GC Chromatogram and Mass Spectrum of Policosanols and Long Chain

B. GC Chromatogram and Mass Spectrum of Policosanols and Long Chain

Aldehydes Figure 12 shows the typical chromatogram of standard mixture and sample extracts. All compounds of standard mixture were completely separated to every single peak Figure 12a. The retention time of three synthesized aldehyde compounds hexacosanal, octacosanal, and triacontanal were detected 1 minute faster than their corresponding alcohol compounds. Quantification of each policosanol and aldehyde compounds in extracted samples was determined base on the retention time and peak height of each referred standard. Derivatization is primarily performed to modify an analyte’s functionality in order to enable chromatographic separations. The formation of chemical derivatives to facilitate meaningful analysis has long been a common practice in gas chromatography. The use of MSTFA as silylation reagent for derivatization has been reported in policosanol determination using GC-FID by some researchers Adhikari et al. 2006; Morrison et al. 2006, however, the use of MSTFA in the pre-study was found in less effective for policosanol determination comparing to direct analysis. Besides this, the use of internal standard i.e. octacosanoic acid was avoided due to a number of closed peaks in interest area of chromatogram of samples Figure 12b and 12c. Policosanol and long chain aldehyde contents in analyzed samples were determined based on the relation between peak height and calibration curve of each standards. An example of standard curve of octacosanol is shown in Figure 13, it has a linear equation of y = 24006x + 173.2. Figure 12 Typical gas chromatogram of a policosanol and long chain aldehyde standards, b Kokuto A, c sugarcane rind of Ni 15 cultivar. The chromatograms were obtained by GC-FID. Peak 1 : C22-OH Peak 2 : C24-OH Peak 3 : C26-OH Peak 3 a Peak 4 : C28-OH Peak 4 : C26-CHO a Peak 5 : C30-OH Peak 5 : C28-CHO a : C30-CHO 0.0 10.0 20.0 30.0 40.0 min 50.0 b 4 5 a 5 3 3 a 2 1 4 a 0.0 10.0 20.0 30.0 40.0 4 a 4 5 a 5 min 3 3 a 2 1 a 0.0 10.0 20.0 30.0 40.0 min 50.0 c 4 5 a 5 3 3 a 2 1 4 a Figure 13 Standard curve of octacosanol. Figures 14 and 16 show the mass spectrum of trimethylsilyl ether of C28, derivate of octacosanol, and C28 aldehyde octacosanal as standards. The policosanol and aldehyde compounds of the samples Figures 15 and 17 were identified by direct comparison of their chromatographic retention times and mass spectra with those of their respective standards. The mass spectra of policosanol were also confirmed with NIST 2005 Mass Spectral Library by GCMSsolution software Shimadzu, however the mass spectra of aldehydes were not available yet. Identification of policosanol was recognized from the mass fragment pattern of its trimethylsilyl derivate as the target ion. For example, mass fragment of mz 467 was a specific target ion of trimethylsilyl ether of C28 and the qualifier ions were mz 103; 468; and 469 Figures 14 and 15, Table 6. Mass fragment of mz 103, ion of ·CH 2 OSiCH 3 3, was found in all policosanol fragments. Splitting of 2 × ·CH 3 group from main chain policosanols were recognized in the target ion and qualifier ions of each analyzed compounds Table 6. y = 24006x + 173.22 R² = 0.9951 2,000 4,000 6,000 8,000 10,000 12,000 14,000 0.00 0.10 0.20 0.30 0.40 0.50 0.60 P ea k H ei g h t Concentration mgml Mass fragment pattern of C28 aldehyde C 28 H 56 O of sugarcane rind of Ni 15 cultivar are shown in Figure 17. The fragmentation of this compound, even though weak, was clearly recognized from the mass fragment of mz 362 M-46, loss of CH 2 =CH 2 and H 2 O from C 28 H 56 O + ; mz 364 M-44, loss of ion CH 2 =CH-O + from C 28 H 56 O + , mz 390 M-18, loss of H 2 O from C 28 H 56 O + ; and mz 408 M + , C 28 H 56 O + ion which are characteristic fragments of aldehyde Figure 16 and 17, Table 6. Similar policosanol and aldehyde fragmentations were identified in previous reports Irmak et al. 2006; Pérez-Camino et al. 2003. Figure 14 Mass spectrum of trimethylsilyl ether of C28 of standard. Figure 15 Mass spectrum of trimethylsilyl ether of C28 of Kokuto. 50 100 150 200 250 300 350 400 450 20 40 60 80 100 120 467 75 57 468 43 83 103 451 208 O Si mz 100 125 467 75 57 468 43 83 103 451 25 50 75 O Si 50 100 150 200 250 300 350 400 450 mz Figure 16 Mass spectrum of C28 aldehyde of standard. Figure 17 Mass spectrum of C28 aldehyde of sugarcane rind of Ni 15 cultivar. Table 6 Mass fragmentation pattern of policosanols and long chain aldehydes Compound Target ion mz Qualifier ions mz Docosanol C22-OH 383 103, 384, 385 Tetracosanol C24-OH 411 103, 412, 413 Hexacosanol C26-OH 439 103, 440, 441 Octacosanol C28-OH 467 103, 468, 469 Triacontanol C30-OH 495 103, 496, 497 Hexacosanal C26-CHO 380 336, 334, 362 Octacosanal C28-CHO 408 362, 364, 390 Triacontanal C30-CHO 436 390, 392, 418 290 300 310 320 330 340 350 360 370 380 390 400 mz 10 390 362 335 306 292 320 348 408 392 364 336 C O H 310 320 330 340 350 360 370 380 390 400 mz 10 390 362 334 306 320 348 333 408 322 394 C O H

C. Policosanols and Long Chain Aldehydes in Sugarcane Rind