H.J.E.M. Reeuwijk

Simultaneous determination of furosemide and amiloride in plasma using high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method using fluorescence detection for the simultaneous determination of furosemide and amiloride is described. The chromatographic system is based on reversed-phase ion-pair chromatography with sodium dodecylsulphate as ion-pairing agent. The same counter-ion is used for the ion-pair liquid-liquid extraction to ethyl acetate. The minimum detectable concentration amounts to 0.3 ng of furosemide and 0.03 ng of amiloride per ml of plasma. The applicability of the method is demonstrated by the analysis of plasma samples taken from volunteers receiving both drugs.

Liquid chromatographic determination of β-cyclodextrin derivatives based on fluorescence enhancement after inclusion complexation

A liquid chromatographic method using fluorescence detection for the determination of beta-cyclodextrin (beta CD) and its derivatives is presented. The chromatographic system is based on size-exclusion chromatography with the addition of the fluorophoric compound 1-naphthol to the mobile phase. Detection is based on fluorescence enhancement caused by the formation of inclusion complexes. By incorporating 10(-4) M 1-naphthol in the mobile phase, detection limits of 90, 27, 370 and 37 pmol were obtained for beta CD, hydroxypropyl-beta CD, trimethyl-beta CD and dimethyl-beta CD, respectively. The method was applied to the determination of dimethyl-beta CD in urine: the minimum detectable concentration was 0.2 microgram/ml after preconcentration of 10 ml of urine.

Bioanalysis of fluorouracil applying liquid chromatography and valve switching

SummaryFluorouracil has been analyzed with a reversed-phase ion-pair liquid chromatographic system applying PRP-1 as the stationary phase. A valve switching technique was applied for the separation of the analyte and some other chemotherapeutic agents. The described procedure gives a rapid screening for the analyte in urine and plasma samples without interferences from late eluting solutes. Biological samples are pretreated by means of a liquid-liquid extraction resulting in a recovery of about 52% for plasma and urine with minimum detectable amounts of about 5ng/ml. The sensitivity and the selectivity of the procedure allows the monitoring of fluorouracil in body fluids and subsequent pharmaco-kinetic studies of the fluoropyrimidine.

Bioanalysis of suramin in human plasma by ion-pair high-performance liquid chromatography.

A liquid chromatographic method is described that can be used for the determination of suramin in plasma samples from cancer patients treated with this drug. The chromatographic system is based on the use of tetrabutylammonium bromide as an ion-pairing agent, while ultraviolet detection is applied. The sample pretreatment is a simple deproteination step by an organic solvent. The same counter-ion as used in the phase system is added in order to increase the recovery of the almost complete protein-bound suramin. The minimum detectable concentration in plasma is ca. 0.1 microgram/ml, thus allowing the monitoring of patients treated with this drug. One example of a plasma concentration-time course after administration of suramin is given.

Reversed-phase ion-pair analysis of 5-fluorouracil in pig bile, liver homogenate, plasma and urine after administration by isolated liver perfusion

5-Fluorouracil is at present one of the most administered cytostatic drugs in cancer chemotherapy. However, due to its high toxicity, local administration of the drug, e.g. by isolated liver perfusion, may be advantageous. A bioanalytical procedure, suitable for the routine analysis of 5-fluorouracil in pig bile, liver homogenates, plasma and urine is described using reversed-phase ion-pair chromatography with absorbance detection at 268 nm. Using a protein precipitation procedure with acetonitrile, a determination limit of 3500 ng g-1 was achieved for liver samples, and 5 ng ml-1 for plasma samples using a liquid-liquid extraction step.

Analysis of quaternary ammonium drugs by thermospray liquid chromatography - mass spectrometry using a resin-based stationary phase

Abstract The analysis of quaternary ammonium drugs by thermospray liquid chromatography-mass spectrometry (LC-TSP-MS), using a resin-based stationary phase, is described. The use of such a stationary phase eliminates the need of using an ion-pairing agent, which often is incompatible with mass spectrometric detection. Gradient elution has been used to improve the separation and to reduce the analysis time. Detection limits in selective ion monitoring mode as well as in selective reaction monitoring, are in the order of 50 pg for each compound. The usefulness of the method has been demonstrated with the bioanalysis of antrenyl in plasma, using mepenzolate as an internal standard, showing a limit of determination of 1 ng ml−1.

High-performance liquid chromatographic analysis of isoxazolylpenicillins in plasma and urine samples

SummaryA simple and fast liquid chromatographic method is described, applicable to the routine analysis of isoxazolylpenicillins (cloxacillin, dicloxacillin, flucloxacillin) in biological fluids (plasma, urine). The method is based on a simple dilution step employed to destroy the protein binding, which is over 95%, and allows the detection of concentrations down to 10µg/ml. In order to analyze concentrations of less than 10µg/ml, a liquid-liquid extraction with dichloromethane must be executed prior to the reversed-phase analysis with absorbance detection at 206nm. The minimum detectable amounts of the isoxazolylpenicillins with this procedure are between 2.5 and 5.1 ng in 100µl plasma samples. The stability of the penicillin samples in aqueous solutions (stock solutions, eluents) was investigated and no significant degradation was observed during the storage and analysis of the samples. Furthermore, the degree of protein binding was established by using a suitable ultrafiltration technique, and the usefulness of the developed procedures in pharmacokinetic studies was demonstrated.

Sensitive and selective bioanalytical assay for the determination of dobutamine in rat plasma samples

SummaryDobutamine is one of the synthetic catecholamines acting directly onβ1-receptors. For the analysis of dobutamine in rat plasma samples, a selective and sensitive liquid chromatographic method is described. After a simple liquid-liquid extraction, separation of the analyte was performed using a reversed-phase ion-pair system with an octyl modified silica column. The solute was detected by fluorescence detection, applying an excitation wavelength of 285nm and an emission wavelength of 313nm. The (im)possibilities of the application of the normally used assays for the isolation, concentration and quantitation of catecholamines are discussed. By the addition of a minimum amount of modifier to the mobile phase, the selectivity of the system was increased significantly. With this method the detection limit is 9ng/ml in 0.2ml plasma samples. The application of the method is shown in rat plasma samples by measuring the concentration-time curves to establish plasma level-effect relationships for this drug.