LC determination of leuprolide component amino acids in injectable solution by phanquinone pre-column derivatization labelling procedure

A sensitive LC method for the determination of leuprolide acetate component amino acids in injectable solution with fluorogenic pre-column derivatization has been developed. The derivatization reaction with phanquinone was optimised by a series of experiments. Histidine, arginine, serine, tryptophan, glutamic acid, tyrosine, methionine, isoleucine, leucine and phenylalanine were separated on a reversed-phase ODS column using as eluent a binary mixture of triethylammonium phosphate buffer-methanol, under gradient elution conditions. The derivatives were eluted in 30 min with good reproducibility. The hydrolysis reaction of the peptide was carried out at reflux with 12N hydrochloric acid for 2 h 30 min. The intra-day accuracy of the entire procedure (hydrolysis, derivatization, LC separation) ranged from 80.5 to 109.5% of the nominal concentration of leuprolide acetate and the precision (%R.S.D.) was less than 5.8%; the inter-day accuracy was in the range 81.5-107.2% and corresponding R.S.D. values were less than 4.6%. The detection limits (signal-to-noise ratio = 3) for the adducts are 30-800 fmol.