Analysis of drugs in the presence of serum albumin by liquid chromatography with eluents containing surfactants.

Surfactant-containing eluents are evaluated for possible application to direct serum injection using conventional reverse-phase columns. Serum albumin was quantitatively eluted at the column void volume by using surfactant concentrations below or above the critical micelle concentration, and organic solvents could be used in proportions as high as 40% (w/w). Surfactant choice, pH, and salt effects were also evaluated.

Analysis of carbamazepine and its 10,11-epoxide in serum by direct sample injection using surfactant containing eluents and column switching.

Column switching is used in conjunction with surfactant containing mobile phases and traditional reverse phase LC columns to provide a highly reproducible and accurate analytical procedure for carbamazepine and its 10,11-epoxide in serum. This approach eliminates the tedious sample preparation steps commonly used in the analysis of drugs by HPLC, while providing a high degree of protein removal prior to the final analysis. Various pre-columns were investigated and a pellicular reverse phase column was found suitable for optimum concentration of drugs and removal of serum proteins. A variety of standard reverse phase columns could be used for the analytical separation. The separation of the drugs could be accomplished with a high degree of reproducibility. Tandem pre-column operation was demonstrated to give a sample throughput of 10 h-1.

Analysis of carbamazepine in serum by liquid chromatography with direct sample injection and surfactant-containing eluents.

Surfactant-containing eluents are evaluated for the analysis of carbamazepine in serum with conventional reversed-phase columns. Bovine serum was quantitatively eluted at the column void volume using surfactant concentrations in conventional reversed-phase eluents. The effect of pH, guard columns and column switching was evaluated with respect to separating and detecting clinical levels of the drug and its primary metabolite. Column lifetime was also investigated.

Polymeric benzotriazole reagent for the off-line high-performance liquid chromatographic derivatization of polyamines and related nucleophiles in biological fluids.

A polymeric benzotriazole reagent containing a 9-fluorenylmethyleneoxycarbonyl (FMOC) group has been synthesized, characterized, and its derivatizations, off-line, for three polyamines, have been optimized with regard to solvent, time, and temperature. An authentic FMOC derivative of cadaverine has been prepared, characterized, and used as the external standard for quantitation of off-line derivatizations and identification of final derivatives. Actual percent derivatizations have been determined, rather than just percent disappearance of starting material. The polyamines in urine or other biological fluids can be derivatized without organic solvent or solid phase extraction, but rather in situ by the simple addition of the polymeric reagent to the fluid, incubation for a few minutes at room or elevated temperature, filtration and direct injection. Derivatizations could also be performed by transferring a small volume of the hydrolyzed and filtered biological fluid to a disposable pipette containing the polymeric reagent. Derivatization was then followed by elution, filtration, and direct injection onto the high-performance liquid chromatographic (HPLC) system. Automation of the overall polymeric derivatization, filtration, HPLC injection, separation, detection, quantitation, and data acquisition-interpretation is suggested. The polymeric reagent has been utilized for the qualitative and quantitative determination of cadaverine and putrescine, normally occurring polyamines, in human urine. These levels were compared with the corresponding literature values for healthy human subjects, and the values were found to be in excellent agreement. This novel derivatization approach, though off-line, provides for a much simpler, more rapid, and more efficient conversion of these and related polyamines or nucleophiles to derivatives having vastly improved chromatographic detection properties in HPLC. The final derivatives contain the FMOC group, making them extremely chromophoric and fluorophoric, and providing trace detection at ppb (microgram/l) and sub-ppb levels. The overall approach is recommended for these and other biologically occurring polyamines, in fluids and tissues, as well as related bioorganic and biologically active nucleophiles, including drugs and their metabolites.

New polymeric benzotriazole reagents for off-line derivatizations of amines and polyamines in HPLC.

New polymeric reagents are synthesized, based on a polystyrene-bound benzotriazole containing an o-acetylsalicyl or 9-fluorenyl labelling moiety. This is used in an off-line mode, prior to high performance liquid chromatography (HPLC), for derivatizations of trace primary and secondary amines, polyamines, and related compounds in connection with HPLC. Standards are prepared, characterized by physical and spectral properties, and then used as external standards to determine percent derivatizations. The polymeric reagents are characterized by elemental analyses and loading determinations. The feasibility and applicability of this reagent for derivatization of nucleophiles is confirmed with a number of amines under optimized conditions. The activated labelling moiety, bonded to the polymeric support, makes the derivatization reactions extremely rapid and efficient under mild reaction conditions. This alone provides significant advantages over the analogous solution derivatizations for the same amines. A comparison of solution and solid phase derivatization reactions is reported. The limits of detection are 1 to 2 pmol for polyamines, such as cadaverine, putrescine, and 1,7-diaminoheptane, using the benzotriazole fluorenyl reagent followed by fluorescence detection.

A new, rapid, high-sensitivity analysis of amino acids in food type samples.

A new approach to the analysis of free amino acids and amino acids from hydrolyzed foods is described. The method is based on reaction of the free amino acids with phenylisothiocyanate to form stable derivatives which are subsequently separated by liquid chromatography. Sample preparation procedures are described and results are compared with conventional ion exchange results. Reproducibility of the new method has been determined on a typical food type sample.

PITC derivatives in amino acid analysis.

Refined methods for separating PTC-amino acids on reverse phase columns may pose a challenge to traditional ion exchange techniques.

Rapid analysis of amino acids using pre-column derivatization.

A new approach to the pre-column derivatization and analysis of amino acids is described. The method is based upon formation of a phenylthiocarbamyl derivative of the amino acids. The derivatization method is rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The liquid chromatographic system allows for the rapid, bonded-phase separation with ultraviolet detection of the common amino acids with 12-min analysis time and a 1-pmol sensitivity.

Bare silica as a reverse-phase stationary phase: liquid chromatographic separation of antihistamines with buffered aqueous organic mobile phases.

Unbonded silica gel is an effective support for reverse-phase liquid chromatographic separations of lipophilic amines. Simple buffered aqueous-organic mobile phases provide rapid isocratic separations of antihistamines. Phenylpropanolamine hydrochloride, chlorpheniramine maleate, and dextromethorphan hydrobromide are separated in 8 min on bare silica with a mobile phase of 75% methanol and 25% water which is 0.01 M in (NH4)2HPO4. Quantitation is reproducible. A wide variety of additional compounds may be separated using the same mobile phase.