lc-ms analysis of botanicals - university of alabama at ......in the kudzu root culture extract [a]...
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LC-MS Analysis of Botanicals
Jeevan K. Prasain, Ph.D.Department of Pharmacology & Toxicology, UABPurdue-UAB Botanicals Center for Age-related
Disease
Botanical workshopUAB, Sept 11, 2006
Applications of mass spectrometry Applications of mass spectrometry in botanical researchin botanical research
Identification, characterization and quantitation of chemical components by LC-MS and MS-MS
Purity assessment and quality control of dietary supplements/neutraceuticals
In vitro & in vivo metabolism, bioavailability, toxicity and pharmacokinetics of drugs and their metabolites in biological samples
Molecular target profiling - proteomics/metabolomicsand chemical imaging of analytes
Kudzu rootKudzu root (Radix Puerariae) as a dietary supplementas a dietary supplement
• Different brands are available
O
O
HO
O
HO
OH
OH
CH2OH
O
Puerarin (PN)
O
OH
OH
OH
OOH
HO
Quercetin
The fruit of the American cranberry is a rich source of dietary flavonoids and considered to be
beneficial for Urinary Tract Infection
Grape seeds are one of the richestsources of Proanthocyanidins (B
type)
O
OH
OH
OH
HO
OH
O
OH
OH
OH
HO
OH
O
OH
OH
OH
HO
OH
OH
HO
O
OH
OOH
OH
OH
HO
O
O
OH
OH
OH
HO
OH
Catechin n
extension subunit
terminal subunit
Catechin gallate
Isoflavonoids in KudzuDietary Supplements/root
culture extract
TIC of reverseTIC of reverse--phase LCphase LC--MS of MS of kudzu dietary supplement (KDS) kudzu dietary supplement (KDS)
isoflavonesisoflavones
0 4 8 12 16
Time (min)
100
%
0
4
3
2
1
6
puerarin
daidzin
genistin
daidzein
formononetin
MS/MS spectra of protonated O- and C-glycosides of daidzein (m/z 417)
220 280 300 320 360 380 m/z0
%
0
100
%
255.05
297.04
267.03
281.05307.06
321.04
363.04335.06351.04 381.05
100
240 260 340
[A]
[B]
Daidzein (O-Glc)daidzin
Daidzein (C-Glc)puerarin
-162 Da
-120 Da
Neutral losses of 162 and 120 Da are characteristics
O
OOH
HO
O
OOH
HO
O
O
OOH
O
O
CH2OHOH
HO
HO
O
OOH
OH+
H+
m/z 417
O
m/z 267
[M+H-150]+HOH2C
HO
HO
HO
H+
Puerarin
269
297
O
OOH
O
8
321
Yo+
H+
m/z 297
Base Peak[M+H-120]+
327
H+
Yo+
Proposed fragmentation mechanisms of protonated puerarin and daidzin
Daidzin
m/z 417
Possible product ions of puerarin in ESIPossible product ions of puerarin in ESI--MS/MSMS/MSin negative ion modein negative ion mode
Prasain et al. J Agric Food Chem. 51, 4213, 2003.
O
O-
HO
O
OH
O
O-
HO
O
O
O-
HO
O
HO
HO
O
O-
HO
O
OHOCH2OH
OHHO
m/z 415
m/z 325
-90 Dam/z 295
-120 Da
m/z 267
-28 Da
m/z
100
0
Rel
. Int
. (%
)
m/z
100
0
Rel
. Int
. (%
)
200 300 400 500 600 700 800m/z
100
0.0
Rel
. Int
. (%
)
255 360
417
447475 549579
257
417
447
289 365
417
447 475 579
433
[A]
[B]
[C]
Neutral loss scans 120 and 162 can be used to detectC-and O-glycosides, respectively in a crude mixture
Full scan ESI-MS of kudzu extract
Neutral loss scan 120 LC-MS/MS
Neutral loss scan 162 LC-MS/MS
m/z
100755025
0
Rel
.Int
. (%
)
m/z
12.69.56.33.20.0
Rel
.Int
. (%
)
100 200 300 400
20.215.110.1
5.00.0
Rel
.Int
. (%
)
149 177 239 269283
311
341353
431
268
283
293
311
323
431
283
311
340431
-120 Da
-120 Da
-120 Da
Hydroxylated daidzein C-glucosides were detected in the kudzu root culture extract
[A] 2.6 min
[B] 3.5 min
[C] 6.5 min
Prasain et al. Phytochemical Analysis, in press
LC-MS analysis of cranberryfruits/concentrate
Cranberry fruits (100 g)
Homogenised and extractedWith acetone and filtered
Acetone soluble fractionResidueSolvent EvaporatedUnder reduced pressure
Acetone extract
Extracted with EtOAc
Ethyl acetate extractWater sol. residue
MS analysis
Extraction Flow Chart of an ethyl acetate extract from cranberry fresh fruits
LC-MS and HPLC chromatographic conditions:
LC-MS column: 4.6 x 100 mm, RP-300 columnGradient A : 10% ACN + 10 mM NH4OAc
B : 40% ACN + 10 mM NH4OAc
Linear gradient increased of solvent B 100% over 40 minESI-MS in the negative ion mode of a PE-Eciex API IIITriple quadrupole mass spectrometer.
HPLC analysis:Column : Brownlee (22 cm x 4.6 mm I.d.) C8-columnGradient A : 10% aq. ACN/0.1% TFA
B : 90% aq. ACN/0.1% TFAFlow rate : 1.5 ml with a linear gradient increase of
solvent B 100% over 35 min
12.2 24.4 36.6 48.8Time (min)
100
50
0
Rel
ativ
e In
tens
ity (%
)
M/z 289
catachins
QC-hexosideMC-hexoside
QC-pentosideMC-pentoside
QC m/z 301,MC m/z 317
Me-QC
QC = Quercetin
MC = Myricetin
LC-MS chromatogram of the EtOAc extract obtained from Cranberry fresh fruits
100
50
0
Rel
. Int
. (%
)
100 200 300m/z
100
50
0
Rel
. Int
. (%
)
65
107
151
163179
201 228 245 272299
65 83
107
125
137
151
164
179
192271 299
317
O
O
HO
OH
OH
OH
OH
OH
O
O
OH
OH
OH
HO
OH Quercetin
Myricetin
ESI-MS/MS spectra of the ions at m/z 301 and317 in the cranberry extract
100
50
0
Rel
. Int
. (%
)
100 300 400
m/z
100
50
0
Rel
. Int
. (%
)
151 179270 299
315, 316
477-478
150 178
270287
315,316
447, 448
200
-162 Da
132 Da
Pentose and hexose sugar derivative of Myricetin were detected the cranberry extract
Myricetin-hexoside
Myricetin-pentoside
Time (min)0 5 10 15 20 25
mAU
0
10
20
30
401.
895
2.90
6
4.35
2
6.13
16.
562 7.83
8
10.8
82
13.7
97
18.7
20
0.00
0.50
1.00
1.50
mM ofQuercetin
Cranberry concentrate
Quercetin
HPLC Chromatogram of a commercially available cranberry concentrate
Conclusions• Quercetin and its glycosides are the major
flavonoids in the cranberry fresh fruit, and commercially available cranberry concentrates.
• Catechin was detected but its oligomers(proanthocyanidins) were not detected in the ethyl acetate extract of the samples.
• Quercetin, myricetin and their glycosides produced predominant radical anions in the ms/ms experiments.
Analysis of Grape Seed Extracts (GSE)
Grape Seed Powder (2 g)Dissolved with 25 ml MeOH�Precipitated with 125 ml CHCl3�Filtered through filter paper
Filtrate Precipitate
Dried under dessicator
Dried GSP precipitate (1.35 g)
Fractionation of monomeric catechins fromoligomers
HPLC slides
min0 5 10 15 20 25 30
mAU
0
2
4
6
8
10
DAD1 A, Sig=262,4 Ref=380,40 (062404A\047-0801.D)
4.6
92
8.5
87
10.
589
12.
406
13.
569
16.
039
20.
088
min0 5 10 15 20 25 30
mAU
0
2
4
6
8
10
DAD1 A, Sig=262,4 Ref=380,40 (062404A¥047-0801.D)
4.69
2
8.58
7
10.5
89
12.4
06 13.5
69
16.0
39
20.0
88
GSE
min5 10 15 20 25 30
mAU
0
50
100
150
200
250
300
DAD1 A, Sig=262,4 Ref=380,40 (062404A¥048-0901.D)
4.69
6
7.31
0 8.56
2
9.58
8
10.4
98
11.5
43
12.3
95
13.4
73
14.3
26
15.9
70
20.0
21
GS-CHCl3
min0 5 10 15 20 25 30
mAU
012345678
DAD1 A, Sig=262,4 Ref=380,40 (062404A¥049-1001.D)
GS-PPT
Catechin
Catechin
Catechin
proanthocyanidins
GSE is a complicated mixture of polyphenolsAnd other secondary metabolites
500.0 1000.2 1500.4 2000.6 2500.8 3001.0Mass (m/z)
0
565.3
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
601.2336889.3218
904.4994 1177.4237
579.2272
559.2101
1465.5048541.2063
1193.3955517.2194 753.27151329.4680545.2691 697.2598
1057.3399520.2241 1754.5915681.2176 847.3339 1345.39571633.5731620.1546 2041.7108907.4548 2635.5249
1133.4034 2330.5866 2813.00481484.3190 1758.6862794.3445 951.2909 1312.47891139.3794 2284.92581690.4340962.2295
MALDI-TOF mass spectrum in positive linear mode showinga pronathocyanidin series [M+Na]+ from the dimer m/z 600
to oligomers
1999.0 2799.4 3599.8 4400.2 5200.6 6001.0Mass (m/z)
0
1310.4
0
10
20
30
40
50
60
70
80
90
100
% In
tens
ity
2047.11
2063.75
2200.14
2351.832335.99
2489.282078.85
2503.172368.25
2624.41
2792.732026.552656.94 3066.662178.85
2809.182385.75 3217.562103.92
3522.062427.43 3202.572117.48 2893.302677.75 3812.962267.73 3487.773113.44 4083.364386.702703.53 3743.463445.63 4827.534009.08 4307.33 5254.75 5648.34
Equation to detect poly-galloyl-polyflavans in GSP
290 + 288c + 152g + 23290 = mol wt. of the terminal catechin/epicatechinC = degree of plymerizationG = unit of galloyl esters; 23 = weight of sodium
+288
+152
300 500 700 900 1100 1300 1500 1700 m/z0
100
%
577.229
289.097
245.095
425.148
333.091
865.383
729.287675.239
796.319
1153.5441017.455
963.3911084.991
1305.5941441.710 1730
Mono-
Pento- Hexo-
GSE precipitate in ESI-MS QTOF
Dimer-
Trimer-
Tetro--gallate
-gallate
LCMS and MSMS analysis of the methanolic extract of a Grape Seed Dietary Supplement with ionization polarity
switchTIC
Negative survey scan in trap)
DimerCatechinmonomer
TrimerTetromer
Proanthocyanidin gallates
EPI (Enhanced Product Ion Scan of dimer m/z 577 in Trap)
Metal ion adducts are common in Positive ModeTIC
EMS (positive survey scan in trap)
Dimer [M+H]+
EPI (Enhanced Product Ion Scan of m/z 579 in Trap)
Dimer [M+Na]+Trimer
0.0 2.0 4.0 6.0 8.0 10.0Time (min)
100
0
455
100
0
210
100
0
2005
1001755
Negative ion mode provides better sensitivity fordimer than monomeric catechin
289/137
577/407
577/289
289/121
Gradient: 10 min from 0-60% MeOH in 5 min in 0.1% HCOOH
291/139
291/123
579/289
867/577
0.0 2.0 4.0 6.0 8.0 10.0Time (min)
100500
1534
100500
908
100500
254
100500
309
Monomeric catechin is better detected in positive ion mode
Conclusions
A sensitive LC-MS/MS method was developed to analyze GSP in biological samples. In mass spectrometric analysis, monomeric catechins are more sensitive in positive ion mode, while catechins dimers are in negative ion mode.
GSP is a complex mixture of catechins and non-polyphenols.
Thanks toThanks to……Dr. S. BarnesDr. Mary Ann LilaDr. E. MeezanDr. J. M. WyssDr. N. PengDr. Connie WeaverKenneth JonesRay MooreNancy BrissieM. Kirk
Funding-NCCAM Sponsored Purdue-UAB Botanicals center for Age-Related DiseasesP50 AT-00477