Development and Validation of a High-Performance Liquid Chromatography−Mass Spectrometry Assay for Determination of Amphetamine, Methamphetamine, and Methylenedioxy Derivatives in Meconium

Abstract

A procedure based on liquid chromatography-mass spectrometry (LC-MS) is described for determination of amphetamine, methamphetamine, and methylendioxy derivatives in meconium, using 3,4-methylendioxypropylamphetamine as internal standard. The analytes were initially extracted from the matrix by 17 mM methanolic HCl. Subsequently, a solid-phase extraction with Bondelut Certify columns was applied. Chromatography was performed on a C(18) reversed-phase column using a linear gradient of 10 mM ammonium bicarbonate, pH 9.0-methanol as a mobile phase. Analytes were determined in LC-MS single ion monitoring mode with an atmospheric pressure ionization-electrospray interface. The method was validated in the range 0.005-1.00 microg/g using 1 g of meconium per assay. Mean recoveries ranged between 61.1 and 87.2% for different analytes. The quantification limits were 0.005 microg/g meconium for amphetamine, methamphetamine, and 4-hydroxy-3-methoxymethamphetamine and 0.004 microg/g meconium for 3,4-methylenedioxyamphetamine, 3,4-methylenedioxymethamphetamine, 3,4-methylenedioxyethylamphetamine, and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine. The method was applied to analysis of meconium in newborns to assess eventual fetal exposure to amphetamine derivatives.

Development and validation of a liquid chromatography–atmospheric pressure chemical ionization-tandem mass spectrometry method for simultaneous analysis of 10 amphetamine-, methamphetamine- and 3,4-methylenedioxymethamphetamine-related (MDMA) analytes in human meconium

“…A previous LC-MS method employing electrospray ionization and selective ion monitoring achieved LLOQs of 4 ng/g for MDA, MDEA and MDMA and 5 ng/g AMP, MAMP and HMMA; upper limits of quantification (ULOQ) were 1,000 ng/g. [23] The LLOQs observed in this method using the 10 µL injection were lower than those for MDEA and HMMA, but higher for AMP, MAMP, MDMA and MDA. The higher LLOQs for these analytes were associated with low level interferences present within blank meconium.…”

“…[23,48] Additional meconium from neonates of self-reported heavy MAMP abusing mothers were analyzed by Pichini et al using a reduced specimen aliquot, 0.3 -0.5 g rather than 1 g. [23] High concentrations of AMP (38 -1,019 ng/g) and MAMP (226 -1,157 ng/g) were identified in these specimens, but no methylenedioxy-derivatives were detected, with the exception of one specimen with 12 ng/ g MDMA. The advantage of the current method is that analyte concentrations up to 10,000 ng/ g can be quantified simultaneously with lower concentrations of minor metabolites.…”

“…Possibly, unidentified MAMP or AMP metabolites, other sympathomimetic amines, or endogenous meconium compounds cross-react with the immunoassay, triggering positive responses. The presence of MAMP and AMP metabolites in meconium cannot always be detected with current confirmatory analytical methods that focus on parent compounds, AMP, MAMP, MDMA, MDA, MDEA and N-methyl-1-(3,4-methylenedioxyphenyl)-2-butanamine (MBDB) [15,22,23]; therefore MAMP-exposed neonates could be misidentified and denied interventional measures. Additionally, identification and quantification of other MAMP and AMP metabolites in meconium may provide valuable data regarding the maternal/fetal transfer of AMP-related drugs.…”

“…AMP and MAMP concentrations exceeding 1,000 ng/g have been previously identified in meconium [23]; therefore, analysis of AMP-related analytes requires simultaneous quantification of high parent compound concentrations (that is, greater than 1,000 ng/g) and low minor metabolite concentrations in quantity-limited meconium specimens. To accommodate the large analytical range, two calibration curves were constructed using different injection volumes.…”

Designer drugs: aspectos analíticos e biológicos

“…122 CG/EM e CLAE/EM foram utilizados para a detecção de aminas simpatomiméticas em mecônio, principalmente na análise de anfetaminas e metanfetaminas e, mais recentemente, MDMA, MDEA, MDA e MBDB. 123 Existem outras matrizes alternativas, apesar de pouco utilizadas na rotina em análises toxicológicas, como é o caso do líquido pericárdico e do músculo esquelético, utilizados principalmente em situações post-mortem.…”