Measuring Isoflavonoids in Foods and Biological Fluids by … · HPLC/PDA of soy foods (260 nm)...
Transcript of Measuring Isoflavonoids in Foods and Biological Fluids by … · HPLC/PDA of soy foods (260 nm)...
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
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