Botanical workshop UAB, Sept 11, 2006
LC-MS Analysis of Botanicals
Jeevan K. Prasain, Ph.D. Department of Pharmacology & Toxicology, UAB Purdue-UAB Botanicals Center for Age-related Disease ApplicationsApplications ofof massmass spectrometryspectrometry inin botanicalbotanical researchresearch
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/metabolomics and chemical imaging of analytes KudzuKudzu rootroot (Radix Puerariae) asas aa dietarydietary supplementsupplement • Different brands are available
CH2OH
O OH
OH O HO
HO O
O Puerarin (PN) The fruit of the American cranberry is a rich source of dietary flavonoids and considered to be beneficial for Urinary Tract Infection
OH
OH
HO O
OH
OH O
Quercetin Grape seeds are one of the richest sources of Proanthocyanidins (B type)
OH HO O OH OH HO O OH OH OH OH extension OH OH subunit HO O OH Catechin n OH OH OH OH HO O HO OH O OH
O OH OH OH O terminal subunit OH
HO OH Catechin gallate Isoflavonoids in Kudzu Dietary Supplements/root culture extract TICTIC ofof reversereverse--phasephase LCLC--MSMS ofof kudzukudzu dietarydietary supplementsupplement (KDS)(KDS) isoflavonesisoflavones
1 puerarin 100
%
3 genistin
0 0 4 8 12 16 Time (min) MS/MS spectra of protonated O- and C- glycosides of daidzein (m/z 417)
[A] 100 255.05 Daidzein (O-Glc) daidzin
% -162 Da
[B] 0 100 297.04 Daidzein (C-Glc) 267.03 puerarin
% 321.04 -120 Da 307.06 351.04 281.05 335.06 381.05 363.04 0 m/z 220 240 260 280 300 320 340 360 380 Neutral losses of 162 and 120 Da are characteristics Proposed fragmentation mechanisms of protonated puerarin and daidzin
O 327 321 OH H+ H+ HO CH2OH 297 HO O O HO 269 O HO 8 O OH m/z 297 Base Peak O + OH [M+H-120] m/z 417 H+
Puerarin O O
O OH H+ m/z 267 + HOH2C [M+H-150] + HO O Yo H+ O O O O HO
HO O m/z 417 OH O OH Daidzin Yo+ PossiblePossible productproduct ionsions ofof puerarinpuerarin inin ESIESI--MS/MSMS/MS inin negativenegative ionion modemode
OH HO CH2OH HO O OH
HO O -120 Da HO O
O O-
O- m/z 415 O m/z 295 -90 Da
HO -28 Da
HO HO O HO O
O O- O O- m/z 325 m/z 267
Prasain et al. J Agric Food Chem. 51, 4213, 2003. Neutral loss scans 120 and 162 can be used to detect C-and O-glycosides, respectively in a crude mixture
[A] 100 417 Full scan ESI-MS of kudzu extract Rel. Int. (%) Rel. Int.
255 360 447 549 433 475 579 0 [B] 100
417 Neutral loss scan 120 LC-MS/MS Rel. Int. (%) Rel. Int.
257 447 0 [C] 100 Neutral loss scan 162 417 LC-MS/MS Rel. Int. (%) Rel. Int.
289 365 447 475 579 0.0 200 300 400 500 600 700 800 m/z Hydroxylated daidzein C-glucosides were detected in the kudzu root culture extract
100 311 75 [A] 2.6 min -120 Da 50 283 431 Rel. Int. (%) 25 341 149 177 239 269 353 0
12.6 311 9.5 -120 Da 283 6.3 [B] 3.5 min 431
Rel. Int. (%) 3.2 268 293 323 0.0
20.2 311 15.1 [C] 6.5 min -120 Da 10.1 283
Rel. Int. (%) 5.0 431 340 0.0 100 200 300 400 m/z Prasain et al. Phytochemical Analysis, in press LC-MS analysis of cranberry fruits/concentrate Extraction Flow Chart of an ethyl acetate extract from cranberry fresh fruits Cranberry fruits (100 g) Homogenised and extracted With acetone and filtered
Residue Acetone soluble fraction Solvent Evaporated Under reduced pressure Acetone extract Extracted with EtOAc
Water sol. residue Ethyl acetate extract MS analysis LC-MS and HPLC chromatographic conditions:
LC-MS column: 4.6 x 100 mm, RP-300 column Gradient A : 10% ACN + 10 mM NH4OAc B : 40% ACN + 10 mM NH4OAc
Linear gradient increased of solvent B 100% over 40 min ESI-MS in the negative ion mode of a PE-Eciex API III Triple quadrupole mass spectrometer.
HPLC analysis: Column : Brownlee (22 cm x 4.6 mm I.d.) C8-column Gradient A : 10% aq. ACN/0.1% TFA B : 90% aq. ACN/0.1% TFA Flow rate : 1.5 ml with a linear gradient increase of solvent B 100% over 35 min LC-MS chromatogram of the EtOAc extract obtained from Cranberry fresh fruits
100 QC = Quercetin MC = Myricetin QC-hexoside MC-hexoside
50 QC-pentoside M/z 289 MC-pentoside
Relative Intensity (%) Relative Intensity catachins QC m/z 301, MC m/z 317 Me-QC
0 12.2 24.4 36.6 48.8 Time (min) ESI-MS/MS spectra of the ions at m/z 301 and
317 in the cranberry extract OH
OH 151 100 HO O
107 OH O 50 OH Rel. Int. (%) 179 Quercetin 65 299 163 201 228 245 272 0 OH 100 151 OH
137 HO O OH 107 50 179 Rel. Int. (%) OH Myricetin O 65 83 164 192 OH 317 125 271 299 0 100 200 300 m/z Pentose and hexose sugar derivative of Myricetin were detected the cranberry extract
100 315, 316
Myricetin-hexoside 50 -162 Da Rel. Int. (%) 270 299 477-478 151 179 0 100 315,316 Myricetin-pentoside
50 132 Da Rel. Int. (%) 270 287 150 178 447, 448 0 100 200 300 400
m/z HPLC Chromatogram of a commercially available cranberry concentrate mAU 1.895 40 1.50
1.00 mM of Quercetin 0.50 30 0.00
4.352 Cranberry concentrate
20 18.720
10.882 Quercetin 7.838 2.906 6.131
10 13.797 6.562
0 0 5 10 15 20 25 Time (min) 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) Fractionation of monomeric catechins from oligomers
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) GSE is a complicated mixture of polyphenols And other secondary metabolites
DAD1DAD1 A, Sig=262,4 A, Sig=262,4Ref=380,40 (062404A\047-0801.D) Ref=380,40 (062404A¥047-0801.D) mAU
10 10.589 GSE 10.589 Catechin 8 HPLC slides 6 13.569 13.569 6 16.039 20.088 16.039 20.088
4 4.692 12.406
4 4.692 12.406 8.587 8.587 2
0 0 5 5 10 10 15 15 20 20 25 25 30 min30 min DAD1 A, Sig=262,4 Ref=380,40 (062404A¥048-0901.D) mAU 300 10.498 Catechin 250 GS-CHCl
200 3 13.473
150 12.395 4.696 15.970
100 20.021 8.562 50 14.326 9.588 7.310 11.543 0 5 10 15 20 25 30 min DAD1 A, Sig=262,4 Ref=380,40 (062404A¥049-1001.D) mAU 8 7 Catechin 6 5 GS-PPT 4 3 2 1 proanthocyanidins 0 0 5 10 15 20 25 30 min MALDI-TOF mass spectrum in positive linear mode showing a pronathocyanidin series [M+Na]+ from the dimer m/z 600 to oligomers
100 601.2336 565.3 889.3218 90
80
70
60 904.4994 1177.4237 50 % Intensity
579.2272 40 559.2101 541.2063 30 1465.5048 517.2194 753.2715 1193.3955 545.2691 1329.4680 20 697.2598 520.2241 1057.3399 681.2176 847.3339 1345.3957 1754.5915 620.1546 907.4548 1633.5731 2041.7108 2635.5249 2813.0048 10 1133.4034 2330.5866 794.3445 951.2909 1484.3190 1758.6862 1139.3794 1312.4789 2284.9258 962.2295 1690.4340 0 0 500.0 1000.2 1500.4 2000.6 2500.8 3001.0 Mass (m/z) Equation to detect poly-galloyl-polyflavans in GSP
290 + 288c + 152g + 23 290 = mol wt. of the terminal catechin/epicatechin C = degree of plymerization G = unit of galloyl esters; 23 = weight of sodium
2047.11 100 1310.4 2063.75 90 +288 80 2200.14 70 2351.83 60 2335.99 +152 2489.28 50 % Intensity 2078.85 2624.41 40 2368.25 2503.17 2792.73 30 2026.55 2656.94 2178.85 3066.66 2385.75 2809.18 20 2103.92 .56 2117.48 2427.43 2893.30 3217 2677.75 3202.57 3522.06 2267.73 3487.77 3812.96 10 2703.53 3113.44 3445.63 3743.46 4083.364386.70 4009.08 4307.33 4827.53 5254.75 5648.34 0 0 6001.0 1999.0 2799.4 3599.8 Mass (m/z) 4400.2 5200.6 GSE precipitate in ESI-MS QTOF
Dimer- 100 577.229
Trimer- Mono- 865.383 289.097
%
-gallate Tetro-
-gallate 425.148 729.287 1153.544 Pento- 675.239 Hexo- 245.095 1017.455 1305.594 333.091 963.391 1441.710 1730 796.319 1084.991 0 m/z 300 500 700 900 1100 1300 1500 1700 LCMS and MSMS analysis of the methanolic extract of a Grape Seed Dietary Supplement with ionization polarity switch TIC Proanthocyanidin gallates
Negative survey scan in trap) Catechin monomer Dimer Trimer Tetromer
EPI (Enhanced Product Ion Scan of dimer m/z 577 in Trap) Metal ion adducts are common in Positive Mode
TIC
EMS (positive survey scan in trap) Dimer [M+H]+ Dimer [M+Na]+ Trimer
EPI (Enhanced Product Ion Scan of m/z 579 in Trap) Negative ion mode provides better sensitivity for dimer than monomeric catechin
455 100 289/137
0 210 289/121 100
0 577/407 2005 100
0 577/289 1755 100
0.0 2.0 4.0 6.0 8.0 10.0 Time (min) Gradient: 10 min from 0-60% MeOH in 5 min in 0.1% HCOOH Monomeric catechin is better detected in positive ion mode
291/139 1534 100 50 0 291/123 908 100 50 0 579/289 254 100 50 0 867/577 309 100 50 0 0.0 2.0 4.0 6.0 8.0 10.0 Time (min) 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. ThanksThanks toto……
Dr. S. Barnes Dr. Mary Ann Lila Dr. E. Meezan Dr. J. M. Wyss Dr. N. Peng Funding- Dr. Connie Weaver NCCAM Sponsored Purdue-UAB Kenneth Jones Botanicals center for Age- Ray Moore Related Diseases P50 AT-00477 Nancy Brissie M. Kirk