112011/02011/055// 2323

Development of Development of SPE SPE

for urine samplefor urine sample

222011/02011/055// 2323

hydrophobic interaction type hydrophobic interaction type SPESPE

hydrophobic interaction Interaction between hydrophobic chemicals Aromatic compounds dissolve in organic solvent

effective for hydrophobic chemicals widely used for collection of the chemicals in water

not effective for ionic, hydrophilic chemicalsnot effective for ionic, hydrophilic chemicals

modify polymer with multifunctional group for ionic chemicals ionic group for hydrophilic chemicals hydrophilic

group

332011/02011/055// 2323

highly-functional highly-functional solid phase extraction(SPE) solid phase extraction(SPE)

polymerpolymer

CH2

CH2

CH2

CH2

CH2

CH2

CH2

CH3

CO NH2

SO O

O-

NCH2 CH2

C COO

OH OHN

+CH3 CH3

CH3

OH

Hydrophobicinteraction

Hydrophilicinteraction

Ion-exchangeinteraction

Complex formationCation exchange

Anion exchange

－interaction

Basel resin polymerBasel resin polymer

New functional group

442011/02011/055// 2323

Chemical structure of SPE Chemical structure of SPE (reverse phase ion exchange (reverse phase ion exchange

type)type)

NO

N+

R1R1

R2

hydrophilic

N O

CH3

O

O

OH

O

N+

R1R1

R2ion exchange

hydrophobic hydrophobic

polar

552011/02011/055// 2323

Chemical structure of SPE Chemical structure of SPE for urine sample for urine sample

CHCH2 CH2C

CHCH2

OO

CH3

OH

N+CH3

CH3

OH

CHCH2 CH2C

CHCH2

COO

CH3

OH

N CH3

CH3

CHCH2 CH2C

CHCH2

COO

CH3

CHCH2

CN

CH3

CH3

OH

OH

O

RP-SAX RP-WAX

RP-OH

662011/02011/055// 2323

Property of SPE Property of SPE

樹脂名樹脂名 単位単位 RP-SAXRP-SAX RP-WAXRP-WAX RP-OHRP-OHmedian diameter µm 68 68 58specific surface area m2/g 550 560 250micropore diameter nm 8.8 9.7 14micropore capacity mL/g 0.66 0.59 0.67

ion exchange capacity meq/g 0.33 0.50 －

unit capacity (dry) mL/g 2.42 2.52 3.40unit capacity(swelling) mL/g 3.04 3.23 4.19degree of swelling － 1.26 1.28 1.24ion exchange － － N(CH3)2C2H4OH － N(C2H5)2 －

772011/02011/055// 2323

Treatment Treatment for urine samplefor urine sample

urine sample add. x1, 20mM Phosphate buffer (pH 4.0 )

add. surrogate ： OH-PCB 13C mix (100ppb) 20uL,

Solid PhaseExtraction （ RP-WAX.)

Concentration

elution, 8 mL 0.1% NH3 in MeOH

adjust pH 4.0 by 10% HCOOH aq.

add. 5mL, 20mM Phosphate buffer (pH 7.0 )add. 20 μL β-Glucuronidase / Aryl sulfatase,

Enzymegenation

Extraction

Centrifuge 2600rpm. 10min

Concentration

add. x 9 CH3CN

Concentration

LC/MS analysis

by N2 gas, 1 mL

by N2 gas, 1 mL

by CH2Cl2, 2 mL x 2

37 ℃, 1.5 hrs

dryness by N2 gas,

GC/MS analysis

methylation

9 2011/05/23

LC/MS Analysis

Time7.00 8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

7.00 8.00 9.00 10.00 11.00 12.00 13.00

%

0

100std_06_0 1: TOF MS ES-

374.829 0.05Da1.25e5

10.5410.2510.08

std_01_3 1: TOF MS ES- 374.829 0.05Da

1.25e5x200

4-OH-146

3-OH-138

4-OH-153100 ppb

0.5 ppb

Time7.00 8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

7.00 8.00 9.00 10.00 11.00 12.00 13.00

%

0

100std_06_0 1: TOF MS ES-

340.868+408.79 0.05Da2.27e5

9.10

10.999.53

std_01_3 1: TOF MS ES- 340.868+408.79 0.05Da

2.27e5x200

11.019.54

4-OH-1874-OH-107 4-OH-172

100 ppb

0.5 ppb

dominant OH-PCB in human blood sample

Compound name: 4-OH CB-187Correlation coefficient: r = 0.999882, r^2 = 0.999763Calibration curve: 144.295 * x + -29.3967Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

0

5000

10000

Compound name: 4-OH-CB-107Correlation coefficient: r = 0.999818, r^2 = 0.999637Calibration curve: 66.5632 * x + -13.6398Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

0

2000

4000

6000

Compound name: 4'-OH CB-146Correlation coefficient: r = 0.999130, r^2 = 0.998261Calibration curve: 51.9021 * x + -9.84197Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Res

pons

e

0

1000

2000

S/N ratio of 0.5ppb 4-OH-CB-54 on selected ion chromatogram(mass window: 30mDa)

min8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

F1:TOF MS,ES-374.829

sample__b_0

1.300e+003

8.157.24 12.9510.668.34

8.99 10.0810.94

min8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

F1:TOF MS,ES-374.829

std_02_0

1.300e+00310.08

8.487.27 7.75

8.709.789.28

4'-OH CB-15310.54

10.8212.8011.76 12.09

min8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

F1:TOF MS,ES-374.829

sample_u_spk

1.300e+0034'-OH CB-153

10.5110.20

7.80 8.37 9.638.77 9.39 10.7011.92 12.47

min8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

F1:TOF MS,ES-374.829

sample__u2_0

1.300e+003

4'-OH CB-15310.61

10.30

9.75

9.539.358.898.207.46 12.8211.09

Std 2.0 ppb

urine blankSpike 2.0 ppb

urine sample

urine blank

4-OH-1463-OH-138

4-OH-153

4-OH-1533-OH-138

ND

Std.2.0 ppb

Urea blankSpike 2.0 ppb

Urea sample

Urea blank

min8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

F1:TOF MS,ES-340.868

std_02_0

1.715e+003

4-OH-CB-107

8.497.86 10.16 12.1110.9612.51

min8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

F1:TOF MS,ES-340.868

sample_u_blk0

1.715e+003

10.8510.23

min8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

F1:TOF MS,ES-340.868

sample_u_spk

1.715e+003

4-OH-CB-107

8.427.58 7.96 10.06 10.49 11.02 11.78

min8.00 9.00 10.00 11.00 12.00 13.00

%

0

100

F1:TOF MS,ES-340.868

sample__u2_0

1.732e+003

4-OH-CB-107

8.53

7.779.588.70

10.15

12.0411.37

12.82

Std 2.0 ppb

urine blank+ 2.0 ppb

urine sample

urine blank

4-OH-107

4-OH-107

4-OH-107

Compounds / Samples urine 1 urine 2 urine blank urine Spike* yield 4-OH CB-187 N.D. N.D. N.D. 2.0 100 4-OH-CB-107 0.7 0.9 N.D. 1.9 954-OH CB-146 N.D. N.D. N.D. 1.7 853-OH CB-138 0.8 1.1 N.D. 1.9 953-OH CB-153 0.9 1.9 N.D. 1.8 904-OH-CB-172 N.D. N.D. N.D. 2.0 100

Compounds mean (area) s %RSD 4-OH CB-187 99.04 2.37 2.39 4-OH-CB-107 48.12 1.93 4.014-OH CB-146 37.32 0.82 2.193-OH CB-138 29.76 1.47 4.953-OH CB-153 40.14 0.82 2.044-OH-CB-172 61.95 2.21 3.57

urine urine 4-OH CB-187 0 0 4-OH-CB-107 4.7 4.54-OH CB-146 0.0 03’-OH CB-138 5.3 5.53-OH CB-153 6 9.54-OH-CB-172 0 04'-OH-CB-165 0 0

OH-PCB concentration in urine sample (ng/mL)

Compound name: 3-OH CB-H138Correlation coefficient: r = 0.999568, r^2 = 0.999137Calibration curve: 51.2725 * x + -14.3414Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Res

pons

e

0

1000

2000

Compound name: 4'-OH CB-153Correlation coefficient: r = 0.997974, r^2 = 0.995952Calibration curve: 57.0245 * x + -7.55719Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0 45.0 50.0

Res

pons

e

0

1000

2000

3000

Compound name: 4'-OH-CB-172Correlation coefficient: r = 0.999243, r^2 = 0.998486Calibration curve: 79.3337 * x + -8.89529Response type: External Std, AreaCurve type: Linear, Origin: Exclude, Weighting: 1/x, Axis trans: None

Conc0 10 20 30 40 50 60 70 80 90 100

Res

pons

e

0

2000

4000

6000

PCB & PFCsCa metabolism

blood sampling

PFHxA/PFOA anethetize

swelling 1924g

kidney brain

livergallbladder

0

1

2

3

4

5

6

7

Plas

ma

Ca

conc

. (m

g/dl

)

Initial

1 day

PFOA PFHxA

chiral PCB Enantioselective

analysis

What is enantiomer?What is enantiomer?

enantiomerenantiomer is is one of two one of two stereoisomers that are that are mirror images of each other of each other that are not identical, that are not identical, much as one's left and right much as one's left and right hands are are "the same" but opposite"the same" but opposite..

Enantiomer Fraction (EF)

EF = --------------E(+)

E(+)+ E(-)E(+) : (+) enantiomer peak areaE(-) : (-) enantiomer peak area

technical products

EF = 0.5

metabolism

EF > 0.5

racemates

EF < 0.5

non-racemic residues

The EF variation may distinguish POPs transfer of current use and that of past applications.

POPs in the oceans

(+) (-) (+)

(-) (+)

(-)

To identify the behavior of POPsTo identify the behavior of POPs

The results of Enantioselective analysis were The results of Enantioselective analysis were shown using Enantiomer Fraction (EF).shown using Enantiomer Fraction (EF). for tracking and apportioning chemical movement between environmental compartments for investigating microbial degradation processes.

Enantiomeric compositions of POPs in seawater were investigated

0.00

0.25

0.50

0.75EF

Enantiomer Fraction(EFEnantiomer Fraction(EF ））EF=

E(+)

E(+) + E(-)E(+): amount of (+)enantiomerE(-) : amount of (- ） enantiomer

(+)

(-) (+)

(-)

(+)(-)(+)

(-)

(+)

(-)

EF=

E1

E1 + E2E1: amount of the first enantiomerE2: amount of the second enantiomer

oror

Enantiomer Fraction(EF)Enantiomer Fraction(EF)Changes in physicochemical process,

It is possible to distinguish between newly caused pollution and old pollution by monitoring EF

the EF value does not change.

Changes in metabolic process,

the EF value changes.

Enantioselective determination of chiral PCBs in Spanish breast milk samples by heart-cut MDGC, Luisa R Bordajandi, Maria Jose Gonzalez (2005 ) : Organohalogen Compounds. 67, 1361-

DISTRIBUTION OF CHIRAL PCBs IN SELECTED TISSUES IN THE LABORATORY RAT , Hans-Joachim Lehmler, et al (2004): Organohalogen Compounds. 66, 443-

　 PCB-91 PCB-95 PCB-149Chlorofen n.d. 0.5 0.5

soil 0.46 0.5 0.5Adipose 0.73, n=1 0.66, n=2 0.50±0.04

　 (0.62, n=1) (0.53, n=1) (0.48, n=2)Brain n.d. n.d. n.d.Heart n.d. n.d. n.d.

　 　 　 (0.53,n=1)Kidney n.d. n.d. 0.52±0.04

　 (0.55,n=2) 　 　

Liver n.d. 0.47,n=1 0.52±0.04　 (0.54,n=2) 　 　

Lung n.d. n.d. 0.47,n=2Skin n.d. 0.54,n=1 0.49±0.04

　 　 (0.41,n=1) 　

Spleen n.d. 0.51,n=2 0.54±0.03　 　 (0.56,n=1) (0.54±0.04)

WholeBlood n.d. n.d. 0.52,n=1Serum n.d. n.d. 0.06,n=2

Enantiomeric fraction of PCB 91, 95 and 149 in tissues and blood from soil extract-treated animals

30 2011/05/23

GC/MS Analysis

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40Retention Time (min)

0

200000

400000

600000

800000

1000000

1200000

1400000

Inte

nsity

2PM

121

4PH

104

3PM

982M

832M

111

4PM

121

3M12

12P

M11

24P

M93

2M10

93M

119

3M84

4PM

101

3M11

32P

M11

42P

M10

64P

M12

04P

M86

4PM

112

4PM

107

3M11

84M

109

3M11

04M

974P

M12

74P

M10

6

OH-PCB （ 5Cl ）のクロマトグラム（ STD ）

10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40Retention Time (min)

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

110000

Inte

nsity

2PM

121

4PH

104

3PM

982M

832M

111

4PM

121

3M12

12P

M11

24P

M93

2M10

93M

119

3M84

4PM

101

3M11

32P

M11

42P

M10

64P

M12

04P

M86

4PM

112

4PM

107

3M11

84M

109

3M11

04M

974P

M12

74P

M10

6

OH-PCB （ 5Cl ）のクロマトグラム（尿試料）

OH-PCB （ 6Cl ）のクロマトグラム（ STD ）

20 22 24 26 28 30 32 34 36 38 40 42 44Retention Time (min)

0

100000

200000

300000

400000

500000

600000

700000

Inte

nsity

4M13

4

4PM

165

4M14

6

3PM

138

4PM

130

4M16

3

4PM

159

4M16

2

OH-PCB （ 6Cl ）のクロマトグラム（尿試料）

20 22 24 26 28 30 32 34 36 38 40 42 44Retention Time (min)

0

10000

20000

30000

40000

50000

60000

70000

80000

90000

100000

Inte

nsity

4M13

4

4PM

165

4M14

6

3PM

138

4PM

130

4M16

3

4PM

159

4M16

2