Liquid Chromatography Fingerprint Analysis of Released Compounds in Plasma Samples of Stroke Patients after Thrombolytic Treatment

Abstract

Plasma samples obtained from stroke patients treated with recombinant tissue-type plasminogen activator (rt-PA) and not treated with rt-PA were evaluated with different HPLC methodologies to obtain information about the possible release of small molecules as a result of the thrombolytic treatment. Plasma samples, without derivatization and derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), were evaluated with a HPLC gradient method, which consisted of a mobile phase of 10 mM ammonium acetate buffered solution (pH = 5.3) and acetonitrile. Three different detection methods were applied: UV, fluorescence, and ESI-MS. The results obtained showed that a group of new highly hydrophilic compounds appeared in most samples analyzed from treated patients, just after the administration of rt-PA. These compounds appeared shortly after the administration of the drug and were detected during the first 24 h after treatment, disappearing from plasma after this time. These new compounds were not detected either in controls or in non-treated stroke patients, which suggests that they were released into the plasma as a consequence of the thrombolytic effect of the drug. Our results suggest that these new compounds might be free glycans. The use of AQC as a derivatizing reagent has demonstrated that the new compounds detected cannot contain primary or secondary amine groups in their structure. The molecular mass determined by ESI-MS (821 Da) suggests that if these compounds are free glycans they might be a high-mannose type.