J.J.M. Holthuis

High-performance liquid chromatographic determination of amantadine in urine after micelle-mediated pre-column derivatization with 1-fluoro-2,4-dinitrobenzene

Cationic micelles have been used for the derivatization of the anti-Parkinson drug amantadine with the chromophore 1-fluoro-2,4-dinitrobenzene in urine. In the presence of 90 mM cetyltrimethylammonium bromide (CTAB), the conversion of amantadine into its derivative is complete within 4 min at 60 degrees C and pH 11. Such a short reaction time allows a fully automated pre-column derivatization of amantadine in an on-line combination with reversed-phase high-performance liquid chromatography. This cannot be attained when using purely aqueous derivatization mixtures because then the reaction takes some 20 min at the same temperature. Without the use of an internal standard, the repeatability of the automated determination at the 0.5 microgram ml-1 level is ca. 6%, whilst the detection limit is 75 ng ml-1 (S/N = 3). The present study clearly demonstrates that micellar systems can be beneficially used for the on-line precolumn derivatization of amines in urine.

Solid-phase extraction of vinblastine and vincristine from plasma and urine: variable drug recoveries due to non-reproducible column packings

A sensitive and selective high-performance liquid chromatographic (HPLC) method for the determination of vinblastine and vincristine in plasma and urine is described. The drugs are isolated from 1.0 ml of the biological fluid with a solid-phase extraction column (Bond-Elut Diol). The HPLC method was combined with electrochemical detection at +850 mV versus an Ag/AgCl reference electrode. The detection limit is 100 pg for vinblastine and 250 pg for vincristine with a signal-to-noise ratio of 3, which permits the determination of these compounds in biological fluids at the nanogram level. Evaluation of the isolation method revealed that the drug recoveries and the reproducibility of the extraction procedure depend on the batch number of the solid-phase extraction column used.

Automated high-performance liquid chromatographic determination of plasma free fatty acids using on-line derivatization with 9-bromomethylacridine based on micellar phase-transfer catalysis

The on-line use of micellar phase-transfer catalysis is described for the automated reversed-phase high-performance liquid chromatographic (RP-HPLC) determination of free fatty acids in plasma; minimum manual sample handling is involved. After diluting plasma ten-fold with the aqueous micellar system, which contains 25 mM of the non-ionic surfactant, Arkopal N-130 and 6 mM of the ion-pair agent tetrakis(decyl)ammonium bromide, the reaction of the fatty acids with the fluorophore 9-bromomethylacridine is complete within 5 min at 60 degrees C. Prior to RP-HPLC separation, interfering proteins are removed using an on-line filter and a column-switching unit. More than 100 samples can be injected onto a single pre-column. The detection limit is ca. 300 nM; the precision is better than 3% using an internal standard.

Derivatization of carboxylic acids with 9-bromomethylacridine using micellar phase-transfer catalysis

SummaryMicellar phase-transfer catalysis (MPTC) offers the opportunity to derivatize carboxylic acids directly in an aqueous matrix without prior extraction of the acids into a suitable aprotic solvent. The currently developed MPTC system consists of a non-ionic surfactant, Arkopal N-130, an ion-pair agent, tetrakis-(decyl)-ammonium bromide, and a novel fluorescence reagent, 9-bromomethylacridine. The MPTC system can be applied to the derivatization of many types of carboxylic acids. The reaction rate is affected by the lipophilicity of the acid and by the presence of other functional groups. For lipophilic carboxylic acids the reaction is complete within 5 min at 60°C and pH 7.0.

Automated monitoring of amino acids during fermentation processes using on-line ultrafiltration and column liquid chromatography: application to fermentation medium improvement

Abstract An automated system for the monitoring of amino acids during fermentation processes is described and evaluated. The system is based on the on-line combination of a hollow-fibre ultrafiltration module, for the removal of cellular and macromolecular material from the broth, and ion-exchange chromatography with post-column o -phthalaldehyde derivatization, for determination of amino acids in the filtrate. As the complete sampling system can be steam-sterilized together with the fermenter, use of the monitoring system does not increase the risk of infection. The method is used to study the amino acid metabolism of a recombinant Escherichia coli , cultured on a complex nitrogen source. Monitoring of the 24-h fermentation can be performed unattendedly and the analysis frequency of once per hour provided detailed information with regard to the consumption of 15 amino acids. Subsequent improvement of the fermentation medium led to an increase in productivity of ca. 35%. In addition, preliminary results about the monitoring of peptides during this fermentation are reported.

Mechanistic study on the derivatization of aliphatic carboxylic acids in aqueous non-ionic micellar systems

Abstract A model is proposed for the derivatization mechanism of carboxylic acids with a fluorescent label, 4-bromomethyl-7-methoxycoumarin, in an aqueous non-ionic micellar system with the use of a tetraalkylammonium ion-pairing cation. Using experimentally obtained extraction constants and partition coefficients for aliphatic carboxylic acids in the micellar solution, the model was compared with the observed derivatization rate constants of these acids; this gave a satisfactory correlation. Owing to its similarity with phase-transfer catalysis, the present micelle-mediated derivatization is termed micellar phase-transfer catalysis.

On-line method for the generation of electrochemical reagent generation for liquid chromatography with luminol-based chemiluminescence detection

Abstract An on-line method for the generation of electrochemical reagent for liquid chromatography, with luminol-based chemiluminescence detection, has been developed. An ESA Coulochem guard cell, equipped with a porous graphite working electrode, operated at − 600 mV and inserted after the column, produces an oxidative reagent for the luminol-based reaction. This method has been compared with the conventional method with post-column addition of hydrogen peroxide as the oxidative reagent. With this novel method a detection limit of 0.15 pmol of ibuprofen (labelled with an isoluminol derivative) can be obtained, and a good alternative for post-column addition of hydrogen peroxide is presented.

Study of the derivatization of n-alkylamines with 1-fluoro-2,4-dinitrobenzene in the presence of aqueous cetyltrimethylammonium bromide micelles

The use of aqueous cetyltrimethylammonium bromide micelles in the derivatization of n-alkylamines with 1-fluoro-2,4-dinitrobenzene was investigated systematically. The rate constants of derivatization of the n-alkylamines (C1-C8) were analysed using liquid chromatography. Up to butylamine the micellar rate enhancement depends on the electrostatic interactions between the amines and cetyltrimethylammonium bromide, and beyond C4 it depends mainly on the hydrophobic interactions. The reaction rates are also enhanced by a micelle-induced decrease of the pKa of the amines, but to a lesser extent. The derivatization rates for the longer alkylamines are comparable with those in dipolar aprotic solvents. Pharmaceutical and biomedical science is likely to benefit from the use of micellar systems in pre-column derivatization reactions in aqueous solutions.

Comparative study on the determination of the anti-neoplastic drug teniposide in plasma using micellar liquid chromatography and surfactant-mediated plasma clean-up

The potential of micellar liquid chromatography and of an on-line surfactant-mediated sample cleanup, which involves column-switching prior to conventional reversed-phase high-performance liquid chromatography, has been evaluated for the determination of the anti-neoplastic drug teniposide in plasma by using electrochemical detection. A major advantage of surfactant-mediated techniques is that they allow fully automated processing of plasma samples, because protein precipitation is prevented by the addition of the surfactant sodium dodecylsulphate. With the automated column-switching technique, a degree of sample enrichment and of selectivity can be attained, which is similar to that for the conventional procedure which, however, involves a labour-intensive off-line isolation of teniposide, using liquid-liquid extraction prior to chromatography. An inherent drawback of automated micellar liquid chromatography is that no sample clean-up or preconcentration can be carried out, which results in only a moderate detection limit and selectivity. The linearity, reproducibility and recovery of the surfactant-mediated techniques are similar to those of the conventional procedure. Based on the presented results, it was concluded that the surfactant-mediated column-switching technique is a highly attractive sample enrichment technique with respect to simplicity, speed and cost.

THE STABILITY OF ANTINEOPLASTIC VINCA ALKALOIDS IN PLASMA AND URINE

Vinblastine and vincristine (Fig. 1) are naturally occurring vinca alkaloids present in the Madagascar Periwinkle plant. Vindesine is a semi-synthetic vinca alkaloid, derived from vinblastine. The vinca alkaloids are active against various types of cancer, e.g. Hodgkin’s disease, lymphomas and testicular carcinoma [1].