Evaluation of capillary electrophoretic techniques towards systematic toxicological analysis

Chapter 15 - Capillary electrophoresis

Two capillary electrophoresis (CE) methods were developed for the analysis of 16 antineoplastic drugs contained in injectable pharmaceutical formulations. A capillary zone electrophoresis (CZE) method coupled to UV was developed with a background electrolyte (BGE) made of a 100 mM phosphate buffer at pH 2.5 containing 50% v/v of acetonitrile and dynamic coating of capillaries with Ceofix®. This method allowed the analysis of doxorubicin, epirubicin, idarubicin, daunorubicin, irinotecan, topotecan, vincristine, vindesine, vinblastine, and vinorelbine in less than 8 min. A micellar electrokinetic chromatography (MEKC) method coupled to UV was also developed for the determination of methotrexate, pemetrexed, etoposide, etoposide phosphate, fludarabine phosphate, and 5-fluorouracil. A run time of 16min was obtained with a BGE made of 50mM borate buffer at pH 9.2 with 80mM of sodium dodecyl sulfate (SDS) and 20% v/v of acetonitrile. For both methods, the applied voltage was 30kV and the sample injection was performed in the hydrodynamic mode. All analyses were carried out in fused silica capillaries with an internal diameter of 50μm and a total length of 64.5cm. Both methods were validated and trueness values between 99.4 and 101.3% were obtained with repeatability and intermediate precision values of 0.5-1.8% for all drugs. These methods were found appropriate for controlling injectable pharmaceutical formulations containing antineoplastic drugs and successfully applied in quality control.

Chapter 10 Separation of nucleic acids and oligonucleotides and sequencing of DNA Fragments by capillary electrophoresis

Nine xanthones from a Chinese traditional medicine, Securidaca inappendiculata Hassk, were separated by different capillary electrophoresis (CE) modes, including capillary zone electrophoresis (CZE), micellar electrokinetic chromatography (MEKC), microemulsion electrokinetic capillary chromatography (MEEKC), and capillary electrochromatography (CEC). The comparison of separation selectivity and efficiency for xanthones by using these CE methods indicated that different CE modes greatly varied in the separation selectivity and efficiency for xanthones; their advantages and disadvantages are discussed. Moreover, compared with traditional chromatographic methods, e.g., high performance liquid chromatography (HPLC), CE shows higher separation ability and analytical speed for these xanthones.

Determination of illicit and/or abused drugs and compounds of forensic interest in biosamples by capillary electrophoretic/electrokinetic methods

Chromatographic techniques are basic tools in systematic toxicological analysis. Extensive data bases with retention parameters of known drugs to aid in the identification of substances found are available or in preparation. For a search in such a data base the computer is indispenable. The commonly used window search has some disadvantages which can be overcome by a search based on the statistical concept, the mean list length. The latter retrieval system gives for each candidate in the identification process a probability value. It is shown that these probability values are highly influenced by the reproducibility of the retention parameters of the analytical systems used. Explanations for these phenomena are given.

Separation of stereoisomers of aminoglutethimide using three capillary electrophoretic techniques

Capillary electromigration methods for the study of collagen

Influence of organic solvents on the separation selectivity in capillary electrophoresis

Capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with sodium dodecyl sulfate (SDS) as an anionic carrier were applied to the analysis of nine principal components of eye lotion. Simultaneous separation of these components except cyanocobalamin (VB12), which has the same migration time with that of electroosmotic flow, was achieved by CZE using a 72 cm×50 μm i.d. fused silica capillary tube filled with 20 mM borate buffer (pH 10) at 25 kV DC voltage with UV detection at 200 nm. Under these conditions, the peak of chondroitin sulfate (CS) was overlapped with that of aspartic acid in case the latter was contained in the sample as a co-existing component. However, when pH was decreased to 9, CS was separated from other components and also from aspartic acid, and gave a more shapely peak. VB12 could be separated from other components by MEKC with 20 mM borate buffer (pH 10) containing 40 mM SDS. The applicability to the determination of nine principal components of eye lotion is demonstrated.

Various modes of capillary electrophoresis offer numerous possibilities for biopharmaceutical analysis. Chapter 2 studied capillary zone electrophoresis (CZE) for characterization of glycoprotein erythropoietin by its isoform distribution. The CZE method for identification of recombinant human erythropoietin described in the European Pharmacopoeia has shown poor reproducibility. In this study, it was found that the root cause of the irreproducibility was due to inadequate capillary conditioning. The method was optimized to make it more robust and suitable for quality control laboratory for analysis of epoetin alfa. Further, a procedure for removal of polysorbate 80 from formulated epoetin alfa was developed, allowing the material to be analyzed using the modified CZE method. Chapter 3 describes a micellar electrokinetic chromatography (MEKC) method for the determination of residual cell culture media components including folic acid, hypoxanthine, mycophenolic acid, nicotinic acid, riboflavin, and xanthine. Acceptable limits of detection and quantitation were obtained. The MEKC method was compared to the corresponding CZE method using the same running buffer containing no sodium dodecyl sulfate (SDS). The effect of SDS concentration on separation, the pH of the running buffer, and the detection wavelength were studied and optimal MEKC conditions were established. Chapter 4 describes a capillary SDS gel electrophoresis method for purity and impurity analysis of monoclonal antibodies. The study investigated the effect of sample buffer pH, incubation temperature and duration, alkylation conditions with iodoacetamide, and reduction conditions with 2-mercaptoethanol. It was observed that the sample buffer at slight acidic conditions (pH 5.5-6.5) greatly decreased thermally induced fragmentation of non-reduced CNTO3157. As such, a citrate-phosphate buffer at pH 6.5 was used for sample preparation to replace the commonly used Beckman sample buffer (pH 9.0). Chapter 5 details an investigation of the effect of sample injection length on efficiency and detection sensitivity enhancement in MEKC using the sweeping technique under conditions of suppressed electroosmotic flow. The relationship between injection length and retention factor was derived and experimentally confirmed. A method for predicting the optimal injection length for a given analyte was developed.

In capillary electrophoresis, head-column field-amplified sample stacking (FASS) provides the largest sensitivity enhancement of all electrokinetic concentration techniques (Zhang, C.-X.; Thormann, W. Anal. Chem. 1996, 68, 2523). Application of head-column FASS to the analysis of closely related opioids by capillary zone electrophoresis in binary systems with ethylene glycol is described. It is shown that sample condensation is further increased about 2-fold by introduction of a preinjection plug, i.e., introduction of a short solution plug of high conductivity, high pH, and high viscosity at the capillary tip prior to injection. The preinjection plug acts as a temporary trap for solutes. Its effective length is shown to be limited to a few millimeters. The highest sample stacking efficiency in head-column FASS is obtained via optimization of the electric field strength and the effective electrophoretic mobility of the solutes within the sample and the adjacent zones and is thus strongly dependent on the compositions of both the sample matrix and the preinjection plug. Binary sample solutions of low conductivity and low viscosity containing small amounts of a weak acid are demonstrated to be most effective for the stacking of positively charged opioids. The procedure developed for capillary zone electrophoresis of opioids in binary systems with ethylene glycol and UV absorbance detection is documented to provide a 3 orders of magnitude sensitivity enhancement, exhaustive sample injection from an external reservoir of up to 20 microL (i.e., from a volume that is more than 20-fold the volume of the capillary employed), and a lowest detectable concentration of 0.1 ng/mL (S/N = 3). Using internal calibration, typical intraday and interday imprecisions of solute concentrations between 3 and 10 ng/mL and between 0.5 and 1.5 ng/mL are < or = 5% and < or = 15%, respectively. The stacking approach has been successfully applied to the analysis of dihydrocodeine in extracts of 20 microL of human plasma and is shown to permit the precise (imprecision < or = 10%) determination of dihydrocodeine plasma levels of pharmacological interest (3-300 ng/mL or 10-1000 nM). Furthermore, using a modified protocol for micellar electrokinetic chromatography, head-column FASS is shown to provide a 400-fold sensitivity enhancement for a number of opioids. The stacking procedure is based on insertion of a surfactant-free preinjection plug and temporary application of reversed voltage after electroinjection.

High throughput, outstanding certainty in peptide/protein identification, exceptional resolution, and quantitative information are essential pillars in proteome research. Capillary electrophoresis (CE) coupled to mass spectrometry (MS) has proven to meet these requirements. Soft ionization techniques, such as matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), have paved the way for the story of success of CE-MS in the analysis of biomolecules and both approaches are subject of discussion in this article. Meanwhile, CE-MS is far away from representing a homogeneous field. Therefore the review will cover a vast area including the coupling of different modes of CE (capillary zone electrophoresis, capillary isoelectric foscusing, capillary electrochromatography, micellar electrokinetic chromatography, nonaqueous capillary electrophoresis) to MS as well as on-line preconcentration techniques (transient capillary isotachophoresis, solid-phase extraction, membrane preconcentration) applied to compensate for restricted detection sensitivity. Special attention is given to improvements in interfacing, namely addressing nanospray and coaxial sheath liquid design. Peptide mapping, collision-induced dissociation with subsequent tandem MS, and amendments in mass accuracy of instruments improve information validity gained from MS data. With 2-D on-line coupling of liquid chromatography (LC) and CE a further topic will be discussed. A special section is dedicated to recent attempts in establishing CE-ESI-MS in proteomics, in the clinical and diagnostic field, and in the food sector.

Evaluation of the enantioseparation capability of the novel chiral selector clindamycin phosphate towards basic drugs by micellar electrokinetic chromatography

Systematic toxicological analysis (STA) is the process of using an adequate analytical methodology to detect and identify as many potentially toxicologically relevant compounds as possible in biological samples. STA is an important part of everyday routine work within forensic toxicology, and several methods for STA have frequently been published and reviewed independently. However, the many drugs and other substances involved, as well as the constant emergence of new ones, may pose a major challenge in STA, which often demands a strategy involving multiple analytical methods in parallel. Such strategies have been published and evaluated less frequently despite their relevance in forensic toxicology. This mini-review briefly summarizes commonly applied methods for STA in forensic toxicology, including gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-MS (LC-MS) methods, and highlights some of their potential pitfalls. Second, it provides an overview of previously reported strategies to conduct STA, including a presentation of the STA strategy applied in the authors' laboratory. This involves broad drug screening by LC-high-resolution MS, supported by targeted screening and quantification using LC-tandem MS, headspace (HS)-GC-MS, HS-GC-flame ionization detector and other complementary methods. The STA strategy aims to cover as many potentially relevant drugs as possible and seeks to reduce potential pitfalls arising in forensic casework. The review underlines that not every substance can be identified in all circumstances even with a comprehensive STA strategy.