A Highly Sensitive LC-MS/MS Method for the Quantification of Tetrabenazine and DihydroTetrabenazine in Human plasma
31 Aug 2018
Tetrabenazine is a drug for the symptomatic treatment of hyperkinetic movement disorders. Tetrabenazine metabolises rapidly and extensively by carbonyl reductase to the active metabolites ?-hydroxy tetrabenazine and ?-hydroxy tetrabenazine (Dihydrotetrabenazine as racemic mixture), hence a selective and sensitive method at an LLOQ of about 5 pg/mL for Tetrabenazine and 0.2 ng/mL of dihydrobenazine is required. This was accomplished in the new method developed at our end.
Briefly, the method for the quantification of tetrabenazine and dihydrotetrabenazine involves liquid-liquid extraction using tetrabenazine-d7 and ?-hydroxy tetrabenazine-d7 as internal standards (ISTD). Chromatographic separation was achieved on Venusil AQ C18 3?, 4.6X100 mm column with a mobile phase consisting of 5 mM ammonium acetate buffer and acetonitrile with isocratic time program, and a total run-time of 5.0 min. Detection was carried out in positive electro-spray ionization mode using MRM transitions of 318.400?220.200, 320.400?165.100, 325.400?220.200 and 327.400?165.100 for tetrabenazine, dihydrotetrabenaxine, tetrabenazine d7 and ?-hydroxy tetrabenazine d7, respectively.
The method was validated in terms of linearity, stabilities, carry-over effect, reinjection reproducibility, precision, accuracy, and selectivity. The validated method has a linearity range from 5 to 2500 pg/mL for tetrabenazine and 0.2 to 100 ng/mL for dihydrotetrabenazine, and is found to be stable in plasma under the assay condition. The method exhibited good precision and accuracy with precision better than 10.9% and accuracy greater than 94.9% for tetrabenazine, and 9.4% and 93.9% for dihydrotetrabenazine. Also, there was no significant matrix effect observed in normal human lots.
The validated method is suitable for the analysis of samples from a single oral dose of 25 mg administration to humans under fasting and fed conditions and will be applied to the analysis of study samples.
To conclude, a selective, simple, precise, accurate and highly sensitive LC-MS/MS method is now available for quantification of tetrabenazine and dihydrotetrabenazine in human plasma.