title: Combined derivatization and high-performance liquid chromatography with fluorescence and ultraviolet detection for simultaneous analysis of octreotide and gabexate mesylate metabolite in human pancreatic juice samples.
abstract: A simple and sensitive method based on the combination of derivatization and high-performance liquid chromatography with ultraviolet and fluorimetric detection was developed for the simultaneous determination of octreotide and gabexate mesylate metabolite in human pancreatic juice samples. Parameters of the derivatization procedure affecting extraction efficiency were optimized. The developed method was validated according to the International Conference on Harmonization guidelines. The calibration curves were linear over a range of 0.1-15 µg/mL for octreotide and 0.20-15 µg/mL for gabexate mesylate metabolite. Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The limits of detection were 0.025 and 0.05, respectively, for octreotide and gabexate mesylate metabolite. This paper reports the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-FL) method for the simultaneous analysis of octreotide and gabexate mesylate metabolite in pancreatic juice by protein precipitation using zinc sulfate-methanol-acetonitrile containing the derivatizing reagent, 4-fluoro-7-nitro-[2,1,3]-benzoxadiazole (NBD-F). Derivatized products of octreotide and gabexate mesylate metabolite were separated on a Luna C18 column (4.6 × 250 mm; 5 µm particle size) using a gradient with a run time of 36 min, without further purification. The method was validated over the concentration ranges 0.1-15 and 0.2-15 µg/mL for octreotide and gabexate mesylate metabolite, respectively, in human pancreatic juice. Biphalin and methyl-p-hydroxybenzoate were used as the internal standards. This method was successfully utilized to support clinical studies in humans. The results from assay validations show that the method is selective, sensitive and robust. The limit of quantification of the method was 0.1 µg/mL for octreotide and 0.2 µg/mL for gabexate mesylate metabolite, and matrix matched standard curves showed a good linearity up to 15 µg/mL. In the entire analytical range the intra- and inter-day precision (RSD%) values were respectively ≤5.9% and ≤3.1% for octreotide and ≤2.0% and ≤3.9% for gabexate mesylate metabolite. For both analytes the intra- and inter-day accuracy (bias) values ranged respectively from -6.8 to -2.5% and from -4.6 to -5.7%. This method utilizes derivatization with NBD-F and provides adequate sensitivity for both drugs.
authors.affiliation: Università degli Studi 'G. d'Annunzio' Chieti-Pescara, Dipartimento di Farmacia, via dei Vestini 31, 66100, Chieti, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Carlucci
authors.affiliation: Dipartimento di Scienze Cliniche e Sperimentali, via dei Vestini 31, 66100, Chieti, Italy.
authors.forename: Federico
authors.initials: F
authors.lastname: Selvaggi
authors.affiliation: Dipartimento di Scienze Cliniche e Sperimentali, via dei Vestini 31, 66100, Chieti, Italy.
authors.forename: Sara
authors.initials: S
authors.lastname: Sulpizio
authors.affiliation: Università degli Studi di Verona, Dipartimento di Chirurgia, Ospedale 'G.B. Rossi', 37100, Verona, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Bassi
authors.affiliation: Università degli Studi 'G. d'Annunzio' Chieti-Pescara, Dipartimento di Farmacia, via dei Vestini 31, 66100, Chieti, Italy.
authors.forename: Maura
authors.initials: M
authors.lastname: Carlucci
authors.affiliation: Dipartimento di Scienze Cliniche e Sperimentali, via dei Vestini 31, 66100, Chieti, Italy.
authors.forename: Roberto
authors.initials: R
authors.lastname: Cotellese
authors.affiliation: Università degli Studi 'G. d'Annunzio' Chieti-Pescara, Dipartimento di Farmacia, via dei Vestini 31, 66100, Chieti, Italy.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Ferrone
authors.affiliation: Dipartimento di Scienze Cliniche e Sperimentali, via dei Vestini 31, 66100, Chieti, Italy.
authors.forename: Paolo
authors.initials: P
authors.lastname: Innocenti
authors.affiliation: Università degli Studi 'G. d'Annunzio' Chieti-Pescara, Dipartimento di Farmacia, via dei Vestini 31, 66100, Chieti, Italy.
authors.forename: Marcello
authors.initials: M
authors.lastname: Locatelli
mesh_terms: D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D016670:Gabexate / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D016014:Linear Models; D015282:Octreotide / Q000032:analysis* / Q000737:chemistry; D010189:Pancreatic Juice / Q000737:chemistry*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence
chemical_list: D016670:Gabexate; D015282:Octreotide
keywords: HPLC-diode array detection (DAD)-fluorescence detection (FLD); gabexate mesylate metabolite; human pancreatic juice samples; method development; octreotide; sample preparation