On-line desorption of dried blood spot: A novel approach for the direct LC/MS analysis of micro-whole blood samples.

The aim of this work is to present a new concept, called on-line desorption of dried blood spots (on-line DBS), allowing the direct analysis of a dried blood spot coupled to liquid chromatography mass spectrometry device (LC/MS). The system is based on an inox cell which can receive a blood sample (10 microL) previously spotted on a filter paper. The cell is then integrated into LC/MS system where the analytes are desorbed out of the paper towards a column switching system ensuring the purification and separation of the compounds before their detection on a single quadrupole MS coupled to atmospheric pressure chemical ionisation (APCI) source. The described procedure implies that no pretreatment is necessary in spite the analysis is based on whole blood sample. To ensure the applicability of the concept, saquinavir, imipramine, and verapamil were chosen. Despite the use of a small sampling volume and a single quadrupole detector, on-line DBS allowed the analyses of these three compounds over their therapeutic concentrations from 50 to 500 ng/mL for imipramine and verapamil and from 100 to 1000 ng/mL for saquinavir. Moreover, the method showed good repeatability with relative standard deviation (RSD) lower than 15% based on two levels of concentration (low and high). Function responses were found to be linear over the therapeutic concentration for each compound and were used to determine the concentrations of real patient samples for saquinavir. Comparison of the founded values with those of a validated method used routinely in a reference laboratory showed a good correlation between the two methods. Moreover, good selectivity was observed ensuring that no endogenous or chemical components interfered with the quantitation of the analytes. This work demonstrates the feasibility and applicability of the on-line DBS procedure for bioanalysis.

Intraneuronal angiotensinergic system in rat and human dorsal root ganglia.

To elucidate the local formation of angiotensin II (Ang II) in the neurons of sensory dorsal root ganglia (DRG), we studied the expression of angiotensinogen (Ang-N)-, renin-, angiotensin converting enzyme (ACE)- and cathepsin D-mRNA, and the presence of protein renin, Ang II, Substance P and calcitonin gene-related peptide (CGRP) in the rat and human thoracic DRG. Quantitative real time PCR (qRT-PCR) studies revealed that rat DRG expressed substantial amounts of Ang-N- and ACE mRNA, while renin mRNA as well as the protein renin were untraceable. Cathepsin D-mRNA and cathepsin D-protein were detected in the rat DRG indicating the possibility of existence of pathways alternative to renin for Ang I formation. Angiotensin peptides were successfully detected with high performance liquid chromatography and radioimmunoassay in human DRG extracts. In situ hybridization in rat DRG confirmed additionally expression of Ang-N mRNA in the cytoplasm of numerous neurons. Intracellular Ang II staining could be shown in number of neurons and their processes in both the rat and human DRG. Interestingly we observed neuronal processes with angiotensinergic synapses en passant, colocalized with synaptophysin, within the DRG. In the DRG, we also identified by qRT-PCR, expression of Ang II receptor AT(1A) and AT(2)-mRNA while AT(1B)-mRNA was not traceable. In some neurons Substance P and CGRP were found colocalized with Ang II. The intracellular localization and colocalization of Ang II with Substance P and CGRP in the DRG neurons may indicate a participation and function of Ang II in the regulation of nociception. In conclusion, these results suggest that Ang II may be produced locally in the neurons of rat and human DRG and act as a neurotransmitter.

Jacobus Lombardus et Thomas de Aquino, supra librum de anima (5).

On a ajouté en tête une table des questions de Jean Buridan sur la physique.