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Developments in the fields of protein chemistry, proteomics and biotechnology have increased the demand for suitable analytical techniques for the analysis of intact proteins. In 1989, capillary electrophoresis (CE) was combined with mass spectrometry (MS) for the first time and its potential usefulness for the analysis of intact (i.e. non-digested) proteins was shown. This article provides an overview of the applications of CE-MS within the field of intact protein analysis. The principles of the applied CE modes and ionization techniques used for CE-MS of intact proteins are shortly described. It is shown that separations are predominantly carried out by capillary zone electrophoresis and capillary isoelectric focusing, whereas electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) are the most popular ionization techniques used for interfacing. The combination of CE with inductively coupled plasma (ICP) MS for the analysis of metalloproteins is also discussed. The various CE-MS combinations are systematically outlined and tables provide extensive overviews of the applications of each technique for intact protein analysis. Selected examples are given to illustrate the usefulness of the CE-MS techniques. Examples include protein isoform assignment, single cell analysis, metalloprotein characterization, proteomics and biomarker screening. Finally, chip-based electrophoresis combined with MS is shortly treated and some of its applications are described. It is concluded that CE-MS represents a powerful tool for the analysis of intact proteins yielding unique separations and information.

In a previous study on capillary electrophoresis-atmospheric pressure photoionization mass spectrometry (CE-APPI-MS), it was observed that the formation of gas-phase ions does not always proceed through photon-induced mechanisms (Hommerson, P.; Khan, A. M.; De Jong, G. J.; Somsen, G. W. Electrophoresis 2007, 28, 1444-1453). That is, analyte signals were observed when the VUV excitation source was switched off. The aim of the present study was to further explore this photon-independent ionization (PII) process. Parameters such as MS capillary voltage, compound nature, background electrolyte (BGE) composition, and presence of dopants were studied using a CE-APPI-MS setup. Infusion experiments showed a relatively low MS capillary voltage of approximately 600 V to be the main prerequisite for PII. Quaternary ammonium compounds showed strong responses in PII-MS but could not be observed in dopant-assisted APPI. Basic amines could be ionized by both photoionization (PI) and PII, whereas neutral compounds (steroids) could only be observed using PI. Nonvolatile BGEs appeared to cause substantial ionization suppression in PII, while PI signals remained largely unaffected. Selection of the proper interface and MS settings allowed PI and PII to proceed simultaneously, which broadened the range of compounds that could be analyzed in a single CE-APPI-MS run. Based on the observed characteristics, it is concluded that PII most probably occurs by a liquid-phase ionization mechanism, which appears to arise in the APPI source when specific conditions are selected.

The characterization of recombinant human growth hormone (rhGH; somatropin) by capillary electrophoresis (CE) with UV-absorbance and mass spectrometric (MS) detection using capillaries noncovalently coated with polybrene (PB) and poly(vinyl sulfonic acid) (PVS) is demonstrated. Compared with bare fused-silica capillaries, PB-PVS coated capillaries yielded more favorable migration-time reproducibilities and higher separation efficiencies. Optimal separation conditions for the bilayer-coated capillaries comprised a background electrolyte (BGE) of 400 mM Tris phosphate (pH 8.5) yielding migration-time R.S.D.s of less than 1.0% and plate numbers above 300,000 for intact rhGH. The protein was also analyzed using the CE method described in the European Pharmacopoeia (Ph. Eur.) monograph. The pharmacopoeial method gave much longer analysis times (22 min versus 8 min), lower resolution and plate numbers, and consecutive shifts in migration time for rhGH, indicating possible interactions between the protein and the inner capillary wall. Due to stable migration times obtained with the coated capillaries, reliable profiling and quantification of rhGH and its byproducts in time was possible. Analysis of thermally degraded rhGH revealed the formation of two main degradation products. CE-mass spectrometry (MS) of this sample, using a PB-PVS coated capillary and a BGE of 75 mM ammonium formate (pH 8.5), suggests that these products are desamido forms of rhGH. Analyses of expired rhGH preparations with CE-UV and CE-MS indicated the presence of both deamidation and oxidation products.

Optimisation of peak capacity is an important strategy in gradient liquid chromatography (LC). This can be achieved by using either long columns or columns packed with small particles. Monolithic columns allow the use of long columns at relatively low back-pressure. The gain in peak capacity using long columns was evaluated by the separation of a tryptic bovine serum albumin digest with an LC-UV-mass spectrometry (MS) system and monolithic columns of different length (150 and 750 mm). Peak capacities were determined from UV chromatograms and MS/MS data were used for Mascot database searching. Analyses with a similar gradient slope for the two columns produced ratios of the peak capacities that were close to the expected value of the square root of the column length ratio. Peak capacities of the short column were 12.6 and 25.0 with 3 and 15 min gradients, respectively, and 29.7 and 41.0 for the long column with 15 and 75 min gradients, respectively. Protein identification scores were also higher for the long column, 641 and 750 for the 3- and 15-min gradients with the short column and 1,376 and 993 for the 15- and 75-min gradients with the long column. Thus, the use of long monolithic columns provides improved peptide separation and increased reliability of protein identification.

The performance of atmospheric pressure photoionization (APPI) and ESI for CZE was compared using a set of seven drugs (basic amines, quaternary amines and steroids) and four different BGEs. The influence of volatile and nonvolatile BGEs of acidic and neutral pH on the MS responses of test compounds was evaluated by infusion of test solutions into the respective ion sources, and by actual CZE-MS experiments. The infusion experiments indicate that sodium phosphate buffers cause ionization suppression in ESI-MS, although for the amines the suppression was modest (25-60% signal reduction). By contrast, APPI-MS responses were not affected by nonvolatile BGEs. With phosphate buffers, ESI-MS responses for the basic amines were still a factor 3-13 higher than the APPI-MS signals, whereas the steroids yielded similar responses in ESI-MS and APPI-MS. The quaternary amines could readily be detected in ESI-MS, but detection in APPI-MS required specific interface conditions. Using typical CZE-APPI-MS settings, quaternary amines remained undetected. Remarkably, the S/Ns observed in CZE-ESI-MS for the test compounds, were generally similar when using volatile and nonvolatile BGEs. For basic compounds, the S/Ns obtained in CZE-ESI-MS were a factor 2-5 higher than in CZE-APPI-MS, whereas steroids yielded equal S/Ns in both methods. Overall, it is concluded that when using relatively low BGE concentrations, the sensitivity of ESI-MS detection in CZE is more favorable than APPI-MS detection, even when nonvolatile BGEs are employed.

An on-line SPE-CE-MS system has been developed for the analysis of peptides. Analytes are preconcentrated using a C(18) microcolumn (5 x 0.5 mm id), and then introduced into the CE system via a valve interface. The CE system with a Polybrene-poly(vinylsulfonate) bilayer coated capillary is combined with an ion-trap mass spectrometer via ESI using a coaxial sheath-liquid sprayer. The on-line coupling of the SPE and CE step by the valve interface is advantageous because it allows an independent functioning of the system parts. Optimization of the SPE-CE system was performed using UV detection. Subsequently, the SPE-CE system has been coupled to the ion-trap mass spectrometer. Test solutions with enkephalin peptides (50 ng/mL) were used for evaluation of system performance. Repeatability of effective mobility and peak area ratio of the two enkephalins were within 1.2% and 9% RSD, respectively. The analysis of 1:1 v/v diluted cerebrospinal fluid samples spiked with enkephalin peptides showed detection limits (S/N = 3) in the range of 1.5-3 ng/mL (around 5 nM), which were similar to those obtained for enkephalin test solutions. Moreover, the potential of the on-line SPE-CE-MS system was demonstrated by the analysis of a cytochrome C digest. Some hydrophilic peptides did not show sufficient retention on the SPE column, and were lost during preconcentration. Nonetheless, positive identification of the protein was achieved, indicating the feasibility of the system for proteomics.

The feasibility of using noncovalently bilayer-coated capillaries for capillary electrophoresis-mass spectrometry (CE-MS) of acidic proteins was investigated using background electrolytes (BGEs) of medium pH. The capillary was coated by successively rinsing the capillary with solutions of the oppositely charged polymers polybrene (PB) and poly(vinyl sulfonic acid) (PVS). Volatile BGEs containing ammonium formate and/or N-methyl morpholine were tested at pH 7.5 and 8.5. Overall, these BGEs provided relatively fast protein separations (analysis times of ca. 12 min) and showed high efficiencies (70,000-300,000 plates) when the ionic strength was sufficiently high. Migration-time reproducibilities were very favorable with RSDs of less than 1.0%. Infusion experiments showed satisfactory MS responses for studied proteins dissolved in ammonium formate (pH 8.5), however, high concentrations of N-methyl morpholine appeared to seriously suppress the MS protein signals. Evaluation of the CE-MS system was performed by analyzing a mixture of intact proteins yielding efficient separations and good-quality mass spectra. CE-MS analysis of a reconstituted formulation of the biopharmaceutical recombinant human growth hormone (rhGH) which was stored for a prolonged time, revealed one degradation product which was provisionally identified as desamido rhGH. Based on the MS responses the amount of degradation was estimated to be ca. 25%.

Organic acids in cerebrospinal fluid (CSF) are potential diagnostic markers for neurological diseases and metabolic disorders. A capillary electrophoretic (CE) method for the direct analysis, i.e., without any sample preparation, of six organic acids in CSF was developed. A capillary coating consisting of a triple layer of charged polymers (polybrene-dextran sulfate-polybrene) was used in combination with a negative separation voltage, providing fast and efficient analysis of acidic compounds. Separation conditions, such as background electrolyte (BGE) concentration and pH were optimized, and the influence of albumin and sodium chloride was systematically studied using a set of test compounds. With injection volumes of ca. 44 nL, plate numbers of up to ca. 150,000 were obtained with a BGE of 200 mM sodium phosphate (pH 6.0). It appeared that high sodium chloride concentrations in the sample hardly affected the peak width and shape of the organic acids, most probably due to transient isotachophoresis effects occurring in the sample zone. Adverse effects of CSF proteins, which frequently compromise the CE performance, could be effectively minimized by the triple layer coating in combination with rinses of 0.1 M hydrochloric acid. Overall, the developed CE system allowed direct injections of CSF samples, yielding good separation efficiencies and stable migration times (RSDs<2%) for organic acids. Validation of the method with artificial and real CSF samples showed good linear responses (r>0.99), and LODs for the organic acids were in the range of 2-8 microg/mL when applying UV detection. RSDs for migration times and peak areas were <2% and <7%, respectively. The applicability of the CE system is shown for the determination of organic acids in CSF samples.

Previously, we have presented a system hyphenating continuous micellar electrokinetic chromatography (MEKC) with electrospray ionization mass spectrometry (ESI-MS). Here we evaluate this technique for its applicability in impurity profiling of drugs using galantamine and ipratropium as test samples. A background electrolyte (BGE) of 10mM sodium phosphate (pH 7.5), 12.5-15% acetonitrile and 20mM sodium dodecylsulfate (SDS) was used for the MEKC-MS analysis of a galantamine sample containing a number of related impurities, and a heat-treated solution of ipratropium containing a number of unknown degradation products. MEKC provided efficient separation of all sample constituents. Despite the presence of non-volatile BGEs, all impurities in the galantamine sample could be detected by ESI-MS in their respective extracted ion traces (XICs) with a detection sensitivity in the sub-microg/ml range (full-scan mode). MS/MS detection provided useful product spectra allowing the structural characterization of the respective galantamine impurities. With the MEKC-MS/MS system, two degradation products could be revealed and identified in the heat-stressed ipratropium sample. The presented method shows good potential for the detection and structure elucidation of minor impurities in drug substances.

The on-line coupling of sequential injection analysis (SIA) and capillary electrophoresis (CE) via an in-line injection valve is presented. The SIA system is used for automated derivatization of amino acids and peptides. Dichlorotriazinylaminofluorescein serves as the derivatization agent, thus enabling sensitive laser-induced fluorescence detection of the derivatized analytes. The SIA procedure includes the following steps: (a) introduction of reagent and sample zones in a holding coil, (b) sample and reagent mixing in a reaction coil, (c) stop-flow step for increase of the reaction time, and (d) delivery of derivatized sample into the loop of the micro-valve interface. A small portion of the analyte zone is introduced electrokinetically in the separation capillary via the valve interface and CE analysis is performed. Factors affecting the CE separation, such as pH, the borate and sodium dodecyl sulphate concentration of the background electrolyte have been optimized. The derivatization conditions have been studied to obtain a high reaction yield in a relative short time. The transfer of a part of the reaction plug into the loop of the valve interface has been optimized. Using des-Tyr(1)-[Met]-enkephalinamide as test compound, it is demonstrated that after automated derivatization, on-line electrophoretic analysis could be achieved. Glycine has been selected as the internal standard in order to correct for variations in reaction time and filling of the injection loop. For the enkephalin, good reproducibility (RSD<4.5% calculated by the ratio of the peak areas) and linearity (0.5-5 microg mL(-1), R(2)>or=0.994) are obtained with a detection limit of 30 ng mL(-1) (S/N=3).

This paper describes the on-line sample pretreatment and analysis of proteins and peptides with a poly(methylmethacrylate) (PMMA) microfluidic device (IonChip). This chip consists of two hyphenated electrophoresis channels with integrated conductivity detectors. The first channel can be used for sample preconcentration and sample clean-up, while in the second channel the selected compounds are separated. Isotachophoresis (ITP) combined with zone electrophoresis (CZE) was used to preconcentrate a myoglobin sample by a factor of about 65 before injection into the second dimension and to desalt a mixture of six proteins with 100 mM NaCl. However, ITP-CZE could not be used for the removal of two proteins from a protein/peptide sample since the protein zone in the ITP step was too small to remove certain compounds. Therefore, we used CZE-CZE for the removal of proteins from a protein/peptide mixture, thereby injecting only the peptides into the second CZE separation channel.

An on-line size exclusion chromatography (SEC)-solid-phase extraction (SPE)-capillary electrophoresis (CE) system using a Tee-split interface has been developed for the analysis of peptides in biological fluids. The SEC column fractionates the sample by molecular size and the low-molecular-weight fraction, which contains the peptides, is directed to a C(18) SPE microcolumn, where the peptides are trapped and concentrated. The SPE column is desorbed with 425 nL acetonitrile and the effluent is sent to the Tee-split interface, which hydrodynamically splits (1:40) the flow and, thus, allows appropriate injection of analytes into the CE system. The performance of the system is investigated by the analysis of enkephalins in cerebrospinal fluid (CSF). It is demonstrated that the SEC step efficiently removes potentially interfering proteins, permitting reproducible SPE and CE. The total system provides efficient separations of the enkephalins with plate numbers up to 100,000. Concentration limits of detection (S/N = 3) for the peptides are about 100 ng/mL for injection of 20 microL spiked CSF samples. Plots of enkephalin peak areas versus concentration showed good linearity over the 0.25-10 microg/mL range (R2 > or = 0.985). Repeatability of migration time and peak area was within 2% and 10% R.S.D., respectively.

A method was developed for the fast separation of a myoglobin digest using a monolithic RP 18 silica capillary column of 100 microm I.D. The results were compared with those obtained with a particulate RP 18 silica capillary column of 100 microm I.D. at a flow-rate between 0.6 and 1.2 microl/min. The digest was analyzed at the monolithic column at a flow-rate up to 2.8 microl/min. This high flow-rate could not be applied to the particulate column due to the high back-pressure. When the starting composition of the gradient was changed from 0 to 20% and a gradient steepness of 16%/min was used, the analysis time was less than 4 min. A positive Mascot identification score of 115 was achieved for the MS-MS data. When a lower gradient steepness was employed, the chromatographic resolution and the peak capacity did not increase for most compounds. The intraday repeatability for the retention time of the monolithic column was better than 1.5% at 2.8 microl/min and even less than 0.5% using a flow-rate of 0.6 or 1.0 microl/min. For the particulate column, it was between 0.5 and 1.4% for a flow-rate of 0.6 microl/min, probably due to the high column back-pressure. The interday reproducibility for the retention time of the monolithic column was less than 0.9% using a flow-rate of 1.0 microl/min.

The potential of capillaries noncovalently coated with a bilayer of oppositely charged polymers for the analysis of peptides by CE-MS was investigated. Bilayer coatings were produced by subsequently rinsing fused-silica capillaries with a solution of Polybrene (PB) and poly(vinyl sulfonate) (PVS). The PB-PVS coating showed to be fully compatible with MS detection causing no ionization suppression or background signals. The bilayer coating provided a considerable EOF at low pH, thereby facilitating the fast separation of peptides using a BGE of formic acid (pH 2.5). Under optimized CE-MS conditions, for enkephalin peptides high separation efficiencies were obtained with plate numbers in the range of 300,000-500,000. It is demonstrated that both the cancellation of the hydrodynamic capillary flow induced by the nebulizer gas and a sufficiently high-data acquisition rate are crucial for achieving these efficiencies. The overall performance of the CE-MS system using PB-PVS-coated capillaries was evaluated by the analysis of a tryptic digest of cytochrome c. The system provided an efficient separation of the peptide mixture, which could be effectively monitored by MS/MS detection allowing identification of at least 13 peptides within a time interval of 1.5 min. In addition, the PB-PVS coating proved to be very consistent yielding stable CE-MS patterns with highly favorable migration time reproducibilities (RSDs < 1% over a 3-day period).

Analyses of statistical variance were applied to evaluate the precision and practicality of a CD-based NACE assay for R-timolol after enantiomeric separation of R- and S-timolol. Data were collected in an interlaboratory study by 11 participating laboratories located in Europe and North America. General qualitative method performance was examined using suitability descriptors (i.e. resolution, selectivity, migration times and S/N), while precision was determined by quantification of variances in the determination of R-timolol at four different impurity levels in S-timolol maleate samples. The interlaboratory trials were designed in accordance with the ISO guideline 5725-2. This allowed estimating for each sample, the different variances, i.e. between-laboratory (s2(Laboratories)), between-day (s2(Days)) and between-replicate (s2(Replicates)). The variances of repeatability (s2r) and reproducibility (s2R) were then calculated. The estimated uncertainty, derived from the precision estimates, seems to be concentration-dependent above a given threshold. This example of R-timolol illustrates how a laboratory can evaluate uncertainty in general.

The characteristics of automated on-line solid phase extraction with liquid chromatography-mass spectrometry (SPE-LC-MS) are very amenable for flexibility and throughput in therapeutic drug monitoring (TDM). We demonstrate this concept of automated, on-line SPE-LC-MS for the analysis of clozapine and metabolites (desmethylclozapine and clozapine-N-oxide) in serum. Method development, optimisation and validation are described and a comparison with previously published methods for the determination of clozapine and metabolites in serum and plasma is made. Optimisation of chromatographic and SPE conditions for increased throughput resulted in SPE-LC-MS cycle times of only about 2.2 min, demonstrating the great potential of automated on-line SPE-LC-MS for TDM. The new method is shown to be clearly favourable, in particular in terms of ease of sample handling, throughput and detection limits. Recovery is essentially quantitative. Detection limits are at about 0.15-0.3 ng ml(-1), depending on the ionisation source used. Calibration follows a quadratic model for clozapine and its N-oxide and a linear model for the desmethyl metabolite (all cases: R > 0.99). Accuracy, evaluated at three concentration levels spanning the whole therapeutic range, shows that bias is less than 10%. Precision (intra - and inter assay) ranges from about 5% R.S.D. at the high end of the therapeutic range (700-1,000 ng ml(-1)) to about 20% R.S.D. (OECD defined limit) at the lower limit of quantitation ( approximately 50 ng ml(-1)). The lower limit of quantitation is well below the low end of the therapeutic range at 350 ng ml(-1).

The usefulness of the on-line coupling of nonaqueous capillary electrophoresis (NACE) with electrospray ionization (ESI) mass spectrometry (MS) using heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-cyclodextrin (HDAS-beta-CD) was demonstrated for the enantioselective determination of low concentrations of salbutamol in human urine. After optimization of several parameters, such as sheath-liquid composition and flow rate, nebulizing gas pressure, CE counter-pressure and position of the CE capillary outlet, a limit of quantification of 18 and 20 ng/ml was obtained for salbutamol enantiomers. Moreover, the relative standard deviation values for repeatability at a concentration of 30 ng/ml were below 7% for both enantiomers. Typical regression lines obtained after application of a simple linear regression model revealed a good relationship between peak area and analyte concentration (with 0.9988 and 0.9966 as coefficients of determination). This paper proposes an easy to use and sensitive NACE-MS method to determine enantiomers of a basic chiral drug in biological fluids preceded by solid-phase extraction as sample cleanup.

In this paper, augmentation has been applied to data matrices, which originate from hyphenated methods that share the same mode of detection, but use different separation methods, HPLC-DAD and MEKC-DAD. A novel method, wavelength shift eigenstructure tracking (WET), has been proposed for the alignment between the wavelength scale of both detectors. WET proves to be suitable for the detection as well as correction of wavelength shift between both detectors. After correction of the wavelength scale, data obtained on both systems have been augmented and submitted to iterative target transformation factor analysis. Augmented curve resolution provides significantly better estimates of the chromatographic and electrophoretic profiles and spectra than the use of non-augmented curve resolution on HPLC and MEKC data separately. It is particularly useful when the pure fraction of a chromatographic peak is less than 0.10. Finally, the relative weight of MEKC versus HPLC in augmentation may be increased using intensity and noise normalisation. However, since noise normalisation and its accompanying decrease in signal-to-noise ratio leads to a loss of information, and, since intensity normalisation may cause a failure of the augmented curve resolution algorithm, benefits and drawbacks of normalisation should be weighed on a case-by-case basis.

A study was initiated to investigate the use of surface plasmon resonance (SPR) for the detection in plasma of a high pI model protein, recombinant human interferon-gamma (IFN-gamma). Initially a number of self-assembled monolayers (SAMs) and hydrogel-derivatised SAM-coatings were characterised for the adsorptive and desorptive properties of plasma components. Next a monoclonal anti-IFN-gamma antibody, MD-2, was covalently attached to dextran-modified mercaptoundecanoic acid surfaces that performed best. On coatings consisting of carboxyl-modified dextran (CMD) a difference in interaction behaviour was observed when IFN-gamma was injected in either buffer or diluted plasma. During the injection of IFN-gamma in buffer, an acceleration of the interaction process was observed and the signal continued to increase after the injection plug had passed. Upon injection of diluted plasma spiked with IFN-gamma, the response increased without acceleration of the binding process. After the injection was finished, some of the bound material desorbed as expected, resulting in a signal decrease. On non-charged dextrans, the interaction between the antibody-modified surface and IFN-gamma in either plasma or buffer was similar. During sample injection the response increased with a binding rate depending on the concentration of IFN-gamma present in solution. When the injection was finished, some of the bound material was washed away from the surface and only a minor contribution of non-specific adsorbed plasma components was noticeable. From the coatings tested, the non-modified dextran-coated SPR sensor disks prove to be best suited for the detection of IFN-gamma in complex matrices like plasma. The interaction of IFN-gamma in both diluted plasma and buffer is comparable and concentrations of IFN-gamma of 250 ng ml-1 and higher can be detected in both buffer and 100x-diluted plasma. The non-specific adsorption of plasma components is low, whereas the specific IFN-gamma response is relatively high.

LC-ESI-MS is applied frequently in pharmaceutical analysis. The sample amount is generally not restricted, however with LC-ESI-MS, a lack of sensitivity may still be observed with standard-bore LC columns in isocratic mode. Therefore, it was investigated whether increased sensitivity could be achieved by using miniaturized LC-ESI-MS. Seven columns ranging from 0.1 to 4.6 mm ID were tested using several instrument setups. For proper comparison, a sensitivity gain factor (SGF) was introduced. The SGF expresses the extra sensitivity that may be obtained on top of the normal increase of peak concentration, which can be expected when the column ID is reduced. Desogestrel, mirtazapine, and sugammadex sodium were used as test compounds. For desogestrel and sugammadex sodium, the SGF increased up to a factor of 5-13 when the column ID was reduced, indicating enhanced ionization efficiencies at lower flow rates. Optimum sensitivity was found for the 0.3 mm column coupled in combination with a microinjection valve and a dedicated low flow rate interface. For mirtazapine, no increase of SGF was observed when the column ID was decreased. Apparently, the ionization efficiency of this compound is not affected by the flow rate and the spray quality.

Atmospheric pressure photoionization (APPI) is presented as a novel means for the combination of micellar electrokinetic chromatography (MEKC) and mass spectrometry (MS). The on-line coupling is achieved using an adapted sheath flow interface installed on an orthogonal APPI source. Acetone or toluene is added as dopant to the sheath liquid to enhance analyte photoionization. It is demonstrated that with APPI signal suppression and interferences by the surfactant sodium dodecyl sulfate (SDS) and nonvolatile buffers can be circumvented. This implies that MEKC conditions can be selected independently from MS detection. Moreover, it is shown that both polar and apolar compounds can be photoionized, thereby also facilitating the analysis of compounds that are not amenable to electrospray ionization. Consequently, the MEKC-APPI-MS system can provide effective separation and detection of compounds of diverse character in one run using background electrolytes containing up to 50 mM SDS. Concentration limits of detection derived from extracted-ion traces (full scan mode) of test compounds were approximately 1 microg/mL, and the detection sensitivity remained unaffected during 1 day of continuous use. Overall, the system features are very favorable for applications such as drug impurity profiling as is illustrated by the analysis of mebeverine and related compounds (both charged and neutral) at the 0.25% (w/w) level.

The suitability of noncovalently bilayer-coated capillaries for the analysis of proteins by capillary electrophoresis (CE) at medium pH was investigated. Fused-silica capillaries were coated simply by successively flushing with a polybrene (PB) and a poly(vinyl sulfonate) (PVS) solution. A protein test mixture was used to evaluate the performance of the coated capillaries. Comparisons with bare fused-silica capillaries were made. Several background electrolytes (BGEs) were tested in combination with the PB-PVS coating, showing that optimum performance was obtained for the proteins using high BGE concentrations. With a 300 mM Tris phosphate buffer (pH 7.0), good plate numbers (150,000-300,000), symmetrical peaks, and favorable migration-time repeatabilities (RSDs below 0.8%) were obtained for the proteins. Using bare fused-silica capillaries, the protein peaks were significantly broadened and the migration-time RSDs often exceeded 5%. It is concluded that the PB-PVS coating effectively minimizes adverse protein adsorption and provides a very stable electroosmotic flow (EOF). We also investigated the potential of a commercially available bilayer coating (CEofix) for protein analysis. It is demonstrated that with this coating, good plate numbers and peak symmetries for proteins can be achieved when the CEofix BGE ("accelerator") is replaced by a common BGE such as sodium or Tris phosphate. Apparently, the negatively charged polymer present in the "accelerator" interacts with the proteins causing band broadening. The utility of the bilayer coatings is further illustrated by the separation of proteins such as interferon-alpha 2b, myoglobin and carbonic anhydrase, by the analysis of a degraded insulin sample in time, and by the profiling of the glycoprotein ovalbumin. In addition, it is demonstrated that even in the presence of concentrations of human serum albumin in the sample of up to 60 mg/mL, the PB-PVS coating still provides reproducible protein separations of good performance.

The research on our flow-injection, label-free, non-faradaic impedimetric immunosensor for interferon-gamma (IFN-gamma) has been extended. The sensor is prepared by immobilization of anti-IFN-gamma antibodies on a self-assembled monolayer (SAM) of acetylcysteine, deposited on polycrystalline gold. A multi-frequency impedance method is described, which allows time-resolved measurement of Nyquist plots. To these plots, an equivalent circuit was fitted, which is discussed in terms of a two-layer structure (inner and outer layer) of the interfacial region. Because binding of IFN-gamma mainly causes a decrease of Q (a constant-phase element), this element is considered as the outer layer. Several aspects of the impedimetric sensor response are studied, including the dependence on detection frequency, target concentration and applied dc potential. For quantitative detection of IFN-gamma, an optimum of the signal-to-noise (S/N) ratio of the out-of-phase impedance component (Z'') was found at about 100 Hz. At a dc-potential of +0.2 V versus a saturated calomel reference electrode, the sensor response is higher than at 0.0 V. Logarithmic dose-response curves of IFN-gamma in the concentration range of 10(-18) to 10(-9) M were obtained using two procedures: by successive injections over a single electrode, and by using freshly prepared electrodes for each measurement. Using the latter method, the repeatability is impaired. The need for in situ complementary techniques for a correct interpretation of the studied parameters is discussed.

Peptides and proteins are gaining increasing attention in biosciences and, consequently, in analysis. This overview highlights the different approaches to couple on-line various separation techniques for the determination of proteins and peptides. The first section discusses the liquid chromatography (LC)-LC coupling, the second one reviews the on-line LC-capillary electrophoresis (CE) coupled systems and the third section summarizes the strategies for on-line CE-CE. The advantages, disadvantages, most relevant difficulties and particular systems for on-line coupling are discussed. Special attention is paid to the interface between the two dimensions. Applications are summarized in tables and a few typical examples are discussed. Many multidimensional separation methods are available, and it is demonstrated that peptide and protein mapping, or quantitation of proteins or peptides in various samples (aqueous solutions, cells, plasma) require different coupled systems. For mapping a semi-quantitative detection is often sufficient, while comprehensiveness is very important. For quantitation of a certain peptide or protein at a low concentration level a validated method should be used, while a heart-cut transport of the first dimension to the second one can offer sufficient selectivity. The combination with mass spectrometry as part of the total system is stressed and illustrated.

The on-line coupling of capillary electrophoresis (CE) and mass spectrometry (MS) via atmospheric pressure photoionization (APPI) is demonstrated. To achieve CE-APPI-MS, an adapted coaxial sheath-flow interface was combined with an ion-trap mass spectrometer equipped with an APPI source originally designed for liquid chromatography-MS. Effective photoionization of test compounds was accomplished after optimization of several interface and MS parameters, and of the composition and flow rate of the sheath liquid. Further enhancement of the ionization efficiency could be achieved by adding a dopant, such as acetone or toluene, to the sheath liquid to aid indirect ionization. Acetone significantly increased the ionization of the polar test compounds by proton transfer, while toluene was more useful for the enhanced formation of molecular ions from nonpolar compounds. The effect of several common CE background electrolytes (BGEs) on the APPI-MS response of the analytes was also studied. It appeared that in contrast with electrospray ionization, nonvolatile BGEs do not cause suppression of analyte signals using APPI. Therefore, in CE-APPI-MS, a variety of buffers can be chosen, which obviously is a great advantage during method development. Remarkably, also sodium dodecyl sulfate (SDS) did not affect the photoionization of the test compounds, indicating a strong potential of APPI for the on-line coupling of micellar electrokinetic chromatography (MEKC) and MS.

Various types of mutations exist that exert an effect on the normal function of a gene. Among these, exon/gene deletions often remain unnoticed in initial mutation screening. Until recently, no fast and efficient methods were available to detect this type of mutation. Molecular detection methods for gene copy number changes included Southern blot (SB) and fluorescence in situ hybridisation, both with their own intrinsic limitations. In this paper, we report the development and application of a fast, sensitive and high-resolution method for the detection of single exon or larger deletions in the VHL gene based on real-time quantitative PCR (Q-PCR). These deletions account for approximately one-fifth of all patients with the von Hippel-Lindau syndrome, a dominantly inherited highly penetrant familial cancer syndrome predisposing to specific malignancies including phaeochromocytomas and haemangioblastomas. Our VHL exon quantification strategy is based on SYBR Green I detection and normalisation using two reference genes with a normal copy number, that is, ZNF80 (3q13.31) and GPR15 (3q12.1). Choice of primer sequences and the use of two reference genes appears to be critical for accurate discrimination between 1 and 2 exon copies. In a blind Q-PCR study of 29 samples, all 14 deletions were detected, which is in perfect agreement with previously determined SB results. We propose Q-PCR as the method of choice for fast (within 3.5 h), accurate and sensitive (ng amount of input DNA) exon deletion screening in routine DNA diagnosis of VHL disease. Similar assays can be designed for deletion screening in other genetic disorders.

Solid-phase extraction (SPE) and capillary electrophoresis (CE) are on-line coupled via a Tee-split interface, which provides hydrodynamic injection of the SPE eluate by flow splitting. The interface allows sample preconcentration independently from the CE separation and prevents sample matrix and washing solvents from entering the separation capillary. The effect of the Tee-split interface on the CE efficiency was examined using enkephalin peptides as model compounds. Most favorable plate numbers were obtained using a split ratio of 1:40. Breakthrough volume, desorption efficiency and elution volume for the C18 micro SPE column (5 mm x 0.5 mm i.d.) were found to be 750 microL, 65% and 1 microL, respectively. The performance of the complete system was demonstrated by the preconcentration and separation of an enkephalin mixture. Plate numbers up to 120,000 were obtained using a sample volume of 250 microL and a split ratio of 1:40. Enkephalin peak areas were linear (R2 = 0.996) over the 10-1000 ng/mL range. UV absorbance concentration limits of detection (SIN = 3) were about 5 ng/mL. For 250 microL injections of 100 ng/mL, the relative standard deviation (n = 5) of peak area was lower than 10%.

A preconcentration-capillary electrophoresis (CE) system using a small precolumn in combination with an in-line injection valve is presented. The advantage of the present design is the ability to perform the sample preconcentration fully independently from the CE separation and to prevent sample matrix and washing solvents from entering the CE capillary. With a micro injection valve, sample could be effectively introduced into the CE system in an in-line fashion without seriously affecting the CE separation efficiency. Breakthrough volume, desorption efficiency, and elution volume for the C18 microcolumn (5 x 0.5 mm i.d.) were established, yielding values of 750 microL, 70%, and 0.9-1.1 microL, respectively, using enkephalin peptides. The time between the start of the desorption of the analytes from the precolumn and the injection into the CE system was also studied in order to achieve optimal sensitivity and separation efficiency. The performance of the complete system was demonstrated by the preconcentration and separation of an enkephalin mixture. Using a sample volume of 250 microL and a CE injection voltage of -15 kV for 12 s, linearity was observed over 2 orders of magnitude, and detection limits (S/N = 3) were in the 5-10 ng/mL range. A 1000-fold sensitivity enhancement is obtained using this setup, as compared to a regular CE setup. For 100 ng/mL samples, repeatabilities (RSDs) of migration time and peak area were 1.2 and 11%, respectively.

An automated method for the optimisation of high-performance liquid chromatography is developed. First of all, the sample of interest is analysed with various eluent compositions. All obtained data are combined into one augmented data matrix. Subsequently, augmented iterative target transformation factor analysis performs the integrated tasks of curve resolution and peak tracking. Since this type of curve resolution processes all data at once, it can deal with strong peak overlap and reveal the correspondence of compounds between runs, i.e. peak tracking. The retention time and peak width at half height for each component of the sample are determined for every eluent composition. Next, models are built for the retention time and the peak width at half height. These models are used to predict the resolution and the analysis time for each point in factor space. Finally, a multi-criterion decision-making method, Pareto optimality, is used to find the optimum. The method completes all calculations within a few minutes and without user intervention. By means of this procedure, a mixture of three benzodiazepines is successfully separated using a ternary mobile phase. There are two requirements for the automated optimisation method to work correctly. Firstly, all components of the sample must have sufficiently different spectra. Secondly, each compound should have the same spectrum under all experimental conditions.

The usefulness of a noncovalent capillary coating consisting of two layers of oppositely charged polymers for the separation of peptides with capillary electrophoresis (CE) was studied. Capillaries were coated simply by subsequently flushing with solutions of 1% m/v Polybrene and 1% v/v poly(vinylsulfonate) (PVS) forming a bilayer, which showed to produce a strong and highly reproducible electroosmotic flow (EOF) at low pH. Using this coating in combination with a background electrolyte (BGE) containing sodium phosphate (pH 2.5) and 0.01% v/v PVS, initially broadened and overlapping peaks were obtained for some test peptides. By omitting the PVS from the BGE, the peak width and shape of the peptides improved resulting in baseline separation. A systematic study of the influence of the BGE composition showed that considerable further enhancement of the separation efficiency was achieved by increasing the ionic strength of the BGE. Using a BGE of 200 mM tris(hydroxymethyl)aminomethane (Tris)-phosphate (pH 2.5) plate numbers for the peptides were in the 300 000-600 000 range and the relative standard deviation of the peptide migration times was less then 0.3% (n = 5). The use of Tris-phosphate instead of sodium phosphate allowed the current to stay within acceptable limits when 30 kV was used as separation voltage. Overall, the bilayer coating showed a remarkable EOF repeatability, as well as long-term stability. Compared to bare fused-silica capillaries the intraday and interday repeatability of migration times was very favorable and coated capillaries could be used for over a month performing analyses with low and high ionic strength BGEs without any performance deterioration. The usefulness of the bilayer-coated capillaries for the analysis of positively charged peptides was demonstrated by the fast and efficient separation of various closely related enkephalins and the baseline separation of an isomeric peptide/peptoid couple exhibiting efficiencies of over 550 000 plates.

The combination of capillary electrophoresis (CE) and electrospray ionization-mass spectrometry (ESI-MS) via a triaxial interface was studied as a potential means for the characterization of intact proteins. To evaluate the possibility to use a nonvolatile electrolyte for CE, the effect of sodium phosphate and ammonium borate on the MS signal of the proteins insulin, myoglobin, and bovine serum albumin (BSA) was investigated by employing infusion experiments, and compared to the effect of ammonium formate and formic acid. The study shows that with formic acid (50 mM, pH 2.4) the most intense protein signals were obtained, while the use of sodium phosphate buffer (5 and 10 mM, pH 7.5) almost completely diminished the MS response. Ammonium formate and ammonium borate (up to 100 mM, pH 8.5) also caused protein ion suppression, but especially with the borate buffer significant MS intensity remained. MS analysis of myoglobin revealed the loss of the heme group when an acidic CE electrolyte was used. Using a background electrolyte containing 25 mM ammonium borate (pH 8.5), it is demonstrated that a CE separation of a protein test mixture can be monitored with ESI-MS without degrading the MS performance allowing molecular weight determinations of the separated compounds. In the presence of borate, detection limits were estimated to be 5-10 microM (ca. 100 fmol injected). The usefulness of the CE-MS system employing a borate buffer is indicated by the analysis of a stored sample of BSA revealing several degradation products. A sample of placental alkaline phosphatase (PLAP), a potential therapeutic agent, was also analyzed by CE-MS indicating the presence of a protein impurity. Probably due to insufficient ionization of the PLAP (a complex glycoprotein), no MS signals of the intact protein were observed.

Solid-phase extraction (SPE) was directly coupled to mass spectrometry (MS) to assess the feasibility of the system for the rapid determination of prednisolone in serum. A C(18) stationary phase allowed washing of the cartridge with 25% methanol. Elution was performed by switching the methanol percentage from 25% in the washing step to 50% during elution. The high flow-rates during the extraction (5.0 ml/min) combined with ion-trap MS detection resulted in a total analysis time of 4 min. Some tailing of the prednisolone peak was observed. However, the tailing was found acceptable, since by this elution procedure most matrix compounds were prevented from eluting from the cartridge. Some matrix interference was still observed with a triple-quadrupole MS, even in the multiple reaction monitoring mode. This resulted in a detection limit (LOD) of about 10 ng/ml. The matrix interference and the LOD were similar for atmospheric pressure chemical ionisation and atmospheric pressure photo ionisation. Applying an ion-trap MS in the MS-MS mode resulted in cleaner chromatograms. Due to extensive fragmentation of prednisolone, the LOD was not lower than about 5 ng/ml prednisolone in serum, and a limit of quantitation of about 10 ng/ml (relative standard deviation <15%) was observed.

An on-line method for the coupling of micellar electrokinetic chromatography (MEKC) and mass spectrometry (MS) is presented which allows conventional MEKC conditions to be employed without further modification. The MEKC system is coupled directly to electrospray ionization (ESI) MS using a triaxial interface. A systematic study of the influence of the surfactant concentration, the nature and concentration of buffer salts and presence of organic modifier on the interface performance indicated the feasibility of the MEKC-MS approach. Effective interfacing of MEKC was achieved with both single quadrupole and ion-trap MS instruments. Using a background electrolyte containing 20 mM sodium dodecyl sulfate (SDS) and 10 mM sodium phosphate buffer, it is demonstrated that full MEKC runs of test mixtures of mebeverine and related compounds can be monitored by ESI-MS with satisfactory sensitivity. Sub-microg/ml levels of the analytes can still be detected in full scan mode, while detection limits are in the 10-50 ng/ml range when selected ion monitoring is applied. It is shown that such sensitivity would allow full-scan MS detection of 0.1% (w/w) levels of potential impurities in mebeverine. With the ion-trap instrument successful MEKC-MS/MS experiments were carried out providing information-rich MS spectra of the related compounds. Repeated MEKC-MS analyses proved that in the course of 1 day the migration time of mebeverine remained fairly constant while the MS-signal intensity only gradually decreased to approximately 65% of its original value. Once-a-day cleaning of the first part of the ion source, which takes only 5 min, suffices to preserve an optimal interface performance for a prolonged period of time.

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebrospinal fluid (CSF) and tissues. The present study shows the feasibility of a recently developed system, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on an RP18 microcolumn with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE) for the quantification of structural-related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. After SEC separation of the enkephalins from large proteins present in CSF a heart-cut of 200 nuL, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution of the enkephalins with 80% acetonitrile, a fraction of the eluate is electrokinetically injected into the CZE system, where stacking and separation is achieved. The degradation of the peptides, caused by endogenous peptidases in the matrix, is sufficiently inhibited with imipramine HCl. The assay has a satisfactory linearity and intraday (9.70-16.3%) precision considering the complexity of this multidimensional separation system. The sensitivity of the method, with a concentration limit of quantification of 2.5 nug/mL, is comparable with other CZE assays for peptides and sufficient for the quantification of peptide drugs in biological matrices.

Since biologically active peptides usually exhibit their effects in low concentrations, the development of sensitive analytical methods has become a challenge. In this paper, a multidimensional system is presented, consisting of a size-exclusion chromatographic (SEC) separation followed by a trapping procedure on a 4 mm x 3 mm ID reversed-phase C18 (RP18) column with subsequent elution of the trapped fraction and separation by capillary zone electrophoresis (CZE). The system has been tested with mixtures of six enkephalins and albumin, mimicking biological matrices such as plasma and cerebrospinal fluid. After separation of albumin from the enkephalins in the SEC dimension a heart-cut of 200 micro L, containing the enkephalin peak, is taken, concentrated on the RP18 microcolumn and, after elution with a 20 micro L plug of 80% acetonitrile, electrokinetically injected into the CZE system, where stacking and separation is achieved. While validation shows generally good linearity and reproducibility, the quantitation limit with UV detection is acceptable (2.5 micro g/ mL with an injection volume of 50 micro L).

Alkaline phosphatase (AP) is a potential therapeutic agent in the treatment of sepsis. In this paper the potential of capillary zone electrophoresis (CZE) for the monitoring of the degradation of placental alkaline phosphatase (PLAP) was investigated. To induce degradation PLAP samples were exposed to high temperatures, low and high pH and freeze-drying. The samples were then analyzed by CZE and enzymatic activity assay. Upon exposure to temperatures above 65 degrees C, PLAP lost its activity exponentially over time, while CZE revealed both a linear decrease of the area of the main peak and a rise of degradation products. At acidic pH the enzyme appeared to lose its activity. CZE revealed a decrease of the area of the main peak, but no degradation products could be detected. At pH 12 the enzymatic activity and the area of the main peak both decreased linearly over time and, in addition, formation of degradation products could be detected by CZE. Activity and CZE profile of PLAP remained unchanged upon freeze-drying in the presence of inulin. Prolonged storage of freeze-dried samples at room temperature caused a slight decrease of enzymatic activity, while the potential formation of oligomers was revealed by CZE analysis. The examples in this study show that, in combination with activity assays, CZE can provide useful complementary information, especially on the status of the protein and the presence of degradation products.

Ion suppression effects were observed during the determination of clenbuterol in urine with solid-phase extraction/multiple-stage ion-trap mass spectrometry (SPE/MS(3)), despite the use of atmospheric pressure chemical ionisation. During SPE, a polymeric stationary phase (polydivinylbenzene) was applied. Post-cartridge infusion of analyte to the SPE eluate after the extraction of blank urine was performed to obtain a profile of the suppression. Single and multiple-stage MS were performed to provide insight in the suppressing compounds. The ion suppression was mainly ascribed to two m/z values, but still no identification of the compounds was achieved from the multiple-stage MS data. No ionisable and non-ionisable complexes and/or precipitation of clenbuterol with matrix compounds were observed. A concentration dependence of the percentage of suppression was observed. Up to 70% of the signal was suppressed upon post-cartridge infusion of 0.22 microg/mL (at 5 microL/min) clenbuterol into the eluate, and this decreased to about 4% at infusion of 22 microg/mL clenbuterol. Molecularly imprinted polymers were used to enhance the selectivity of the extraction. Although matrix components were still present after extraction, no interference of these compounds with the analyte was observed. However, the bleeding of the imprint from the polymer (brombuterol) caused significant ion suppression., (Copyright 2002 John Wiley & Sons, Ltd.)

The aim of this study was to investigate the formulation of sugar glass stabilised alkaline phosphatase from bovine intestine (BIAP) into tablets. Two major subjects of tablet formulation were investigated. First, the compaction behaviour of the inulin sugar glass was investigated. Secondly, the effect of the compaction process on the physical stability of sugar glass stabilised BIAP was investigated, comparing inulin and trehalose glass. The tabletting properties of freeze-dried inulin without BIAP were studied first. Freeze-dried inulin conditioned at either 20 degrees C/0% relative humidity (RH) or 20 degrees C/45% RH was compacted at various pressures. As expected, the yield pressure of the material conditioned at 0% RH was higher (68 MPa) than after conditioning at 45% RH (39 MPa). Tablets made of the material stored at 0% RH showed severe capping tendency, especially at high compaction pressures. In contrast, material conditioned at 45% RH gave tablets without any capping tendency and a friability of less than 1%. Sugar glasses of BIAP and either inulin or trehalose were prepared by freeze-drying (BIAP/sugar 1/19 (w/w)). The material was subsequently compacted. Tablets and powders were stored at 60 degrees C/0% RH. The activity of the incorporated BIAP was measured at various time intervals. It was found that inulin was by far superior to trehalose as stabiliser of BIAP in tablets. The poor stabilising capacities of trehalose after compaction are explained by crystallisation of trehalose induced by the compaction process and moisture in the material. The results clearly show that inulin is an excellent stabiliser for BIAP. The tabletting properties are adequate, showing sufficient tablet strengths and low friability. Furthermore, the good (physical) stability of inulin glass with respect to exposure to high relative humidities makes it practical to work with.

Ceruloplasmin is a multicopper oxidase essential for normal iron homeostasis. To elucidate the mechanisms of copper incorporation into this protein, holoceruloplasmin biosynthesis was examined by immunoblot analysis and (64)Cu metabolic labeling of Chinese hamster ovary cells transfected with cDNAs encoding wild-type or mutant ceruloplasmin. This analysis reveals that the incorporation of copper into newly synthesized apoceruloplasmin in vivo results in a detectable conformational change in the protein. Strikingly, despite the unique functional role of each copper site within ceruloplasmin, metabolic studies indicate that achieving this final conformation-driven state requires the occupation of all six copper-binding sites with no apparent hierarchy for copper incorporation at any given site. Consistent with these findings a missense mutation (G631R), resulting in aceruloplasminemia and predicted to alter the interactions at a single type I copper-binding site, results in the synthesis and secretion only of apoceruloplasmin. Analysis of copper incorporation into apoceruloplasmin in vitro reveals that this process is cooperative and that the failure of copper incorporation into copper-binding site mutants observed in vivo is intrinsic to the mutant proteins. These findings reveal a precise and sensitive mechanism for the formation of holoceruloplasmin under the limiting conditions of copper availability within the cell that may be generally applicable to the biosynthesis of cuproproteins within the secretory pathway.

On-line coupled analytical techniques can be advantageous in the assay of smaller peptides in complex biological matrices such as plasma, cerebro-spinal fluid (CSF) and tissues. The present study shows the feasibility of the recently reported on-line coupled size exclusion chromatography (SEC) and reversed phase liquid chromatography (RP-LC) separation system for the quantitation of structural related peptides in biological matrices, as demonstrated for a number of enkephalins in CSF. The degradation of the peptides, caused by endogenous peptidases in the matrix, could sufficiently be inhibited with imipramine HCL to allow an assay with satisfactory linearity and intraday (0.70-4.9%) precision. The sensitivity of the method, with a concentration limit of quantitation (LOQ) of 2 microgram/ml is comparable with other kinds of assays for peptides and sufficient for the quantitation of peptide drugs with higher therapeutic ranges in biological matrices. However, for the assay of low concentrations of peptides, such as endogenous components of a biological matrix, the sensitivity may need improvement. The LOQ cannot be improved by increasing the sample amount, because of interference of other endogenous components of the CSF. This indicates that a larger selectivity is desired. The LOQ may be improved by using more sensitive and selective detection methods such as mass spectrometry or fluorescence after post-column derivatization. Miniaturization of the system, combined with on-line trapping may also contribute to a better sensitivity.

This paper describes the characterisation of reversed-phase liquid chromatography (RPLC) columns using thermodynamic measurements. Retention versus 1/T data were used to construct Van't Hoff plots. The slope of these plots indicates the standard enthalpy of transfer of the analyte from the mobile to the stationary phase. The standard entropy can be calculated from the intercept. Van't Hoff plots were linear for the investigated RPLC columns, meaning that for basic analytes over the temperature range studied no changes in the retention mechanism occurred. Enthalpies and entropies of transfer of basic analytes from the mobile to the stationary phase revealed information about the types of interaction of protonated and neutral compounds with the stationary phases. However, a clear view using the present set of basic compounds on how these thermodynamic data may explain the observed substantial differences in peak symmetry cannot be given. It is considered that addition of N,N-dimethyloctylamine (DMOA) to the eluent will results in a dynamically coating of the stationary phase. Addition of DMOA to the eluent resulted for protonated basic compounds in a reduction of both enthalpy and entropy. In practice, with DMOA in the eluent symmetrical peaks were obtained. It is assumed that this is due to blocking residual silanols and/or ion exclusion effects.

The use of capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection for the rapid determination of apolipoprotein E (apoE) genotypes was studied. High resolution and sensitive detection of the concerned DNA restriction fragments was achieved using CE buffers with hydroxypropylmethylcellulose (HPMC) as sieving polymer and ethidium bromide (EB) as fluorescent intercalating agent. In order to achieve adequate resolutions in short analysis times, parameters such as concentration of HPMC and EB, separation voltage, and length and coating of the capillary were evaluated. Using a separation buffer with 0.8% (w/w) HPMC and 7 microM EB, characteristic DNA-fragment profiles could be obtained for all common apoE genotypes at an overall rate of ten samples per hour. The method allows direct injection of untreated PCR samples and the use of standard fused-silica capillaries which are effectively coated following a short, one-step rinse procedure. With a simple computerized algorithm based on migration-time ratios for pattern assignment, highly reliable apoE genotyping was achieved. Overall, in terms of speed, ease of use and objectivity the presented method provides a significant improvement over previously reported CE-based procedures for apoE genotyping., (Copyright 2002 Elsevier Science BV.)

Spherical molecularly imprinted polymer particles obtained via precipitation polymerization, were introduced as a pseudostationary phase in capillary electrophoresis (CE) to study molecular recognition. Analyses were performed via a partial filling technique using (+)-ephedrine-imprinted microspheres (100-200 nm) which were polymerized from methacrylic acid and 1,1,1-Tris(hydroxymethyl)propanetrimethacrylate using acetonitrile as the solvent. The influence of pH and the modifier content on the separation was investigated. A 0.1% w/v suspension in an aqueous 10 mM phosphate buffer (pH 2.5 with 40% acetonitrile) was hydrodynamically injected into the CE system (80% of the effective capillary length) and led to full baseline separation of racemic ephedrine within 10 min.

On-fiber derivatization was used for solid-phase microextraction (SPME) in order to increase the detectability and extractability of drugs in biological samples. Amphetamine, which was used as a model compound, was derivatized with pentafluorobenzoyl chloride (PFBCl) and subjected to gas chromatography with electron capture or mass spectrometric detection. Extraction was performed by direct immersion of a 100 microm polydimethylsiloxane-coated fiber into buffered human urine. On-fiber derivatization was performed either after or simultaneously with extraction. The former procedure gave cleaner chromatograms but the latter turned out to be superior with respect to linearity and repeatability. For the on-fiber derivatization of amphetamine an excess of reagent is required. Because a considerable part of the PFBCl loaded on to the fiber is used up by reaction with matrix compounds and water, a reagent loading time of 5 min was needed to obtain a linear range (r = 0.9756) from 250 pg mL(-1) to 15 ng mL(-1). Due to an interfering matrix compound, the limit of detection was also found to be dependent on the reagent loading time, i.e., the limit of detection for a PFBCl loading time of 5 min is 250 pg mL(-1) whereas that for a 1 min loading time it is 100 pg mL(-1). The relative standard deviation (n = 7) of the method was about 11% at an amphetamine concentration of 1 ng mL(-1). The applicability of the method for the determination of drugs in biological samples is shown.

The application of immunoaffinity chromatography for the purification of Taxus plant and cell extracts prior to the HPLC analysis is described. Polyclonal antibodies raised against 10-deacetylbaccatin III (10-DAB III), paclitaxel's main precursor in plant, were characterised by enzymed-linked immunosorbent assay. Immunoglobulins from selected antisera were immobilised on CNBr-activated Sepharose 4B. The immunoaffinity column was used for the purification of plant and plant cell culture extracts prior to their analysis by HPLC. Immunoaffinity chromatography enabled the selective concentration of taxoids and enhanced sample clean-up.

To determine sub-ppb levels of drugs in biological samples, selective, sensitive and rapid analytical techniques are required. This work shows the possibilities for high-throughput analysis of solid-phase microextraction (SPME) directly coupled to an ion-trap mass spectrometer equipped with an atmospheric pressure chemical ionisation source. As no chromatographic separation is performed, the SPME procedure is the time-limiting step. Direct immersion SPME under non-equilibrium conditions permits the determination of lidocaine in urine within 10 min. After a 5 min sorption time with a 100 microm polydimethylsiloxane-coated fibre, the extraction yield of lidocaine from urine is about 7%. When applying 4 min desorption, using a mixture of ammonium acetate buffer (pH 4.5) and acetonitrile (85 + 15 v/v), about 10% of the analyte is retained on the fibre. An extra cleaning step of the fibre is therefore used to prevent carry-over. By use of tandem MS, no matrix interference is observed. The detection limit for lidocaine is about 0.4 ng ml(-1) and the intraday and interday reproducibility are within 14% over a concentration range of 2-45 ng ml(1).

Solid-phase extraction-pipette tips (SPE-PTs) were used for micro solid-phase extraction of lidocaine and diazepam from plasma. Off-line extraction was followed by on-line desorption. On-line desorption was carried out by direct coupling of the SPE-PTs with the liner of the programmed-temperature vaporiser. This coupling only required shortening of the liner by maximally 16 mm, cutting the SPE-PT, and equipping the remaining part with two O-rings. Due to the heating of the injector the SPE-PTs were heated as well, which resulted in a significant amount of impurities. Pre-heating and pre-washing was performed prior to the extraction to reduce the impurity level. The internal coupling device was applied successfully for the analysis of plasma samples with gas chromatography (GC) and mass-selective detection. Detection limits of 0.75 ng/ml and 2.5 ng/ml were obtained for lidocaine and diazepam, respectively, using 200 microl plasma. Recoveries for both compounds were about 80%. Although it is possible, the internal coupling device was not developed to be used as such. The main goal of this coupling was to show the feasibility of the integration of SPE-PTs with GC and to realize an important step to new automated SPE-GC systems.

The reversed-phase liquid chromatographic analysis of basic pharmaceuticals can be problematic. Both the properties of the eluent and the stationary phase can influence the chromatographic performance. Therefore selection of suitable experimental conditions for the analysis of basic compounds can be difficult. This paper shows that the organic modifier and the nature of the buffer influence the eluent properties. Moreover, the nature and amount of modifier also influence the basicity of the analytes. Investigations showed that the nature of the buffer can have a significant influence on retention and peak shape of basic compounds. Test procedures using basic analytes as test probes provided relevant information with respect to selecting columns to analyse basic pharmaceutical compounds. Test procedures using compounds like aniline, phenol and benzene were found to be less suitable.