Rokus A. de Zeeuw

Recent innovations in the use of charged cyclodextrins in capillary electrophoresis for chiral separations in pharmaceutical analysis

A review is presented on the use of charged cyclodextrins (CDs) as chiral selectors in capillary electrophoresis (CE) for the separation of analytes in pharmaceutical analysis. An overview is given of theoretical models that have been developed for a better prediction of the enantiomeric resolution and for a better understanding of the separation mechanism. Several types of charged CDs have been used in chiral capillary electrophoretic separation (anionic, cationic, and amphoteric CDs). Especially the anionic CDs seem to be valuable due to the fact that many pharmaceutically interesting compounds can easily be protonated (e.g., amine groups). For that reason several anionic CDs are now commercially available. Cationic and amphoteric CDs are less common in chiral analysis and only a few are commercially available. Attention is paid to the most common synthesis routes and the characterization of the CDs used in chiral capillary electrophoretic separations. The degree of substitution in the synthesized CDs may vary from one manufacturer to another or even from batch to batch, which may have a detrimental effect on the reproducibility and ruggedness of the separation system. In Sections 4, 5, and 6 the applications of anionic, cationic, and amphoteric CDs for the chiral separation in CE are described. Many interesting examples are shown and the influence of important parameters on the enantioselectivity is discussed.

Solid-phase extraction procedures in systematic toxicological analysis.

In systematic toxicological analysis (STA) the substance(s) present is (are) not known at the start of the analysis. In such an undirected search the extraction procedure cannot be directed to a given substance but must be a general procedure where a compromise must be reached in that the substances of interest are isolated at a yield as high as possible and the interfering substances from the biological material are removed. When using solid-phase extraction (SPE) it is desirable to have procedures using just one column. An overview of screening procedures using diatomaceous earth, polystyrene-divinylbenzene copolymer and mixed-mode bonded silica as column material in SPE is given. The latter type of sorbent is most popular at the moment and the critical steps in the procedure are outlined in more detail. Recent developments of SPE disks look very promising for STA.

Cannabinoids with a propyl side chain in cannabis: occurrence and chromatographic behavior

Neutral cannabinoids with a pentyl side chain—for example, cannabidiol, tetrahydrocannabinol, and cannabinol—are generally accompanied by homologs with a propyl side chain, of which at least one has psychotropic activity. Samples of hashish and marihuana from Asia especially sometimes have abundant amounts of propyl cannabinoids, the quantities being of the same order as that of the accompanying pentyl cannabinoids. Detection and identification of the propyl and pentyl cannabinoids in gas chromatography and thin-layer chromatography is discussed.

Δ1-Tetrahydrocannabinolic acid, an important component in the evaluation of cannabis products*

The occurrence and some analytical properties of Δ1‐etrahydrocannabinolic acid have been investigated, including its chromatographic behaviour in the presence of other cannabinoids. The acid is inactive but is converted on smoking into the active tetrahydrocannabinol. The acid is present in abundant amounts in various cannabis samples, marihuana in particular, and these will be more active on smoking than when administered by injection or orally. A method for the separate determination of tetrahydrocannabinol and its acid is also described.

Enantioseparation of ofloxacin in urine by capillary electrokinetic chromatography using charged cyclodextrins as chiral selectors and assessment of enantioconversion

A method was developed for the enantioseparation of ofloxacin, a member of the fluoroquinolones, using an anionic cyclodextrin‐derivative with or without combination with a neutral cyclodextrin‐derivative, as the chiral selector (s) in an electrokinetic chromatography system. The best results were obtained with 0.35 mM sulfated β‐cyclodextrin dissolved in a 50 mM phosphate buffer, pH 2.5, and at 15°C. Under these conditions, a resolution of 2 was readily achieved. Furthermore, under adequate separation conditions, studies were performed in order to assess possible in vitro and in vivo enantioconversion of levofloxacin. The current method allows detection of 2 μg R‐(+)‐ofloxacine/mL diluted urine without the necessity of sample cleanup.

ANALYSIS OF QUATERNARY AMMONIUM-COMPOUNDS AND BASIC DRUGS BASED ON ION-PAIR ADSORPTION HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

Abstract A general approach to the ion-pair adsorption high-performance liquid chromatography of basic drugs and quaternary ammonium compounds ins decribed in which suitable counter ions such as Br − and ClO 4 − are dissolved in the eluent. The columns are packed with silica ( e.g. , Lichrosorb and LiChrosfer). The systems thus obtained showed high efficiencies and stability in which th ecapacity ratios were found to be exponentially dependent on the concentration of the counter ion. This indicates that the separation mechanism is dominated by adsorption processes. The degree of retention and the separation order can be varied by the nature and the concentration of the counter ion, the sorbent and the composition of the eluent.

Selectivity in capillary electrokinetic separations

This review gives a survey of selectivity modes in capillary electrophoresis separations in pharmaceutical analysis and bioanalysis. Despite the high efficiencies of these separation techniques, good selectivity is required to allow quantitation or identification of a particular analyte. Selectivity in capillary electrophoresis is defined and described for different separation mechanisms, which are divided into two major areas: (i) capillary zone electrophoresis and (ii) electrokinetic chromatography. The first area describes aqueous (with or without organic modifiers) and nonaqueous modes. The second area discusses all capillary electrophoretic separation modes in which interaction with a (pseudo)stationary phase results in a change in migration rate of the analytes. These can be divided in micellar electrokinetic chromatography and capillary electrochromatography. The latter category can range from fully packed capillaries, via open‐tubular coated capillaries to the addition of microparticles with multiple or single binding sites. Furthermore, an attempt is made to differentiate between methods in which molecular recognition plays a predominant role and methods in which the selectivity depends on overall differences in physicochemical properties between the analytes. The calculation of the resolution for the different separation modes and the requirements for qualitative and quantitative analysis are discussed. It is anticipated that selectivity tuning is easier in separation modes in which molecular recognition plays a role. However, sufficient attention needs to be paid to the efficiency of the system in that it not only affects resolution but also detectability of the analyte of interest.

Screening for the presence of drugs in serum and urine using different separation modes of capillary electrophoresis

Capillary electrophoresis (CE) is a modern separation technique that has some distinct advantages for toxicological analysis, such as a high efficiency, fast analysis, flexibility, and complementary separation mechanisms to chromatographic methods. CE can be applied in various modes, which each have a different separation mechanism or selectivity. The most common mode is capillary zone electrophoresis (CZE), in which charged analytes migrate in a buffer under the influence of an electric field. In micellar electrokinetic chromatography (MEKC), micelles are added to the buffer which interact with the analytes. MEKC can also be used for the separation of neutral compounds. In non-aqueous CE (NACE), the aqueous buffer is replaced by a background of electrolytes in organic solvents. A sample that needs to be screened can easily be analyzed subsequently by these CE modes using the same instrumentation. The aim of the study was to develop procedures for the analysis of basic and acidic drugs in serum and urine using CZE, MEKC, and NACE. A test mixture that consisted of six basic and six acidic compounds was used to study the separation behavior of five CE methods. The results showed that three methods (based on CZE, MEKC, and NACE) were suitable for the analysis of basic compounds and three methods (based on CZE and MEKC) for the analysis of acidic compounds. For the extraction of analytes from serum and urine, a solid-phase extraction (SPE) and a liquid-liquid extraction (LLE) method were compared. Both SPE and LLE methods provided clean extracts after extraction of the basic compounds from serum and urine. The extracts of acidic compounds contained more matrix interferences, especially for urine. The SPE method had some advantages compared to LLE, as it lead to cleaner extracts and higher peaks, and as it elutes basic and acidic compounds in one fraction. The potentials and pitfalls of the various methods for screening purposes in analytical toxicology are discussed.

Potential and pitfalls of chromatographic techniques and detection modes in substance identification for systematic toxicological analysis

The potential and the constraints of thin-layer chromatography (TLC), gas chromatography (GC) and high-performance liquid chromatography (HPLC) towards substance identification, together with their detection modes, are considered. The latter include colour reactions on the plate, molecular masses through chemical ionization mass spectrometry (MS) and diode-array UV spectrophotometry. Evaluations are carried out by the mean list length approach. Not surprisingly, GC-MS and HPLC-diode array detection qualify as the two most powerful combinations. However, one does not necessarily need to have access to these sophisticated detection modes: the identification power of TLC and colour reactions plus GC or HPLC retention indices is high and even a suitable combination of TLC and colour reactions remains a valuable tool. After analysis, the findings for the unknown substance(s) must be matched against databases containing the behaviour of reference substances. The search process for the computerized retrieval of potential candidates must allow the handling of all possible combinations of identification techniques applied.

Intra- and interinstrument reproducibility of migration parameters in capillary electrophoresis for substance identification in systematic toxicological analysis.

The intra‐ and interinstrument reproducibilities of four capillary electrophoresis instruments were studied for identification purposes in systematic toxicological analysis (STA). A test set of 20 acidic test compounds and 5 reference compounds were analyzed for five days on each instrument using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). The buffers consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate and 50 mM sodium dodecyl sulfate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries at an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA. To deal with the poor reproducibility of the migration time, we recently introduced the corrected effective mobility. In this study, we investigated the intra‐ and interinstrument reproducibility of the migration time, the effective mobility, and the corrected effective mobility. Large differences in intra‐instrument reproducibility were found when the migration time was used. The calculation of the effective mobility and the corrected effective mobility diminished these differences and enhanced the interinstrument reproducibility roughly by a factor 3. For (corrected) effective mobilities, intrainstrument reproducibilities were between 0.8—2.6% and interinstrument reproducibilities were between 3.2—3.9%.