Quantitative detection of bisphenol A, 4- T - octylphenol , and

1. Quantitative detection of bisphenol A, 4-T-octylphenol, and 4-nonylphenol in human blood serum by liquid chromatography–electrospray tandem mass spectrometry Alexandros G. Asimakopoulos and Nikolaos S. Thomaidis Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, PanepistimioupolisZografou, 15771 Athens, Greece. E-mail: ntho@chem.uoa.gr ABSTRACT Alkylphenols are a group of compounds widely used in industry, and are collectively known as endocrine disrupting compounds (EDCs). A rapid method was developed for the simultaneous identification and quantitation of three alkylphenols, bisphenol A (BPA), 4-nonylphenol (4-NP), and 4-t-octylphenol (4-t-OP) in human blood serum. These compounds were determined by liquid chromatography negative electrospray ionization - tandem mass spectrometry (LC–ESI(-)MS/MS) after enzymatic deglucuronidation by E. Coli K12 β-glucuronidase. Due to the fact that these compounds exhibit unstable ionization and low sensitivity with this technique, their response was increased significantly by optimizing the parameters of electrospray ionization thoroughly with the use of full factorial design. The extraction was achieved by Hybrid SPE-PPT, a new sample pretreatment technique, that is considered a breakthrough in the field of solid phase extraction and generally speaking in bioanalysis. This method will assist in elucidating potential associations between human exposure to these compounds and adverse health effects. EXPERIMENTAL PROCEDURES Table 1. Precursor & Fragment Ions. Table 2. Screening Design. Full factorial experimental design is applied as a mathematical tool for the optimization of electrospray (ESI) parameters, so that potential interaction between the variables can be identified (Screening Design), and the optimal values of these variables can be located performing additional multilevel experiments (Response Surface Design). The optimum values can be represented graphically with a desirability plot. The SIM mode was applied for monitoring the signals of the deprotonated molecules. The developed method made use of the Single Reaction Monitoring (SRM) scan. The method was validated using serum pool sample from ten individuals. Fig .1. Pareto Charts from Screening Design. Fig .2. Main Effects Plots from Screening Design. Fig .3. Correlation of Responses from Screening Design. Table 3. Response Surface Design. Fig .4. Main Effects Plots from Resp. Surf. Design. Fig .5. Desirability Plot. METHOD PROTOCOL METHOD VALIDATION Table5.St. Addition Curves in serum. 150 μL serum Table4.Instrumental Parameters of method. Addition of 1.5μL solution Ε.coli K12 (β-D-Glucuronosideglucuronosohydrolase, EC 3.2.1.31) and incubation at 400C for 3 hours Addition of 450 μL methanol Table6.Validation Data. Vortex for 1 min Centrifugation for 10 min at 4000 rpm LODmethod = 3 * SD / b   where b: slope of standard addition curve SD: Standard deviation of 6 spiked samples with2 μg/L Transfer supernatant fluid to a Hybrid SPE-PPT cartridge CONCLUSIONS 500 μL extraction solvent - evaporation and redissolution with 150 solvent (37.5 μL water - 107.5 μL methanol – 5 μL acetone) • The causes of instability in response of the three alkylphenols in electrospray • were located and faced effectively. • A rapid and low-cost method was developed and validated, with the use of • the new technique “Hybrid SPE-PPT”. LC–MS/MS Fig .6. TIC and SRM chromatograms in spiked serum sample.