Determination of polyamines in rat intestinal epithelial cell line IEC-6 by RP-HPLC

Aim To establish a method for determination of polyamines in rat intestinal epithelial cell line(IEC-6) in researches of pathology,physiology and pharmacology.Methods Pre-column derivatization reversed-phase high performance liquid chromatography with ODS-C18 column was adopted,with the polyamines(putrescine,spermidine and spermine) as indices,and benzoyl chloride as derivative reagent,to investigate and optimize conditions of the derivatization and the chromatography.Results When derivatization reaction was conducted at 50℃ for 8h,each of the polyamine derivatives had a bigger peak area in HPLC;given mobile phase as methanol-water(45 ∶ 55),flow rate 1.0 ml·min-1 and detection wavelength 234 nm,each peak of the derivatives was separated in HPLC.Linear ranges: putrescine 0.054~1.35 nmol,spermidine 0.046~1.15 nmol,spermine 0.080~2.00 nmol,r2≥0.9995;intra-day precision(RSD)of high,medium and low concentration: putrescine 1.5%~3.2%,spermidine 1.2%~4.5%,spermine 1.3%~4.1%;inter-day precision(RSD)of high,medium and low concentration:putrescine 1.2%~2.7%,spermidine 1.0%~3.5%,spermine 0.8%~3.9%;method recoveries of high,medium and low concentration: putrescine 85%~110%,spermidine 88%~110%,spermine 90%~108%.Conclusion The pre-column derivatization RP-HPLC method developed in this experiment can be used for determination of polyamines in intestinal epithelial cell line.