Sensitive isotope dilution liquid chromatography/tandem mass spectrometry method for quantitative analysis of bumetanide in serum and brain tissue

Abstract

We have developed and validated a simple and sensitive stable isotope dilution liquid chromatography/tandem mass spectrometric (LC-MS/MS) method for the quantification of bumetanide in human serum. Samples were prepared with a simple acetonitrile based protein precipitation. The supernatant was then analyzed directly using LC-MS/MS. Chromatographic separation was achieved on a C18 reversed phase column using a methanol and water gradient. The detection was performed in selected reaction monitoring (SRM) mode via a positive electrospray ionization (ESI) interface. The method had a lower limit of quantification (LLOQ) of 1 ng/mL, linearity up to 1250 ng/mL, intra- and inter-day precision less than 10%, and accuracy within ±10%. This method was also demonstrated to be suitable for the analysis of bumetanide in rat serum and brain tissue. Bumetanide concentrations in rat serum and brain were determined for samples collected at several intervals following intraperitoneal (i.p.) injection of bumetanide, and were used to calculate bumetanide permeability through the blood-brain barrier.

Copyright © 2011 Elsevier B.V. All rights reserved.

Figures

Fig. 1. Representative LC-MS/MS chromatograms

SRM transitions 365.3 > 240.2 (bumetanide) and 370.3 > 244.2 (IS) are represented by the solid and dotted lines, respectively. (a) and (b) were the human serum and rat brain samples prepared with the respective assay procedures where IS was omitted from the IS working solutions; (c), bumetanide spiked human serum sample at 312 ng/mL; (d) and (e) were rat serum samples with measured bumetanide concentration of 2 and 210 ng/mL, respectively; and (f) was rat brain sample with measured bumetanide concentration of 0.24 ng/mL. The inserts in (d) and (f) are the zoomed in chromatograms of bumetanide SRM transition.

Fig. 2

Bumetanide concentration in rat serum and brain after a single dose of bumetanide (0.3 mg/kg). Each point is the average of two rats, with standard deviation shown as the error bars.