Application of Solid-Phase Extraction and High-Performance Liquid Chromatography with Fluorescence Detection to...

Amniocentesis involves taking a sample of the amniotic fluid in order to perform a karyotype test and diagnose any genetic defects that may affect the fetus. Amniotic fluid has been collected from patients with an indication for amniocentesis in the 15-26th week of pregnancy. A simple and sensitive high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method for identification and quantification of eleven selected bisphenols in amniotic fluid samples is proposed. The proposed method involved protein precipitation using acetonitrile, and next the extraction and concentration of analytes by solid-phase extraction (SPE). The solid-phase extraction (SPE) procedure with application of Oasis HLB SPE columns performed well for the majority of the analytes, with recoveries in the range of 67-121% and relative standard deviations (RSD%) less than 16%. The limits of detection (LODs) and quantification (LOQs) of all the investigated analytes were in the range of 0.8-2.5 ng mL^-1 and 2.4-7.5 ng mL^-1 (curves constructed in methanol) and 1.1-5.2 ng mL^-1 and 3.2-15.6 ng mL^-1 (curves constructed in the amniotic fluid), respectively. The method was validated at the following two concentration levels: 10 ng mL^-1 (2 × LOQ) and 20 ng mL^-1 (4 LOQ). The results confirm the validity of the SPE procedure and HPLC-FLD method for identification and quantification of bisphenols in amniotic fluid samples collected during an amniocentesis. The result obtained show that HPLC-FLD is a useful method for determination of bisphenol residues at nanogram per milliliter concentrations in amniotic fluid samples. Residues of five analytes (BADGE·2H₂O, BPAF, BADGE, BADGE·H₂O·HCl and BADGE·2HCl) were detected in amniotic fluid samples. Additionally, the harmfulness of bisphenols as potential pathogens that may cause karyotype disorders and contribute to preterm birth was estimated.