Simultaneous determination of catecholamines and polyamines in PC-12 cell extracts by micellar electrokinetic capillary chromatography with ultraviolet absorbance detection.
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 100mM boric acid buffer containing 10mM SDS and 10mM 18-crown-6 at a pH of 9.5. The detection limit ranged from 1.0 x 10(-7) to 9.0 x 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.
Liu
Guanshu
G
Department of Chemistry, University of Missouri-Rolla, MO 65409, USA.
Chen
Junnan
J
Ma
Yinfa
Y
D000818:Animals; D002021:Buffers; D002457:Cell Extracts / Q000737:chemistry*; D020374:Chromatography, Micellar Electrokinetic Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D016716:PC12 Cells; D051381:Rats; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D002021:Buffers; D002457:Cell Extracts