High-performance liquid-chromatographic tandem-mass spectrometric methods for atropinesterase-mediated enantioselective and chiral determination of R- and S-hyoscyamine in plasma.

S-hyoscyamine (S-hyo) is a toxic tropane alkaloid from plants of the solanacea family, which is extracted for pharmaceutical purposes thereby undergoing racemization (atropine). Merely the S-hyo enantiomer acts as an antagonist of muscarinic receptors (MR). Nevertheless, racemic atropine is clinically administered in e.g. ophthalmology and for symptomatic therapy of acute poisoning with organophosphorus compounds (OPCs, e.g. pesticides, nerve agents). However, very limited data are available of comparative pharmacokinetics of S- and R-enantiomers in humans or other species. Therefore, we developed an enantioselective LC-ESI-MS/MS assay making use of rabbit serum containing atropinesterase (AtrE, EC 3.1.1.10) which is suitable for stereospecific hydrolysis of S-hyo into tropine and tropic acid while R-hyo is unaffected. For sample preparation plasma was incubated with human serum (not containing AtrE, procedure A) and with rabbit serum (procedure B). Afterwards, hyoscyamines were quantified by a validated previously published non-chiral LC-ESI-MS/MS method. Following procedure A the concentration of total hyo and following procedure B remaining R-hyo were determined. S-hyo was calculated by the difference between these concentrations. This assay design allowed reproducible, precise (RSD 2-9%), accurate (93-101%) and selective determination of total and individual hyoscyamines. Potential therapeutics for OPC poisoning (carbamates, oximes) and thiono-pesticides did not interfere with the assay whereas some oxon-pesticides inhibited S-hyo hydrolysis. A control experiment was designed allowing to be aware of such interferences thus avoiding the use of false results. To validate this assay, results were compared to those from a novel isocratic chiral LC-ESI-MS/MS method. Separation of S-hyo (t(R) 31.1 ± 0.2 min) and R-hyo (t(R) 33.4 ± 0.2 min) was achieved on α-glycoprotein (AGP) chiral stationary phase at 40°C (selectivity factor α 1.07). Ammoniumformate (0.01 M, pH 8.0) with 3.75% (v/v) acetonitrile served as mobile phase (300 µL min(-1)). Hyoscyamines were detected in the positive multiple reaction monitor mode. The enantioselective assay was applied to the analysis of atropine degradation in diluted rabbit serum in vitro as well as to human in vivo plasma samples from a pesticide-poisoned patient treated with atropine.

Intoxicaciòn por inhibidores de la enzima acetilcolinesterasa / Intexication by inhibitos of the acetil cholinesterase enzyme

Con el objeto de determinar la incidencia de las intoxicaciones por inhibidores de la enzima acetil colimerasa, su distribuciòn de acuerdo a la edad y sexo, las causas, los sìntomas màs frecuentes, el tiempo promedio en que estos pacientes llegaron al Hospital a partir del momento de la ingestiòn y determinar el grado de gravedad de acuerdo a los sintomas presentados, es que se revisaron retrospectivamente los expedientes clìnicos de pacientes internados con este diagnòstico en el Hospital del niño "Dr. Ovidio Aliaga Uria" de la ciudad de La Paz desde enero de 1993 a diciembre de 1994. Se encontro que la intoxicacion por inhibidores de la enzima acetil colinesterasa representò el 0,68 porciento de los ingresos al Hospital; 62,5 porciento de ellos fueron varones, las edades fluctuaron entre 11 dìas y 13 años (promedio de 6.75 años), el 50 porciento de ellos llegaron al Hospital dentro de las primeras dos horas de haber ingerido el veneno 20 porciento de ellos recibieron remedios caseros antes de su ingreso; el 50 porciento fue manejado en el servicio de terapia intensiva; 6 pacientes se complicaron con broncoaspiraciòn y un paciente fallecio. El nùmero promedio de dosis de atropina utilizada como antidoto de la intoxicaciòn fue de 10,7 con una desviaciòn estàndar en 6,66. Se consideraron intoxicaciòn graves a 13 (32,5 porciento), moderados a 25 (62, 5 porciento) y leves a 2 (5 porciento). El tiempo de hospitalizaciòn promedio fue de 4.28 dìas