Brian A. Bidlingmeyer

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.

Pre-chromatographic derivatization of primary and secondary amines with a polymeric anhydride for improved high-performance liquid chromatographic detection

Abstract A polymeric anhydride containing o-acetylsalicyl as the labelling moiety was utilized as a derivatization reagent in conjunction with high-performance liquid chromatography for primary and secondary amines. The derivatization reactions were performed off-line, before the chromatographic separation. Standards were prepared, characterized by melting point, UV, IR, NMR, mass spectrometry, and elemental analysis, and these were then used as external standards to determine the percent reaction. The derivatives are easily chromatographed on a reversed-phase high-performance liquid chromatographic system and can be monitored by a UV detector at 196 nm, or an electrochemical detector in the oxidative mode, with or without post-column photolysis. No pH suppression of the eluent was needed. The minimum detection limits of underivatized and derivatized amines were determined. There was a decrease of 3 to 4 orders of magnitude in minimum detection limits as a result of these off-line derivatizations. The minimum amount of amine that can be detected through derivatization is also reported.

Derivatization of alkyl halides and epoxides with picric acid salts for improved high-performance liquid chromatographic detection

Abstract The use of picric acid (2,4,6-trinitrophenol)_salts present in solution or on a solid support for use as leabelling reagents for alkyl halides and epoxides is discussed. These derivatization reactions were performed off-line before the chromatographic separation. Labelled standards were prepared in solution, and characterized by melting point, IR, NMR, mass spectrometry and elemental analysis. These were then used as external standards to quantitate the percent derivatization under a variety of reaction conditions. The labelled derivatives can be monitored with a UV detector set at 220 nm, ane elctrochemical detector in the reductive mode, or with an electrochemical detector in the oxidative mode after post-column photolysis.

Polymeric benzotriazole reagent for the off-line high-performanceliquid 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.

Solid Phase Derivatizations in HPLC: Borohydride/Silica Reductions for Carbonyl Compounds

Abstract Sodium borohydride adsorbed onto silica gel has now been utilized for on-line, pre-and post-analytical column chemical derivatizations via reductions of various organic carbonyl compounds. These on-line reactions have been performed using normal phase HPLC conditions, involving conventional silica gel packings, organic mobile phases, and commercially available HPLC equipment and instrumentation. This approach for on-line HPLC derivatizations has been evaluated for a large number of organic carbonyl compounds, at a variety of temperatures for aldehydes and ketones. The overall rates of such carbonyl reductions via sodium borohydride/silica are sufficiently different as a function of temperature of the reaction to permit for compound/class identifications. Analyte identification can be both qualitative and quantitative, even wherein an analyte co-elutes with a non-carbonyl compound. In-house prepared borohydride/silica gel reactors can be characterized via standard iodine titration procedures along...

Amino Acid Analysis of Submicrogram Hydrolyzate Samples

Understanding the mechanisms by which diseases occur is a complex process, and often involves the isolation and characterization of minute quantities of rare proteins from living tissue. Amino acid analysis is a vital tool for the elucidation of the structure of such molecules, but today’s researchers must operate at or beyond the utmost limits of conventional methods. In February, 1984 (1) we reported on a new approach to precolumn derivatization and analysis of amino acids which appeared to satisfy all the needs of protein chemists. The approach was based upon the formation of a phenylthiocarbamyl (PTC) derivative of the amino acids (2). The method was shown to be rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydrolyzates. The method allows for the rapid, bonded phase separation with ultraviolet (UV) detection at 254 nm of the common amino acids with 12 minute analysis time and a one picomole sensitivity for a standard amino acid sample.

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.

On the need for “Wide-Pores” in the Reversed-Phase Liquid Chromato- Graphic Separation of Proteins

Abstract Pore size information for 13 commercially available reversed-phase columns is determined by application of the GPC technique described by Halasz and Martin. In most cases, the experimental median pore diameter (MPD) is in good agreement with the nominal pore size for the packing material, although the discrepancies are large in a few cases. The measured MPDs are used as a basis for identifying whether pore size is a major contributor to resolution, peak width and sample capacity in the reversed-phase separation of proteins on commercially available columns.

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.

Determination of low molecular weight molecules in creams and ointments by gel permeation chromatography

Abstract The analysis of low molecular weight compounds can frequently be achieved by small molecule gel permeation chromatography (SMGPC). In the SMGPC mode, large molecules are excluded, while separation of the analytes is based on the effective size of the compounds of interest in solution. It is possible to analyze two compounds whose molecular weight differ by 10% or more by the judicious selection of the mobile phase. For instance, the separation of tolnaftate (the active ingredient of an anti-fungal preparation) and BHT (which was present as an antioxidant), was accomplished using methylene chloride, a non-hydrogen bonding solvent. For this assay, sample preparation is very simple and the overall analysis takes only 12 minutes.