ISOLATION OF THE 9-FLUORENYLMETHYL CHLOROFORMATE DERIVATIVE IN THE DETERMINATION OF SULFAMETHAZINE

Derivatisation of amine-containing analytes with 9-fluorenylmethyl chl oroformate (FMOC) to form fluorescent adducts requires a large excess of FMOC. This excess hydrolyses to form FMOC-OH, which is also fluores cent. Solvent extraction has been investigated as a means of isolating the sulphamethazine (SMZ) adduct (FMOC-SMZ) from the hydrolysis produ ct in order to perform rapid spectrophotometric or spectrofluorimetric assays. However, even under the most favourable pH conditions possibl e, FMOC-OH was not totally removed. Attempts to enhance the separation by reaction of FMOC-OH with 1-ethoxy-4-dichloro-S-triazinylnaphthalen e (EDTN) or by acetylation were also unsuccessful. On the other hand, reaction of FMOC with mixed substrates, followed by two pentane extrac tions to remove the excess FMOC and direct injection into an HPLC prov ides the desired separations on a reversed phase column (RPLC) with me thanol-modified, (pH 3.5) phosphate buffers. FMOC-SMZ is readily separ ated from FMOC-OH under all elution conditions, from the FMOC-amino ac ids (under gradient conditions or isocratically up to 75% methanol), a nd from other FMOC-sulphonamides and FMOC-dihydrofolate reductase inhi bitors (isocratically up to 70% methanol). Hence conversion to the FMO C derivatives permits SMZ to be separated from all of the potential in terferants tested by isocratic elution with 70% methanol in RPLC. Anal ysis for the amino acid derivatives of FMOC may be done without interf erence from SMZ in samples.