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