Luteinizing hormone-releasing hormone (LH-RH) is the blood-borne messenger between the hypothalamus and the anterior pituitary which controls reproductive function. The therapeutic usefulness of LH-RH would be limited because of its short biological half-life and also because of rapid inactivation in vivo. Rat ovulation studies revealed Leuprolide to be 50-80 times more active than LH-RH. Leuprolide is chemically defined as: p-Glu-L-His-L-Trp-L-Ser-L-Tyr-D-Leu-L-Leu-L-Arg-L-Pro-ethylamide. An HPLC method was developed to analyze component amino acids in Leuprolide acetate (except Pro) using phanquinone derivatization and fluorometric detection. The amino acid analysis, indeed, presents difficulty because a weak detectability of the compounds. A fluorogenic derivatization prior to HPLC separation constitutes an effective and convenient technique to overcome the problem and to perform sensitive analyses. In previous studies (1,2) phanquinone reagent, devoid of significant native fluorescence, was found to react selectively with the primary amino function to give stable and highly fluorescent iminoquinols without degradation product forming. The peptide was hydrolyzed with HCl 37% at reflux for 3 h and the resulting solution was derivatized and analyzed. The amino acid was found to react in 45 min at 50°C using 0.1 M phosphate buffer (pH 7). The RP-HPLC separations of the amino acid adducts were performed at 33±2°C on a Phenomenex Prodigy 5u ODS (250 x 3.2 mm i.d.) with gradient elution using a mobile phase consisting of a mixture A/B where A=0.05 M triethylamine phosphate buffer (pH 3) and B=methanol, at a flow rate of 0.4 ml/min. The fluorescence intensity at 460 nm (excitation at 400 nm) was monitored. The proposed method, proved to be selective, sensitive, linear, precise and accurate and was applied with satisfactory results to the determination of Leuprolide acetate amino acids in commercial injection solution. (1) R. Gatti, M.G. Gioia, A.M. Di Pietra, Anal. Chim. Acta 474 (2002) 11-20. (2) R. Gatti, M.G. Gioia, P. Andreatta, G. Pentassuglia, J. Pharm. Biomed. Anal. (2003) in press.
M.G. Gioia, R. Gatti (2004). LC ANALYSIS OF LEUPROLIDE COMPONENT AMINO ACIDS IN INJECTABLE SOLUTION BY PHANQUINONE PRE-COLUMN DERIVATIZATION LABELLING PROCEDURE. FLORENCE : s.n.
LC ANALYSIS OF LEUPROLIDE COMPONENT AMINO ACIDS IN INJECTABLE SOLUTION BY PHANQUINONE PRE-COLUMN DERIVATIZATION LABELLING PROCEDURE
GIOIA, MARIA GRAZIA;GATTI, RITA
2004
Abstract
Luteinizing hormone-releasing hormone (LH-RH) is the blood-borne messenger between the hypothalamus and the anterior pituitary which controls reproductive function. The therapeutic usefulness of LH-RH would be limited because of its short biological half-life and also because of rapid inactivation in vivo. Rat ovulation studies revealed Leuprolide to be 50-80 times more active than LH-RH. Leuprolide is chemically defined as: p-Glu-L-His-L-Trp-L-Ser-L-Tyr-D-Leu-L-Leu-L-Arg-L-Pro-ethylamide. An HPLC method was developed to analyze component amino acids in Leuprolide acetate (except Pro) using phanquinone derivatization and fluorometric detection. The amino acid analysis, indeed, presents difficulty because a weak detectability of the compounds. A fluorogenic derivatization prior to HPLC separation constitutes an effective and convenient technique to overcome the problem and to perform sensitive analyses. In previous studies (1,2) phanquinone reagent, devoid of significant native fluorescence, was found to react selectively with the primary amino function to give stable and highly fluorescent iminoquinols without degradation product forming. The peptide was hydrolyzed with HCl 37% at reflux for 3 h and the resulting solution was derivatized and analyzed. The amino acid was found to react in 45 min at 50°C using 0.1 M phosphate buffer (pH 7). The RP-HPLC separations of the amino acid adducts were performed at 33±2°C on a Phenomenex Prodigy 5u ODS (250 x 3.2 mm i.d.) with gradient elution using a mobile phase consisting of a mixture A/B where A=0.05 M triethylamine phosphate buffer (pH 3) and B=methanol, at a flow rate of 0.4 ml/min. The fluorescence intensity at 460 nm (excitation at 400 nm) was monitored. The proposed method, proved to be selective, sensitive, linear, precise and accurate and was applied with satisfactory results to the determination of Leuprolide acetate amino acids in commercial injection solution. (1) R. Gatti, M.G. Gioia, A.M. Di Pietra, Anal. Chim. Acta 474 (2002) 11-20. (2) R. Gatti, M.G. Gioia, P. Andreatta, G. Pentassuglia, J. Pharm. Biomed. Anal. (2003) in press.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
