Abram Hulshoff

Fluorescence detection in high performance liquid chromatography

Abstract The principles and applications of fluorescence detection and fluorescence introducing reagents and methods in HPLC are reviewed. The design and requirements for fluorescence detectors, flow cells and excitation sources and the conversion of non-fluorescent compounds into fluorescent products by pre-column and post-column derivatization reactions are discussed. For the applications the emphasis is on drug analysis, where possible in biological fluids (serum, urine, etc.). The last paragraphs are divided in a number of sections in which newly developed and some scarcely used reagents are mentioned shortly; a more complete treatment is given of the reagents and labels most frequently used in the derivatization of certain functional groups. In this discussion the methods of derivatization as well as the selectivity, stability, fluorescence behaviour of the reagents/labels and derivatives and the reaction conditions are included. An up-to-date survey of the applications of fluorescence detection in l...

Rapid, sensitive and specific derivatization methods with 9-(hydroxymethyl)anthracene for the fluorimetric detection of carboxylic acids prior to reversed-phase high-performance liquid chromatographic separation

Three derivatization procedures are described for the pre-column fluorescence labelling of carboxylic acids. The methods are based on esterification with 9-(hydroxymethyl)anthracene. The carboxylic acid function is activated with 2-bromo-l-methylpyridinium iodide, N,N′-carbonyldiimidazole or N-ethyl-N′-(3-dimethylanopropyl)carbodiimide hydrochloride, respectively. Benzoic acid was completely converted into the corresponding ester with all three methods. About 100 fmol of the acid could be detected after high-performance liquid chromatographic analysis of the derivative. The methods are well suited for the analysis of carboxylic acids in plasma.

High-performance liquid chromatographic analysis of basic compounds on non-modified silica gel and aluminium oxide with aqueous solvent mixtures

A comparison is made between the use of aluminium oxide and non-modified silica gel as cation-exchange materials for the separation of basic drugs (amines) with aqueous solvent mixtures. The retention behaviour of the amines is studied and appears to be controlled predominantly by the pH and the concentration and nature of the modifier; the nature and concentration of the competing ions and the buffer components of the mobile phase also exert some influence on the retention. Preparations with imidazoline and tetracycline derivatives have been analysed as examples of the application of these ion-exchange systems on non-modified silica gel and aluminium oxide in the analysis of pharmaceutical formulations.

A reversed-phase thin-layer chromatographic method for the determination of relative partition coefficients of very lipophilic compounds

A reversed-phase thin-layer chromatographic method has been developed for the determination of partition coefficients. A support phase has been chosen, following investigation of the lack of adsorptive properties, which has a minimal effect on the pH of the buffer system. A stationary phase has been chosen to give deltaRm values of the same magnitude as Hansch pi values for a series of phenothiazines. The method can be applied to molecules of a wide range of lipophilicity following preliminary investigations of suitable phase-volume ratios and of the pH and composition of the binary mobile phase, providing adsorption on the support phase is excluded.

Micro determination of gentamicin in serum by high-performance liquid chromatography with ultraviolet detection

A procedure for the high-performance liquid chromatographic determination of gentamicin in serum is described using pre-column derivatisation and UV detection. The serum proteins are precipitated with acetonitrile and the gentamicin components in the supernatant are derivatized with 1-fluoro-2,4-dinitrobenzene. The reaction products are chromatographed on a microparticulate C18 reversed-phase column and detected at 365 nm. Sample volumes of 50 microliters are sufficient for the determination of gentamicin concentrations in, and well below, the therapeutic range.

Micro-determination of tobramycin in serum by high-performance liquid chromatography with ultraviolet detection.

A procedure for the high-performance liquid chromatographic determination of tobramycin in serum is described using pre-column derivatisation with 1-fluoro-2,4-dinitrobenzene and subsequent chromatographic analysis on a reversed-phase column with ultraviolet detection. Gentamicin is used as the internal standard. The sensitivity is 0.5 mg/l with 50-microliters samples. Precision, expressed as the coefficient of variation, is 3% or better in the concentration range 0.5-16 mg/l. The absolute recovery of tobramycin is 41%. The analyses of serum samples obtained in an in vivo experiment correlated well with the results from a microbiological assay. The influence of variation of derivatisation conditions and the implications for the reliability of the internal standardisation were studied. The 2,4-dinitrophenyl tobramycin derivative was synthesized and its structure was proved to be the fully derivatized tobramycin. Side-products of the derivatisation reaction were isolated.

Ion-exchange phenomena and concomitant pH shifts on the equilibration of reversed-phase packings with ion-pairing reagents

Abstract Breakthrough patterns of eluents containing tetrabutylammonium or cetyltrimethylammonium ions (two frequently used ion-pairing agents in high-performance liquid chromatography) have been studied, using reversed-phase columns with octadecyl chains permanently bonded to 10-μm silica particles. The occurrence of pH shifts either before or after the breakthrough of the ion-pairing agents has been demonstrated. An explanation for the breakthrough patterns is offered and evidence for two different binding mechanisms of these agents is presented.

Alkylation with alkyl halides as a derivatization method for the gas chromatographic detemination of acidic pharmaceuticals

Abstract The various types of alkylation reactions with alkyl halides and their application in the gas chromatographic analysis of acidioc compounds of pharmaceutical interest are reviewed. An extensive survey of the use of these methods for the analysis of various (classes of) compounds is given, with special reference to their determination in biological matrices.

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.

Improved detectability of barbiturates in high-performance liquid chromatography by pre-column labelling and ultraviolet detection

2-Naphthacyl derivatives of barbiturates are formed in acetone at 30° within 30 min, in an essentially quantitative manner, using caesium carbonate as a catalyst. These derivatives absorb UV radiation strongly at 254 nm. Using a variable-wavelength detector set at 249 nm, 1 ng of N,N-dinaphthacylphenobarbital could be detected after chromatography using a high-performance liquid chromatograph equipped with a microparticulate reversed-phase column. This derivatization technique has the potential of allowing the determination of barbiturates, in small plasma or serum samples, in concentrations well below the therapeutic range.