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Supplementary Information Metabolite profiling and fingerprinting of commercial cultivars of Humulus lupulus L. (hop) a comparison of ms and nmr methods in metabolomics


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Supplementary Information

Metabolite profiling and fingerprinting of commercial cultivars of Humulus lupulus L. (hop) - a comparison of MS and NMR methods in metabolomics
Mohamed A. Farag, Andrea Porzel, Jürgen Schmidt, Ludger A. Wessjohann*

Leibniz Institute of Plant Biochemistry, Department of Bioorganic Chemistry, Weinberg 3, D 06120 Halle (Saale), Germany; wessjohann@ipb-halle.de



Supplementary file 1



NMR quantification

For the quantification of total - and -bitter acids, xanthohumol, -humulene and linalool using NMR spectroscopy, the peak area of selected proton signals belonging to the target compounds, and the peak area of the IS (HMDS) were integrated manually for all the samples. The following equation was applied for the calculations.

mT mass of the target compound in the solution used for 1H NMR measurement [g]

MT molecular weight of target compound [g/mol]

IT relative integral value of 1H NMR signal of the target compound

ISt relative integral value of 1H NMR signal of the standard compound

xSt number of protons belonging to the 1H NMR signal of the standard compound

xT number of protons belonging to the 1H NMR signalof the target compound

cSt concentration of standard compound in the solution used for 1H NMR measurement [mmol/L]

vSt volume of solution used for 1H NMR measurement [mL]


Supplementary Table 1




HSQC assignment of metabolites identified in Hop resin HHT

Cross peak Compound Assignment


1 adhumulone CH3,

adlupulone CH3, 4´

2 adlupulone CH3, 5´

3 adhumulone CH3, 5´

4 H CH3,16

5 xanthohumol CH3,4´´

6 H+L CH3,11

L CH3,16 & 21

7 cohumulone CH3,3´



colupulone CH3, 3´& 4´

8 cohumulone CH3, 4´

9 humulone + lupulone CH2,7a

10 humulone + lupulone CH2,7b

11 lupulone CH3, 4´& 5´

humulone CH3.4´

humulone CH3.5´

12 xanthohumol CH3, 5´´

13 L CH3,15 & 20

14 H CH3,15

15 H+L CH3,10

humulone CH,3´

cohumulone CH, 2´

16 L CH2,12a

17 L CH2,12b

18 adhumulone CH,2´

19 H CH2,12

20 humulone CH2,2´a

21 humulone CH2,2´b

lupulone CH2,2´

22 xanthohumol CH2,1´´

23 xanthohumol OCH3

24 xanthohumol CH, 5´

25 xanthohumol CH, 3 & 5

26 H CH, 13

27 L CH, 13 & 18

28 L CH, 8

29 H CH, 8

30 xanthohumol CH, 2 & 6

31 xanthohumol CH, 

32 xanthohumol CH, 
H series = adhumulone, cohumulone, humulone

L series = lupulone, adlupulone, colupulone


Supplementary Table 2


Relative quantification of metabolites identified in Hop cone extract normalized to the amount of umbelliferone (IS) for comparison.

No. rt (min) Identification ATHM CZAG EHM ENB HHE HHM HHS HHT HPE HSE HTU TPE


1 2.05 Maltose 0.6 2.2 0.4 1.7 2.2 1.0 1.0 2.3 1.7 2.5 0.9 1.0

2 8.18 Rutin tr. 0.1 tr. tr. tr. tr. tr. tr. tr. 0.1 tr. tr.

3 8.76 Isoquercitrin 1.3 5.8 0.7 2.3 6.8 1.9 3.1 2.7 2.3 2.8 1.3 2.0

4 9.44 Isoquercitrin malonate 0.9 2.8 0.2 1.4 1.1 0.8 0.4 2.2 1.6 1.8 0.5 1.3

5 10.01 Astragalin tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr.

6 10.55 Unknown glucoside tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr.

7 10.76 Astragalin malonate 1.1 4.9 0.5 2.8 2.5 1.0 0.4 3.7 2.5 4.6 0.6 1.8

8 14.27 Isoxanthohumol tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr.

9 17.35 8-Prenylnaringenin tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr.

10 18.23 Cohulupone 40.5 21.4 23.3 35.6 203.4 191.1 207.6 122.4 142.7 106.3 190.5 37.4

11 18.9 Humulinone tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr.

12 19.75 Unknown 7.5 6.9 0.6 27.3 340.2 217.5 136.4 111.0 124.4 98.2 252.5 16.5

13 20.69 Desmethylxanthohumol 41.1 42.2 32.1 17.1 6.8 26.0 15.3 18.2 22.8 12.6 8.7 18.4

14 21.92 Unknown 200.5 81.2 145.3 109.3 5.5 79.9 56.4 35.9 57.8 10.5 26.2 101.2

15 22.34 Xanthohumol 481.5 651.7 447.0 471.2 115.1 365.4 719.3 320.1 490.9 502.4 545.9 485.0

16 24.53 Lupulone E 103.9 110.1 115.0 41.0 6.3 76.3 49.2 54.2 48.3 10.0 47.8 72.8

17 24.85 Posthumulone 49.0 71.8 45.6 33.3 2.0 41.9 76.2 18.7 32.8 4.8 79.0 44.0

18 25.19 Diprenyl naringenin 28.0 40.5 31.6 30.2 4.6 23.3 9.1 11.6 11.2 6.2 4.0 32.4

19 25.36 Unknown tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr. tr.

20 25.9 Unknown 17.7 19.3 22.2 18.5 57.0 44.5 33.6 33.1 33.9 29.5 36.8 22.2

21 26.32 Cohumulone 2064.7 1934.8 1696.2 1188.1 267.8 1683.8 2628.5 851.3 1355.4 915.3 1489.9 1287.5

22 27.2 4-Deoxycohumulone 128.8 99.7 101.0 77.7 9.2 49.9 28.0 41.7 58.5 57.2 7.7 122.0

23 & 24 27.6 Humulone 4151.5 4572.6 3667.8 2456.5 839.7 3562.6 4196.8 1895.6 2705.4 2650.0 3500.0 2496.8

28.1 Adhumulone

25 28.36 4-Deoxyhumulone 179.2 177.2 158.2 130.4 29.7 106.2 34.3 99.9 108.5 166.8 16.5 165.2

26 29.09 Adprehumulone

27 29.29 Postlupulone 151.4 176.6 163.7 137.1 16.9 133.7 133.7 129.6 181.9 36.8 95.1 180.7

28 29.83 Prehumulone 204.2 320.4 200.0 112.9 22.1 171.0 147.7 84.9 172.6 56.2 218.9 190.6

29 30.48 Unknown 35.1 14.9 40.1 18.6 9.1 45.0 8.6 11.3 33.6 22.0 6.6 25.7

30 30.51 Unknown 15.4 28.4 16.3 36.1 13.1 13.7 17.9 31.5 52.5 37.1 15.9 47.1

31 30.81 Colupulone 2630.3 2341.4 2384.9 1859.1 1125.1 2295.6 2308.7 1921.5 2689.6 3286.3 1331.6 2218.3

32 & 33 31.99 Lupulone 3059.8 3141.7 3076.7 2150.6 1760.1 2837.9 2269.1 2308.5 2905.6 4500.1 1773.6 2363.0

32.21 Adlupulone

34 32.95 Adprelupulone 245.4 218.0 248.3 143.6 24.2 212.1 159.4 50.1 109.1 50.9 167.3 111.8

35 33.35 Prelupulone 227.6 237.1 232.8 135.2 70.1 198.5 89.0 154.6 216.2 136.6 88.1 206.6

36 36.15 Adlupone 37.8 19.2 38.8 28.5 5.4 39.1 54.3 11.1 38.5 13.3 22.9 29.6

37 36.38 Unknown 75.5 54.6 84.8 62.6 22.2 80.8 72.0 26.9 66.8 54.2 51.0 55.8

38 38.72 Unknown 1.0 1.4 2.0 1.5 10.3 19.8 5.8 7.0 11.4 25.6 16.4 3.1

39 39.55 Unknown 1.2 0.6 1.5 1.2 0.3 2.5 0.8 0.8 2.1 0.7 0.9 1.7



N.B. Due to peak overlap, humulone and adhumulone quantifications were summed together; same for lupulone and adlupulone peaks. tr = trace amount


Supplementary Fig. 1



1H-NMR spectra of hop cones extracted with acetone-d6, ethanol-d6 and methanol-d4. The expansion of the spectral region from 2.4 to 3 ppm shows that acetone (inset A) improves the resolution of CH2 protons in bitter acids compared with ethanol (inset B) and methanol (inset C).


Supplementary Fig. 2


Expanded region of the 1H-13C HSQC experiment (HHT resin) in methanol-d4 of the unique cross peaks assigned to the methyl group at 5´ position in adhumulone and adlupulone. A detailed description of cross peaks is provided in supplementary Table 1.




Supplementary Fig. 3

Expanded region of the 1H-13C HSQC experiment of hop HHT resin in methanol-d4 showing resonance signals 1-22. A detailed description of cross peaks is provided in supplementary Table 1.

Note the colour code: blue = CH2; red = CH or CH3

Expanded region of the 1H-13C HSQC experiment of the resonances sginals 23-32.

A detailed description of cross peaks is provided in supplementary Table 1.



Supplementary Fig. 4

TIC chromatograms of methanol (trans-) esterified hop HHT resin acquired by GC-MS showing the presence of palmitic, linoleic and linolenic acid as major fatty acids.






Supplementary Fig. 5

Comparative display of CZAG resin extract as viewed by different spectroscopic techniques. Both NMR in methanol-d4 and negative ESI-LCMS shows that the humulone series (H) is more abundant than lupulone series (L) as evident from signal integrations at 1.51 and 1.55 in NMR, and peak areas in LCMS. In contrast, negative ion ESI-FTICR-MS displays higher signals for L-series peaks (m/z 399 & 413) than for H-series peaks (m/z 347 & 361), likely because lupulones compete more efficiently for ionisation in FTICR-MS.

H series = adhumulone, cohumulone, humulone

L series = lupulone, adlupulone, colupulone









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