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Hydroxylated PCBs:OH -PCBs

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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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slide1

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

slide2

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
slide3

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 ?

slide4

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
slide5

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

slide6

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

slide9

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
slide10

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

slide11

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

slide12

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

slide13

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

slide14

Repeatability

(STD 1 pg/uL X 5 injections)

Penta-Chloro/OH-CB

Hexa-Chloro/OH-CB

Hepta-Chloro/OH-CB

slide15

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

slide16

Urine Sample Chromatogram

4-OH-CB 187

Urine 1

Urine 2

4-OH-CB 107

Urine 1

Urine 2

slide17

Urine Sample Chromatogram

4-OH-CB 146

Urine 1

Urine 2

3’-OH-CB 138

Urine 1

Urine 2

slide18

Urine Sample Chromatogram

3-OH-CB 153

Urine 1

4’-OH-CB 165

Urine 2

4-OH-CB 172

Urine 1

Urine 2

slide20

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

slide21

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.
slide22

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
slide23

Thank you for your attention !

E-mail : [email protected]

Waters

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