Bioanalytical method development and validation of milnacipran in rat plasma by LC-MS/MS detection and its application to a pharmacokinetic study

Abstract

A simple, sensitive and specific liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of milnacipran (MC) in rat plasma by using the liquid-liquid extraction method. Milnacipran-d10 (MCD10) was used as an internal standard (IS). Chromatographic separation was achieved on Zorbax SB-CN (4.6 mm×75 mm, 3.5 µm) column with an isocratic mobile phase composed of 10 mM ammonium acetate (pH 4.0) and methanol in the ratio of 25:75(v/v), at a flow-rate of 0.7 mL/min. MC and MCD10 were detected with proton adducts at m/z 247.2→230.3 and m/z 257.2→240.4 in multiple reaction monitoring (MRM) positive mode respectively. The method was validated over a linear concentration range of 1.00-400.00 ng/mL with a correlation coefficient (r²)≥0.9850. This method demonstrated intra- and inter-day precision within 5.40-10.85% and 4.40-8.29% and accuracy within 97.00-104.20% and 101.64-106.23%. MC was found to be stable throughout three freeze-thaw cycles, bench top and postoperative stability studies. This method was successfully applied to a pharmacokinetic study of rats through i.v. administration.

Keywords: LC–MS/MS; Milnacipran; Pharmacokinetics; Rat plasma.

Fig. 1

Chemical structures of milnacipran and milnacipran-d10.

Fig. 2

Mass spectra of (A) milnacipran parent ion, (B) milnacipran product ion, (C) milnacipran-d10 parent ion and (D) milnacipran-d10 product ion.

Fig. 3

Chromatogram of blank rat plasma.

Fig. 4

LLOQ chromatograms of milnacipran and milnacipran-d10.

Fig. 5

Mean plasma concentrations versus time graph of milnacipran after intravenous administration of 0.9 mg/200 g in male rats.