Biogenic amines and their metabolites in mouse brain tissue: development, optimization and validation of an analytical HPLC method
Faculty of Pharmaceutical, Biomedical and Veterinary Sciences . Biomedical Sciences
Journal of chromatography : B: analytical technologies in the biomedical and life sciences. - Amsterdam, 2002, currens
, p. 3003-3014
University of Antwerp
A simple and fast HPLC method based on an isocratic, reversed-phased ion-pair with amperometric end-point detection for simultaneous measurement of noradrenergic (MHPG/NA and A), dopaminergic (DOPAC, HVA/DA) and serotonergic (5-HIAA/5-HT) compounds in mouse brain tissue was developed. In order to improve the chromatographic resolution (Rs) with an acceptable total analysis time, experimental designs for multivariate optimization of the experimental conditions were applied. The optimal conditions for the separation of the eight neurotransmitters and metabolites, as well as two internal standards, i.e., DHBA and 5-HMT, were obtained using a mixture of methanolphosphatecitric buffer (pH 3.2, 50 mM) (9:91, v/v) containing 2 mM OSA as mobile phase at 32 °C on a microbore ALF-115 column (150 mm × 1.0 mm, 3 μm particle size) filled with porous C18 silica stationary phase. In this study, a two-level fractional factorial experimental design (½ 2K) was employed to optimize the separation and capacity factor (k′) of each molecule, leading to a good separation of all biogenic amines and their metabolites in brain tissue. A simple method for the preparation of different bio-analytical samples in phosphatecitric buffer was also developed. Results show that all molecules of interest were stabilized for at least 24 h in the matrix conditions without any antioxidants. The method was fully validated according to the requirements of SFSTP (Société Française des Sciences et Techniques Pharmaceutiques). The acceptance limits were set at ±15% of the nominal concentration. The method was found accurate over a concentration range of 42000 ng/ml for MHPG, 1450 ng/ml for NA, 1700 ng/ml for A, 1300 ng/ml for DOPAC, 1300 ng/ml for 5-HIAA, 1700 ng/ml for DA, 42800 ng/ml for HVA and 1350 ng/ml for 5-HT. The assay limits of detection for MHPG, NA, A, DOPAC, 5-HIAA, DA, HVA and 5-HT were 2.6, 2.8, 4.1, 0.7, 0.6, 0.8, 4.2 and 1.4 pg, respectively. It was found that the mean inter- and intra-assay relative standard deviations (RSDs) over the range of standard curve were less than 3%, the absolute and the relative recoveries were around 100%, demonstrating the high precision and accuracy, and reliability of the analytical method described to apply in routine analysis of biogenic amines and their metabolites in brain tissue.