Structural elucidation of amino amide-type local anesthetic drugs and their main metabolites in urine by LC-MS after derivatization and its application for differentiation between positional isomers of prilocaine.

The demand for clinical toxicology analytical methods for identifying drugs of abuse and medicinal drugs is steadily increasing. Structural elucidation of amino amide-type local anesthetic drugs and their main metabolites by GC-EI-MS and LC-ESI-MS/MS is of great analytical challenge. These compounds exhibit only/mostly fragments/product ions representing the amine-containing residue, while the aromatic amide moiety remains unidentified. This task becomes even more complicated when discrimination between positional isomers of such compounds is required. Here, we report the development of a derivatization procedure for the differentiation and structural elucidation of a mixture of local anesthetic drugs and their metabolites that possess tertiary and secondary amines in water and urine. A method based on two sequential "in-vial" instantaneous derivatization processes at ambient temperature followed by LC-ESI-MS/MS analysis was developed. 2,2,2-Trichloro-1,1-dimethylethyl chloroformate (TCDMECF) was utilized to selectively convert the secondary amines into their carbamate derivatives, followed by hydrogen peroxide addition to produce the corresponding tertiary amine oxides. The resulting derivatives exhibited rich fragmentation patterns, enabling improved structural elucidation of the original compounds. The developed method was successfully applied to the differentiation and structural elucidation of prilocaine and its four positional isomers, which all possess similar GC and LC retention times and four of them exhibit almost identical EI-MS and ESI-MS/MS spectra, enabling their structural elucidation in a single LC-ESI-MS/MS analysis. The developed technique is fast and simple and enables discrimination between isomers based on different diagnostic ions/fragmentation patterns.

Formation of formaldehyde adducts from various drugs by use of methanol in a toxicological screening procedure with gas chromatography-mass spectrometry.

Use of methanol as a solvent in a toxicological screening procedure with gas chromatography-mass spectrometry may be associated with artifact formation. Artifacts with a molecular ion of [M + 12]+ are formed from various drugs, such as amphetamine, propafenone, flecainide, beta-blockers and prilocaine. The mechanism of artifact formation was studied by mass spectral techniques, labelling and nuclear magnetic resonance spectroscopy. It was shown that the artifacts were generated by the addition of formaldehyde and subsequent loss of water. Formaldehyde is probably formed by thermal dehydrogenation of methanol in the injection port of the gas chromatograph.