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
description
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 ?. - PowerPoint PPT Presentation
Transcript of 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
Hydroxylated PCBs: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
Why OH-PCBs ?
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 165 is difficult in
GC-HRMS with methoxy-derivatization.— Methoxy metabolized PCB and methoxy-derivatization OH-
PCB is difficult to be separated.
GC/MS? or LC/MS?
MetaboliteMeO-PCBs
MetaboliteOH-PCBs
Biological sample
Derivatization
GC-MS sampleMetaboliteMeO-PCBs
DerivedMeO-PCBs
From metabolized? or derived ?
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
Previous study and Aim
Major 6 components of OH-PCBs in human blood — purchased from Wellington Laboratories Inc.(Guelph, On, Canada)— Penta-chloro/OH-CB 4-OH-CB 107— Hexa-chloro/OH-CB 3-OH-CB 153 4-OH-CB 146 3’-OH-CB 138— Hepta-chloro/OH-CB 4-OH-CB 187 4-OH-CB 172
Material
OH
ClCl
ClCl
Cl
Cl
4-OH-CB 146
Cl
OHCl
Cl
Cl
Cl
Cl
3’-OH-CB 138
Cl
OHCl
ClCl
Cl
Cl
3-OH-CB 153
OH
ClCl
ClClCl
Cl
Cl
4-OH-CB 187
OH
ClCl
Cl
Cl
Cl
4-OH-CB 107
OH
ClCl
ClCl
Cl
Cl
Cl
4-OH-CB 172
Another OH-PCBs— Hexa-chloro/OH-CB 4’-OH-CB 165
4’-OH-CB 165
OH
Cl
ClCl
Cl
Cl
Cl
Sample preparationUrine(100 ～ 200 mL)
Add X1, 20mM Phosphate buffer (pH 4.0)
Extraction2 mL of CH2Cl2 X 2 times
Adjust pH at 4.0 with 10%-HCOOH aq.
Solid Phase Extraction(RP-WAX:240 mg)
Elute with 8 mL of 0.1%-NH3 in methanol
ConcentrationLess than 1 mL by N2 gas
Add. 5mL of 20mM-Phosphate buffer (pH 7.0 )
Add. 20 μL β-Glucuronidase/ Aryl sulfatase,
Enzyme reaction at 37 degree for 1.5 hr
Centrifugation2600 rpm. for 10 min
Concentration1 mL by N2 gas
Concentration1 mL by N2 gas
Add. X 9 CH3CN
LC/MS analysis
Analytical Condition LC condition
UPLCInstrument: ACQUITY UPLCColumn: BEH C18 2.1ID X 150 mm, 1.7umFlow rate: 0.5 mL/min.Column heater: 60 degree centigradeMobile Phese A: 5mM CH3COONH4 aq.Mobile Phese B: THF/CH3CN (v/v: 1/4)
Gradient :Time %A %BInitial 75 25
17 min. 25 7518 min. 1 99
18.5 min. 75 25Total Run Time: 20 min
Analytical Condition MS condition
MSInstrument: Xevo G2 Q-TOFIonization mode: ESI negativeCapillary: 1.5 kVSampling Cone: 40 VSource Temp: 120 degree centigradeDesolvation Temp: 600 degree centigradeCone Gas Flow: 20 L/hr.Desolvation Gas Flow:800 L/hr.Resolving Power: <20,000
Selected Ion:Penta-chloro/OH-CB m/z=340.8675Hexa-chloro/OH-CB m/z=374.8286Hepta-chloro/OH-CB m/z=408.7896
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
Mass Spectra of OH-PCBs50
m/z335 340 345 350
%
0
100
340.868
338.871 342.865
344.862346.861
50
m/z365 370 375 380 385
%
0
100
374.829
372.832
376.826
378.823
380.821
50
m/z400 405 410 415 420
%
0
100
408.790
406.793
410.787
412.784
Penta-Chloro/OH-CB Hexa-Chloro/OH-CB
Hepta-Chloro/OH-CB
35Cl537Cl0
35Cl437Cl1
35Cl337Cl2
35Cl237Cl3
35Cl637Cl0
35Cl537Cl1
35Cl437Cl2
35Cl337Cl3
35Cl737Cl0
35Cl637Cl1
35Cl537Cl2
35Cl437Cl3
m/z=340.8675 m/z=374.8286
m/z=408.7896
High Resolution MS Chromatogram2
Time6.00 8.00 10.00 12.00 14.00 16.00
%
0
100 9.56
2
Time6.00 8.00 10.00 12.00 14.00 16.00
%
0
100 9.56
2
Time6.00 8.00 10.00 12.00 14.00 16.00
%
0
100 9.56
500 mDa
50 mDa
5 mDa
２ pg/uL Standard solution
4-OH-CB 187
4-OH-CB 187
4-OH-CB 187
LC Separation of OH-PCBs2
Time8.00 9.00 10.00 11.00 12.00 13.00 14.00
%
0
100
9.56
4-OH-CB 187
4-OH-CB 172
4-OH-CB 107
3’-OH-CB 1383-OH-CB 153
4-OH-CB 146
4’-OH-CB 165
Calibration Curve(STD 0.5 to 50 pg/uL)
4-OH-CB 187
4-OH-CB 172
4-OH-CB 107
3’-OH-CB 138 3-OH-CB 153
4-OH-CB 146Conc
0.0 10.0 20.0 30.0 40.0 50.0
Res
pons
e
0
2000
4000
6000
Conc0.0 10.0 20.0 30.0 40.0 50.0
Res
pons
e
0
1000
2000
3000
Conc0.0 10.0 20.0 30.0 40.0 50.0
Res
pons
e
0
1000
2000
Conc0.0 10.0 20.0 30.0 40.0 50.0
Res
pons
e
0
1000
2000
3000
Conc0.0 10.0 20.0 30.0 40.0 50.0
Res
pons
e
0
1000
2000
Conc0.0 10.0 20.0 30.0 40.0 50.0
Res
pons
e
0
1000
2000
3000
4000
R^2=1.000 R^2=0.999 R^2=0.998
R^2=0.999 R^2=0.996 R^2=0.998
Repeatability(STD 1 pg/uL X 5 injections)
Compounds mean (area) S.D. %RSD
4-OH CB 187 99.04 2.37 2.39 4-OH-CB 107 48.12 1.93 4.01 4-OH CB 146 37.32 0.82 2.19 3’-OH CB 138 29.76 1.47 4.95 3-OH CB 153 40.14 0.82 2.04 4-OH-CB 172 61.95 2.21 3.57
1
Time8.00 10.00 12.00
%
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100
1
Time8.00 10.00 12.00
%
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100
1
Time8.00 10.00 12.00
%
0
100 9.13
Penta-Chloro/OH-CB
Hexa-Chloro/OH-CB Hepta-Chloro/OH-CB
Matrix Effect(Urine blank + spike 2 pg/uL)
Compounds Urine blank
Urine Spike yield
4-OH-CB 187 N.D. 2.00 100 4-OH-CB 107 N.D. 1.90 954-OH-CB 146 N.D. 1.70 853'-OH-CB 138 N.D. 1.90 953-OH-CB 153 N.D. 1.80 904-OH-CB 172 N.D. 2.00 100
2
Time8.00 10.00 12.00
%
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8.00 10.00 12.00
%
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100
8.00 10.00 12.00
%
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100
2
Time8.00 10.00 12.00
%
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8.00 10.00 12.00
%
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100
8.00 10.00 12.00
%
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100
2
Time8.00 10.00 12.00
%
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8.00 10.00 12.00
%
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100
8.00 10.00 12.00
%
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Penta-Chloro/OH-CB
Hexa-Chloro/OH-CB Hepta-Chloro/OH-CB
Blank
STD 2 pg/uL
Spike 2 pg/uL
Blank
STD 2 pg/uL
Spike 2 pg/uL
Blank
STD 2 pg/uL
Spike 2 pg/uL
Urine Sample Chromatogram4-OH-CB 187
4-OH-CB 107urea1
Time6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
0
100
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
0
100 Urine 1
Urine 2
Urine 2
urea1
Time6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
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100
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
0
100 Urine 1
urea1
Time6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
0
100
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
0
100
Urine Sample Chromatogram
3’-OH-CB 138
4-OH-CB 146
Urine 1
Urine 2
Urine 1
Urine 2
urea1
Time6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
0
100
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
0
100
Urine Sample Chromatogram
4-OH-CB 172
3-OH-CB 153
Urine 1
Urine 2
Urine 1
Urine 2
urea1
Time6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
0
100
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
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100
urea1
Time6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
0
100
6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00
%
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100
4’-OH-CB 165
Urine Sample Level and Ratio
Compounds Urine 1 Urine 2
4-OH-CB 187 N.D. N.D. 4-OH-CB 107 4.7 pg/mL 4.5 pg/mL4-OH-CB 146 N.D. N.D.3‘-OH-CB 138 5.3 pg/mL 5.5 pg/mL3-OH-CB 153 6.0 pg/mL 9.5 pg/mL4-OH-CB 172 N.D. N.D.4’-OH-CB-165 N.D. N.D. Urine 1 Urine 2
0.00
1.00
2.00
3.00
4.00
5.00
6.00
7.00
8.00
9.00
10.00
4'-OH-CB 1073'-OH-CB 1383-OH-CB 153
Comparison blood and urine
Japanese 4HO-CB 107 > 4HO-CB 187 > 4HO-CB 146Belgian 4HO-CB 107 > 4HO-CB 146 > 4HO-CB 187Romanian 4HO-CB 187 > 4HO-CB 146 > 3'HO-CB 138
Blood sample
Urine 1 3HO-CB 153 > 3’HO-CB 138 > 4HO-CB 107Urine 2 3HO-CB 153 > 3’HO-CB 138 > 4HO-CB 107
Urine sample
Blood 4HO-CB 146 > 4HO-CB 107 > 3'HO-CB 138 > 4HO-CB 187Urine 1 3HO-CB 153 > 3’HO-CB 138 > 4HO-CB 107
Japanese sample
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 derivatization is 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.
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
Thank you for your attention !
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