Figure 7 Gas chromatographic separation of pairs of C24 epimeric sterol trimethylsilyl ethers on SP-2340 at 195°C (elution time from left to right). Peaks are identified in the figure. Instrument: Hewlett-Packard model 5840 gas chromatogaph equipped with flame ionization detector. Column: glass capillary (115m×0.25mm) coated with SP-2340, a cyanosilicone phase (Quadrex, New Haven, CT) providing 230,000 effective theoretical plates. Injector, 280°C; detector, 300°C; column, 185 or 195°C. Carrier gas, H at 20–34 psi.

The common plant sterols are tentatively identified based on the GLC retention times especially when the GC separations are preceded by a TLC or AgNO3-TLC separation. More complex mixtures require confirmation by MS (66). Beveridge et al. (32) identified cholesterol, campesterol, stigmasterol, sitosterol, β-amyrin, sitostanol, lupeol, and erythrodiol by comparing their RRf values and their MS fragments with those of authentic samples. Beveridge et al. (32) have prepared a summary of the fragmentation ions obtained for selected 4-desmethyl, 4-monomethyl, and 4,4-dimethyl sterols as obtained under GC/MS conditions (Table 2). The TMS ether derivatives of spinasterol, ∆5-avenasterol, ∆7-sitosterol, and ∆7-avenasterol were identified by comparing the GC/MS and GC/FID values reported in the literature (66-68) and MS data (69-71). Clerosterol, ∆5,25 (27), was identified on the basis of its retention time relative to sitosterol acetate (71,72) and free sterol (73,74), and the characteristics of the allylic cleavage in ∆5,25(27) sterols (75). The peak for sitosterol contained a minor peak identified on basis of retention time and an uncharacteristic mass spectra at m/z 343 as the ∆7 isomer of sitosterol. The

Main fragment ions, m/z (Relative intensity)aRRTb

M+ M-15 M-90 M-105 M-129 Others Squalene 0.36 410(1) 69c

Oxidosqualene 0.45 426(1) 69c

4-Desmethylsterols

Campesterol 0.84 472(18) 457(3) 382(42) 367(21) 343(53) 129c

Stigmasterol 0.89 484(13) 469(3) 394(18) 379(7) 355(9) 83c

Clerosterol 0.96 484(18) 469(1) 394(15) 379(9) 355(15) 55c

Sitosterol 1.00 486(15) 471(5) 396(41) 381(20) 357(50) 129c

∆5-Avenasterol 1.03 484(4) 469(3) 394(1) 379(4) 355(8) 55c

∆5,24(25)-Stigmasterol 1.09 484(4) 469(4) 394(5) 379(9) 355(4) 55c

∆7-Sitosterol 1.12 486(57) 471(16) 396(3) 381(14) 255c

∆7-Avenasterol 1.16 484(1) 469(5) 394(1) 379(4) 343c

4-Monomethylsterols

Citrostadienol 1.38 498(tr) 483(5) 408(1) 393(6) 357c

4,4,-Dimethyl triterpene alcohols

β-Amyrin 1.02 498(1) 483(tr) 408(tr) 393(1) 218c

Lupeol 1.11 498(9) 483(2) 408(3) 393(6) 73c

24-Methylene cycloartenol

1.26 586(1) 496(3) 481(tr) 73c

a Intensity relative to base peak (percent); tr indicates trace. b retention times relative to sitosterol TNS ether (35.9min) using a DB-5 60-m column. The internal standard 5α-cholestane elutes with a relative retention time of 0.40. c Base peak (relative intensity=100%). The peak for sitosterol also contained spinasterol; the peak for β-amyrin also contained sitostanol; the peak for ∆5avenasterol also contained butyrospermol; and the peak for citrostadienol also contained erythrodiol, as minor components. Source: Adapted from Ref. 32.