Robust and cost-effective capillary electrophoresis-mass spectrometry interfaces suitable for combination with on-line analyte preconcentration.

This paper describes several successful cost-effective attempts to couple capillary electrophoresis (CE) and mass spectrometry (MS) without make-up flow or nebulizing gas. An in-depth analysis of several interfaces using conductive spray tips was performed as well as an easy-to-prepare T-junction with direct electrode contact, the latter being the most robust interface. No coating is necessary and the spray voltage is applied through a gold wire positioned at the gap between the separation and spray capillaries. The T-junction interface is made by puncturing a small piece of transparent rubber. The on-line preconcentration CE-MS system allows immunoassay sensitivity, as is demonstrated by a calibration plot in the picomolar range for angiotensin II and gonadorelin. It also shows good reproducibility and has the ability of excellent automation. The secure electrical contact gives a constant spray quality, even with 100% aqueous separation buffers. The described setup has a wide applicability as is demonstrated by the analysis of larger peptides, such as insulin and cytochrome c. Detailed information is given on critical factors in the preparation of the described interfaces.

Capillary electrophoretic bioanalysis of therapeutically active peptides with UV and mass spectrometric detection after on-capillary preconcentration.

An earlier developed capillary electrophoresis (CE) system with an on-capillary adsorptive phase is investigated for its suitability to quantitate low concentrations of angiotensin II and gonadorelin in plasma. An off-line solid-phase extraction is used for sample preparation. The on-line preconcentration CE system allows multiple capillary volumes of sample solution to be injected, increasing the concentration sensitivity of CE with 3-4 orders of magnitude. Furthermore, possible influence of matrix salts can be ruled out by employing a rinsing step after sample application. Using short-wavelength UV detection, reproducibility and linearity in the low nanomolar range were satisfactory. The capillary could be efficiently regenerated using a programmed between-run rinsing procedure, allowing 20-30 large injections of sample extracts. Coating of the capillary improved the robustness of the method. Mass spectrometric detection via a previously reported sheathless interface increased the selectivity and sensitivity substantially. Recommendations are provided for the sample preparation process, the most critical part of the system. Further purification of the sample is required to allow the loading of larger sample volumes and to optimize the system's robustness.

On-line sample preconcentration in capillary electrophoresis, focused on the determination of proteins and peptides.

This overview highlights the possibilities of on- or in-line preconcentration procedures in combination with a CZE separation, focused on the determination of peptides and proteins. The discussed methods, including sample stacking, field-amplified injection, isotachophoresis, solid phase extraction, membrane preconcentration, electroextraction, supported liquid membranes, hollow fibers, immunoaffinity, and molecularly imprinted polymers technology preconcentration are categorized in electrophoresis-based and chromatography-based preconcentration. The chromatography-based preconcentration is subdivided in low-specificity and high-specificity methods. A number of preconcentration methods are available, however, this paper demonstrates that various compounds in different media (aqueous solutions, urine, and plasma) require different preconcentration systems. The preconcentration techniques of first choice in general seem to be solid-phase extraction and membrane preconcentration, because of their high concentration ability, multiapplicability, relative simplicity and clean-up capability. For the future, hollow fibers seem to hold a great potential as preconcentration technique, yielding high concentration factors, using simple designs. New techniques, such as hollow fibers, molecularly imprinted polymers technology and supported liquid membranes may have the potential to supersede the conventional preconcentration techniques in some cases. The larger the arsenal of preconcentration techniques becomes, the more efficiently peptides and proteins may be analyzed in the future. These techniques, in some cases, require pre-cleanup procedures, to ensure the purity of the samples to concentrate.

Degradation kinetics of aplidine, a new marine antitumoural cyclic peptide, in aqueous solution.

The degradation kinetics of aplidine were investigated using reversed-phase high-performance liquid chromatography combined with UV detection. Aplidine consists of at least two isomers that undergo interconversion at a low rate. Influences of pH, temperature, buffer ions and ionic strength on the degradation kinetics were studied. The log kobs) -pH profile can be divided into three parts, a proton, a solvent and a hydroxyl-catalysed section. The stability-indicating properties of the used analysis technique as well as the identities of the main degradation products were checked using gradient liquid chromatography and mass spectrometric detection. The overall degradation rate constant as a function of the temperature under acidic and alkaline conditions obeys the Arrhenius equation. No catalytic influences were observed with phosphate and carbonate buffers and, in addition, the ionic strength showed no substantial effect on the stability, as expected. Results from gradient LC-MS indicated that hydrolysis of the ester groups present in the ring structure was the main degradation route. There is no difference in degradation rate constants for the individual isomers.

Formation and efficacy of vancomycin group glycopeptide antibiotic stereoisomers studied by capillary electrophoresis and bioaffinity mass spectrometry.

The conformational stability of vancomycin group antibiotics (i.e., vancomycin and avoparcin) in aqueous solution has been studied. These complex glycopeptide antibiotics contain many chiral centers allowing the potential formation of stereoisomers. Using capillary electrophoresis these stereoisomers could be separated and detected by UV and/or mass spectrometry. Fresh aqueous samples of both vancomycin and avoparcin already contained a plethora of stereoisomers. Thermal degradation of the antibiotics was studied as well. For vancomycin thermal degradation led primarily to the formation of CDP-I and aglycons. In the case of avoparcin thermal degradation led mainly to the interconversion between stereoisomers. These antibiotic stereoisomers may exhibit different antibacterial efficacy. Solution-phase association constants of fresh and heated samples of these antibiotics and their bacterial cell wall mimicking receptors were determined by bioaffinity mass spectrometry and revealed that the heated samples exhibited, in general, a lower affinity. Minimum inhibitory concentrations (Micrococcus flavus) were determined and confirmed the decrease in antibacterial efficacy upon heating.

Quantitative analysis of pharmaceutically active peptides using on-capillary analyte preconcentration transient isotachophoresis.

An on-capillary adsorptive phase in combination with capillary electrophoresis (CE), frequently referred to as preconcentration CE, for quantitative analysis of low peptide concentrations was developed. The capillary containing the on-line analyte preconcentrator can be constructed within 5 min from commercially available extraction disks. These disks contain poly(styrenedivinylbenzene) adsorbent particles incorporated in a matrix of inert Teflon, creating a mechanically stable sorbent. Therefore, no frits are needed in the capillary to hold the stationary phase in place. Several parameters, such as the required minimal elution volume, required elution strength, sample application speed or ionic strength, and the capacity were investigated and special interest was given to the quantitative properties of the method. Instead of nL injections, volumes up to a least 25 microL are possible, yielding improvements in detection limits of 3-4 orders of magnitude. The observed limit of detection for both model peptides was 20 pg, corresponding to a 20 microL injection of a 1 ng/mL solution of both model peptides. Using low-wavelength UV detection, reproducibility and linearity in the low nanogram range were satisfactory. No influence of matrix salt concentrations was observed, extending the use of CE to all kinds of samples.

Separation and detection techniques for peptides and proteins in stability research and bioanalysis.

In this paper, a brief overview of the most commonly used methods for the separation and analysis of peptides and proteins in stability and bioanalysis studies is presented. To investigate the physical stability of peptides and proteins, size-exclusion chromatography and electrophoretic separation techniques are being used, apart from several other methods. To determine the chemical stability of these compounds, separation systems are also important, with informative detection modes, such as various spectroscopic detections, electrochemical detection and mass spectrometric detection. For the bioanalysis of peptides, separation is the most important factor, while the detection must be done at the highest possible level of sensitivity.

Derivatization trends in capillary electrophoresis.

This survey gives an overview of recent derivatization protocols, starting from 1996, in combination with capillary electrophoresis (CE). Derivatization is mainly used for enhancing the detection sensitivity of CE, especially in combination with laser-induced fluorescence. Derivatization procedures are classified in tables in pre-, on- and postcapillary arrangements and, more specifically, arranged into functional groups being derivatized. The amine and reducing ends of saccharides are reported most frequently, but examples are also given for derivatization of thiols, hydroxyl, carboxylic, and carbonyl groups, and inorganic ions. Other reasons for derivatization concern indirect chiral separations, enhancing electrospray characteristics, or incorporation of a suitable charge into the analytes. Special attention is paid to the increasing field of research using on-line precapillary derivatization with CE and microdialysis for in vivo monitoring of neurotransmitter concentrations. The on-capillary derivatization can be divided in several approaches, such as the at-inlet, zone-passing and throughout method. The postcapillary mode is represented by gap designs, and membrane reactors, but especially the combination of separation, derivatization and detection on a chip is a new emerging field of research. This review, which can be seen as a sequel to our earlier reported review covering the years 1991-1995, gives an impression of current derivatization applications and highlights new developments in this field.

Comparison between transient isotachophoretic capillary zone electrophoresis and reversed-phase liquid chromatography for the determination of peptides in plasma.

Low levels of peptide drugs in human plasma can be determined employing off-line solid-phase extraction, followed by capillary zone electrophoresis with UV detection. A bioanalytical procedure is presented, using gonadorelin and angiotensin II in human plasma as model compounds. The solid-phase extraction method, based on a weak cation exchange mechanism, is able to remove interfering endogenous components from the plasma sample, extract the model peptides quantitatively, and give a possibility of concentrating the sample at the same time. Transient isotachophoretic conditions were kept to increase the sample loadability by about two orders of magnitude. Up to about 70% of the capillary was filled with the reconstituted extract, whereafter the peptides were selectively concentrated during the first 15 min. Subsequently, the concentrated sample zones were separated under capillary zone electrophoresis conditions, showing the technique's high resolution. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility was observed in the 20-100 ng/mL concentration range. A more extensive washing procedure permits quantitation of gonadorelin at the 5 ng/mL level. In comparison with a liquid chromatography analysis, superior mass sensitivity and separation are obtained with the transient isotachophoretic capillary zone electrophoresis method. Moreover, in this case equivalent sensitivity is achieved when it is directly compared with a liquid chromatography method with UV detection, keeping in mind that 60 times more sample is needed for the latter method. A further gain in sensitivity can be obtained when the analysis is combined with native fluorescence detection, as is demonstrated by combining liquid chromatography separation with fluorescence detection.

Capillary electrophoresis as a versatile tool for the bioanalysis of drugs--a review.

This review article presents an overview of current research on the use of capillary electrophoretic techniques for the analysis of drugs in biological matrices. The principles of capillary electrophoresis and its various separation and detection modes are briefly discussed. Sample pretreatment methods which have been used for clean-up and concentration are discussed. Finally, an extensive overview of bioanalytical applications is presented. The bioanalyses of more than 200 drugs have been summarised, including the applied sample pretreatment methods and the achieved detection limits.

Development and validation of transient isotachophoretic capillary zone electrophoresis for determination of peptides.

Although capillary zone electrophoresis (CZE) is known for its high resolution power and low mass detection limits, the concentration detection limits are rather poor when ultraviolet absorbance detection is used. To overcome this limitation, several on-column transient isotachophoresis (tITP) protocols have been developed and validated for the determination of both cationic and anionic model peptides, separately. Using this preconcentration method, up to 72% of the capillary can be filled with sample solution, without any loss in resolution. Thus, without any modification of the hardware set-up, the sensitivity is increased about two orders of magnitude. For the model cationic peptides (gonadorelin, angiotensin II) good linearity and reproducibility is observed in the 20 to 100 ng/mL concentration range. For the anionic peptides (N-t-Boc-Pentagastrin and two related peptides), a tITP method was developed using a dynamically coated capillary. The coating was prepared by adding Fluorad FC-135 to the leading electrolyte buffer. In this way a positively charged bilayer was formed on the inside of the capillary, producing an electroosmotic flow towards the outlet using reversed polarity conditions. In this way, acceptable analysis times were achieved. Using the developed tITP method, up to 72% of the capillary can be filled with sample solution as well. The anionic peptides are separated even better than when using CZE conditions. Linearity and reproducibility in the 20-100 ng/mL range proved to be excellent.

Pre-, on- and post-column derivatization in capillary electrophoresis.

This survey gives a short overview of the various reagents and procedures that can be used for pre-, post- and on-column derivatization in capillary electrophoresis. First there is an introduction about capillary electrophoresis as an analytical technique; this is followed by a discussion of the pros and cons of the various modes of derivatization and a comparison with liquid chromatography. In the following paragraphs the reagents for a number of functional groups are discussed. The emphasis is on derivatization of the amino group. Most of the information on the reagents and derivatization procedures is listed in tables together with information on the detection mode, analytes, sensitivity and samples. In addition to the amino group, information is given on labeling of aldehyde, keto, carboxyl, hydroxyl and sulfhydryl groups.

Reversed-phase high-performance liquid chromatography and capillary electrophoresis in the stability study of the neuropeptide growth factor antagonist [Arg6,D-Trp7,9,MePhe8]-substance P (6-11): a comparative study.

Reversed-phase high-performance liquid chromatography and capillary zone electrophoresis are widely used in protein and peptide analysis. Degradation of the basic peptide [Arg6,D-Trp7,9,MePhe8]-substance P (6-11) (antagonist G) was monitored with reversed-phase high-performance liquid chromatography, free capillary zone electrophoresis, and capillary zone electrophoresis with a capillary cationic coating. Capillary zone electrophoresis with a dynamically coated capillary provided better separation between antagonist G and its degradation products (formed at pH/Hv 13) than high-performance liquid chromatography and free zone capillary electrophoresis. Rate constants of the alkaline degradation of antagonist G measured with reversed-phase high-performance liquid chromatography and capillary zone electrophoresis with a dynamic coated capillary wall are similar whereas the values measured with free zone capillary electrophoresis are lower. Rate constants for the degradation of antagonist G in acidic media are comparable for the three techniques. It is concluded that capillary zone electrophoresis using a dynamic coating with Fluorad is the most suited of the above-mentioned techniques in analyzing antagonist G and its degradation products.