Simultaneous Determination of Catecholamines and Polyamines in PC-12 Cell Extracts by Micellar Electrokinetic Capillary Chromatography with Ultraviolet Absorbance Detection

Abstract

A method for simultaneous determination of polyamines and catecholamines in cell extracts by micellar electrokinetic capillary chromatography with UV detection at 254 nm was established at the first time. The polyamines (putrescine, spermidine and spermine) and catecholamines (dopamine, serotonin, norepinephrine and epinephrine) were extracted from PC-12 cells and were derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Different derivatization conditions such as temperature, ratio of derivatization reagents and incubation time were investigated to find the best reaction condition which gave the highest detection sensitivity for polyamines and catecholamines. The influence of running buffer and additives on the separation such as pH, sodium dodecyl sulfate (SDS) concentrations and various additives was also investigated. Separation was achieved within 20 min with good repeatability in a 100 mM boric acid buffer containing 10 mM SDS and 10 mM 18-crown-6 at a pH of 9.5. The detection limit ranged from 1.0×10-7 to 9.0×10-7 M, which is sufficient for determination of polyamines and catecholamines in many cell extracts. This technique can be easily applied to polyamine-related anticancer drug studies or clinical follow-ups after each dosage of these anticancer drugs, since these drugs not only have great inhibition on polyamine levels in blood, but also have a large influence on catecholamine levels in blood.

Department(s)

Chemistry

Keywords and Phrases

Blood; Derivatives; Drug Therapy; Micelles; PH Effects; Ultraviolet Detectors; Catecholamines; Micellar Electrokinetic Capillary Chromatography; Polyamines; UV Detection; Amines; 6 Aminoquinolyl N Hydroxysuccinimidylcarbamate; Adrenalin; Carbamic Acid Derivative; Catecholamine; Cell Extract; Crown Ether Derivative; Dodecyl Sulfate Sodium; Dopamine; Noradrenalin; Polyamine; Putrescine; Serotonin; Spermidine; Spermine; Tumor Marker; Unclassified Drug; Accuracy; Analytic Method; Animal Cell; Cancer Cell; Controlled Study; Derivatization; Disease Marker; Drug Targeting; Incubation Time; Micellar Electrokinetic Chromatography; Nonhuman; PH; Priority Journal; Protein Analysis; Protein Determination; Protein Expression; Protein Function; Protein Synthesis; Protein Targeting; Rat; Separation Technique; Temperature Dependence; Ultraviolet Spectroscopy; Animals; Buffers; Cell Extracts; Chromatography, Micellar Electrokinetic Capillary; Hydrogen-Ion Concentration; PC12 Cells; Rats; Reproducibility Of Results; Sensitivity And Specificity; Spectrophotometry, Ultraviolet; Polyamines

International Standard Serial Number (ISSN)

1570-0232

Document Type

Article - Journal

Document Version

Citation

File Type

text

Language(s)

English

Rights

© 2004 Elsevier, All rights reserved.

Publication Date

01 Jun 2004

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