title: Precolumn o-phthalaldehyde-N-acetyl-L-cysteine derivatization followed by RP-HPLC separation and fluorescence detection of sitagliptin enantiomers in rat plasma.
abstract: An indirect reversed-phase high-performance liquid chromatographic separation and fluorescence detection of sitagliptin enantiomers in rat plasma was developed and validated. Deproteinized rat plasma containing racemic sitagliptin was derivatized with o-phthalaldehyde and N-acetyl-L-cysteine under alkaline conditions, converted to diastereomers, and separated on a Lichrospher 100 RP-18e column using 20 mM phosphate buffer and methanol (45:55 v/v) as a mobile phase under isocratic mode of elution at a flow rate of 1.0 mL/min. Fluorescence detection was performed at 330 and 450 nm as excitation and emission wavelengths, respectively. The method was linear in the range of 50-5000 ng/ mL for both enantiomers. The intra- and interday accuracy and precision were within the predefined limits of ≤15% at all concentrations. The method was successfully applied to a pharmacokinetic study of sitagliptin after 5 mg/kg oral administration to Wistar rats. Robustness of the method was evaluated using design of experiments.
authors.affiliation: Analytical Chemistry Division, Discovery Laboratory, Indian Institute of Chemical Technology, Tarnaka, Hyderabad, India.
authors.forename: R
authors.initials: R
authors.lastname: Nageswara Rao
authors.forename: B
authors.initials: B
authors.lastname: Sravan
authors.forename: K
authors.initials: K
authors.lastname: Ramakrishna
authors.forename: Shaik
authors.initials: S
authors.lastname: Saida
authors.forename: Raju
authors.initials: R
authors.lastname: Padiya
mesh_terms: D000111:Acetylcysteine / Q000737:chemistry*; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004355:Drug Stability; D005456:Fluorescent Dyes / Q000737:chemistry*; D057230:Limit of Detection; D015394:Molecular Structure; D011719:Pyrazines / Q000097:blood* / Q000737:chemistry; D051381:Rats; D000068900:Sitagliptin Phosphate; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014230:Triazoles / Q000097:blood* / Q000737:chemistry; D009764:o-Phthalaldehyde / Q000737:chemistry*
chemical_list: D005456:Fluorescent Dyes; D011719:Pyrazines; D014230:Triazoles; D009764:o-Phthalaldehyde; D000068900:Sitagliptin Phosphate; D000111:Acetylcysteine
keywords: diabetes mellitus; enantiomer separation; fluorescence detection; precolumn derivatization; rat plasma; sitagliptin