Simultaneous Determination of Different Polyamines and their Mono-Acetylated Derivatives in Gastric Tissue by HPLC with Post-Column Derivatization

摘要

A simple and highly sensitive procedure is described enabling the simultaneous determination of biogenic polyamines (PAs) and their related monoacetyl derivatives in stomach tissue. The method is based on HPLC using octane sulfonate as an ion-pairing agent employed in acetate buffers at pH 4.5. The application is accompanied with fluorescence detection followed by post-column derivatization with o-phthaldialdehyde at room temperature (20±0.5°C). N¹- and N⁸-acetylspermidines (ASPD) can be determined with this method in the same run without performing any special procedures or pre-purification in concentrations exceeding 8.5 pmoles. The variability in reproducibility of the day-today precision and duplicate determination, and simultaneous determination of standard mixture and biological samples were found < 2%. The mean (± s.e.mean) retention times (n=12) for putrescine (Put), N¹-ASPD, N⁸-ASPD, spermidine (Spd) and spermine (Spm) are 8.97±0.025; 17.64±0.063; 18.99±0.133; 28.20±0.070 and 39.81±0.098 min, respectively. The method was applied to determine PAs and specifically N¹- and N⁸-ASPD in glandular part of stomach tissue of fasting rats (STFR) without any interference with endogenous aminoacids, histamine, and other reactive moieties. PAs and both mono-ASPD have been successfully determined in the STFR and the values are as follows: Put 37.2±10.1; N¹-ASPD 5.88±0.48; N⁸-ASPD 4.43±0.94; Spd 750.7±22.7 and Spm 618.2±37.4 nmole/g of wet tissue. Information on gastric tissue polyamines and their acetylated derivatives may be useful in understanding the mechanism of drugs or agents that play some part in gastric ulcer production or its repair mechanisms.