Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris Preissii

J. Mass Spectrom. Soc. Jpn. Vol. 58, No. 6, 2010

Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii

Shuichi Y6B6BDID,1῎ Robert E. CDM,2 and Bernd R. T. S>BDC:>I3, 4

1 Department of Environmental Engineering for Symbiosis, Faculty of Engineering, Soka University, Hachioji, Tokyo, JAPAN 2 24 Fancis Street, Blackburn, Victoria, AUSTRALIA 3 Department of Chemistry, Oregon State University, Corvallis, OR 97331, U.S.A. 4 COGER, King Saud University, 11451 Riyadh, SAUDI ARABIA

The total extract of resin from Callitris preissii was analyzed by gas chromatography/mass spectrometry and 34 lignans, underivatized and as trimethylsilyl ether derivatives, were identified by interpretation of the mass spectra. The lignans in the resin include 13 new lignans, i.e., four secoisolariciresinol derivatives, four lariciresinol derivatives, 3῏,4῏-methylenedioxy-7῏-hydroxylariciresinol, 3,4-methylenedioxypinoresinol isomer, and two matairesinol derivatives. Furthermore, 17 lignans of the resin are characterized by methylenedioxy substitution on either or both guaiacylpropyl units or the syringylpropyl unit: i.e., five secoisolariciresinol derivatives, three lariciresinol derivatives, 3῏,4῏-methylenedioxy-7῏-hydroxylariciresinol, three pinoresinol derivatives, and five matairesinol derivatives, have methylenedioxy structures. Eleven lignans, including two secoisolariciresinol derivatives, four lariciresinol derivatives, two pinoresinol derivatives, and three matairesinol derivatives, contain a syringyl moiety.

(Received June 28, 2010; Accepted August 12, 2010)

of their mass spectra. Here we report many other and 1. Introduction novel lignans in the resin of C. preissii by GC/MS Lignans are synthesized via the shikimate pathway analysis of total underivatized and silylated extracts. and their carbon skeletons are derived from oxidative We present their mass spectra with interpretations of coupling of two phenyl propane units.1), 2) The lignans the fragmentation patterns of the underivatized com- are widely distributed in the plant kingdom and pounds and of their trimethylsilyl (TMS) derivatives. known for about one thousand species.3) They are of 2. Experimental utility in development of new drugs, e.g., cytotoxins of enzymes, and cancer chemotherapy,2), 3) as well as bio- 2.1 Sample marker tracers for plants in the environment.4), 5) Mass The resin was collected as hardened extrusions on spectrometric data for such biomarker tracers are cru- the bark of a C. preissii (Cypress pine) tree in the Royal cial to advances in environmental sciences. The Botanic Gardens Melbourne, Australia. Pinales, i.e., Araucariaceae, Cupressaceae, Pinaceae, 2.2 Extraction and derivatization of extracts Podocarpaceae, and Taxaceae, are the major source The resin from C. preissii was crushed and sonicated plants of lignans. Many lignans have been character- three times with dichloromethane : methanol (1 : 1; v/ ized especially in the Cupressaceae (9 genera, 29 spe- v) for 15 min. The total extracts were combined and cies) and Pinaceae (6 genera, 31 species).2), 3), 6), 7) Howev- filtered through a glass fiber filter (Whatman GF/A). er, although Callitris preissii (Cypress pine) belongs to The filtrate was first concentrated with a rotary evap- the Cupressaceae, the chemical constituent of Callitris orator and then with a stream filtered nitrogen. An preissii (Cupressaceae) has not been investigated in aliquot (50 mL) of the total extract was converted detail. This is the first survey of the lignan composi- to trimethylsilyl (TMS) derivatives by reaction with tion in resin of C. preissii based on the lignan mass N,O-bis-(trimethylsilyl)trifluoroacetamide and pyridine spectra. for 3 hrs at 80῍. The excess reagent was removed by Although absolute structures of unknown lignans blow-down using a dry nitrogen stream and the tri- cannot easily be determined by NMR analysis in mix- methylsilylated extract dissolved in n-hexane for GC/ tures, gas chromatography/mass spectrometry (GC/ MS. MS) analysis of such mixtures is very useful to deter- 2.3 Gas chromatography/mass spectrometry mine compositions and possible structures of various GC/MS analyses of the underivatized and deriva- lignans by interpretation of the fragmentation patterns tized extracts were carried out using an Agilent model 6890 GC coupled to an Agilent model 5973 quadrupole ῎ Correspondence to: Shuichi Y6B6BDID, Department of MSD. A fused silica capillary column (Agilent DB-5MS, Environmental Engineering for Symbiosis, Faculty of 30 mῌ0.25 mm i.d., film thickness 0.25 mm) was used Engineering, Soka University, 1῍236 Tangi-cho, Hachioji, with helium as the carrier gas. The oven temperature Tokyo 192῍8577, JAPAN, e-mail: [email protected] was programmed as follows: temperature hold at 65῍

ῌ195ῌ S. Yamamoto, R. E. Cox, and B. R. T. Simoneit

Fig. 1. GC/MS traces (TIC) of the total extract (a) underivatized, and (b) TMS-derivatives of the extract of Callitris preissii resin. Peak annotation, see Table 1.

ῌ196ῌ Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii for 2 min, increase from 65 to 300ῌ at a rate of 6ῌ penoids are not significant components in the resin, minῌ1, and with a final isothermal hold at 300ῌ for 20 because they comprise only 12.7 (4.2῍), whereas the min. The samples were injected in the splitless mode lignans, consisting of thirty-four compounds comprise with the injector temperature at 300ῌ. The mass 268 (88.1῍), are the major resin components. The spectrometer was operated in the electron ionization lignans include the dibenzylbutanediol type, i.e., mode at 70 eV ionization energy and scanned from m/z secoisolariciresinol (1, Table 1) and the derivatives 3,3῎, 50 to 650. GC/MS data were acquired and processed 4,4῎-dimethylenedioxysecoisolariciresinol-9-methyl with the Agilent Chemstation software. ether-9῎-acetate (2), secoisolariciresinol-9-methyl ether- Identifications of compounds were based on compar- 9῎-acetate (3), 3,3῎,4,4῎-dimethylenedioxysecoisolaricire- isons with standards, literature mass spectra, Wiley sinol (4), 3,4-methylenedioxysecoisolariciresinol 275 library data, and interpretation of mass spectro- (5), secoisolariciresinol-9῎-methylether (6), 3,4-meth- metric fragmentation patterns. The relative abun- ylenedioxy-5῎-methoxysecoisolariciresinol (7), and dance of each compound was calculated by using its 3,4-methylenedioxy-5῎-hydroxysecoisolariciresinol (8). peak area in the total ion current (TIC) chromatogram Their total relative abundance is 22.6, which is not of TMS derivatives (Fig. 1b), and the relative abun- a dominant portion of the extract (Table 1). The dance detected in only the underivatized extract TIC tetrahydrofuran type lignans are comprised of the fol- (Fig. 1a) was normalized by the peak area of pinore- lowing classes (Table 1): shonanin (9); lariciresinol sinol which is the maximum compound in both derivatives: lariciresinol (10), its isomer (11), 3῎,4῎-meth- analyses. When two compounds overlapped, each area ylenedioxylariciresinol (12), 3῎,4῎-methylenedioxy- was allocated proportionally by the area of each base 5-hydroxylariciresinol (13), 3῎,4῎-methylenedioxy-5- peak. methoxylariciresinol (14), 5-methoxylariciresinol (15), and 5-methoxylariciresinol isomer (16); and 7῎-hy- 3. Results and Discussion droxylariciresinol derivatives: 7῎-hydroxylariciresinol The GC/MS (TIC) traces of the underivatized and (17), 7῎-hydroxylareciresinol isomer (18), and 3῎,4῎- TMS derivatized total extract from the C. preissii resin methylenedioxy-7῎-hydroxylariciresinol (19). The tetra- are shown in Fig. 1. The lignans identified and their hydrofuran type lignan including lariciresinol is a relative abundances versus the pinoresinol TMS deriv- major component for the TIC area in the resin, amount- ative on the basis of TIC area are listed in Table 1. The ing to 101.1 (33.2῍). The seven pinoresinol derivatives numbers in Table 1 refer to the chemical structures of (Table 1) are furofuran types and comprise: pinoresinol the lignans, which are shown in the plots of the mass (20), epi-pinoresinol (its isomer) (21), sesamin (22), 3,4- spectra (Figs. 2῍6), and the classification of the lignans methylenedioxypinoresinol (23), 3,4-methylenedioxy- based on chemical structure is according to Castro et epi-pinoresinol (an isomer) (24), 5-hydroxypinoresinol al.2) (25), and 5-methoxypinoresinol (26). The furofuran 3.1 Resin composition class including pinoresinol is also a dominant compo- The underivatized analysis of the resin extract in- nent for the TIC area in the resin, comprising a total of cludes twenty lignans, three sesquiterpenoids and four 129.2 (42.4῍). Matairesinol derivatives, which are diterpenoids (Fig. 1a and Table 1). On the other hand, lignanolides, consist of: matairesinol (27), hinokinin twenty-five lignans were identified in the analysis of (28), 3,4-methylenedioxymatairesinol (29), 3῎,4῎-meth- the TMS derivatives of the resin extract besides ylenedioxymatairesinol (30), 3῎,4῎-methylenedioxy-5- vanillic acid (35) and four diterpenoids (Fig. 1b methoxymatairesinol-4-methyl ether (31), 3῎,4῎-meth- and Table 1). The following eight lignans: 3,3῎,4,4῎- ylenedioxy-5-methoxymatairesinol (32), and 5-hydroxy- dimethylenedioxysecoisolariciresinol-9-methyl ether- matairesinol (thujaplicatin) (33). The class is not a 9῎-acetate (2), secoisolariciresinol-9-methyl ether-9῎- major component, with total of 14.2 (4.7῍). Isolaricire- acetate (3), shonanin (9), sesamin (22), 3,4-methyl- sinol (34), which is the cyclolignan type, is a trace (0.9) enedioxy-epi-pinoresinol (24), hinokinin (28), 3῎,4῎- component of the extracts. methylenedioxymatairesinol (30), and 3῎,4῎-methyl- 3.2 Interpretation of the mass spectra enedioxy-5-methoxymatairesinol-4-methyl ether (31), We already reported many mass spectra of lignans in and three sesquiterpenoids dihydrocolumellarin (36), the resin from Araucaria angustifolia, including secoiso- callitrisin (37), and its isomer (38) are not detected in lariciresinol derivatives, shonanin, lariciresinol deriva- the TMS derivative analysis. That may be due to their tives, 7῎-hydroxylariciresinol derivatives, pinoresinol high polarities with no derivatizable functional group, derivatives, and isolariciresinol derivatives, with the relatively low concentrations, or coelution with larger exception of matairesinol derivatives, as well as the peaks. interpretation of their mass spectrometric fragmenta- Vanillic acid which is a component of conifer lignin tion patterns.11) Although many of the lignans in the comprises 23.6 (7.8῍) to the TIC area of pinoresinol resin of A. angustifolia occur as acetates, it is notewor- and is a major component of the resin. Dihydro- thy that the lignans in the resin of C. preissii are columellarin, callitrisin and its isomer, which have comprised of 17 compounds with a methylenedioxy been reported in C. columellaris,8), 9) may possibly be group on one or both guaiacylpropyl units. They are specific for the Callitris genus. Although diterpenoids secoisolariciresinol derivatives (2, 4, 5, 7, and 8), laricire- are common to conifers, sandaracopimaric acid, and sinol derivatives (12, 13, and 14), 3῎,4῎-methylenedioxy- especially callitrisol and callitrisic acid in the resin are 7῎-hydroxylariciresinol (19), pinoresinol derivatives not widely distributed in coniferous plants10) and may (22, 23, and 24), and matairesinol derivatives (28, 29, 30, also be specific for the Callitris genus. These diter- 31, and 32) (Table 1). The mass spectra presented here

ῌ197ῌ S. Yamamoto, R. E. Cox, and B. R. T. Simoneit

Table 1. Lignan Derivatives Analyzed in the Resin of Callitris preissii by GC/MS

Relative abundance Num- Compound Com- Composition MW MW (as TIC Max areaῌ ber (Chemical structure) position (as TMS) (TMS) 100 in TMS deriv.) Dibenzylbutanediol derivatives Secoisolariciresinols: 1 Secoisolariciresinol C20H26O6 362 C32H58O6Si4 650 14.5 23,3῍,4,4῍-Dimethylenedioxysecoisolariciresinol-9-methyl C23H26O7 414 0.6 ether-9῍-acetate (Ia) 3 Secoisolariciresinol-9-methyl ether-9῍-acetate (Ib)C23H30O7 418 0.9 43,3῍,4,4῍-Dimethylenedioxysecoisolariciresinol (Dihydro- C20H22O6 358 C26H38O6Si2 502 1.2 cubebin) (Ic) 5 3,4-Methylenedioxysecoisolariciresinol (Id)C20H24O6 360 C29H48O6Si3 576 1.1 6 Secoisolariciresinol-9῍-methyl ether (Ie)C21H28O6 376 C30H52O6Si3 592 2.8 7 3,4-Methylenedioxy-5῍-methoxysecoisolariciresinol (If)C21H26O7 390 C30H50O7Si3 606 0.9 8 3,4-Methylenedioxy-5῍-hydroxysecoisolariciresinol (Ig)C20H24O7 376 C32H56O7Si4 664 0.6 Total 22.6 Tetrahydrofurans Shonanin: 9 Shonanin C20H24O5 344 0.2 Total 0.2 Lariciresinols: 10 Lariciresinol C20H24O6 360 C29H48O6Si3 576 62.1 11 Lariciresinol isomer C20H24O6 360 1.8 12 3῍,4῍-Methylenedioxylariciresinol (IIa, IIa῍)C20H22O6 358 C26H38O6Si2 502 9.8 13 3῍,4῍-Methylenedioxy-5-hydroxylariciresinol (IIb)C20H22O7 374 C29H46O7Si3 590 4.4 14 3῍,4῍-Methylenedioxy-5-methoxylariciresinol (IIc)C21H24O7 388 C27H40O7Si2 532 3.6 15 5-Methoxylariciresinol (IId, IId῍)C21H26O7 390 C30H50O7Si3 606 2.6 16 5-Methoxylariciresinol isomer C21H26O7 390 C30H50O7Si3 606 1.9 Total 86.3 7῍-Hydroxy lariciresinols: 17 7῍-Hydroxylariciresinol C20H24O7 376 C32H56O7Si4 664 9.0 18 7῍-Hydroxylariciresinol isomer C20H24O7 376 C32H56O7Si4 664 2.9 19 3῍,4῍-Methylenedioxy-7῍-hydroxylariciresinol (III)C20H22O7 374 C29H46O7Si3 590 2.7 Total 14.6 Furofurans Pinoresinols: 20 Pinoresinol C20H22O6 358 C26H38O6Si2 502 100.0 21 Epi-pinoresinol (Pinoresinol isomer) C20H22O6 358 C26H38O6Si2 502 6.2 22 Sesamin (IVa)C20H18O6 354 11.4 23 3,4-Methylenedioxypinoresinol (IVb, IVb῍)C20H20O6 356 C23H28O6Si 428 6.9 24 3,4-Methylenedioxy-epi-pinoresinol (3,4-Methylenedioxy- C20H20O6 356 1.4 pinoresinol isomer) 25 5-Hydroxypinoresinol (IVc)C20H22O7 374 C29H46O7Si3 590 2.4 26 5-Methoxypinoresinol C21H24O7 388 C27H40O7Si2 532 1.0 Total 129.2 Lignanolides Matairesinols: 27 Matairesinol (Va, Va῍)C20H22O6 358 C26H38O6Si2 502 8.3 28 Hinokinin (Vb)C20H18O6 354 0.1 29 3,4-Methylenedioxymatairesinol (Vc, Vc῍)C20H20O6 356 C23H28O6Si 428 0.3 30 3῍,4῍-Methylenedioxymatairesinol (Vd)C20H20O6 356 0.3 31 3῍,4῍-Methylenedioxy-5-methoxymatairesinol-4-methyl C22H24O7 400 2.4 ether (Ve) 32 3῍,4῍-Methylenedioxy-5-methoxymatairesinol (Vf, Vf῍)C21H22O7 386 C24H30O7Si 458 0.6 33 5-Hydroxymatairesinol (Thujaplicatin) (Vg)C20H22O7 374 C29H46O7Si3 590 2.2 Total 14.3 Non-lactone naphthalene derivatives Isolariciresinol: 34 Isolariciresinol C20H24O6 360 C32H56O6Si4 648 0.9 Total 0.9 Others 35 Vanillic acid C8H8O4 168 C14H24O4Si2 312 23.6 36 Dihydrocolumellarin C15H22O2 234 0.6 37 Callitrisin C15H20O2 232 2.4 38 Callitrisin isomer C15H20O2 232 1.0 39 Unknown (Resin acid) C20H30O2 302 C23H38O2Si 374 2.1 40 Sandaracopimaric acid C20H30O2 302 C23H38O2Si 374 2.6 41 Callitrisol C20H30O 286 C23H38OSi 358 0.8 42 Callitrisic acid C20H28O2 300 C23H36O2Si 372 3.2 Total 36.3 Total all compounds 304.3

ῌ198ῌ Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii

Fig. 2. Mass spectra of the secoisolariciresinol derivatives.

ῌ199ῌ S. Yamamoto, R. E. Cox, and B. R. T. Simoneit

Fig. 3. Mass spectra of the lariciresinol derivative.

ῌ200ῌ Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii

Fig. 4. Mass spectrum of 3῎,4῎-methylenedioxy-7῎-hydroxylariciresinol-diTMS.

Fig. 5. Mass spectra of pinoresinol derivatives. include 13 new lignans, namely, secoisolariciresinol ously. Although the mass spectra of underivatized and derivatives (2, 3, 5, 7, and 8), lariciresinol derivatives TMS-derivatized matairesinols, and hinokinin and (13, 14, 15, and 16), 3῎,4῎-methylenedioxy-7῎-hydroxyl- sesamin have already been reported by a few au- ariciresinol (19), 3,4-methylenedioxy-epi-pinoresinol thors,12)῍14) the mass spectra of the lignans in this study (24), and matairesinol derivatives (30 and 32) (Fig. 2 to have not been described. Since the fragmentation pat- Fig. 6) and the interpretations of their mass spectra terns of the mass spectra of hinokinin and sesamin, (Fig. 7 to Fig. 11) which have not been reported previ- both with dimethylenedioxy groups, and under-

ῌ201ῌ S. Yamamoto, R. E. Cox, and B. R. T. Simoneit

Fig. 6. Mass spectra of the matairesinol and its derivatives.

12), 13) ivatized matairesinol have been interpreted, we (If)or349(Ig) by loss of CH2C6H3(OCH3)(OR1)or refer to those reports for the interpretations of the CH2C6H3(OCH3)(OR2). In the case of R4ῌMe and R8ῌ related lignans. Furthermore, the fragmentation pat- Ac (Ia and Ib), the M-32 or M-60 ions are not detected. terns of the mass spectra of the secoisolariciresinol, Breakdown by path (b) yields the fragment Ih at m/z lariciresinol, 7῍-hydroxylariciresinol, and pinoresinol 135 (Ia, Ic, Id, If,andIg), 137 (Ib), 209 (Id and Ie), 239 derivatives are interpreted on the basis of our previous (If)or297(Ig, m/z 298 by adding a proton) with a high report.11) intensity. The intense ion at m/z 135 is characteristic 3.3 Secoisolariciresinols for the guaiacylpropyl unit with the methylenedioxy The mass spectra of the secoisolariciresinol deriva- group and is often the base peak. Furthermore, frag- tives are shown in Fig. 2 and their fragmentation path- mentation gives m/z 107 (Ib), 179 (Id and Ie), 209 (If), or ways in Fig. 7. The four patterns from (a) to (d) are 267 (Ig) by loss of OCH2 from Ih, but this does not occur mainly as follows: Ia, Ib, Ie,andIf have intense molec- from ion Ih at m/z 135 with the methylenedioxy struc- ῍ῌ ular ions (M ) and only Ig has a trace M-CH3. Frag- ture (Ia, Ic, Id, If,andIg). In the case of Ig and If,the mentation of M-92 (Ia and Ib), M-180 (Ic, Id, If,andIg), m/z 179 ion for Ig is derived from loss of OCH2 after or M-122 (Ie) proceed by loss of HOR4 (R4ῌMe [methyl] elimination of OTMS and m/z 179 forms for If by or TMS [(CH3)3Si]) and HOR8 (R8ῌAc [acetyl], TMS, or further loss of OCH2. Cleavage of the molecular ion by Me). After that, fragmentation gives the ions of m/z path (c) forms fragments (Ij, m/z 220, 250, 266, 324, 187 (Ia, Ic, Id, If,andIg), 189 (Ib), 261 (Id and Ie), 291 354, and 412 for each case, respectively), which are

ῌ202ῌ Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii

Fig. 6. (continued) unfavored and weak. Fragment Ik at m/z 161 (Ia, Ic, Id, quently, the rather intense ion IIf at m/z 223 (IIa), 151 If,andIg), 163 (Ib), 235 (Id and Ie), 265 (If), or 323 (Ig) (IIa῍), 311 (IIb), 253 (IIc and IId), or 181 (IId῍) forms by arises from subsequent loss of OR4 or OR8. In the case splitting o# C4H6. Fragment IIf then gives m/z 193 of underivatized compound Ib, breakdown by path (d) (IIa), 281 (IIb), 223 (IIc and IId), or 151 (IId῍) after loss of yields the fragment Il at m/z 122. OCH2 and furthermore, the m/z 223 fragment from IIg 3.4 Lariciresinols yields the m/z 193 (IIc and IId) ion by elimination of

The mass spectra of the lariciresinol derivatives (Fig. another OCH2. In the case of the underivatized com- 3) are interpreted by the fragmentation pathways of (a) pound (IIa῍ and IId῍), fragment IIg occurs at m/z 122 by to (e) as shown in Fig. 8. They all have intense molec- the same elimination of OCH2. On the other hand, ular ions. For path (a) the M-121 or M-49 fragments, fragment IIh from the B-ring gives m/z 135 (IIa, IIa῍, m/z 381 (IIa), 309 (IIa῍, very weak), 469 (IIb), 411 (IIc), IIb, IIc), 209 (IId)or137(IId῍), and subsequently IId

485 (IId), or 341 (IId῍), occur after initial loss of M-90 or yields the ion at m/z 179 by cleaving o# OCH2. M-18 as HOR3 (R3ῌTMS or H) from the molecular ion Cleavage of the molecular ion by path (c) leads to the and subsequent loss of OCH3. For path (b) the molecu- fragments IIi and IIl. Fragment IIi with the di#erent lar ion cleaves at the b-position of the phenyl group (B) R1,R2, and R3 substituents appears at m/z 324 (IIa), 180 to form the fragments IIe and IIh with concurrent loss (IIa῍), 412 (IIb), 354 (IIc and IId), or 210 (IId῍), respec- of HOR3. Thus fragment IIe derived from ring-A is at tively. The ion IIi becomes fragment IIj at m/z 235 m/z 277 (IIa), 205 (IIa῍), 307 (IId), or 235 (IId῍). Subse- (IIa), 323 (IIb), or 265 (IIc and IId) by loss of OR3.

ῌ203ῌ S. Yamamoto, R. E. Cox, and B. R. T. Simoneit

Fig. 7. The MS fragmentation pathways of secoisolariciresinol derivatives.

ῌ204ῌ Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii

Fig. 8. The MS fragmentation pathways of lariciresinol derivatives.

ῌ205ῌ S. Yamamoto, R. E. Cox, and B. R. T. Simoneit

Fig. 9. The MS fragmentation pathway of 3῎,4῎-methylenedioxy-7῎-hydroxy-lariciresinol-diTMS.

Furthermore, fragment IIk from IIj occurs at m/z 209 loss of C4H6 and produces IIIc at m/z 193 by splitting (IIa), 137 (IIa῎), 297 (IIb), 239 (IIc and IId), or 167 (IId῎) o# OCH2. Moreover, it is suggested that fragment IIId after splitting o# C2H2. Since the compounds of IIc, IId, at m/z 194 arises by elimination of C4H5 and CO from and IId῎ have a syringyl unit, they lose OCH2 (῍R2ῌH) IIIa. Fragment IIIe forms the base peak at m/z 223 to m/z 209 (IIc and IId) or 137 (IId῎). In the case of the (IIIf) by loss of a proton. Fragment IIIe also produces TMS-derivatives of IIc and IId, the ion at m/z 179 is IIIg at m/z 135 by losing OTMS. Breakdown of the derived from an additional loss of OCH2. Breakdown of compound by path (c) yields the fragment IIIh at m/z the compounds by path (d) yields the fragment IIm at 324. This fragmentation path yields weak ions, where m/z 161 (IIa, IIa῎, IIb, IIc,andIId῎)or235(IId) after loss IIIj at m/z 209 arises from loss of C2H2,bywayofIIIi of a proton. In a few cases, the compounds cleave at the which is due to elimination of OTMS from IIIh. Fur- a-position of phenyl group (B) by path (e) to produce thermore, IIIj produces m/z 179 (IIIk) by a loss of the fragment IIn at m/z 291 (IIa), 219 (IIa῎), or 249 (IId῎) OCH2. after loss of HOR3. 3.6 Pinoresinols 3.5 7῎-Hydroxylariciresinols The pinoresinol derivatives have intense molecular 3῎,4῎-Methylenedioxy-7῎-hydroxylariciresinol-diTMS ions, with most as base peak for all compounds (Fig. 5), (III) has a relatively intense molecular ion (Fig. 4) and and their fragmentation pathways of (a) to (e) are pre- the three fragmentation pathways (a) to (c) are shown sented in Fig. 10. In pathway (a) the M-31 fragment is in Fig. 9. First the minor fragments at m/z 500 and 410 entirely due to elimination of OCH3 and in path (b) are derived from the successive loss of two HOTMS. cleavage of the molecular ion produces fragments IVd The molecular ion cleaves at the b-position of phenyl and IVf. Since fragment IVd has two possible origins, group (B) by the second path (b) to produce fragments ﬁrst with R1,R2, and R3 substituents or second with R1῎, (IIIa, m/z 277) and (IIIe, m/z 224) by loss of OTMS. R2῎, and R3῎, the fragment ion for the opposite part of Subsequently, fragment IIIa gives IIIb at m/z 223 by each molecule is given in parentheses. Thus, the frag-

ῌ206ῌ Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii

Fig. 10. The MS fragmentation pathways of pinoresinol derivatives (parentheses indicate the opposite ion). ment ions at m/z 203 (203) of IVa, m/z 203 (277) of IVb, group, i.e., IVa, IVb,andIVb῍, a moderately intense ion m/z 203 (205) of IVb῍,orm/z 365 (277) of IVc arise from at m/z 122 arises by elimination of CO from fragment fragment IVd by loss of a proton. Subsequent cleavage IVe. However, in the other cases fragment IVh is a of fragment IVe at the dashed line gives the intense ion weak ion. When fragment IVf does not have methyl- IVg at m/z 149 (149) of IVa, m/z 149 (223) of IVb, m/z enedioxy group it yields fragment IVi, i.e., IVb and IVc. 149 (151) of IVb῍,orm/z 311 (223) of IVc. Moreover, The ion IVi at m/z 194 is formed from fragment IVe by fragment IVg yields various weak ions, at m/z 119 loss of OCH2 and IVi in turn becomes fragment IVj at (119) of IVa, m/z 119 (193) of IVb, m/z 119 (121) of IVb῍, m/z 166 by elimination of CO. or m/z 281 (193) of IVc by elimination of the other In the cases of paths (c), (d), and (e), direct cleavage

OCH2. On the other hand, fragment IVf can break from the respective molecular ions produces fragments down further to various ions. The intense fragment IVk at m/z 161 (161), 235 (161), 163 (161), or 235 (323), IVg is also produced from IVf by loss of a proton as in IVl at m/z 135 (135), 209 (135), 137 (135), or 209 (297), pathway (b). When fragment IVf has methylenedioxy and IVm at m/z 178 (178), 252 (178), 180 (178), or 252

ῌ207ῌ S. Yamamoto, R. E. Cox, and B. R. T. Simoneit

Fig. 11. The MS fragmentation pathways of matairesinol and its derivatives.

(340), respectively. Although fragments IVk and IVl Vf῍), respectively. The other fragment Vi is intense and are quite intense, fragment IVk is weak. Furthermore, often the base peak at m/z 209 (Va), 137 (Va῍, Vd), 135 the intense ion IVl at m/z 209 or 297 forms the m/z 179 (Vb, Vc, Vc῍), 181 (Ve), 239 (Vf), 167 (Vf῍), or 297 (Vg). or 267 fragments by loss of OCH2 as in the case of IVb Fragment Vi then loses OCH2 to various ions at m/z and IVc. 179 (Va), 107 (Va῍), 105 (Vb, Vc, Vc῍), 151 (Ve), 209 (Vf), 3.7 Matairesinols 137 (Vf῍), or 267 (Vg) and subsequently produces IIIc at

Matairesinol and its derivatives all have intense mo- m/z 121 (Ve) or 179 (Vf) by another loss of OCH2.The lecular ions, some as base peaks, as shown in Fig. 6. molecular ion cleaves at the b-position of phenyl group Their mass spectra are interpreted by the fragmenta- (B) by fragmentation path (b) to produce fragments Vj tion pathways (a) to (e) as shown in Fig. 11. b-Cleavage and Vk. Although fragment ion Vj is weak, the other of phenyl group (A) in the molecular ion by path (a) fragment Vk is relatively intense at m/z 209 (Va, Vc, occurs to the fragments Vh and Vi. Fragment Vh with and Vg), 137 (Va῍ and Vc῍), or 135 (Vb, Vd, Ve, Vf,and the di#erent R4 and R5 substituent scarcely appears at Vf῍), and is often also the base peak similarly as frag- m/z 293 (Va), 221 (Va῍), or 219 (Vb, Vc, Vd, Ve, Vf,and ment Vi. Fragment Vk then yields various ions at m/z

ῌ208ῌ Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii

179 (Va, Vc,andVg), 107 (Va῍ and Vc῍)or105(Vb, Vd, vatives, two pinoresinol derivatives, and three mataire-

Ve, Vf,andVf῍) by loss of OCH2. Breakdown by path sinol derivatives. (c) yields the fragments Vl and Vm, when the former Vl Acknowledgements has weak ion at m/z 236 (Va), 164 (Va῍ and Vd), 162 (Vb, Vc,andVc῍), 266 (Vf), 208 (Ve), or 194 (Vf῍), and the S. Yamamoto thanks to the board of Soka University latter Vm also has a low ion at m/z 194 (Va῍ and Vc῍)or for providing support and the opportunity to visit to 192 (Vb, Vd, Ve, Vf,andVf῍). Oregon State University. We thank the director and In some cases, the molecular ions cleave at the a- Dr. Peter Syme of the Royal Botanic Gardens Mel- position of phenyl group (A) by path (d) to produce the bourne for permission to acquire samples of resin. fragments Vn or Vo, according to the chemical substi- References tuent on the phenyl group. That is, while the ion at m/z 121 (Vb, Vc,orVc῍) arises in the case of Vo with a 1) K. Nabeta, M. Hirata, Y. Ohki, S. W. A. Samaraweera, methylenedioxy phenyl group, the fragments at m/z and H. Okuyama, Phytochemistry, 37, 409 (1994). 122 (Va῍ or Vd), 166 (Ve), or 152 (Vf῍) are observed for 2) M. A. Castro, M. Gordaliza, J. M. Miguel Del Corral, and the other substitution of Vn. Fragment Vf῍ produces A. San Felciano, Phytochemistry, 41, 995 (1996). 3) O. R. Gottlieb and M. Yoshida, “Natural Products of the m/z 122 ion by loss of OCH2. Path (e) is the opposite case of path (d). The fragments Vp or Vq are at m/z 122 Woody Plants I,” Springer-Verlag, Heidelberg (1989), p. 439. (Va῍ and Vc῍)orm/z 121 (Vb, Vd, Ve,andVf῍), respec- 4) B. R. T. Simoneit, W. F. Rogge, M. A. Mazurek, M. A. tively. Standley, L. M. Hildemann, and G. R. Cass, Environ. Sci. 4. Conclusion Technol., 27, 2533 (1993). 5) B. R. T. Simoneit, Appl. Geochem., 17, 129 (2002). In this study, 34 lignans were identified for the first 6) J. R. Cole and R. M. Wiedhope, “Chemistry of Lignans,” time in the extract of resin from C. preissii by GC/MS ed. by C. B. S. Rao, Andhra University Press, Waltair analyses and revealed the following points: (1) Thirteen (1978), p. 39. new lignans were found in the resin. They are com- 7) V. S. Parmar, A. Jha, K. S. Bisht, P. Taneja, S. K. Singh, A. prised of four secoisolariciresinol derivatives, four Kumar, J. R. Poonam, and C. E. Olsen, Phytochemistry, lariciresinol derivatives, 3῍,4῍-methylenedioxy-7῍-hy- 50, 1267 (1999). droxylariciresinol, 3,4-methylenedioxy-epi-pinoresinol, 8) J. B. Douglas and M. C. Raymond, Tetrahedron Lett., 1, and two matairesinol derivatives. (2) 30 novel mass 73 (1978). spectra of lignans are presented, and their MS fragmen- 9) J. B. Douglas and M. C. Raymond, Aust. J. Chem., 32, 2455 (1979). tation patterns have been interpreted in the same 10) A. Otto and V. Wilde, Bot. Rev., 67, 141 (2001). manner despite the various derivatives. (3) 17 lignans 11) S. Yamamoto, A. Otto, and B. R. T. Simoneit, J. Mass have the methylenedioxy group on one or both guai- Spectrom., 39, 1337 (2004). acylpropyl units. They include five secoisolariciresinol 12) A. M. Du$eld, J. Heterocycl. Chem., 4, 16 (1967). derivatives, three lariciresinol derivatives, 3῍,4῍-methyl- 13) A. Pelter, J. Chem. Soc. (C) Organics, 15, 1376 (1967). enedioxy-7῍-hydroxylariciresinol, three pinoresinol de- 14) R. Ekman, Holzforschung, 30, 79 (1976). rivatives, and five matairesinol derivatives. Their mass spectra are characterized by the m/z 135 key ion. (4) Keywords: Callitris preissii, Cypress pine, Lignan, GC/MS, Eleven lignans have a syringyl unit and comprise two Mass spectra of TMS-derivatives secoisolariciresinol derivatives, four lariciresinol deri-

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