Measuring IsoflavonoidsMeasuring Isoflavonoidsin Foods and Biological Fluids in Foods and Biological Fluids by LC/PDA and ion trap MSby LC/PDA and ion trap MS

Adrian A. Franke, Cancer Research Center of Hawai‘i

Honolulu

Soy, Isoflavones & Breast CancerSoy, Isoflavones & Breast Cancer

Cline 2004 pers.commun.

0.50.5

Wu 2004

plasma or urinary IFL as biomarkerIFL as biomarker

Japan@nucl.sites

China

Holland

Asian Americans

CaucasiansChina

ChinaAsian Americans

China

China

Japan (miso, IFL)

Australia

Japan

NON-Asian Americans

Yan&Spitznagel 2004 (meta, n=14)(meta-post, n=7)(meta-urine, n=4)

BIOACTIVITYBIOACTIVITY

EXPOSUREEXPOSURE BIOAVAILABILTYBIOAVAILABILTY

Value ofValue ofDietaryDietary

PhytochemicalPhytochemical

How potent?

How much consumed? How much absorbed &in target tissue?

High Pressure Liquid Chromatography(HPLC)

Analytes1. Pump�

data processing�

3. PDA or mass detector

Liquid

Solid 2. column

Typical HPLC conditions for IFL

•SOLID PHASE Hydrobond C18 (100 x 3.0 mm; 5µm)Beta Basic-8 (100 x 2.1 mm, 3 µm)

•LIQUID PHASE MeOH/MeCN/aq HForm or HOAcgradient 40%-90% organicflow ~0.25 mL/min

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

589 nmNa=D-line

486 nmH2=F-line

656 nmH2=C-line

Molecules interact with light

QuickTime™ and aGIF decompressor

are needed to see this picture.

470 nmCarotenoids

Abs:450-470 nmAppear:620 nm

Principle of UV/VIS detectionvia photo-diode array (PDA)

Detector

420 nm

580 nm

PDA Optical Diagram

Flow Cell

MirrorBeam

Shaper

Fixed Grating1 x 512

Photodiode Array

Focusing Lens

Deuterium Lamp

Beam Combiner Filter Wheel

Tungsten-halogen Lamp

QuickTime™ and aPhoto - JPEG decompressor

are needed to see this picture.

+ O

OH

HO

OH

OH

OHC

2B

A

ANTHOCYANIDINStrans beta- CAROTENE

OHOH2C

OHHO

OH

HO

Ascorbic acid (vitamin C)

O

O

OCH3

R7O

OH

OH2

A

FLAVONOIDS

C

B

O

O

HO

OHOH

3

7

B5

CA

ISOFLAVONOIDSα-Tocopherol (a vitamin E)

O

HO

260nm

300nm 257nm

320nm

Glycitein

UV Scan as Diagnostic Tool

O

O

HO

OH

3

4'

7O

O

HO

OH

63

7

H3CO4'

Daidzein

330nm

258nm

290nm

Genistein

O

O

HO

OHOH

3

7

54'

UV Scan as Diagnostic Tool

CH3OH

O

HO

OH

7

4'

280nmEquol

320nm275nm

DMA

O

O

HO

OH

3

4'

7

Hesperetin

323nm

273nm

274nm

Enterolactone

Enterodiol

Naringenin

323nm

UV Scan as Diagnostic Tool

289nm 289nm

7 2O

O

OCH3

HO

OH

OH

4'

3'7 2O

O

OH

HO

OH

H

4'

3'

HO

CH2OH

CH2OH

OH

HO

OH

O

O

HPLC/PDA of soy foods (260 nm)

Franke 1998; 1999

Glycitein

Daidzein

Genistein

6''

OO

HO

HOHO

O

OO

HO

6''

OO

HO

HOHO

OH3C

O

6''

OO

HO

HOHO

HO

(b) malonate

(a) acetate

(c) glucoside

O

O

O

R5 OH

R6

3

4'

7

HPLC/PDA trace of Urine

Abs

orba

nce

at 2

80 n

m

Abs

orba

nce

at 2

60 n

m

DE

DE

GLYE

GE

DHGE

DHDE

GE

EQ

DMAFlav (i.s.)

0 42[MINUTES]

O

O

HO

OHOH

3

7

4'5

GENISTEINO

O

HO

OH

H3CO

GLYCITEIN

4'

3

7

6

O

O

HO

OH

3

DAIDZEIN

4'

7

3

7

4'

OHO

OHEQUOL

3

4'

CH3OH

O

HO

OHDMA

O

O

FLAVONE internal standard

HPLC Trace of Urine after Soy Intake

HPLC/PDA of a Random Urine Sample

GE

DE

40MINUTES

isoflavonoids are distinguishable from flavonoids

15

NE HT

HESPERETIN289

323

289

323

NARINGENIN

PDA trace at 280nm from urine extract

mA

U

8.0 9.0 10.0 11.0 12.0 13.0 14.0

25

50

75

100

125

150

175

200

225

250

275

ED

DE GLYENE

EL

EQHTGE

DMA

Retention Time (minutes)

FOR (i.s.)DHDE DHGE

Q

Bottom line isSIGNAL/noise ratio

(resolution from interference)should be sufficient

PDA works for urine and foods, sometimes for blood

Quantitate diagnostic ions

Mass Spectrometry (MS) higher selectivity!

Multiple Reaction Monitoring(MRM)

Daidzein (MW 254.1)

m/z 225

m/z 253

m/z 197+

O

O

O

3

7 2

A C

BOH

OO

3

7 2

A C

BOH

OO 7 2

A C

-CO

Charged droplet formation, desolvation, disintegration and ion evaporation

LIQUID

GAS

Principle of mass detectiona) liquid to gas by electrospray (ESI)

Electrospray Process

Principle of mass detectionb) ion trap mass spectrometry (itMS)

0 2 4 6 8 10 120

20406080

100020406080

100020406080

100020406080

100020406080

10002040

6080

100

0 2 4 6 8 10 120

20

40

60

80

100

Rel

ativ

e A

bund

ance

20

40

60

80

100

Rel

ativ

e A

bund

ance

20

40

60

80

100

Rel

ativ

e A

bund

ance

0

20

40

60

80

100

Rel

ativ

e A

bund

ance

50000

100000

Inte

nsity

O

O

HO

OH

3

4'

O

O

HO

OH

H3CO 6

3

7

4'

O

O

HO

OHOH

3

7

54'

O

O

HO

OH OH

3

7

54'

CH 3OH

O

HO

OH4'

3

OHO

OH

3

4'

OH

O

HO

OH

3

4'

***

Equol

DMA

13C3-Equol

13C3Daidzein

13C3-Genistein

IFL by neg.ESI-itMS (MRM)

DH-Daidzein

DH-Genistein

13C3-DMA

Daidzein

Glycitein

Genistein

OHO

OH

*

4'

3**

O

O

HO

OH

*4'

* 3

*

O

O

HO

OH4'

3

OH

** *

O

O

HO

OH

3

4'

Run 1 (metabolites)

Franke 2002; Blair 2003

Run 2 (non-metabolites, parents)

MW 272.1

MW 242.1

MW 258.1

MW 256.1

MW 245.1

MW 261.1

MW 254.1

MW 284.1

MW 270.1

MW 257.1

MW 273.1

Minutes Minutes

m/z 257.1

m/z 109.2

m/z 268.1

m/z 283.3

m/z 149

m/z 255.1 m/z 253.4

m/z 196-198, 208-210, 223-226

m/z 271.3

m/z 165

m/z 269.3

m/z 241.3

m/z 121.2, 135.2

m/z 244.3

m/z 123.2, 138.2

m/z 272.3

m/z 182-184, 198-205, 226-229, 244

m/z 198-201, 210-213, 226-229

m/z 256.4

m/z 260

m/z 109.2, 242.3

m/z 180-184, 194-202, 224-227, 241

•Hydrobond C18 (100 x 3.0 mm;5µm)•MAWacid 40%-90%, 0.25 mL/min

0 5 10 15 20

Time (minutes)0

20406080

1000

20406080

1000

20406080

1000

20406080

1000

20406080

100

0

50

100

0

50

100

0

50

1000

50

100

PDA: 280nm

mA

U

8.0 9.0 10.0 11.0 12.0 13.0 14.0

25

50

75

100

125

150

175

200

225

250

275

ED

DE GLYENE

EL

EQHTGE

DMA

Analysis of Flavonoids, Isoflavonoids & Lignans

MS: neg. ESI, ms2 Retention Time (minutes)

m/z 301

m/z 253, 283

FOR (i.s.)

ED

m/z 271

m/z 151, 177

m/z 297

m/z 189, 253

m/z 301

m/z 242, 257, 283 m/z 301

m/z 151,179

Hesperetinm/z 302.1

Enterolactonem/z 298.1

HO

CH2OH

CH2OH

OH

Enterodiolm/z 302.1

HO

OH

O

O

O

O

OH

HO

OH

7 2

Naringeninm/z 272.1

O

O

OCH3

HO

OH

7 2OH

NE

HT

EL

HT

Rel

ativ

e A

bund

ance

Run 1 Run 2DE

DHDE

GE m/z 269.3

m/z 180-184, 194-202, 224-227, 241

m/z 257.1

m/z 109.2

O

O

HO

OH

3

4'

O

O

HO

OHOH

3

7

54'

CH3OH

O

HO

OH4'

3

DMA

Daidzeinm/z 254.1

Dihydro-Daidzeinm/z 256.1

Genisteinm/z 270.1

O-DMAm/z 258.1

DHDE DHGE

Q

Q

Quercetinm/z 302.1

O

O

OH

HO

OH

7 2OH

OH

m/z 271.3

0

50

Time (minutes)

Dihydro-Genisteinm/z 272.1

DHGEm/z 165

100

4 5 10 15 20

0

50

100 GLYEm/z 268.1

m/z 283.3Glyciteinm/z 284.1

O

O

HO

OH

H3CO 6

3

7

4'

m/z 196-198, 208-210, 223-226

m/z 253.4

m/z 149

m/z 255.1

PDA combined with MS easy , complementary

1 2conc.

1000

2000

real point

AU

1500

calibration curvesa) ion trap MS b) PDA

good at: low levels medium-high levels10 10

DELPELCYA

Minutes

Abs

orb a

nce

0 5 10 15 20 25

at 525 nm

O 2

4'

3'

35

7+

A C

B

ANTHOCYANIDINPelargonidin PEL OH-3,3',5,7 Cyanidin CYA OH-3,3',4',5,7 Delphinidin DEL OH-3,3',4',5,5',7

Flavonoid containing foods by PDA

Franke 2004

L

M

AQ

K

at 370 nm

O

O

2

4'

3'

35

7A C

B

FLAVON(OL) Apigenin A OH-4',5,7 Luteolin L OH-3',4',5,7 Kämpferol K OH-3,4',5,7 Quercetin Q OH-3,3',4',5,7 Myricetin M OH-3,3',4',5,5',7

Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

mA

U

0

10

20

30

40

50

mA

U

0

10

20

30

40

50

Minutes

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

mA

U

-5

0

5

10

15

20

25

30

35

40

45

mA

U

-5

0

5

10

15

20

25

30

35

40

45

4 50

n m

coen

zym

e Q

10

coen

zym

e Q

10

spiked

neat

spiked

neat

HPLC/PDA of lipophilicplasma micronutrients

carotenoidscarotenoids

tocopherols

295

nm

MW

Genistein 270.0528Apigenin 270.0528Estrone 270.1620Estriol-water 270.1620

DHGE 272.0685Naringenin 272.0685Estradiol 272.1776

Masses as Diagnostic Tool some uncertainty

Applications in food analyses

a) Soy foods: PDA is sufficient

Franke 1999

b) Foods with soy addition:PDA + itMS

Umphress 2005

Applications in clinical and

epidemiologic studies

Maskarinec 1998

Soy Intake & Urinary IFLfoods by PDAurine by itMS

01234567

Chinese Filipino NativeHawaiian

Japanese Caucasian

Soy

prot

ein

inta

ke (g

/day

)

0

200

400

600

800

Urin

ary

isof

lavo

nes

(nm

ol/h

)Soy protein intake Urinary isoflavones

0

1000

2000

3000

4000

5000

DEGE

GLYE

Tot. IFLEQ

Tok

yoB

angk

ok

Lex

ingt

on

heal

thy

Fanti 2003

Plasma IFLs in Dialysis Patients serum by itMS

Seru

m le

vels

(nM

)

Soy Intake & Urinary, Plasma, Milk IFLfoods by PDA

urine, plasma, milk by itMS

0

50

100

150

200

250

0 100 200 300 400

B

BU (pmol/mg)

0

50

100

150

200

250

0 100 200 300 400

A

MU (nmol/mg)

0

50

100

150

200

250

300

0 50 100 150 200 250 300 350BU (pmol/mg)

C

0

500

1000

1500

2000

0 200,000 400,000 600,000

D r=0.975; p<0.001(n=14)

BU (pmol/mg)

-2

3

8

4 6 8 10 12 14

ln B

P

r=0.863; p<0.001(n=13)

Franke 2006

r=0.661; p=0.004(n=16)

r=0.775; p=0.002(n=12)

MM=Mother’s Milk MU=Mother’s Urine, BU=Baby’s Urine; BP=Baby’s Plasma

How to determine stock solution concentrations

accurately

L

Transmission: I1 / I0 (%) ~ ε(α) -c* L

Molecules interact with light=>Lambert-Beer Law

I0 = neat lightI1 = transmitted light L = 1 (cm)c = M (mol/L)

relates light absorption to the material through which the light is travelling: Absorbance ~ Concentration

L

I0 = neat lightI1 = transmitted light L = 1 (cm)c = M (mol/L)

Transmission: I1 / I0 (%) ~ ε(α) -c* L

I1 / I0 = 10- ε * c* L

ε(α)=extinction coefficient (cm-1*mol-1*L)

-lg (I1 / I0 ) = ε * c* L = E (Extinction)

Molecules interact with light=>Lambert-Beer Lawrelates light absorption to the material through which the light is travelling: Absorbance ~ Concentration

c (M) = E / ε

E = 1 => -lg (I1 / I0) =1 => I1 / I0 =10−1 = 10%E = 2 => I1 / I0 =10−2 = 1%

• a stock solution of Daidzeinreads 0.200 absorbance units (AU)in spectrophotometer

a) low ε (20,892)=>0.2 /20,892 = 9.6 µMb) HIGH ε (31,563)=>0.2 /30,892 = 6.3µM

• this stock gives 5 area units in HPLCa) low ε =>5 area units = 9.6 µM “OVER estimate”b) HIGH ε =>5 area units = 6.3 µM “underestimate”

Daidzein solvent λ (nm) ε (L/mol/cm)Ollis 1962 (in Geissman, ed.) 96% EtOH 250 20,893in Wiseman 2002 (5UK labs) 100%EtOH 262! 24,739Hendrich&Murphy 2001 ? 249 31,563Sigma-Aldrich 2006* MeOH 248-249 27,100-27,200Walz 1931 80%MeOH 250 27,542

cv = 15%max/min=+51%

GenisteinOllis 1962 (in Geissman, ed.) 96% EtOH 263 37,154in Wiseman 2002 (5UK labs) 100%EtOH 262 35,842Hendrich&Murphy 2001 ? 263 35,323Sigma-Aldrich 2006* EtOH 263 35,000 -38,400Merck Index 10th ed. 96% EtOH 262.5 37,291Williams&Harborne 1989 ? 261 33,113Walter 1941 85% MeOH 261 24,435

cv = 4%max/min=+13%

Molar Extinction coefficients (ε)

* private communications

Molar Extinction coefficients (ε)Glycitein solvent λ (nm) ε (L/mol/cm)

Kelly 1993 alcohol 256 22,387 Hendrich&Murphy 2001 ? 256 25,388

cv = 9 %max/min =+13%

DaidzinHendrich&Murphy 2001 ? 249 26,830 Univ.od St.Andrews* 23,749 Purina/Nestle MeOH/Water 250 28,561

cv =9%min/max=+20%

GenistinNestle/Purina 2006* MeOH/Water 262.5 39,129 Hendrich&Murphy 2001 ? 262.5 35,323 Sigma-Aldrich 2006* 85%aq EtOH 262 39,000-40,000

cv =6%min/max=+12%* private communications

Molar Extinction coefficients (ε)

λ (nm) ε (L/mol/cm)

Daidzein 249 31,563 Daidzin 249 26,830 Acetyldaidzin 256 29,007 Malonyldaidzin 258 26,830 Genistein 263 35,323 Genistin 263 30,895 Acetylgenistin 261 38,946 Malonylgenistin 260 29,895 Glycitein 256 25,388 Glycitin 259 26,713 Acetylglycitin 260 29,595 Malonylglycitin 260 26,313

Hendrich&Murphy 2001

Molar Extinction coefficients (ε)

Franke et al. 2002; 2004

in alcoholAnalyte λ (nm) ε (L/mol/cm) Reference

Daidzein 250 20,893 Ollis 1962Dihydro-daidzein 277 13,600 Wähälä et al 1998*Glycitein 256 22,387 Kelly et al. 1993Genistein 263 37,154 Ollis 1962Dihydro-genistein 290 18,300 Wähälä et al 1998*O-Desmethyl-angolensin 280 12,023 Wähälä et al 1998*

Equol 281 6,761 Wähälä et al 1994*Formononetin 256 29,512 Ollis 1962Biocahanin-A 263 27,542 Ollis 1962

* private communications

see details: Wang, Prasain, Barnes 2002; Hendrich 2002; Lampe 2002

PDA ion trap MS triple quad MSSelectivity + +++ +++

SensitivityLOD* (nM) neat 50 (EQ150) 5 (EQ15) 0.05 (EQ0.1)

plasma 500 (EQ1500) 5 (EQ15) 0.05 (EQ0.1)

Range >104 102 104

Linearity +++ - (quadratic) ++

Robustness +++ + ++

Analytes/peak 1 2-3 many

Analyte Destruction no! yes yes

*Limit of Detection at 3x noise, 20µL injection

for isoflavonoid analysesEvaluation of Methods

PDA ion trap MS triple quad MSCost of instrument $25k $150k $300k

Cost/sample $$ $$+50% $$+87.5%

Time /sample 30-40’ 10-20’ 5-10’

Skills low medium-high high

for isoflavonoid analysesEvaluation of Methods (cont’d)

PDA itMS triple quad MSAnalyte levels high medium low to high

wide range narrow range wide range

Applicationsfood yes! at low levels sure, no need urine yes yes! sure plasma no yes suretissue no no yesintervention studies compliance metabol.profile anythingcross sectional studies no ok yes

LC/PDA/MS analysesSUMMARY

GMP needed for calibration (ε)

Acknowledgements

Laurie Custer

NCI #CA71789NCRR #P20 RR11091

CRCH developmental funds

IFL Extraction

HPLC/PDA/MSAGLYCONS (200 µL)

Plasma,Urine: 0.005-0.5 mL•Enzymatic Hydrolysis (37OC; 2-12h)

and internal standard addition

•ethyl ether phase separation

IFL Food Extraction

HPLC/PDA/(MS)Aglycons+Conjugates

lyophilized & homogenized: 0.2 g

•Shake in 80% aq. MeOH (2x2h) with internal standard

Pos. ESI on itMS: Qualitative Info

MW=518

[M+H]+

[M-sugar+H]+

[M-sugar+H]+ =Aglycon (m/z 271)

Sugar (248 amu)

O

O

HO

OHOH

3

7

B5

CA

Genistein

H

[M+H]+

[M-sugar+H]+

O

O

HO

OHOH

3

7

B5

CA

Genistein

H

Quantitate diagnostic ions

Pos. ESI Mass Spectrum: Quantitative Info

m/z 519

m/z 271, 230

Multiple Reaction Monitoring