New luminometric method for quantification of biological sulfur nucleophiles with the participation of 9-cyano-10-methylacridinium salt.

Biologically active compounds containing sulfhydryl groups (RSHs: N-acetyl-l-cysteine, d-penicillamine, glutathione and acetylthiocholine chloride) were used to develop a luminometric method for their quantification. The title substrate capable of chemiluminescence (CL) was isolated in a highly pure state as a chloride salt (99.9% using RP-HPLC) and identified using mass spectrometry (ESI Q-TOF) and H NMR spectroscopy. The cation included in the salt, 9-CMA , underwent oxidation in an alkaline environment containing RSHs by molecular oxygen, generating CL of various intensities, with no need for the use of hydrogen peroxide. The amount of produced light was linearly proportional to the content of investigated analytes in the system over the concentration range ~0.2-2 μM, with the detection limits in the range 0.19-1.73 μM. The mechanism of chemiluminogenic oxidation of 9-CMA in the presence of RSHs and molecular oxygen is proposed, using computational methods at the density-functional theory level. The presence of RSHs in an alkaline medium seems to be crucial to produce hydroperoxide anions ( OOH), which initiate the 'light path' of 9-CMA transformations, ending with the excretion of electronically excited molecules of 10 methyl-acridan-9-one.