980 Eur. J. Lipid Sci. Technol. 2011, 113, 980–984

Research Article Fatty acids of populnea: Mass spectrometry of picolinyl esters of fatty acids*

Gerhard Knothe1, Umer Rashid2, Suzana Yusup2 and Farooq Anwar3,4

1 National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, Peoria, IL, USA 2 Chemical Engineering Department, Universiti Technologi PETRONAS, Bandar Sari Iskandar, Tronoh, Perak, Malaysia 3 Department of Chemistry and Biochemistry, University of Agriculture, Faisalabad, Pakistan 4 Faculty of Food Science and Technology, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

Thespesia populnea belongs to the family of which contain and cyclopropene fatty acids. However, previous literature reports vary regarding the content of these compounds in seed oil. In this work, the content of malvalic acid (8,9-methylene- 9-heptadecenoic acid) in the fatty acid profile of Thespesia populnea seed oil was approximately 7% by GC. Two cyclopropane fatty acids were identified, including dihydrosterculic acid. The methyl and picolinyl esters of Thespesia populnea seed oil were also prepared. The mass spectrum of picolinyl malvalate was more closely investigated, especially an ion at m/e 279, which does not fit the typical series of ions observed in picolinyl esters. It is shown that this ion is caused by cleavage at the picolinyl moiety and contains the fatty acid chain without the picolinyl moiety. This type of cleavage has previously not been observed prominently in picolinyl esters and may therefore be diagnostic for picolinyl esters of cyclopropene fatty acids. The NMR spectra of Thespesia populnea methyl esters are also discussed.

Practical applications: The work reports the fatty acid composition of Thespesia seed oil whose derivatives have not yet been extensively utilized for industrial purposes, for example, biodiesel. Knowing this composition is essential for understanding potential uses and, for example, in case of biodiesel the fuel properties. Besides this issue, some data (mass spectrometry and NMR) crucial for obtaining the composition information are analyzed in detail. The biodiesel properties of methyl esters of Thespesia populnea will be reported separately.

Keywords: GC / MS / Nuclear magnetic resonance / Thespesia populnea Received: January 4, 2011 / Revised: March 24, 2011 / Accepted: April 12, 2011 DOI: 10.1002/ejlt.201100004

1 Introduction malvalic acid (8,9-methylene-8-heptadecenoic acid) in its seed oil [9]. Thespesia populnea belongs to the Malvaceae Several plant families whose oils contain cyclic fatty acids are family and, accordingly, its seed oil is known to contain known. Prominent representatives containing cyclopropene cyclopropane and cyclopropene fatty acids [5, 6, 10]. Both and/or cyclopropane fatty acids are Sterculaceae, malvalic acid and sterculic acid (9,10-methylene-9-octade- Bombacaceae, and Tiliceae, Sapindaceae besides Malvaceae cenoic acid) as well as dihydrosterculic acid (9,10-methylene and others as well as some bacteria [1–7]. Eight cyclopropyl octadecanoic acid) have been reported in Thespesia populnea fatty acids, including several novel compounds, were ident- [10], while an earlier report [11] does not mention the pres- ified by GC–MS in walnut oil [8]. One , Gnetum ence of cyclic fatty acids and another gives them combined as gnemon, was even reported to contain approximately 39% sterculic acid [3].

Correspondence: Dr. Gerhard Knothe, USDA/ARS/NCAUR, 1815 N. Disclaimer: Mention of trade names or commercial products in this University St., Peoria, IL 61604, USA publication is solely for the purpose of providing specific information and E-mail: [email protected] does not imply recommendation or endorsement by the US Department of Fax: þ1-309-681-6524 Agriculture. USDA is an equal opportunity provider and employer.

ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com Eur. J. Lipid Sci. Technol. 2011, 113, 980–984 Fatty acids of Thespesia populnea 981

During our ongoing investigation of potential feedstocks for equipped with a Agilent 5973 mass selective detector. biodiesel [12, 13], defined as the mono-alkyl esters of veg- Components were identified by comparison of retention etable oils or animal fats although other lipid feedstocks can times and mass spectra with authentic samples. also be used, we also became interested in Thespesia populnea. High-resolution MS was carried at the University of The fatty acid profile of a feedstock for biodiesel is generally Minnesota MS Facility (Minneapolis, MN) using a DB-5 identical with that of the biodiesel fuel and is largely respon- column on a Hewlett-Packard Series II 5890 gas chromato- sible for the fuel properties. Therefore, the fatty acid profile is graph and Finnigan MAT 95 double-focusing high-resol- essential when considering suitability of a feedstock for bio- ution mass spectrometer. diesel production. Here we report on the fatty acid profile of NMR spectra were acquired on a on a Bruker (Billerica, Thespesia populnea seed oil, also in light of the aforementioned MA) Avance 500 spectrometer operating at 500 MHz 1 13 inconsistent results. ( H-NMR) or 125 ( C-NMR) with CDCl3 as solvent. The mass spectra of picolinyl esters of cyclopropene fatty acids, namely malvalic and sterculic acids, have been dis- 3 Results and discussion cussed in the literature [14, 15]. However, there are discrep- ancies in this literature regarding some aspects of these Fatty acid profile: Previous literature on the fatty acid profile of spectra. In the course of identifying the various fatty acids Thespesia populnea seed oil shows some disagreements or in Thespesia populnea oil and its methyl esters, we analyzed the variations. While one report [11] does not even mention corresponding picolinyl esters by GC with a FID (GC) and cyclopropene or cyclopropane fatty acids, another report GC–MS. In light of the aforementioned discrepancies in the indicates 2.5% malvalic and 1.6% sterculic acid with 0.9% literature, we also report on the mass spectrum of picolinyl combined dihydrosterculic acid/C18:3v3 [12] while another malvalate. As a third aspect of this work, the essential features 1 13 [3] reports up to 8% cyclopropenoid acids (given as sterculic of the H- and C-NMR spectra of the methyl esters of acid). The fatty acid profile of Thespesia populnea oil as Thespesia populnea are briefly discussed. obtained here is given in Table 1 and shows malvalic acid at approximately 7% (6.8% as determined by GC). 2 Materials and methods Furthermore, two cyclopropane fatty acids were identified here which contrasts with another report [12] in which two The seeds from fully ripened Milo (Thespesia populnea L.) cyclopropene fatty acids (malvalic and sterculic) and one fruits were collected in the vicinity of the University of cyclopropane fatty acid (dihydrosterculic) were reported. Karachi, Karachi, Pakistan. The oil from the seeds was With the exception of a greater amount of palmitic acid found extracted with hexane in a Soxhlet extractor. The oil content here and malvalic acid as discussed above, the fatty acid of the seeds determined from this procedure is approximately profile determined in this work largely agrees with that dis- 20 wt%. cussed in the literature. Thespesia populnea methyl esters (TPME) were synthes- MS: Figure 1 shows the EI mass spectrum of picolinyl ized by a conventional transesterification procedure at 608C malvalate as obtained by GC–MS analysis. It largely agrees with a 6:1 molar ratio of alcohol/vegetable oil and 1% sodium methoxide as catalyst for 1 h. The product was purified by washing with water until the wash water showed a neutral pH, Table 1. Fatty acid profile of Thespesia populnea as determined by removing the methanol by rotary evaporation and drying with GC. Components are given in order of elution. The order of elution is magnesium sulfate. the same for picolinyl esters when using the same GC conditions Picolinyl esters of Thespesia populnea and standard fatty (however, analysis time extended) acids (palmitic, stearic, oleic, linoleic) were synthesized by a procedure available at the Lipid Library [16]. The picolinyl Fatty acid Amount (%) esters of the standard fatty acids were used to verify identity Tetradecanoic (Myristic) 0.5 and retention times of the components of picolinyl esters of Hexadecanoic (Palmitic) 26.8 Thespesia populnea. 9(Z)-Hexadecenoic (Palmitoleic) 0.7 GC for fatty acid profile analysis was carried out with an 9,10-Methylenehexadecanoic 0.9 Agilent 6890 gas chromatograph equipped and a Supelco Octadecanoic (Stearic) 4.1 (Bellefonte, PA) SP–2380 capillary column (30 m 8,9-Methylene-8-heptadecenoic (Malvalic) 6.8 0.25 mm inner diameter 0.2 mm film thickness). The oven 9(Z)-Octadecenoic (Oleic) 15.7 temperature ramp program was 1508C for 15 min, 150– 11(Z)-Octadecenoic (Asclepic) 1.8 2108Cat28C/min, and then ballistic heating to 2208C with 9(Z),12(Z)-Octadecadienoic (Linoleic) 39.2 9.10-Methyleneoctadecanoic (Dihydrosterculic) 1.5 a 5 min hold with He flow rate of 0.8 mL/min and 15 psi Eicosanoic (Arachidic) 0.5 (217.6 bar) pressure. For mass spectrometric analysis by Tetracosanoic (Lignoceric) 0.5 electron ionization (EI) of the picolinyl esters, analysis was Other 1.2 carried out using the same column in an Agilent 6890 GC

ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com 982 G. Knothe et al. Eur. J. Lipid Sci. Technol. 2011, 113, 980–984

92 have been virtually no reports on such a fragment being observed prominently in the mass spectra of the picolinyl esters of fatty compounds. Therefore, the question arises if m/z 279 corresponds to C H NO (cleavage between the Abundance 17 29 2 cyclopropenyl moiety and the terminal CH3)orC18H31O2

108 (cleavage of the picolinyl moiety; in case of picolinyl - 164 þ 55 67 81 late 279 ¼ 371 (M ) – 92). To clarify this issue, high-resol- 272 279 ution MS was carried out on a sample of picolinyl esters of 370 121 151 206 286 328 Thespesia populnea oil. The result of HR-MS analysis (dupli- 220 300 135 178192 258 314 342 230 356 cate determination) was exact masses of 279.2342 and m/z > 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 279.2324. This agrees well with a calculated exact mass of Figure 1. Mass spectrum of picolinyl malvalate obtained from a 279.2324 for the C18H31O2 (C17H29NO2 would be GC–MS run of the picolinyl esters of Thespesia populnea seed oil. 279.2198). Thus, the peak at m/z 279 is unequivocally caused by cleavage at the picolinyl moiety. It may be surmised that the cyclopropene moiety exerts a stabilizing effect on this ion, the reason for which, however, is not immediately clear. Note O CH 279 3 that the mass spectrum of picolinyl sterculate accordingly

O exhibits m/z 293, which corresponds to m/z 279 in the spec- trum of picolinyl malvalate [15]. A prominent Mþ – 92 ion 92 108 164 206 272 328 may be diagnostic for picolinyl esters of cyclopropene fatty acids. MW = 371 N It may be noted that the mass spectrum of methyl mal- valate treated with silver nitrate-methanol has been discussed Figure 2. Mass spectral cleavage pattern of picolinyl malvalate. [17, 18] as has that of the dimethyloxazoline derivative of malvalic acid [19]. The position of the cyclopropene ring in with spectra reported previously except for the observation the fatty acid chain was confirmed by this approach. The that, in contrast to previous results [14, 15], the ion [M-1]þ in mass spectrum of methyl malvalate is nearly identical to that the spectrum of picolinyl malvalate is stronger than Mþ of the methyl esters of C18:2 acids [20–21] thus the presence (m/z 371). and location of the cyclopropene ring is not determinable Some essential aspects of the mass spectral fragmentation from the spectrum. Therefore, derivatization of malvalic acid pattern of picolinyl malvalate (Fig. 2) have been discussed and other cyclopropene fatty acids to compounds yielding a [14, 15]. However, there is disagreement in this literature more useful fragmentation pattern is necessary. Overall, pico- regarding the existence of a peak at m/z 279 (m/z 293 in linyl esters are easily prepared without giving a variety of picolinyl sterculate), with one report [14] not showing this products as the silver nitrate-methanol approach does and peak while the other [15] pointing out its existence. As Fig. 1 show the necessary advantageous fragmentation pattern. shows, m/z 279 was observed in the course of this work, thus NMR: The 1H- and 13C-NMR spectra of TPME were agreeing with [15]. In any case, the location of the cyclo- also recorded. Figure 3 depicts the 1H-NMR spectrum of propene ring can be identified by the gap between m/z 206 and m/z 272. Besides the fragments at m/z 92, 108, 151 and 164 typical for picolinyl esters, the other major fragments with the exception of m/z 279 in the mass spectrum are caused by successive cleavage of CH2 moieties from the chain. Since the ion at m/z 279 does not fit the series of fragments increasing by 14 Da (or diagnostic variations to account for functional groups) typical for picolinyl esters continuously ‘‘migrating’’ down the fatty acid chain from C1, another explanation for its presence in the mass spectrum must be found. Therefore, if m/z 279 should contain the picolinyl moiety it could only be explained by rearrangement of the chain around the cyclopropenyl moiety with cleavage between the cyclopropenyl moiety and the terminal methyl group, although it is not clear how a difference of 7 Da could 6.0 5.0 4.0 3.0 2.0 1.0 0.0 arise. On the other hand m/z 279 can be easily explained if the pyridylmethylene moiety is cleaved, which also leads to m/z Figure 3. 1H-NMR spectrum of the methyl esters of Thespesia 92 prominently observed in picolinyl esters. However, there populnea oil.

ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com Eur. J. Lipid Sci. Technol. 2011, 113, 980–984 Fatty acids of Thespesia populnea 983

1 13 Table 2. Peaks in the H- and C-NMR spectra (in CDCl3) of TPME.

Assignment 1H-NMR 13C-NMR

CH3OOC– 3.67 51.39 CH3OOC– – 174.32, 174.26 CH3OOC–CH2– 2.31 (t), 2.38 (t; weak) 34.11, 34.09 CH3OOC–CH2–CH2– 1.63 (m) 24.935, 24.955 –CH2– 1.25–1.40 (m) (28.92, 28.97), 29.08, 29.11, 29.14, 29.25, (29.28), 29.31, 29.34, (29.40), 29.44, 29.51, 29.58, 29.64, 29.66, 29.27, (29.76)

–CH ¼ CH–CH2–CH ¼ CH– 2.78 25.62 (indicates cis) –CH2–CH ¼ CH–CH2–CH ¼ CH–CH2– 2.02–2.08 (m) 27.19, 27.18, 27.15 (27.37) –CH ¼ CH– 5.30–5.43 (m) 127.90, 128.04, 129.73, 129.98, 130.02, 130.19 –C ¼ C– (cyclopropenyl moiety) – 109.13, 109.52

–CH2–CH2–CH3 31.95, 31.52 –CH2–CH3 22.56, 22.74 –CH3 0.89 (m) 14.04, 14.09 –CH2– (cyclopropene moiety) 0.77 7.36 Malvalic ester 25.92, 26.03 (CH2 a to cyclopropene ring), 27.38 CH2 b to cyclopropene ring)

TPME. The spectrum is typical for methyl esters of a veg- mass spectrum of the picolinyl malvalate acid was analyzed. A etable oil but includes a few features that can be traced to the peak at m/z 279 was found which agrees with the observation more unique fatty acid profile of TPME. A singlet at by Christie [15]. To our knowledge a diagnostic peak not 0.77 ppm in the 1H-NMR spectrum is correlated with a peak containing the picolinyl moiety has not been observed in the at 7.36 ppm in the 13C-NMR spectrum. The peak at mass spectra of picolinyl esters of other fatty acids. The NMR 0.77 ppm in the 1H-NMR spectrum is ascribed to the meth- spectra of TPME are also discussed. The evaluation of ylene moiety of the cyclopropene ring in methyl malvalate in TPME as biodiesel fuel will be reported separately. accordance with other literature including than of cyclopro- pene fatty compounds obtained synthetically [22–24] The The authors thank Dr. Karl Vermillion (USDA/ARS/NCAUR) peak at 7.36 ppm in the 13C-NMR spectrum correlates well for obtaining the NMR spectra, Kevin Steidley (USDA/ARS/ with literature assignments of the methylene moiety of cyclo- NCAUR) for excellent technical assistance as well as Sean Murray propene fatty esters [25]. When integrating the 1H-NMR and Joseph Dalluge of the University of Minnesota MS facility for peak at 0.77 ppm, a concentration of malvalic acid of about obtaining the high-resolution mass spectra. 9% in TPME is obtained (integration value of methyl ester peak ¼ 3, maximum integration value of the peak at The authors have declared no conflict of interest. 0.77 ppm ¼ 2; observed value of 0.18), a value higher than determined by GC. Several peaks in the 13C-NMR spectrum are also split. References

For example, the peaks of C1, the terminal CH3 moiety and CH are split (see Table 2) with no assignments possible to [1] Carter, F. L., Frampton, V. L., Review of the chemistry of 2 cyclopropene compounds. Chem. Rev. 1964, 64, 497–525. which fatty acid species the individual peaks belong. 13 [2] Christie, W. W., in: Vol. 1, Gunstone, F. D. (Ed.), Topics in The C-NMR peaks at 109.13 and 109.52 ppm of the Lipid Chemistry, Wiley–Interscience, New York 1970. unsaturated carbons in methyl malvalate agree well with [3] Cornelius, J. A., Hammonds, T. W., Leicester, J. B., assignments in the literature where 109.16 (C8 of malvalate) Ndabahweji, J. K. et al., New tropical seed oils III. and 109.55 (C9 malvalate) ppm were reported [19] 109.11 Component acids of leguminous and other seed oils (C8 of malvalate; carbon number corrected) and 109.56 (C9 of (Continued). J. Sci. Food Agric. 1970, 21, 49–50. malvalate; carbon number corrected) [26] as well as 109.13 [4] Bohannon, M. B., Kleiman, R., Cyclopropene fatty acids of (C8 of malvalic acid) and 109.59 (C9 of malvalic acid) [27]. selected seed oils from Bombaceae, Malvaceae, and Sterculaceae. Lipids 1978, 11, 270–273. [5] Badami, R. C., Patil, K. B., Structure, and., Occurrence of unusual fatty acids in minor seed oils. Prog. Lipid Res. 1981, 4 Summary and conclusions 19, 119–153. [6] Sebedio, J. L., Grandgirard, A., Cyclic fatty acids: Natural The fatty acid profile of Thespesia populnea was investigated sources, formation during heat treatment, synthesis and bio- and found to contain in the range of 7–8% malvalic acid. The logical properties. Prog. Lipid Res. 1989, 28, 303–336.

ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com 984 G. Knothe et al. Eur. J. Lipid Sci. Technol. 2011, 113, 980–984

[7] Scrimgeour, C. L., Harwood, J. L., in: Gunstone, F. D., [18] Hosamani, K. M., Unique occurrence of unusual fatty acids Harwood, J. L., Dijkstra, A. J. (Eds.), The Lipid Handbook, and their industrial utilization. Ind. Eng. Chem. Res. 1996, 35, Third Edition, CRC Press, Boca Raton 2007. 326–331. [8] Hanus, L. O., Goldshlag, P., Dembitsky, V. M., [19] Spitzer, V., GC–MS characterization (chemical ionization Identification of cyclopropyl fatty acids in walnut (Juglans and electron impact modes) of the methyl esters and oxazo- regia L.) oil. Biomed. Pap. Med. Fac. Univ. Palacky. Olomouc. line derivatives of cyclopropenoid fatty acids. J. Am. Oil Czech Repub. 2008, 152, 41–45. Chem. Soc. 1991, 68, 963–969. [9] Berry, S. K., Cyclopropene fatty acids in Gnetum gnemon (L.) [20] Pawlowski, N. E., Eisele, T. A., Lee, D. J., Nixon, J. E., seeds and leaves. J. Sci. Food Agric. 1980, 31, 657– Sinnhuber, R. O., Mass spectra of methyl sterculate and 662. malvalate and 1,2–dialkylcyclopropenes. Chem. Phys. [10] Gaydou, E. M., Ramanoelina, A. R. P., Cyclopropenoic fatty Lipids 1974, 13, 164–172. acids of malvaceae seed oils by gas-liquid chromatography. [21] Aued–Pimentel, S., lago, J. H. G., Chaves, M. H., Kumagai, Fette Seifen Anstrichm. 1984, 86, 82–84. E. E., Evaluation of a methylation procedure to determine [11] Subbaram, M. R., Analysis, and., Characterisation of the oil cyclopropenoids fatty acids from Sterculia striata St. Hil. from the seeds of thespesia populnea. Proc. Math. Sci. 1954, Et Nauds seed oil, H. Chromatogr. A 2004, 1054, 235–239. 39, 301–304. [22] Hopkins, C. Y., Nuclear magnetic resonance in fatty acids [12] Knothe, G., Krahl, J., Van Gerpen, J. (Ed.), The Biodiesel and glycerides. Prog. Chem. Fats Other Lipids 1966, 8, 215– Handbook, 2nd Edn., AOCS Press, Urbana, IL 2010. 252. [13] Mittelbach, M., Remschmidt, C., Biodiesel– The Comprehensive [23] Gensler, W. J., Pober, K. W., Solomon, D. M., Floyd, M. B., Handbook, publ. by M. Mittelbach, Graz, Austria 2004. Syntheses of methyl malvalate and methyl 5,6–methano–5– undecenoate. J. Org. Chem. 1970, 35, 2301–2307 . [14] Spitzer, V., Marx, F., Maia, J. G. S., Pfeilsticker, K., The mass spectra of the picolinyl ester derivatives of malvalic and [24] Arsequell, G., Fabria´s Gosalbo. L., Camps, F., Synthesis of sterculic acid. Fat Sci. Technol. 1994, 96, 395–396. inhibitors of a delta–11 desaturase in the moth spodoptera littoralis. Chem. Phys. Lipids 1992, 63, 149–158. [15] Christie, W. W., Mass Spectra of Derivatives of Cyclopropyl and Cyclopropenyl Fatty Acids, http:// [25] Gunstone, F. D., 13C–NMR Spectroscopy of Fatty Acids lipidlibrary.aocs.org/ms/ms18/index.htm, http://lipidlibrary. and Derivatives, Cyclic Fatty Acids, http://lipidlibrary.aocs. aocs.org/ms/arch_pic/pi_misc/Pi 0947.htm. org/nmr/nmrcycl/index.htm. [16] Christie, W. W., Preparation of Nitrogen–Containing [26] Gunstone, F. D., High resolution 13C–NMR. A technique Derivatives for Mass Spectrometry of Fatty Acids, http:// for the study of lipid structure and composition. Prog. Lipid lipidlibrary.aocs.org/ms/ms02/index.htm. Res. 1994, 33, 19–28. [17] Ahmad, M. S., Ahmad, M. U., Osman, S. M., Ballantine, [27] Gaydou, E. M., Ramanoelina, A. R. P., Rasoarahona, J. R. J. A., Eriolaena hookeriana seed oil: A rich source of malvalic E., Combres, A., Fatty acid composition of sterculia seeds acid. Chem. Phys. Lipids 1979, 25, 29–38. and oils from Madagascar. J. Agric. Food Chem. 1993, 41, 84–86.

ß 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.ejlst.com