An improved liquid chromatographic method with electrochemical detection for direct determination of serotonin in microdialysates from Caudate-putamen and pineal gland regions of rat brain.

A liquid chromatography method coupled with electrochemical detection has been developed for the direct measurement 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in brain microdialysates. The separation conditions have been optimized to detect only the 5-HT and 5-HIAA in dialysates and elute the other monoamines and their metabolites in the void. Linear regression analysis of chromatographic peak area as a function of concentration in the range 5-1000 pg/mL gave correlation coefficients over 0.995. Sample stability and an assay validation for precision and accuracy were also performed. The limit of detection (S/N = 3) for 5-HT was 12 femtomol/mL. The method has been applied to simultaneously measure extracellular 5-HT and 5-HIAA in brain microdialysates from the pineal gland and caudate-putamen of awake and freely-moving rats under basal conditions.

An automated blood sampler for simultaneous sampling of systemic blood and brain microdialysates for drug absorption, distribution, metabolism, and elimination studies.

INTRODUCTION: A major problem in preclinical drug development where blood sampling from small animals is a routine practice is the time and labor involved in the serial sampling of small blood volumes from small animals such as rats for the duration of pharmacokinetic/pharmacodynamic (PK/PD) studies. The traditional method of manually drawing blood from the animal requires the animal to be anesthetized or restrained with some device, both of which cause stress to the animal. METHODS: An automated blood sampler (ABS) was developed to simultaneously collect blood and brain microdialysate samples at preprogrammed time points from awake and freely moving animals. The samples are delivered to fraction collectors and stored at 4 degrees C until use. The lost blood volume during collection is replaced with sterile saline to prevent fluid loss from the animal. In addition, the system is capable of collecting urine and feces for metabolism studies and monitoring the animal activity for behavioral studies. In the present study, blood samples were collected for 24 h after dosing rats orally with a 5 mg/kg dose of olanzapine (OLAN). Brain dialysates were collected for the same duration from a microdialysis probe implanted in the striatum. RESULTS: The pharmacokinetic parameters, obtained after an oral dose, are in good agreement with reported values in literature. The pharmacodynamic information obtained from brain dialysates data show that OLAN elevates the concentration of dopamine (DA) in the brain and remains in the brain even after it is cleared from the plasma. DISCUSSION: The ABS described here is a very useful tool in drug development to accelerate the pace of preclinical in vivo studies and to simultaneously provide pharmacodynamic and physiological information.