1-pyrenyldiazomethane and 9-diazomethylanthracene

Highly effective separation and highly sensitive detection reagents for clinical chemistry and biochemistry were developed and their applications were investigated. The sensitive detection of carboxylic acids was accomplished using 9-anthryldiazomethane (ADAM) which gave intensely fluorescent derivatives from carboxylic acids without catalysts or heating. 1-Pyrenyldiazomethane was then synthesized and proved to react also readily with carboxylic acid and more sensitive than ADAM. The optical resolution of amino acid enantiomers was achieved using 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (GITC). GITC derivatized enantiomeric amino acids under mild conditions to give highly hydrophobic diastereomers which could be resolved on conventional reversed phase columns and easily detected by the absorption based on the thiourea structure. Then we devised an o-phthalaldehyde-N-acetylcystein reagent (OPA/NAcCys) giving diastereomers which were also resolved on a reversed phase column and detected fluorometrically with excellent sensitivity. OPA/NAcCys was useful for the assay of D-amino acids in the blood of uremic patients. The hypochlorite-thiamine method for the assay of proteins and peptides was established providing sensitive fluorometry, which well reflected the number of peptide groups in the molecule. This principle was applied to the assay using N-chlorodansylamide, designed for the fluorometry of peptides. The alkaline ninhydrin method was applied to the detection of guanidino compounds in the blood of uremic patients. Several fluorometric methods for the simultaneous detection of reducing and non-reducing carbohydrates, and guanidines were found to be useful reagents for this purpose because these compounds were resistant to the oxidation with periodate. Then protamine-bound columns were prepared for the separation of carbohydrates on HPLC, which showed excellent recovery of reducing carbohydrates in comparison with conventional alkylamino columns.

Topics: Amino Acids; Anthracenes; Biochemistry; Carbohydrates; Carboxylic Acids; Chemistry, Clinical; Chromatography, High Pressure Liquid; Fluorometry; Guanidines; Humans; Indicators and Reagents; Isothiocyanates; Peptides; Proteins; Pyrenes; Sensitivity and Specificity

Several derivatization reagents for the conversion of okadaic acid and related DSP toxins to fluorescent derivatives for analysis by liquid chromatography have been examined, viz: 9-anthryldiazomethane (ADAM), 1-pyrenyldiazomethane (PDAM), 4-diazomethyl-7-methoxycoumarin (DMMC), 4-bromomethyl-7-methoxycoumarin (BrMMC), 4-bromomethyl-7,8-benzcoumarin (BrMBC), 4-bromomethyl-7-acetoxycoumarin (BrMAC), and 4-bromomethyl-6,7-dimethoxycoumarin (BrDMC). The ADAM reagent provides the greatest selectivity and sensitivity, but its application on a routine basis has been limited by its instability and cost. Improvement of this method was achieved through the production of ADAM in situ from the stable 9-anthraldehyde hydrazone. A detection limit of 30 ng/g hepatopancreas (equivalent to 6 ng/g whole tissue) was achieved. The other aryldiazomethane reagents were found to have insufficient reactivity. Of the bromomethylcoumarin reagents, BrDMC was found to have the greatest promise. The reagent is inexpensive and has excellent stability and purity. Quantitative derivatization may be achieved in a 2 hour reaction at 45 degrees C with N,N-diisopropylethylamine as a catalyst. Unfortunately, the lower reaction selectivity of BrDMC compared to that of ADAM limits its application to isolated toxins, plankton samples, and shellfish tissues with high levels of DSP toxins. The use of BrDMC for the determination of how toxin levels in shellfish tissues will require development of a more extensive clean-up prior to derivatization. Successful application of the ADAM and coumarin derivatization methods to real-world samples has been demonstrated.

Topics: Animals; Anthracenes; Chromatography, High Pressure Liquid; Coumarins; Dinoflagellida; Ethers, Cyclic; Fluorescent Dyes; Marine Toxins; Okadaic Acid; Pyrans; Pyrenes; Shellfish; Spectrometry, Fluorescence; Temperature; Umbelliferones