Rapid LC–MS/MS method for determination of drotaverine in a bioequivalence study

• An LC–MS/MS method was developed and validated for drotaverine determination.
• An LOQ of 2.24 ng/ml was achieved.
• Sample preparation involves a protein precipitation with methanol.
• Method reproducibility was confirmed by incurred sample reanalysis.
• The method was applied for a bioequivalence study.

A liquid chromatography coupled with tandem mass spectrometry method for the quantification of the antispasmodic drug drotaverine in human plasma was developed and validated according to the current bioanalytical guidelines. The internal standard used was imipramine. The separation was performed on a Kinetex C18 50 × 3 mm, 2.6 μm column under isocratic conditions using a mobile phase of 65:35 (v/v) formic acid 0.2% (v/v) in water and acetonitrile at 40 °C with a flow rate of 0.4 ml/min. The detection of drotaverine and the internal standard was performed in multiple reaction monitoring (MRM) mode using an ion trap mass spectrometer with electrospray ionization, operating in positive mode. The human plasma samples (0.24 ml) were deproteinized with methanol and aliquots of 4 μl from supernatants obtained after centrifugation were directly injected into the chromatographic system. The method shows a good linearity (r2 > 0.997), precision (CV < 6.3%) and accuracy (bias < 5.4%) over the range of 2.24–448 ng/ml drotaverine in plasma. The recovery was between 91 and 98%. The limit of quantification was 2.24 ng/ml. The analysis required only a 3.0 min run. The developed and validated method for the determination of drotaverine in human plasma was successfully applied in a bioequivalence study, for analyzing approximately 1000 subject's samples.

Figure optionsDownload as PowerPoint slide