. This capacity, which is enhanced by the synergetic effect of vitamin C and carotenes, points to a possible application as anti-tumor agent in chemotherapy. There are several isomeric forms, namely alpha-, beta-, gamma-, and delta-tocopherol, having different antioxidative abilities, with alpha-tocopherol being the most biologically active. Using methanol as eluent and a C30 stationary phase, we achieved complete separation of alpha-, beta-, gamma-, and delta-tocopherol and alpha-tocopherol acetate by RP-HPLC within 14 min. Detection was performed by UV and 1H NMR spectroscopy. The advantage of NMR is the possibility of structural identification of chromatographic peaks. Also, coeluting peaks are easily recognized. The enhanced shape recognition of the C30 phase has been attributed to the high order of the alkyl chains of the stationary phase. This has, so far, been proven by solid-state NMR spectroscopy. We now introduce the technique of 13C MAS NMR spectroscopy of suspended stationary phases. The resulting NMR spectra reveal that, in the presence of weak eluents, like methanol, the overall high order of the C30 chains is slightly altered, whereas in stronger eluents, like MTBE, the alkyl chains possess a higher mobility.