LC-MS/MS analysis of acetaminophen and caffeine in amniotic fluid

Abstract

The intake of several substances by pregnant women could be hazardous to the fetus and mother's health: many substances can cross the placenta and reach the fetal compartment, causing adverse outcomes. Consequently, to accurately measure the presence of xenobiotics in fetal matrices, sensitive and specific bioanalytical methods are necessary: this would allow the assessment of fetal exposure to substances which, although licit, can be dangerous for the fetal and child's growth. The aim of this study was to develop and validate a liquid chromatography tandem mass spectrometry method for the simultaneous determination and quantitation of caffeine and acetaminophen in amniotic fluid. Amniotic fluid is a quite complex biological matrix and, as such, it requires a purification step prior to analysis. The extraction method has been optimized by comparing three different commercially available SPE cartridges (Supel™ Select HLB, Phenomenex Strata C18-E, and Agilent ABS Elut-NEXUS), and a liquid/liquid extraction with acetonitrile. A reverse-phase HPLC with a C18 column and gradient elution program was used. MS detection was carried out in MRM mode. Quantitation was performed using the internal standard method. Validation parameters were very satisfactory. The high selectivity and sensitivity of the method (LOQ < 9.5 ng mL⁻¹, and LOD < 3.3 ng mL⁻¹) allowed us to determine target analytes even in small amounts. Precision, matrix effect, and stability were also evaluated. The whole validated method has finally been applied to the analysis of 194 real samples of human amniotic fluid obtained from pregnant women (15–21 weeks of gestation) in order to monitor the effective intake of target analytes: 96% of the examined women consumed caffeine during pregnancy while a lower percentage (20%) showed acetaminophen intake. The whole procedure is simple and easy to perform with minimal sample preparation and short analysis time.