Development of a liquid chromatography-coupled tandem mass spectrometry multi-analyte method for the monitoring of benznidazole and its metabolites in serum

NOELIA A. MARTINEZ; JULIO RABA; MARÍA ELENA MARSON; SOLEDAD CERUTTI; FACUNDO GARCÍA-BOURNISSEN

Rosario

Congreso; Ricifa 2016; 2016

Chagas is a potentially life-threatening disease caused by the protozoan parasite Trypanosoma cruzi. Benznidazole (BNZ) and nifurtimox are the only two drugs approved for human treatment. However, pharmacological properties of these drugs and its derived metabolites have not yet been studied in detail.Although there are some protocols for quantification of BNZ in biological samples, no methodologies have been proposed for determination of its human metabolites: N-benzylacetamide (BAA) and aminobenznidazole (NH2-BNZ). As a consequence, the development of analytical strategies for the simultaneous monitoring of BNZ, BAA, and NH2-BNZ in biological samples is of crucial interest.The present work proposes for first time a novel methodology based on the analysis of spiked serum samples deposited in filter paper for the subsequent liquid extraction of BZN, BAA and NH2-BNZ prior to LC-MS/MS analysis. For the extraction process was employed a solution of H2O:MeOH (70:30) with 0.1% (v/v) of formic acid.The chromatographic separation was performed by employing a C18 column using gradient elution. Under these conditions, the retention times were 2.34, 2.07 and 1.62 minutes for BZN, BAA and NH2-BNZ; respectively. Total run time for the three analytes was under 3 minutes. In addition to the optimization of the extraction and chromatographic conditions, the detection spectrometric parameters were obtained. All the compounds (precursors and fragment ions) were monitored following multiple reaction mode.The methodology was validated through recovery studies. After evaluating the matrix effect on the measured signal for each analyte, the obtained limits of detection were 6.9; 4.5 and 3.6 μg L-1 for BZN, BAA and NH2-BNZ; respectively, while the limits of quantification were 8.2; 4.7 and 5.7 μg L-1 for BZN, BAA and NH2-BNZ; respectively. This new methodology could be a valuable tool for monitoring BNZ and its metabolites in clinical studies.