OPTIMIZATION OF BISPHENOL A, 4-t-OCTYLPHENOL, AND 4-NONYLPHENOL EXTRACTION FROM HUMAN BLOOD SERUM WITH HYBRID SOLID PHASE EXTRACTION – PROTEIN PRECIPITATION TECHNIQUE (HYBRID SPE-PPT) John S. Lignos, Evangelos G. Moschos, Alexandros G. Asimakopoulos and Nikolaos S. Thomaidis Laboratory of Analytical Chemistry, Department of Chemistry, University of Athens, Panepistimioupolis Zografou, Athens, Greece. Bisphenol A (BPA), 4-nonylphenol (4 - NP), and 4 – t - octylphenol (4 – t - OP) are three alkylphenols widely used in industry, and are known as endocrine disrupting compounds (EDCs). For their extraction from human blood serum, a new sample extraction was achieved by hybrid solid phase extraction – protein precipitation technique (Hybrid SPE-PPT), a new sample treatment technique that is considered a breakthrough in the field of solid phase extraction and, generally speaking, in bioanalysis. The technology utilizes a patent-pending zirconia-coated particle, and exhibits selective affinity towards phospholipids while remaining non-selective towards our analytes. An optimized Hybrid SPE-PPT protocol for the extraction of these alkylphenols. This method was thoroughly optimized taking into account the particularities of their determination by liquid chromatography – electrospray ionization - tandem mass spectrometry (LC–ESI(–)MS/MS). A wide range of precipitation agents were examined (acetonitrile, methanol, formic acid and ammonium formate at various concentrations and percentages). The addition of acetone and its effect was thoroughly examined in electrospray ionization, exhibiting an important increase in sensitivity. This sample pretreatment protocol and the LC–MS/MS method were validated and meet the demands of a low-cost rapid sample extraction protocol for routine analysis of serum samples. The internal standards used during optimization were BPA d16, 4 – t - OP d2, and 4 - NP d2. Experiment 1:Optimum quantity of serum Experiment Protocol μL μL – 300 μL serum (Quantities tested). 2. Addition of 300 μL – 600 μL – 900 μL methanol (precipitating agent for proteins) respectively. 3. Vortex for 1 min. 4. Centrifugation for 10 min at 4000 rpm. 5. Transfer supernatant fluid to a Hybrid SPE-PPT cartridge. 6. Extract collected. Experiment 2: Optimum precipitating agent Precipitating agents tested: 1.Methanol, 2.Methanol (0.1 % w / v ammonium formate), 3.Methanol (0.5 % w / v ammonium formate), 4.Methanol (1 % w / v ammonium formate), 5.Methanol (0.1 % v / v formic acid), 6.Methanol (0.5 % v / v formic acid), 7.Methanol (1 % v / v formic acid), 8.Acetonitrile, 9.Acetonitrile (0.1 % w / v ammonium formate), 10. Acetonitrile (0.5 % w / v ammonium formate), 11. Acetonitrile (1 % w / v ammonium formate), 12. Acetonitrile (0.1 % v / v formic acid), 13. Acetonitrile (0.5 % v / v formic acid), and 14. Acetonitrile (1 % v / v formic acid). For each precipitation agent a standard solution, a spiked serum sample solution, and a matrix match solution were prepared. Experiment Protocol μL serum. 2. Addition of 300 μL (precipitating agent for proteins). 3. Vortex for 1 min. 4. Centrifugation for 10 min at 4000 rpm. 5. Transfer supernatant fluid to a Hybrid SPE - PPT cartridge. 6. Extract collected (300 μL). 7. Diluted to 1200 μL so that the final composition of the solution is 1 to 1 Water* : Precipitating agent. 8. LC-MS/MS analysis. 9. Absolute and Relative Recoveries calculated (Fig.1.). *Serum is considered as an aqueous solution Fig. 1. (%) Absolute and Relative Recoveries calculated in the second experiment.  Conclusions: 1.Enhancement of signal observed with formic acid combinations. 2.Suppression of signal observed with ammonium formate combinations. 3.Best precipitating agent Methanol Experiment 3: Optimum ratio Ratio (serum : methanol) tested: 1 to 1, 1 to 2, 1 to 3. Fig. 3. (%) Absolute and Relative Recoveries calculated in the third experiment. For each ratio a spiked serum sample solution, and a matrix match solution were prepared. Experiment Protocol μL serum. 2. Addition of 100 μL (1:1) – 200 μL (1:2) – 300 μL (1:3) methanol. 3. Vortex for 1 min. 4. Centrifugation for 10 min at 4000 rpm. 5. Transfer supernatant fluid to a Hybrid SPE-PPT cartridge. 6. Extract collected (300 μL). 7. Diluted to 1200 μL so that the final composition of the solution is 1 to 1 Water* : Methanol. 8. LC-MS/MS analysis. 9. Absolute and Relative Recoveries calculated (Fig.3.). *Serum is considered as an aqueous solution Experiment 4: Losses during extract evaporation For each ratio a spiked serum sample solution, and a matrix match solution were prepared. Experiment Protocol μL serum. 2. Addition of 300 μL methanol. 3. Vortex for 1 min. 4. Centrifugation for 10 min at 4000 rpm. 5. Transfer supernatant fluid to a Hybrid SPE-PPT cartridge. 6. Extract received (300 μL). 7. Partial (half of extraction) or total evaporation. 8. Diluted to 1200 μL so that the final composition of the solution is 1 to 1 Water* : Methanol. 9. LC-MS/MS analysis. 10. Absolute and Relative Recoveries calculated (Fig.4.) *Serum is considered as an aqueous solution  Conclusion: Losses were observed for 4-t-OP, and 4-NP Experiment 5: Effect of acetone in sensitivity Fig.5. Effect of acetone in response.  150 μL serum.  Addition of 1.5 μL solution Ε.coli K12 (β-D-Glucuronoside glucuronosohydrolase, EC ) and incubation at 40 0 C for 3 hours.  Addition of 450 μL methanol.  Vortex for 1 min.  Centrifugation for 10 min at 4000 rpm.  Transfer supernatant fluid to a Hybrid SPE-PPT cartridge.  500 μL extraction solvent - evaporation and redissolution with 150 solvent (37.5 μL water – μL methanol – 5 μL acetone).  LC-MS/MS analysis.  HILIC interactions were observed between Hybrid SPE-PPT and the three alkyphenols when using acetonitrile as precipitating agent.  The factors that affect recovery were identified.  A rapid method protocol was developed for LC-MS/MS analysis.  Conclusion: Ratio 1 to 3 (serum : methanol) Good values of recoveries and cleaner extract Fig. 2. Retention in cartridge. Fig. 4. (%) Absolute and Relative Recoveries calculated in the fourth experiment.  Conclusion: Cleaner extract 100μL serum