Letter to the Editor

Received: 7 May 2012 Revised: 15 October 2012 Accepted: 17 October 2012 Published online in Wiley Online Library

Rapid Commun. Mass Spectrom. 2013, 27, 276–280

276

(wileyonlinelibrary.com) DOI: 10.1002/rcm.6443

Dear Editor,

Analysis of lathyrane diterpenes using electrospray ionizationquadrupole time-of-flight mass spectrometry

Caper spurge (Euphorbia lathyris L.) seed oil contains a seriesof diterpenoids known as Euphorbia factors, or L-factors,L1–L9. They are esters of several polyols (lathyrol, epoxy-lathyrol, 7-hydroxylathyol, isolathyrol and jolkinol subtypes)and account for about 3%–5% of the oil.[1–5] These lathyranediterpenes have a unique macrocyclic structure, display activ-ities against HIV-1 replication, and vasoconstriction,[6] and areapoptosis inducers and multidrug-resistance modulators.[5–10]

Their unique structures and potential applications necessitatethe development of a method for the reliable, fast and low-costanalysis of this class of compounds.Mass spectrometry (MS), especially tandemmass spectrome-

try (MS/MS), is one of the important physicochemical methodsfor the identification of trace natural products because of itsrapidity and sensitivity, with low levels of sample consump-tion. Some lathyrane polyols and their esters (lathyrol, 7-hydroxylathyrol and epoxylathyrol subtypes) have been analyzedrapidly with high-performance liquid chromatography/electrospray ionizationmass spectrometry (HPLC/ESI-MS).[11]

In our laboratory, a series of lathyrane diterpenes (Fig. 1),including lathyrol, 7-hydroxylathyrol, epoxylathyrol, jolkinoland isolathyrol subtypes, were isolated from the seeds ofEuphorbia lathyris.[5] To our knowledge, this series of lathyranediterpenes has not been studied by ESI-MS/MS. To obtainsufficient information on the structural characteristics of thisclass of compounds, such as their degradation products,metabolites, and biosynthetic intermediates, the detailedfragmentation patterns of the lathyrane diterpenes werestudied with collision-induced dissociation (CID)-MS/MSusing an ESI quadrupole time-of-flight (QTOF) MS/MShybrid instrument in positive-ion mode.HPLC-grade methanol and the mass calibration standard

were purchased from Fisher Scientific (Pittsburgh, PA) andAgilent Technologies (Santa Clara, CA, USA), respectively.The lathyrane diterpenes were isolated from the seeds ofEuphorbia lathyris.[5] MS experiments were performed on aBruker micrOTOF-Q mass spectrometer (Bremen, Germany).The mass resolution was 10 000 FWHM (full width at halfmaximum) at 922 u. Argon gas was used as the collision gasand high-purity nitrogen gas as the nebulizer and dryinggases at flow rates of 0.3 L/min and 4.0 L/min, respectively.The ESI source conditions were as follows: capillary voltage,–4500V; end plate voltage, –4000V; capillary exit voltage,120 V; and drying gas temperature, 180�C. The mass datawere processed with Bruker Compass DataAnalysis 4.0.For the CID-MS/MS analysis, the precursor sodiated

molecules, [M+Na]+, were selected and the product ions wererecorded with the ESI-QTOF-MS/MS instrument. The precursorion [M+Na]+ affords various product ions, mainly with threetypes of CID fragmentation patterns. The precursor [M+Na]+

Rapid Commun. Mass Spectrom. 2013, 27, 276–280

can cause the loss of the different side-chain groups, suchas CH3CO2H, C6H5CO2H, C5H4NCO2H, C8H7CO2H, andC7H7CO2H, and thus produce many high-abundance productions. The cleavage or loss of the three-membered ring moietylinked by C9–11 (Fig. 1) from the precursor ion is another typeof diagnostic CID fragmentation. In the third type of CIDfragmentation, a series of low-abundance ion clusters in the low-mass range can also be formed by the cleavage of the macrocycle.

Euphorbia factor L3 (1), i.e. (2S,3S,4R,5R,9S,11S,15R)-5,15-diacetoxy-3-benzoyloxy-14-oxolathyra-6(17),12E-diene, wasselected as the representative compound for the following dis-cussion of the fragmentation pathways. The precursor sodiatedmolecule, the [M+Na]+ ion at m/z 545, produces the production at m/z 503 with the loss of CH2CO (Fig. 2 and Scheme 1).The product ions at m/z 485, 425, 363 and 303 are formed fromthe precursor ion at m/z 545 by the losses of either CH3CO2Hor C6H5CO2H, or their simultaneous losses. The ion m/z 425produces a product ion atm/z 281with the loss of a C6H5CO2Namolecule. The product ions atm/z 239 and 213 are formed fromthe ion atm/z 281 by the elimination of neutral C3H6 and C5H8,respectively. These processes involve the cleavage or loss of thethree-membered ring moiety. The product ion at m/z 225 isproduced by the elimination of a C4H8 molecule from an inter-mediate resulting from the ion at m/z 281 by 1,3-H rearrange-ment (Scheme 1). The product ions at m/z 253 and 211 areformed by the loss of CO from m/z 281 and 239, respectively.A series of ion clusters was detected in the low-mass range(Fig. 2 and Supporting Information). This indicates that the frag-mentation of the macrocycle is accompanied with H rearrange-ments. The high-abundance product ion at m/z 105 must be thecharacteristic ion of the benzoic acid group. The exact masses ofmajor product ions are provided in Table 1. The MS/MS spec-trumofEuphorbia factor L8 (2) is very similar to that of compound1. The product ions atm/z 146, 124, and 106 in the MS/MS spec-trum of 2 are the characteristic ions of the nicotinic acid group.

For Euphorbia factor L2 (3), i.e. (2S,3S,4R,5R,7R,9S,11S,15R)-5,15-diacetoxy-3,7-dibenzoyloxy-14-oxolathyra-6(17),12E-diene,the CID fragmentation pattern from the precursor [M+Na]+ atm/z 665 (Fig. 3 and Table 2) is similar to those of compounds 1and 2. However, the relative abundances of the product ionsresulting from the same losses of side-chain groups varied.The cleavage and loss of the three-membered ring moiety,corresponding to the losses of C3H6 and C5H8 respectively, isobserved. The loss of a C4H8molecule,with 1,3-H rearrangementfrom m/z 281 in the MS/MS spectrum of compounds 1 and 2,occurs readily, but does not occur readily from compound 3(Supporting Information). This validates the postulated fragmen-tation mechanisms underlying the loss of C4H8. A series of ionclusters in the low-mass range derived from 3 is also observed.The MS/MS spectra of the precursor ions extracted fromcompounds 3 and 4 are very similar, except for the characteristicions of the nicotinic acid group. The fragmentation pattern ofEuphorbia factor L1 (5) is similar to those of compounds 1–4.The relative abundances of the product ions, especially in thelow-mass range, from compound 5 are low compared with

Copyright © 2012 John Wiley & Sons, Ltd.

Figure 1. Laythyrane diterpenes: Euphorbia factor L3 ((2S,3S,4R,5R,9S,11S,15R)-5,15-diacetoxy-3-benzoyloxy-14-oxolathyra-6(17),12E-diene, (1), Mr 522.2618);Euphorbia factor L8 ((2S,3S,4R,5R,9S,11S,15R)-5,15-diacetoxy-3-nicotinoyloxy-14-oxolathyra-6(17),12E-diene, (2), Mr 523.2570); Euphorbia factor L2 ((2S,3S,4R,5R,7R,9S,11S,15R)-5,15-diacetoxy-3,7-dibenzoyloxy-14-oxolathyra-6(17),12E-diene,(3),Mr 642.2829); Euphorbia factor L9 ((2S,3S,4R,5R,7R,9S,11S,15R)-5,15-diacetoxy-3-benzoyloxy-7-nicotinoyloxy-14-oxolathyra-6(17),12E-diene, (4), Mr 643.2781);Euphorbia factor L1 ((2S,3S,4R,5R,6R,11S,15R)-5,15-diacetoxy-3-phenylacetoxy-14-oxolathyra-6(17),12E-diene-6(17)-epoxide, (5), Mr 552.2723); Euphorbia factor L7a((2S,3S,4S,9S,11R,15R)-15,17-diacetoxy-3-cinnamoyloxy-14-oxolathyra-5Z,12E-diene, (6), Mr 548.2774); Euphorbia factor L7b ((2S,3S,4R,5R,9S,11R,15R)-5,15,17-triacetoxy-3-benzoyloxy-14-oxolathyra-6Z,12E-diene, (7), Mr 580.2672).

105.0331

197.1311 225.1267

253.1944

281.1895

303.1711

321.1811

363.1928

425.2080

485.2287

503.2424

545.2512

+MS2(545.0000), 2.2-2.3min #(128-136)

0

2000

4000

6000

Intens.

100 200 300 400 500 600 m/z

119.0846131.0853 145.0984

159.1155169.1008

183.1150

0

1000

120 140 160 180

Figure 2. Product ion scan of the selected precursor [M+Na]+ at m/z 545 (collision energy at 25 eV) forEuphorbia factor L3.

Scheme 1. Major fragmentation patterns of [M+Na]+ forEuphorbia factor L3.

Scheme 2. Major fragmentation patterns of [M+Na]+ forEuphorbia factor L7a.

Letter to the Editor

wileyonlinelibrary.com/journal/rcmCopyright © 2012 John Wiley & Sons, Ltd.Rapid Commun. Mass Spectrom. 2013, 27, 276–280

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Table 1. Elemental constituents of major product ions from [M+Na]+ for Euphorbia factor L3 (collision energy: 25 eV)

Ion Formula Calculated Observed Error (ppm)

[M+Na]+ C31H38O7Na 545.2510 545.2512 �0.5[M+Na–CH2CO]+ C29H36O6Na 503.2404 503.2424 �3.9[M+Na–CH3CO2H]+ C29H34O5Na 485.2298 485.2287 2.5[M+Na–2CH3CO2H]+ C27H30O3Na 425.2087 425.2080 1.6[M+Na–CH3CO2H–C6H5CO2H]+ C22H28O3Na 363.1931 363.1928 0.6[M+Na–CH3CO2H-C6H5CO2H–CH3CO2H]+ C20H24ONa 303.1719 303.1711 2.7[M+Na-2CH3CO2H–C6H5CO2Na]+ C20H25O 281.1900 281.0895 1.6[281–CO]+ C19H25 253.1951 253.1944 2.6[281–C3H6]

+ C17H19O 239.1430 239.1453 �9.6[281–C4H8]

+ C16H17O 225.1274 225.1267 2.9[281–C5H8]

+ C15H17O 213.1274 213.1286 �5.7[281–C3H6-CO]+ C16H19 211.1481 211.1456 11.3[C6H5CO]+ C7H5O 105.0335 105.0331 3.6

Table 2. Elemental constituents of major product ions from [M+Na]+ for Euphorbia factor L2 (collision energy: 25 eV)

Ion Formula Calculated Observed Error (ppm)

[M+Na]+ C38H42O9Na 665.2721 665.2728 �1.0[M+Na–CH3CO2H]+ C36H38O7Na 605.2510 605.2501 1.4[M+Na–2CH3CO2H]+ C34H34O5Na 545.2298 545.2296 0.4[M+Na–C6H5CO2H]+ C31H36O7Na 543.2353 543.2349 0.8[M+Na–CH3CO2H–C6H5CO2H]+ C29H32O5Na 483.2142 483.2138 0.9[M+Na–CH3CO2H–C6H5CO2Na]+ C29H33O5 461.2323 461.2306 3.6[M+Na–CH3CO2H–C6H5CO2H–CH3CO2H]+ C27H28O3Na 423.1931 423.1927 0.9[M+Na–CH3CO2H–2C6H5CO2H]+ C22H26O3Na 361.1774 361.1764 2.8[M+Na–CH3CO2H–C6H5CO2H–CH3CO2H–C6H5CO2H]+ C20H22ONa 301.1563 301.1552 3.4[M+Na–CH3CO2H–C6H5CO2H–CH3CO2H–C6H5CO2Na]+ C20H23O 279.1743 279.1737 2.3[279–CO]+ C19H23 251.1794 251.1787 2.8[279–C3H6]

+ C17H17O 237.1274 237.1275 �0.4[279–C4H8]

+ C16H15O 223.1117 223.1122 �2.2[279–C5H8]

+ C15H15O 211.1117 211.1142 �11.7[C6H5CO]+ C7H5O 105.0335 105.0329 6.0

Table 3. Elemental constituents of major product ions from [M+Na]+ for Euphorbia factor L7a (collision energy: 25 eV)

Ion Formula Calculated Observed Error (ppm)

[M+Na]+ C33H40O7Na 571.2666 571.2675 �1.5[M+Na–CH2CO]+ C31H38O6Na 529.2561 529.2555 1.0[M+Na–CH3CO2H]+ C31H36O5Na 511.2455 511.2457 �0.4[M+Na–2CH3CO2H]+ C29H32O3Na 451.2244 451.2236 1.7[M+Na–CH3CO2H–C8H7CO2H]+ C22H28O3Na 363.1931 363.1926 1.2[M+Na–2CH3CO2H–C8H7CO2H]+ C20H24ONa 303.1719 303.1710 3.0[M+Na–2CH3CO2H–C8H7CO2Na]+ C20H25O 281.1900 281.1900 �0.1[281–CO]+ C19H25 253.1951 253.1939 �4.9[281–C3H6]

+ C17H19O 239.1430 239.1429 0.8[281–C4H8]

+ C16H17O 225.1274 225.1265 3.9[281–C5H8]

+ C15H17O 213.1274 213.1285 �5.1[C8H7CO]+ C9H7O 131.0491 131.0497 �4.4[C8H7CO-CO]+ C8H7 103.0542 103.0544 �1.7

Letter to the Editor

wileyonlinelibrary.com/journal/rcm Copyright © 2012 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 276–280

278

105.0329

171.1151

209.1295

251.1787

279.1737

339.1934

361.1764423.1927

483.2138

543.2349605.2501

665.2728

+MS2(665.0000), 0.8-0.9min #(49-53)

0.0

0.2

0.4

0.6

0.8

1.0

x104Intens.

100 200 300 400 500 600 700 m/z

119.0839133.1008 143.0842

159.1158

171.1151

181.0999

0

500

120 140 160 180

Figure 3. Product ion scan of the selected precursor [M+Na]+ at m/z 665 (collision energy at 25 eV) forEuphorbia factor L2.

103.0544

131.0497

197.1305 225.1265

253.1939 281.1900

303.1710363.1926

451.2236

511.2457

571.2675

+MS2(571.0000), 3.6-3.7min #(213-220)

0.0

0.5

1.0

1.5

2.0

x104Intens.

100 200 300 400 500 600 m/z

119.0857

132.0531

145.1006159.1161 169.1010

183.1154

0

1000

120 140 160 180

Figure 4. Product ion scan of the selected precursor [M+Na]+ atm/z 571 (collision energy at 25 eV) forEuphorbia factor L7a.

Letter to the Editor

those of compounds 1–4, especially in the low-mass range. Thisindicates that the macrocycle of 5 is more stable and difficultto break down. The MS/MS spectra of Euphorbia factor L7a (6)and compound 1 are similar, and the product ions at m/z 131and 103 are the characteristic ions of the cinnamic acid of 6(Fig. 4, Scheme 2, and Table 3). In the high-mass range, theCID fragmentation pathways of Euphorbia factor L7b (7) andcompound 6 are very similar. The product ions in the low-massrange in the MS/MS spectra of compound 3, 4 and 7 arepossibly formed by the same fragmentation pathways.CID fragmentation pathways of seven representative

lathyrane diterpenes were clarified using ESI-QTOF-MS/MSin positive-ion mode. Three types of fragmentation patternswere demonstrated: the loss of the side chain groups, thecleavage or loss of the three-membered ring moiety andthe cleavage of the macrocycle. The information obtainedfrom the fragmentation experiments of the [M+Na]+ precursorions is especially valuable for the rapid identification of thiskind of diterpenes in complex biological systems.

SUPPORTING INFORMATION

Additional supporting information may be found in theonline version of this article.

27

AcknowledgementsThis work was supported by West Light of Foundation ofChinese Academy of Sciences (Y2C6031100) and the NationalNatural Science Foundation of China (No. 21002098).

Copyright © 2012Rapid Commun. Mass Spectrom. 2013, 27, 276–280

Wei Jiao, Dong-Mei Fang, Zhi-JunWu,* Jian-Zhong Chen, Hua-WuShao* and Guo-Lin Zhang

Chengdu Institute of Biology, Chinese Academy of SciencesChengdu 610041, China

*Correspondence to: Z.-J. Wu and H.-W. Shao, Chengdu Instituteof Biology, Chinese Academy of Sciences, Chengdu 610041, China.E-mail: wuzj@cib.ac.cn; shaohw@cib.ac.cn

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