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 2h 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.