Biogenic amines and their metabolites in mouse brain tissue Development, optimization and validation of an analytical HPLC method

A simple and fast HPLC method based on an isocratic reversed-phased ion-pair with amperometnc 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 methanol-phosphate-citric buffer (pH 3 2 50 mM) (9 91 v/v) containing 2 mM OSA as mobile phase at 32 Con a microbore ALF-115 column (150 mm x 1 0 mm 3 mu m particle size) filled with porous C-18 silica stationary phase In this study a two-level fractional factorial experimental design (1/2 2(K)) 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 phosphate-citric buffer was also developed Results show that all molecules of interest were stabilized for at least 24h in the matrix conditions without any antioxidants The method was fully validated according to the requirements of SFSTP (Societe Francaise 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 4-2000 ng/ml for MHPG 1-450 ng/ml for NA 1-700 ng/ml for A 1-300 ng/ml for DOPAC 1-300 ng/ml for 5-HIAA 1-700 ng/ml for DA 4-2800 ng/ml for HVA and 1-350 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 ultra-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 (C) 2010 Elsevier B V All rights reserved