Abstract
A sensitive and specific method for the analysis of oral hypoglycemic biguanides, metformin, buformin, and phenformin, was developed by use of high performance liquid chromatography (HPLC) with fluorometric detection. These biguanide drugs are converted to 2-alkylguanidino-1H-phenanthro[9, 10-d]imidazole derivatives by pre-column fluorescence labeling with 9, 10-phenanthraquinone (PQ) in an alkaline medium. The derivatives are separated on a reversed′phase column with 0.1M phosphate buffer (pH 2.5) containing 75 (v/v)% acetonitrile. Monosubstituted guanidines which exist in biological fluids also react with PQ in the same reaction conditions as the biguanides to form a fluorescent product, 2-amino-1H-phenanthro[9, 10-d]imidazole (API). However, they do not interfere with the analysis of the Biguanides in the elution system. The lower detection limits of biguanides added to serum are 0.2ng for metformin and 0.4ng for both buformin and phenformin, per 10μl of injection volume. The recoveries of 0.1μg biguanides added to 0.5ml of serum are in the range 96-102%.
