Precolumn o-Phthalaldehyde-N-acetyl-L-cysteine Derivatization Followed by RP-HPLC Separation and Fluorescence Detection of Sitagliptin Enantiomers in Rat Plasma

• Published in: Chirality (New York, N.Y.) N.Y.), 2013-12, Vol.25 (12), p.883-889
• Main Authors: Nageswara Rao, R., Sravan, B., Ramakrishna, K., Saida, Shaik, Padiya, Raju
• Format: Article
• Language: English
• Subjects: Acetylcysteine - chemistry; Analytical chemistry; Animals; Chromatography; Chromatography, High Pressure Liquid; diabetes mellitus; Drug Stability; enantiomer separation; fluorescence detection; Fluorescent Dyes - chemistry; Limit of Detection; Molecular Structure; o-Phthalaldehyde - chemistry; Plasma; precolumn derivatization; Pyrazines - blood; Pyrazines - chemistry; rat plasma; Rats; sitagliptin; Sitagliptin Phosphate; Spectrometry, Fluorescence; Time Factors; Triazoles - blood; Triazoles - chemistry
• ISSN: 0899-0042; 1520-636X
• DOI: 10.1002/chir.22229

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. Chirality 25:883–889, 2013. © 2013 Wiley Periodicals, Inc.