2.2.3. HPLC of amines as 9-fluorenylmethyl chloroformate derivatives

9-fluorenylmethyloxycarbonyl chloride, also known as 9-fluorenylmethyl chloroformate (FMOC-Cl), reacts with primary and secondary amines and yields highly fluorescent and stable derivatives. It has been used for the derivatization and analysis of various types of amines including biogenic amines and polyamines; short-chain aliphatic amines; neurotransmitters, including catecholamiens, and metanephrines. FMOC-Cl can also react with the amino groups of a number of drugs to yield the highly fluorescent derivatives for HPLC analysis. This approach is particularly useful to the analytes without chromophore or fluorophore for detection. This chapter reviews the experimental procedures on precolumn derivatization of these amines with FMOC-Cl for fluorescence detection after reversed phase separation. The increase in sensitivity by FMOC derivatization can be more than 3 orders of magnitude, when compared to the usual detection by UV absorbance. Table 1 summarizes the HPLC conditions for the detection of these amines. Despite the high sensitivity of these FMOC derivatives for HPLC analysis, there are some disadvantages associated with this approach, especially in the analysis of biological samples. One of the disadvantages is that many endogenous components in the biological matrix are also reactive towards the derivatizing reagents. Thus, extensive sample clean-up is usually required. Very often, solid phase extraction is employed to extract the analytes from the matrix, followed by derivatization, and in many instances, re-extraction of the derivatized analytes to remove the un-reacted reagent and concentration of the derivatives. As a result, the sample preparation can be very tedious and time-consuming. The second disadvantage of the approach is the interference to the analytes during chromatography from the un-reacted FMOC-Cl and also its hydrolysis product, FMOC-OH. To overcome this problem, an excess of glycine can be added to the reacting mixture, after derivatization of the analytes is complete. By doing this, the excess FMOC-Cl is converted to the relatively hydrophilic glycine derivative that elutes early in the chromatogram, not interfering detection of the much smaller peak of the analyte derivatives. With the advance in column technology, the problem of interference from the derivatizing agent may be further minimized by using the new generation of columns with higher resolution power.