Hypericin Analysis by LC/MS Jeanne B. Li and Michael P. Balogh LC/MS Waters Corporation LC GC, Vol 17, No. 6, June 1999 Introduction to hypericin analysis by LC/MS
Hypericin and Related Compounds O Protohypericin MW 506.45 O O CH 3 * CH 3 CH 3 O CH 3 * CH 2 CH 3 O O Hypericin MW 504.45 O CH 3 CH 3 Protopseudohypericin MW 522.45 O Pseudohypericin MW 520.45 The structures of the compounds of interest in the plant extract are shown in this slide. Note the closing of the ring E on the right-hand side of hypericin and pseudohypericin. This makes them more hydrophobic which effects their elution order in reversed-phase separations. The protohypericin and protopseudohypericin are the biosynthetic precursors that are easily converted to hypericin and psuedohypericin.
Hypericin Analysis Chromatographic Conditions Column: Waters Symmetry C 8, 2.1 mm x 150 mm Mobile Phase: A = 100mM TEA Acetate, ph 7 B = Methanol C = Acetonitrile Gradient: Linear over 15 minutes 30:39:31 to 10:50:40 Flow Rate: 300 µl/min Detection: PDA at 588 nm Mass Spec: Negative Electrospray Ionization (ESI - ) Here are the optimized chromatoghraphic conditions for hypericin analysis. Both PDA and MS detectors were used.
Hypericin UV/Vis Spectra TEA-Acetate vs. TriFluoroAcetate (TFA) AU 0.80 0.70 0.60 0.50 0.40 0.30 0.20 0.10 TEA-TFA TEA-Acetate 588 0.00 250 300 350 400 450 500 550 600 650 700 nm The absorbance spectra of hypericin in trifluoroacetic acid and in triethylammonium acetate are shown in this slide. The unique UV-VIS spectrum of naphthodianthrones has much stronger absorbance at 588 nm in triethylamine acetate than in trifluoroactic acid. This buffer also was conducive to good ionization by electrospray of hypericin and the other compounds in negative mode.
Absorbance 285 328 Hypericin Spectra PDA and ESI - -MS 588 250 300 350 400 450 500 550 600 650 nm 503 Mass Spectrum PDA Spectrum 504 505 547 [M-H] - 250 300 350 400 450 500 550 600 650 A comparison of the UV-VIS spectrum and mass spectrum of the hypericin peak.
Hypericin Standard Chromatograms Photo Diode Array and ESI - -MS Absorbance 588 nm λ Max 503 [M-H] - 0 2 4 6 8 10 12 14 Minutes Extracted chromatograms from the separation of hypericin standard monitored simultaneously by PDA detection at 588 nm and MS detection at 503.
Absorbance Plant Extract Chromatograms PDA & ESI - 1.20 4.79 588 nm 10.67 Hypericin TIC 12.18 11.22 12.84 0 2 4 6 8 10 12 14 Minutes The chromatograms of the plant extract of sample 1 obtained by monitoring aborbance at 588 nm (upper chromatogram) and the total ion current (lower chromatogram). The peak at 10.67 minutes is hypericin.
Plant Extract #1 - Extracted Masses [M-H] - 4.26 Protopseudohypericin 11.52 521 3.12 4.86 Pseudohypericin 519 6.84 9.48 10.86 505 10.74 Hypericin 503 AU 1.20 4.79 10.67 588 nm 0 2 4 6 8 10 12 Minutes 14 Shown here are the extracted mass chromatograms of the plant extract from sample 1 with the UV absorbance chromatogram on the bottom. Note that protohypericin was not detected in this sample.
ESI - Mass Spectra - Plant Extract #1 Protopseudohypericin 139 315 326341 521 522 583 567 584 Peak 4.26 min Intens ity Pseudohypericin 275 385 519 523 543 551 553 4.86 min 9.48 min 279 353 383 555 585 Hypericin 503 417 347349 504 10.74 min 100 150 200 250 300 350 400 450 500 550 600 Shown in this slide are the mass spectra extracted from the chromatographic peaks in sample 1.
Plant Extract #2 - Extracted Masses 4.26 [M-H] - Protopseudohypericin 11.70 521 4.80 Pseudohypericin 519 9.72 Protohypericin 10.92 10.92 Hypericin 505 503 AU 4.72 1.17 10.82 588 nm 0 2 4 6 8 10 12 Minutes 14 Shown here are the extracted mass chromatograms of the plant extract from sample 2 with the UV absorbance chromatogram on the bottom.
ESI - Mass Spectra - Plant Extract #2 Protopseudohypericin 521 52 2 Peak 4. 26 m in Pseudohypericin Protohypericin 505 519 279 179 495506 520 551 553 554 4. 80 m in 9. 7 2 m in 119 Hypericin 471 503 504 10.92 min 100 150 200 250 300 350 400 450 500 550 600 Shown in this slide are the mass spectra extracted from the chromatographic peaks in sample 2.
Comparison of Plant Extracts 4.26 Plant Extract #1 11.5 2 4. 26 Plant Extract #2 11.70 52 1 3.12 4.86 4. 80 51 9 6.84 9.48 10.8 6 9. 72 10.92 50 5 10.7 4 10.92 50 3 AU 0 1.20 4.79 10.6 7 2 4 6 8 10 12 14 0 4. 72 1. 17 10.82 2 4 6 8 10 12 14 58 8 nm Minutes Minutes Comparison of plant extracts 1 and 2 indicate that more of the hypericin compounds were solubilized from the plant powder of sample 2. The hypericin content in plants reportedly changes as a function of climate and season.
In Source Collision-Induced Dissociation of Hypericin 503 High cone voltage Low cone voltage 405 406 433 431 434 459 444 458 487 503 504 504 340 360 380 400 420 440 460 480 500 520 In-source Collision Induced Dissociation (CID) of hypericin in a single-quadrupole mass detector. Shown are the spectra at high (upper spectrum) and low (lower spectrum) cone voltages. The low cone voltage was optimized for sensitivity of the [M-H]- ion at M/Z 503.
Natural Product Analysis Phytoestrogens-Isoflavonoids!Electrospray, is a soft ionization, yielding a single [M+H] + or [M-H] - line!gives some fragmentation for structural identification, but no Libraries available to search!electron Ionization, Particle Beam, yields information rich spectra!can deduce molecular structure and libraries are available for Unknown Identification A group of isoflavonoids commonly called phytoestrogens are found in red clover. Analysis of plant extracts by LC/MS using an electrospray interface, which is a soft ionization technique, yields a single molecular ion with limited fragmentation. The Thermabeam interface, which is an improved form of particle beam interface, is used to generate electron ionization (EI) spectra for LC/MS. Electron ionization, due to it s high ionization energy (about 70 ev), offers a reproducible fragmentation pattern of a molecule, which can be searched against a library for positive compound identification. This type of ionization is well suited for qualitative analysis where extensive structure-informative fragmentation is highly desirable.