ABSTRACT
A novel gradient reverse phase high performance liquid chromatography tandem mass spectrometry (HPLC/MS-MS) was performed as a method for the determination of dobutamine hydrochloride (DOB) in newborn pig plasma samples. It was developed and validated after optimization of sample treatment and various chromatographic and mass spectrometric conditions. Trimethoxydobutamine (TMD) was used as internal standard. Heptafluorobutyric acid (HFBA) and ethyl acetate were used for the treatment of plasma samples. The separation of dobutamine and internal standard was done using a Kinetex F5 (50×2.1mm, 2.6µm, 100Å) analytical column. The mobile phase was a mixture of acetonitrile and HCOOH 0.01%. The column oven temperature was optimized at 40° C and the flow rate was 0.25mL/min. DOB and TMD were detected by multiple reaction monitoring (MRM) mode in ESI+, using a cone voltage (CV) of 25V and a collision energy (CE) of 25eV. The weighted calibration curve (1/x2) was found to be linear over the concentration range of 1-100ng/mL (r2>0.999). The limit of quantification (LLOQ) of the method was 1ng/mL. The values of selectivity, carryover, LLOQ, linearity, accuracy, precision, matrix effect, stability and recovery obtained meet the acceptable range according to European Medicines Agency (EMA) and Food and Drug Administration (FDA) guidelines. The method was efficiently applied to quantify DOB in plasma samples from a pharmacokinetic/pharmacodynamic study in a disease model of newborn piglet.
