OBJECTIVETo develop a high performance liquid chromatography (HPLC) for detection of monoamine neurotransmitters and its metabolites after pre-column derivatization with dansyl chloride.

METHODSThe C(18) chromatograph column (150 mm×4.6 mm×5 µm) was selected for detection, and derived by dansyl chloride (10 mg/ml) under the condition of 50°C water bath by pH11 buffer solution. 20 µl acetic acid acetone solution (1.0 mol/L) was then mixed in for termination of the reaction. Then the solution was cooling to room temperature, 0.1 mol/L acetic acid zinc-acetonitrile-tetrahydrofuran solution was adopted for mobile phrase, with the volume ratio at 62:35:3. The flow rate was 1.0 ml/min between 0-10 min, 2.0 ml/min between 10-35 min. The ultraviolet detection wavelength was 286 nm. The above method separately detected monoamine neurotransmitters and its metabolites and evaluated the limit of detection, accurate degree and accuracy degree.

RESULTSThe linear relations between each component was good in the range of 1 - 20 µg/ml (r = 0.999). The lowest detection limit of norepinephrine, dopamine, 5-hydroxytryptamine and the metabolites 3-methoxy-4-benzoglycols, homovanillic acid and 5-heteroauxin were separately 0.60, 0.80, 0.41, 0.21, 0.19 and 0.1 µg/ml; while the average recovery rates were between 78.5% - 95.9%, and the relative standard deviation (RSD) was 6.62%, 7.64%, 2.98%, 3.60%, 5.09% and 3.09%, respectively. In the process of selection and optimization of the chromatographic conditions, we observed the importance of metal ions to discretion, and discussed the temperature, pH of the buffer solution and dosage of dansyl chloride in derivation. Under the above conditions, the reaction was perfect, and the baseline of the detected materials thoroughly separated.

CONCLUSIONThe method to detect monoamine neurotransmitters and its metabolites by HPLC and pre-column derivatization with dansyl chloride was established; and this method could provide reference for the detection of polyamine by HPLC.

Biogenic Monoamines ; analysis ; metabolism ; Chromatography, High Pressure Liquid ; methods ; Dansyl Compounds ; Neurotransmitter Agents ; analysis ; metabolism

Endothelin systems are believed to play important roles in the emergence and maintenance of functions of various organs during perinatal development, including the kidney. The present study was designed to investigate the roles of endothelin systems on physiologic renal growth and development. Newborn rat pups were treated with either Bristol-Myers Squibb-182874 (30 mg/kg/day), a selective endothelin A receptor (ET(A)R) antagonist, or vehicle for 7 days. To identify cellular changes, kidneys were examined for apoptotic cells by terminal deoxynucleotide transferasemediated nick-end labeling stain and proliferating cell nuclear antigen (PCNA) by immunohistochemical (IHC) stain. To clarify the molecular control of these processes, immunoblots and reverse transcriptase-polymerase chain reaction for Clusterin, Bcl-2, Bcl-X(L), Bax, and p53 were performed. ETAR antagonist treatment resulted in reduced kidney weights, decreased PCNA-positive proliferating cells, and increased apoptotic cells. The protein expressions of renal Bcl-X(L) and Bax in the ETAR antagonist-treated group were significantly decreased, whereas the mRNA expressions of these genes were not changed. There were no significant differences in the expressions of Clusterin, Bcl-2, and p53. In conclusion, inhibition of endogenous endothelins impairs renal growth, in which decreased cellular proliferation, increased apoptosis and decreased expressions of renal Bcl-X(L) and Bax are possibly implicated.
Animals ; Animals, Newborn ; Antihypertensive Agents/*pharmacology ; *Apoptosis ; *Cell Proliferation ; Dansyl Compounds/*pharmacology ; Gene Expression Regulation, Developmental/drug effects ; In Situ Nick-End Labeling ; Kidney/drug effects/*growth & development/*metabolism ; Proliferating Cell Nuclear Antigen/metabolism ; Proto-Oncogene Proteins c-bcl-2/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Receptor, Endothelin A/*antagonists & inhibitors/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Tumor Suppressor Protein p53/genetics/metabolism ; bcl-X Protein/genetics/metabolism