Simultaneous determination of levetiracetam and its acid metabolite (ucb L057) in serum/plasma by liquid chromatography tandem mass spectrometry.

OBJECTIVE

Levetiracetam and its acid metabolite have almost identical MRMs. They therefore need to be separated chromatographically prior to quantitation.

RESEARCH DESIGN AND METHODS

The sample is deproteinized with acetonitrile containing Ritonavir as internal standard, centrifuged and the supernatant diluted with water (1:2 v/v). Sixty microliters of the supernatant is injected into the LC-MS/MS and Levetiracetam (LEV) and LEV metabolite separated chromatographically at room temperature employing a Supelco C(18) column and a 0.1% formic acid methanol gradient at pH of 2.5.

RESULTS

The retention times for LEV metabolite, LEV and Ritonavir were 4.50, 5.38 and 9.18 min, respectively. Calibration curves in spiked plasma were linear over the concentration range of 0-50 microg/mL for LEV and 0.0-5.0 microg/mL for LEV metabolite. Intra- and inter-run imprecision (n=10) gave CVs of 2.3-4.7%, 3.4-8.9% for LEV and 2.9-3.9%, 3.3-7.4% for LEV metabolite. Recoveries of both LEV and LEV metabolite were close to 100%. Results for LEV were compared with those obtained by a commercial reference laboratory (r=0.974).

CONCLUSION

The procedure is reliable, quick, and inexpensive. LEV and LEV metabolite co-elute using C-18 columns at pHs >3.0 and previously published methods employing these conditions could therefore be subject to metabolite interference. In this method LEV and LEV metabolite are separated at pH 2.5. The total run time including the washing step is 10 min/sample, making this method suitable when moderate throughput is needed such as in clinical or commercial reference laboratories.