Using Solid‐Phase Microextraction Gas Chromatography‐Mass Spectrometry and High Performance Liquid Chromatography with Fluorescence Detection to Analyze Fluorescent Derivatives of the Biogenic Amines Creatine and Creatinine

Abstract

To develop a method whereby polar biological metabolites can be analyzed using readily available GC instrumentation and enhance detection limits of small water‐soluble amines as a potential clinical diagnostic method for analyzing both biologically produced amines as well as synthetic pharmaceutical compounds, we have investigated two derivatization techniques for biogenic amines employing fluorescent compounds, namely 9‐fluorenylmethyl chloroformate (FMOC) and 1‐pyrenyldiazomethane (PDAM). Both compounds provide a stable, non‐polar element to the derivatized amines that facilitates the use of GC‐MS methods for analysis as well as allow for the use of fluorescence detection, which increases overall sensitivity. The study presented herein is an application of these derivatization techniques to the simple biogenic amines creatine and creatinine, followed by analysis using GC‐MS with ECD detection and HPLC with both UV and fluorescence detection. Results will show that the coupling of creatine to PDAM and creatinine to FMOC allow both compounds to enter the gas phase to a sufficient extent for gas chromatographic separation and mass spectrometric identification. In terms of fluorescence detection, 0.5 µg creatinine that is derivatized with FMOC is at least seven times more sensitive using fluorescence detection when compared to UV detection of the same compound.