Thin Layer Chromatography-Autography-High Resolution Mass Spectrometry Analysis: Accelerating the Identification of Acetylcholinesterase Inhibitors.

INTRODUCTION: The prevailing treatment for Alzheimer's disease is the use of acetylcholinesterase (AChE) inhibitors. Natural extracts are the principal source of AChE's inhibitors. However, their chemical complexity demands for simple, selective and rapid assays. OBJECTIVE: To develop a strategy for identification of AChE inhibitors present in mixtures employing high resolution mass spectrometry (HRMS) and thin layer chromatography (TLC)-biological staining. METHODOLOGY: The strategy uses an autographic assay based on the α-naphthyl acetate - fast blue B system for the detection of AChE activity. The immobilisation of AChE in agar allowed the extraction of the compounds for analysis by HRMS. Three TLC experiments employing different solvent systems were used in parallel and the mass spectra of the compounds extracted from the inhibition halos, were compared. The analysis was performed under MatLab environment. RESULTS: The strategy was used to detect the presence of physostigmine in an extract of Brassica rapa L. spiked with the inhibitor. Similarly, caffeine was straightforwardly spotted as responsible for the inhibitory properties of an extract of Ilex paraguariensis Saint-Hilaire. Comparison of the HRMS profiles lead to the facile identification of the [M+H](+) and [M+Na](+) of the compounds responsible for the inhibition. CONCLUSION: The proposed methodology, coupling TLC-AChE autography-HRMS, illustrates the feasibility of assigning molecular formulas of active compounds present in complex mixtures directly from autography. The new AChE agar-immobilised assay presented a more homogenous colour and a better definition than direct spraying methods, reducing the cost of the assay and improving its sensitivity.

Mutual contribution by "track" microradioautography method (MRA) and secondary ion mass spectrometry (SIMS) microscopy: prospects in technetium dosimetry at-the cellular level.

The determination of cellular uptake sites of radioligands used for cell labelling for diagnostic purposes is an essential prerequisite for evaluating the radiation dose to the cell nucleus and cytoplasm. The distribution of 99mTc-HMPAO in labelled leukocytes was studied by two microscopic imaging techniques on the same biological material: the "track" microradioautographic method (MRA) and Secondary Ion Mass Spectrometry (SIMS) microscopy. The "track" method used internal conversion electrons of 99mTc, leading to the formation of silver grains in a thick layer nuclear emulsion deposited onto cellular smear. In order to improve the specificity of the "track" detection, a minimum of 5 consecutive silver grains was required. In SIMS Microscopy, mapping with 99Tc "daughter" nuclide of 99mTc (half-life: 2.13.10(5) years) was obtained after sputtering of superficial molecular layers on embedded specimen sections. A mass resolution of about 5,000 was needed to circumvent polyatomic ion interferences. Both methods were able to demonstrate a very heterogeneous distribution of technetium from one cell to another. The sensitivity and signal/noise ratios were excellent for both methods. The lateral resolution of SIMS microscopy (0.5 microns) was far better than that of MRA. Therefore, only SIMS is able to distinguish between nuclear and cytoplasmic localization. On the other hand, quantification was not achieved for SIMS, although semi-quantification is possible with MRA. The field of view of MRA is far larger, allowing a better statistical approach for quantification. Both methods appear to be complementary to determine the distribution of technetium at the cell level. MRA is simpler and better fitted to the study of a cell population or a tissue. The unique spatial resolution of SIMS allows to focus the study on subcellular structures.