J.P. Franke

Multiresidue analysis of β2-agonists in human and calf urine using multimodal solid-phase extraction and high-performance liquid chromatography with electrochemical detection

Abstract Various β2-agonists are used as illegal growth promoters in man and in animals. We developed a multiresidue procedure for the analysis of four β-agonists in human and calf urine. The sample was pre-extracted with an Extrelut column at alkaline pH. The β-agonists were eluted with a mixture of tert.-butylmethyl ether and hexane. Then the extract was further cleaned with a mixed mode SPE column, or with a combination of immunoaffinity chromatography (IAC) and the mixed mode SPE column. The IAC column contained antibodies against salbutamol, which were suitable for multiresidue extractions. The extract was then brought onto a mixed mode SPE column at an acidic pH. The column was washed with 70% methanol in water. Thereafter, the β-agonists were eluted with ammoniated ethanol–hexane. The extract was analysed with an HPLC method with electrochemical detection. The β-agonists were separated on a reversed-phase column using a mobile phase buffered at pH 5.5 and containing an ion-pair reagent. Recoveries were higher when the IAC procedure was not performed (90–105% vs. 65–75%), but the extracts were cleaner when the latter step was included. Detection limits in human and calf urine were in the low ng/ml range. The study indicated that β2-agonists can be analysed in human and calf urine without the selectivity of a mass spectrometer, but that comprehensive clean-up is required to avoid the interference of urine matrix components.

TOXICOLOGICAL ANALYSIS OF WHOLE-BLOOD SAMPLES BY MEANS OF BOND-ELUT CERTIFY COLUMNS AND GAS-CHROMATOGRAPHY WITH NITROGEN-PHOSPHORUS DETECTION

The application of Bond-Elut Certify solid-phase extraction columns to the systematic toxicological analysis of whole blood was evaluated. The reproducibility of the extraction was tested with thirteen drugs varying in physico-chemical properties. Analysis was performed with capillary gas chromatography with nitrogen-selective detection. The recoveries were reproducible, as long as other limiting factors, e.g., chromatographic behaviour or volatility, do not play a significant role. The effect of limiting chromatographic behaviour was studied in more detail with the more sensitive mass spectrometry with selected-ion monitoring after converting the extracted morphine into its ditrimethylsilyl derivative.

Dynamically coated capillaries improve the identification power of capillary zone electrophoresis for basic drugs in toxicological analysis

In systematic toxicological analysis (STA), analytical methods should have a high identification power. This can be suitably expressed by parameters such as mean list length (MLL) or discriminating power (DP). The reproducibility of a method has a great impact on its identification power, and should be as high as possible. In this study, two separation methods based on capillary zone electrophoresis (CZE) were evaluated towards STA applications. Besides a normal phosphate buffer, the commercially available buffer CElixir was used, which is a double-layer dynamic coating system. The coating stabilizes the endoosmotic flow, is independent of the pH, and is claimed to be more reproducible and faster at low pH than with normal buffers. A test set of 73 basic pharmaceutical compounds was analyzed by the two CZE methods. The total analysis time, including rinsing steps, was 8 min when the coating was used and 18 min without the coating. Effective mobilities were calculated and the reproducibilities were a factor of 2 better when the coating was used (between-days SD 0.020 and 0.040 m2/V s with and without the coating, respectively). MLL and DP were calculated for the two CZE methods and for combinations with standardized liquid and gas chromatography systems. CZE with CElixir coating clearly has a high potential for STA applications, as it was shown to have a higher identification power and shorter analysis times than normal CZE.

HEMOPERFUSION-HEMODIALYSIS INEFFECTIVE FOR PARAQUAT REMOVAL IN LIFE-THREATENING POISONING

We report on a patient treated with hemoperfusion-hemodialysis (HP-HD) for severe paraquat poisoning. This procedure was adopted since the combination of adsorption and dialysis may improve overall drug removal. On admission blood paraquat was 15.8 micrograms/ml. He received conventional treatment and combined HP-HD which started within 3 hours after ingestion of the chemical and lasted 5 hours. Blood samples were obtained during and after HP-HD. The samples during HP-HD were taken before the charcoal column, between the charcoal column and the artificial kidney and after the artificial kidney. Blood clearances of paraquat were 116 +/- 32 ml/min (n=6) for the charcoal column (HP), 90 +/- 54 ml/min (n=6) for the artificial kidney (HD) and 151 +/- 37 ml/min (n=6) for the combined systems (HP-HD). After HP-HD a limited rebound of blood paraquat level was seen. One day after admission renal and hepatic failure had developed, and the patient died after 5 days. Tissue paraquat levels (microgram/g wet tissue) were: skeletal muscle 9.4, pancreas 6.0, prostate 5.6, thyroid 4.2, lungs 4.0, bone marrow 4.0, kidney 3.1, spleen 2.9, adrenal 2.9, heart 2.8, liver 2.3, stomach and testis below 1.0. Measurements of blood levels demonstrated the efficient clearances of paraquat with HP-HD from the central (plasma) compartment. However, the present results confirmed those previously reported which suggest that the efficiency of short HP-HD in treating severe paraquat poisoning is questionable since paraquat levels in the peripheral (tissue) compartment remain elevated.

Evaluation of capillary electrophoretic techniques towards systematic toxicological analysis

Two capillary electrophoresis (CE) methods were evaluated for their suitability in systematic toxicological analysis (STA). A test set of 25 barbiturates was analysed using capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC). Buffers used consisted of 90 mM borate set at pH 8.4 (CZE) and 20 mM phosphate, 50 mM sodium dodecyl sulphate set at pH 7.5 (MEKC). All analyses were carried out using fused silica capillaries using an electric field strength of 52.6 kV/m. The use of a reproducible identification parameter is very important in STA as it influences the identification power (IP). To deal with the poor reproducibility of the migration time, we introduced the corrected effective mobility. Inter-day reproducibilities of the latter parameter were < 0.6% for CZE and < 0.5% for MEKC, using daily prepared buffers. The IP of the methods was expressed by calculation of the discriminating power and the mean list length. Data obtained were compared to gas chromatographic and high-performance liquid chromatographic data, and correlations between all methods were calculated. It was shown that little correlation exists between chromatographic and electrophoretic techniques. The results indicated that CE has a good identification power for the application in STA, especially when a combination of methods having a low correlation is used.

DIFFERENTIAL PULSE ANODIC-STRIPPING VOLTAMMETRY AS A RAPID SCREENING TECHNIQUE FOR HEAVY-METAL INTOXICATIONS

This paper describes a relatively simple and rapid analytical procedure capable to carry out systematic screening for 10 heavy metals, namely mercury, bismuth, antimony, copper, lead, tin, thallium, indium, cadmium and zinc.Differential pulse anodic stripping voltammetry was chosen because of its simplicity, speed, low cost, low detection limits, screening potential for a wide selection of metals and the combined qualitative and quantitative output. The method of identification is based on measuring peak potentials in 2 different types of electrolyte solution, using 2 different electrodes. Complexing agents are used to mask certain elements.ZusammenfassungEs wird ein relativ einfaches und schnelles analytisches Verfahren beschrieben, das imstande ist, eine systematische Untersuchung von zehn Schwermetallen auszuführen: Quecksilber, Wismut, Antimon, Kupfer, Blei, Zinn, Thallium, Indium, Cadmium und Zink. Gewählt wurde die inverse Pulsvoltammetrie mit Differenzstrommessung aus nachfolgendem Grunde: Einfachheit, Geschwindigkeit, niedrige Kosten, niedrige Detektionsgrenze, Untersuchungsvermögen für eine Reihe von Metallen und kombinierte qualitative und quantitative Ergebnisse. Die Identifikation ist begründet auf der Messung der Peakpotentiale in zwei verschiedenen Lösungen mit zwei verschiedenen Elektroden und mit oder ohne Komplexbildner.

Feasibility of the direct coupling of solid-phase extraction-pipette tips with a programmed-temperature vaporiser for gas chromatographic analysis of drugs in plasma.

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.

Impact of substituents on the enantioseparation of racemic 2‐amidotetralins on polysaccharide stationary phases. I. chiralcel OD

The direct enantiomeric separation of 32 racemic 2-amidotetralins on the commercially available tris-(3,5-dimethylphenylcarbamate) derivative of cellulose, coated on silica gel (Chiralcel OD), is presented. To date, the selection of a column for the chiral separation of a racemic mixture is done empirically. Studying the impact of small changes in the chemical structure of a series of amidotetralins on the separation behavior may help to give an insight in the chiral recognition mechanism. The amidotetralins differed structurally in three of their substituents, which were never directly located on the chiral carbon atom. The enantiomers of 24 out of 32 amidotetralins could be resolved with a resolution >1.5. Hydrogen bonding and pi-pi interactions are supposed to be the major analyte-chiral stationary phase (CSP) interactions. However, the spatial arrangement of the enantiomers may play an important role too. Increasing the bulkiness of the acyl substituent led to an increase in the resolution (R(s)), whereas a more bulky substituent on the aromatic ring resulted in a very low resolution. The introduction of a chlorine atom into the acyl substituent additionally increased the resolving power

THIN-LAYER CHROMATOGRAPHY UNDER TROPICAL CONDITIONS - IMPACT OF HIGH-TEMPERATURES AND HIGH HUMIDITIES ON SCREENING SYSTEMS FOR ANALYTICAL TOXICOLOGY

The impact of high temperatures (33-38 degrees C) and high relative humidities (80-100%) on the applicability of TLC systems for drug identification was studied during a six month climatologic cycle in Jakarta, Indonesia. In general, the R(F) values as observed on the plates were substantially affected in comparison to values obtained at moderate climates: most substances gave higher R(F) values under the tropical conditions, although exceptions may occur as well. The deviations tended to increase with increasing humidities and could amount easily to 20-30 R(F) units. On the other hand, some TLC systems were more affected than others. Tropical conditions also had a negative effect on the reproducibility of the R(F) values. However, when an R(F) correction procedure was applied, using reference mixtures of standard drugs on each plate, accuracies as well as reproducibilities of the resulting R(F)(c) values were drastically improved and data thus corrected were found to be compatible with existing TLC data bases developed under moderate climatic conditions. These results are in line with earlier studies carried out in a relatively dry tropical climate. In the latter the observed R(F) values tended to be lower than the ones published in the literature, but the R(F) correction procedure was able to correct for this phenomenon.

How to approach substance identification in qualitative bioanalysis

The ultimate goal in qualitative analysis in the biosciences is to demonstrate with acceptable probability that for an unknown constituent in a sample only one substance comes into consideration and that all other substances can be rejected. In the biosciences, identification of relevant substances in complex matrices through database retrieval is frequently required. Yet, despite its importance, the subject has not received much attention, so that progress has been limited and relevant literature is scarce. As a result, one can conclude from many publications and reports that qualitative analysis in practice is often not being addressed properly. In this paper, some fundamental aspects of qualitative analysis will be discussed and a general approach is provided for the correct identification of organic substances in complex matrices through database retrieval. Special attention is given to the choice of proper analytical techniques and their inter-laboratory standard deviations, as well as to match factors and decision criteria based on applying multiple analytical techniques, also if the latter have different dimensions (e.g. retention data and spectral data). In addition, the requirements for suitable databases are outlined and the need for inter-laboratory cooperation is emphasized.