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Hydroxylated PCBs:OH -PCBs

DEVELOPMENT OF HYDROXYLATED POLYCHLORINATED BIPHENIL(OH-PCBs) ANALYTICAL METHOD IN HUMAN URINE WITH UPLC/Q-TOF MS Motoharu Suzuki, Toshihiro Okuno , Chisato Matsumura, Nobutake Sato , Jun Yonekubo , Tatsuya Ezaki, Yoshinori Inoue, Hiromasa Imaishi , Takeshi Nakano. Why OH-PCBs ?.

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Hydroxylated PCBs:OH -PCBs

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  1. DEVELOPMENT OF HYDROXYLATED POLYCHLORINATED BIPHENIL(OH-PCBs) ANALYTICAL METHOD IN HUMAN URINE WITH UPLC/Q-TOF MS Motoharu Suzuki, Toshihiro Okuno, ChisatoMatsumura, NobutakeSato, Jun Yonekubo, Tatsuya Ezaki, Yoshinori Inoue, HiromasaImaishi, Takeshi Nakano

  2. Why OH-PCBs ? • HydroxylatedPCBs:OH-PCBs • -Main metabolite of PCBs in human body, caused by cytochrome P450 monooxygenase • -Some OH-PCBs are carried into the human blood by transthyretin (TTR), carrier protein of thyroxine(T4) • -These OH-PCBs have high retention in the human blood and a few OH-PCB isomers longer half-life than parent compounds • -Competition of OH-PCBs and T4 is the mechanism involved in the disturbance of thyroid hormone(TH) • -Some studies determined the residue levels and patterns of PCB and OH-PCB congeners in human blood

  3. GC/MS? or LC/MS? • GC-HRMS(High Resolution MS) • Need derivatization of OH-PCBs to Methoxy-PCBs because of high polarity of OH-PCBs • Metoxy-derivatization of OH-PCB is needed for analyzing with GC-HRMS • Separation of 3-OH-CB 153 and 4’-OH-CB 165is difficult in GC-HRMS with methoxy-derivatization. • Methoxy metabolized PCB and methoxy-derivatization OH-PCB is difficult to be separated. Biological sample GC-MS sample Metabolite MeO-PCBs Metabolite MeO-PCBs Derivatization Metabolite OH-PCBs Derived MeO-PCBs From metabolized? or derived ?

  4. Previous study and Aim • Previous study • Determined elution order for 51 congeners of OH-PCB without derivatization with UPLC/QTof MS for analyzing human blood sample • Aim of this study • Developing analytical method for quantity to separate mixture of 6 major OH-PCBs in human blood • Applying analytical method to biological sample of human urine

  5. Material • Major 6 components of OH-PCBs in human blood • purchased from Wellington Laboratories Inc.(Guelph, On, Canada) • Penta-chloro/OH-CB4-OH-CB 107 • Hexa-chloro/OH-CB 3-OH-CB 153 4-OH-CB 146 3’-OH-CB 138 • Hepta-chloro/OH-CB4-OH-CB 187 4-OH-CB 172 4-OH-CB 187 4-OH-CB 172 3-OH-CB 153 4-OH-CB 146 3’-OH-CB 138 4-OH-CB 107 • Another OH-PCBs • Hexa-chloro/OH-CB 4’-OH-CB 165 4’-OH-CB 165

  6. Sample preparation Urine(100～200 mL) Add X1, 20mM Phosphate buffer (pH 4.0) Extraction 2 mL of CH2Cl2 X 2 times Adjust pH at 4.0 with 10%-HCOOH aq. Centrifugation 2600 rpm. for 10 min Solid Phase Extraction (RP-WAX:240 mg) Elute with 8 mL of 0.1%-NH3 in methanol Concentration 1 mL by N2 gas Concentration Less than 1 mL by N2 gas Add. X 9 CH3CN Concentration 1 mL by N2 gas Add. 5mL of 20mM- Phosphate buffer (pH 7.0 ) Add. 20 μL β-Glucuronidase / Aryl sulfatase, LC/MS analysis Enzyme reaction at 37 degree for 1.5 hr

  7. Analytical Condition • LC condition

  8. Analytical Condition • MS condition

  9. Result • - Mass Spectra of OH-PCBs • - Selectivity by high resolution MS • - LC separation of 6 major OH-PCBs in human blood • - Calibration curve • - Repeatability • - Matrix effect • - Applying method to urine sample • - Level and ratio of OH-PCB congeners

  10. Mass Spectra of OH-PCBs m/z=340.8675 m/z=374.8286 35Cl437Cl1 35Cl537Cl1 35Cl437Cl2 35Cl537Cl0 35Cl337Cl2 35Cl637Cl0 35Cl337Cl3 35Cl237Cl3 Penta-Chloro/OH-CB Hexa-Chloro/OH-CB m/z=408.7896 35Cl637Cl1 35Cl537Cl2 35Cl437Cl3 35Cl737Cl0 Hepta-Chloro/OH-CB

  11. High Resolution MS Chromatogram ２ pg/uL Standard solution 500 mDa 4-OH-CB 187 50 mDa 4-OH-CB 187 5 mDa 4-OH-CB 187

  12. LC Separation of OH-PCBs 4-OH-CB 107 4-OH-CB 146 3’-OH-CB 138 4-OH-CB 187 3-OH-CB 153 4-OH-CB 172 4’-OH-CB 165

  13. Calibration Curve (STD 0.5 to 50 pg/uL) R^2=1.000 R^2=0.999 R^2=0.998 4-OH-CB 187 4-OH-CB 107 4-OH-CB 146 R^2=0.999 R^2=0.996 R^2=0.998 3’-OH-CB 138 3-OH-CB 153 4-OH-CB 172

  14. Repeatability (STD 1 pg/uL X 5 injections) Penta-Chloro/OH-CB Hexa-Chloro/OH-CB Hepta-Chloro/OH-CB

  15. Matrix Effect (Urine blank + spike 2 pg/uL) Blank STD 2 pg/uL Spike 2 pg/uL Penta-Chloro/OH-CB Blank Blank STD 2 pg/uL STD 2 pg/uL Spike 2 pg/uL Spike 2 pg/uL Hexa-Chloro/OH-CB Hepta-Chloro/OH-CB

  16. Urine Sample Chromatogram 4-OH-CB 187 Urine 1 Urine 2 4-OH-CB 107 Urine 1 Urine 2

  17. Urine Sample Chromatogram 4-OH-CB 146 Urine 1 Urine 2 3’-OH-CB 138 Urine 1 Urine 2

  18. Urine Sample Chromatogram 3-OH-CB 153 Urine 1 4’-OH-CB 165 Urine 2 4-OH-CB 172 Urine 1 Urine 2

  19. Urine Sample Level and Ratio

  20. Comparison blood and urine Blood sample Japanese 4HO-CB 107 > 4HO-CB 187 > 4HO-CB 146 Belgian 4HO-CB 107 > 4HO-CB 146 > 4HO-CB 187 Romanian 4HO-CB 187 > 4HO-CB 146 > 3'HO-CB 138 Urine sample Urine 1 3HO-CB 153 > 3’HO-CB 138 > 4HO-CB 107 Urine 2 3HO-CB 153 > 3’HO-CB 138 > 4HO-CB 107 Japanese sample Blood 4HO-CB 146 > 4HO-CB 107 > 3'HO-CB 138 > 4HO-CB 187 Urine 1 3HO-CB 153 > 3’HO-CB 138 > 4HO-CB 107

  21. Conclusion • Analytical method • 6 major OH-PCBs in human blood is separated by UPLC without derivatization • Moreover, separation of 3-OH-CB 153 and 4’-OH-CB 165 that is difficult in GC-HRMS with derivatizationis enable • Total analytical time is less than 20 min • Urine sample • Developed analytical method is able to be applied to human urine sample • Three of major 6 OH-PCBs in human blood is detected(#107, #138 and #153) and #146, #182 and #172 are not detected in human urine • Concentration of detected three OH-PCBs in urine sample is • #107 > #138 > #153, pattern of detected compound might be different from it in blood sample.

  22. Acknowledgements • This research was partly supported by • Grants-in-Aid for Scientific Research (B)(No. 21310027) from the Ministry of Education, Culture, Sports, Science and Technology, Japan • The Waste Management Research Grant (No. K22037) from the Ministry of the Environment, Japan

  23. Thank you for your attention ! E-mail : nobutake_sato@waters.com Waters THE SCIENCE IS WHAT’S POSSIBLE.TM Xevo G2 Q-Tof

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