Chemical assay of glutaraldehyde incorporation into pericardial tissue

This paper describes the development of a chemical assay to determine the incorporation of glutaraldehyde into pericardial tissue. The procedure is based upon the hydrolysis of the Schiff's bases formed between the glutaraldehyde and the E-amino groups of lysine, the liberated glutaraldehyde being determined as its 2,4-dinitrophenyl-hydrazone by high performance liquid chromatography (HPLC). The HPLC system uses a reverse phase column eluted with a gradient of aqueous acetonitrile (from 60%-70% over 30 minutes). The glutaraldehyde 2,4-dinitrophenylhydrazone is detected at 356 nm. The results show that the assay has sufficient sensitivity (minimum detectable limit 20 pmol of glutaraldehyde 2,4-dinitrophenyl-hydrazone) and reproducibility (+/- 9% at 100 pmol +/- 4% at 1 nmol) to estimate glutaraldehyde incorporation into pericardial tissue. A series of subsequent experiments have documented that temperature and buffer pH are the main determinants affecting glutaraldehyde incorporation into pericardial tissue. Immersion of pericardial tissue in glutaraldehyde solution of pH 9 and 37 degrees C yields an approximately six times greater incorporation than can be achieved if the reaction is performed at 0 degrees C. Further study is underway to relate the amount of Schiff's base bound to that of total incorporated glutaraldehyde.