A validated LC-MS/MS method for the determination of tolterodine and its metabolite in rat plasma and application to pharmacokinetic study

Abstract

Liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was used for simultaneous quantification of tolterodine and its metabolite 5-hydroxy methyl tolterodine in rat plasma. Tolterodine-d6 and 5-hydroxy methyl tolterodine-d14 were used as internal standards (IS). Chromatographic separation was performed on Ascentis Express RP amide (50 mm×4.6 mm, 2.7 μm) column with an isocratic mobile phase composed of 10 mM ammonium acetate and acetonitrile in the ratio of 20:80 (v/v), at a flow-rate of 0.5 mL/min. Tolterodine, tolterodine-d6, 5-hydroxy methyl tolterodine and 5-hydroxy methyl tolterodine-d14 were detected with proton adducts at m/z 326.1→147.1, 332.3→153.1, 342.2→223.1 and 356.2→223.1 in multiple reaction monitoring (MRM) positive mode respectively. The drug, metabolite and internal standards were extracted by liquid-liquid extraction method. The method was validated over a linear concentration range of 20.00-5000.00 pg/mL for tolterodine and 20.00-5000.00 pg/mL for 5-hydroxy methyl tolterodine. This method demonstrated intra- and inter-day precision of 0.62-6.36% and 1.73-4.84% for tolterodine, 1.38-4.22% and 1.62-4.25% for 5-hydroxy methyl tolterodine respectively. This method also demonstrated intra- and inter-day accuracy of 98.75-103.56% and 99.20-104.40% for tolterodine, 98.08-104.67% and 98.73-103.06% for 5-hydroxy methyl tolterodine respectively. Both analytes were found to be stable throughout freeze-thaw cycles, bench top and postoperative stability studies. This method was successfully applied for the pharmacokinetic analysis of rat plasma samples following i.v. administration.

Keywords: 5-Hydroxy methyl tolterodine; LC–MS/MS; Pharmacokinetics; Tolterodine.

Fig. 1

Chemical structures of tolterodine, tolterodine-d6, 5-hydroxy methyl tolterodine, and 5-hydroxy methyl tolterodine-d14.

Fig. 2

Mass spectra of (A) tolterodine parent ion, (B) tolterodine product ion, (c) tolterodine-d6 parent ion and (D) tolterodine-d6 product ion.

Fig. 3

Mass spectra of (A) 5-hydroxy methyl tolterodine parent ion, (B) 5-hydroxy methyl tolterodine product ion, (C) 5-hydroxy methyl tolterodine-d14 parent ion and (D) 5-hydroxy methyl tolterodine-d14 product ion.

Fig. 4

Chromatogram of blank rat plasma: (A) tolterodine, (B) tolterodine-d6, (C) 5-hydroxy methyl tolterodine, and (D) 5-hydroxy methyl tolterodine-d14.

Fig. 5

Chromatogram of tolterodine, tolterodine-d6 and 5-hydroxy methyl tolterodine, 5-hydroxy methyl tolterodine-d14 at LOQ level.

Fig. 6

Mean plasma concentrations versus time graph of tolterodine and 5-hydroxy methyl tolterodine after intravenous administration of 0.072 mg/200 g tolterodine in male rats.