Chemistry and Physics of Lipids 16 (1976) 31-59 © North-Holland Publishing Company
MASS SPECTRAL F R A G M E N T A T I O N OF 5 ~ - H Y D R O X Y S T E R O I D S a,b Furn F. KNAPP, Jr., Martha S. WILSON, and George J. SCHROEPFER, Jr.
Departments of Biochemistry and Chemistry Rice University, Houston, Texas 77001, USA Received September 14, 1975,
accepted November 4, 1975
The mass spectra of a number of C-4a- and C-4fl-alkylatedcholestan-3fl, 5a-diols have been found to contain an intense ion at m/e 332. The corresponding 6fl-alkylated (R) cholestan-3fl, 5a-diols exhibit an abundant ion at role 331 + R. The mass spectra of cholestan-5~-ol and cholestan-3t3,5c~-diolalso show an ion at m/e 332 which, however, is of relatively low abundance. The ion in question appears to arise from fragmentation processes which are characteristic of 5c~-hydroxysteroids. A similar fragmentation has been found to occur in the cases of C-4 and C-6fl-alkylated5a-hydroxy-cholestan-3-ones. The results of isotopic labeling, high resolution and metastable ion defocusing studies are discussed in terms of the origin of several of the ions in the spectra of the various compounds.
I. Introduction A recent report from this laboratory cencerned the mass spectral fragmentation of C-4 alkylated cholesterols [ 1]. The C-4fl alkylated sterols discussed in this study included 4fl-methyl-cholest-5-en-313-ol, [4fl-C2H3]-methyl-cholest-5-en-3fl-ol,and 4fl-ethyl--cholest-5-en-3fl-ol.These sterols were prepared from the analogous 4~3-alkylated cholestan-3fl, 5a-diols. Upon characterization of these 313, 5a-diols by mass spectrometry we noted an abundant ion at role 332 in the spectrum of each compound. Our interest in the origin of this ion resulted in an investigation of the mass spectral fragmentation of a number of 5c~-hydroxysteroids. The presence of an ion at m/e 332 in the mass spectra of cholestan-3fl, 5a-diol and cholestan-4fl, 5a-diol was initially reported by Wyllie [2]. It was suggested that functionalization at C-3 or C-4 is necessary for the formation of this ion since it was not described in the spectrum of cholestan-5a-ol [2]. However, we and others [3,4] have found this ion to be present in the spectrum of the latter compound, suggesting a Presented in part at the 30th Southwest Regional A.C.S. Meeting, Houston, Texas, December 11, 1974. b The configuration of the hydrogen at carbon atom 5 in the various sterols mentioned in this paper is ~. The designation of the configuration as 5a has been omitted throughout the text to conserve space. 31
F.I:: Knapp et al., Massspectra of sterols
32
that the detection and/or formation of this ion is dependent upon the instrument used or the conditions employed in recording the spectra. More recently, the effect of selected 613-substituents on the mass spectral fragmentation of 5a-hydroxysteroids has been studied by Rotman and coworkers [3] who reported that ions analogous to the m/e 332 ion, in the spectrum of cholestan-313, 5a-diol were not found in the spectra of cholestan-5c~, 6/3-diol, 3i3-methoxy-cholestan-5a, 6i3-diol, cholestan-5a, 6a-diol, and 5~3-hydroxy-cholestan-3i3, 6/3-diacetate. It was suggested that substituents at C-6 inhibit the formation of ions analogous to the m/e 332 ion observed for cholestan-313, 5a-diol. In the present study we have noted ions of high abundance corresponding to the ion at m/e 332 (in the spectrum of cholestan-3i3, 5c~-diol) in the spectra of 6i3-alkylated cholestan-3t3, 5a-diols. Others have reported inability to detect metastable transitions for the formation of the ion at m/e 332 in the spectra of cholestan-5a-ol [3] and cholestan-3/3, 5a-diol [2,3]. In addition, no metastable transitions were found for the transition M - ~ M - H 2 0 in the spectrum of cholestan-3/3, 5c~-diol and it was suggested that the loss of water to form an ion at M - 1 8 in the latter compound may result from a thermal process [2]. In the present study we have detected metastable peaks for the formation of the m/e 332 ion (m/e 331 + R in the case of the C-6 alkylated sterol) from both M and M - H 2 0 in the spectra of cholestan-313, 5a-diol, 4a-methyl-cholestan-3i3, 5c~diol, 4~3-methyl-cholestan-313, 5a-diol, and 613-methyl-cholestan-313, 5a-diol. In addition, we have observed the appropriate metastable ions for the transition M -7 M_H20 for these compounds, indicating that the loss of water from the molecular ion is at least partially due to an electron ionization induced fragmentation. Analysis of the mass spectra of [313ASOH]-cholestan-3i3, 5a-diol and [3~-lSOH]-6/3-methyl-cholestan-3i3, 5a-diol indicate that the ions corresponding to M ~ M - H 2 0 contain, within the error limits of the measurements, the oxygen of the 3~-hydroxyl function and none of the oxygen of the 5ahydroxyl group. Detailed studies of the fragmentation of these two 180-labeled diols and of a number of C-4 and C-6 alkylated cholestan-3/3, 5a-diols have permitted the proposal of several mechanisms for the formation of the ion at m/e 332 (331 + R in the cases of the C-6 alkylated sterols). In addition, the mass spectral fragmentations of a number of 5a-hydroxy-cholestan-3-ones have been studied and mechanisms consistent with the formation of characteristic ions in this series of compounds have been proposed.
lI.
Experimental
A. General The C2H31 used in the present experiments was purchased from the ICN Corporation, Irvine, California. The H2180 was 97.42% enriched and was supplied by Miles Laboratories. Thin-layer chromatographic analyses (t.l.c.) were
b:F. Knapp et aL, Mass spectra of sterols
33
performed on 500 micron thick layers of silica gel G using the following solvent systems: S-l, chloroform; S-2, 35% ethyl acetate in chloroform. Spot colors were detected by heating the plates at 80-100°C after spraying the plates with molybdic acid spray. For preparative purposes bands were visualized by ultraviolet irradiation after spraying with a solution of Rhodamine 6G. Bands were then scraped from the plates and the material was eluted from the adsorbent with ether. Gas-liquid chromatography (g.l.c.) was performed using a Hewlett-Packard Model 402 instrument equipped with dual flame ionization detectors. The carrier gas was helium with a flow rate of 66 ml/min. The column temperature was between 240 and 280°C depending upon the column in use. The detector and injector temperatures were normally 20°C higher than the column temperature. Compounds were analyzed on the four stationary phases SE-30 (1%), QF-1 (1%). OV-1 (3%) and OV-17 (3%) all coated on Gas Chrom Q, (100/120 mesh). Nuclear magnetic resonance spectra (n.m.r.) were obtained with a Perkin-Elmer HR-12 instrument at 60 MHz. Samples were run in CDC13 and peaks are reported in p.p.m. (6) downfield from the tetramethylsilane internal standard. Melting points are uncorrected and were determined using a Thomas-Hoover unimelt apparatus. Low resolution mass spectra were routinely obtained using an LKB Model 9000S single focusing mass spectrometer under the following conditions: ionization energy, 70 eV; trap current, 60/JA; source temperature, 230-270CC; probe temperature, 80-120°C. In addition, a few of the low resolution spectra were recorded on a CEC Model 21-110B spectrometer under the following conditions: ionization energy, 70 eV; trap current, 100/~A; source temperature, 200-220°C. Selected spectra were also recorded using a Finnegan Model 3300 spectrometer under the following conditions: ionizing energy, 70 eV; collector current, 500/~A; source temperature, 90°C; probe temperature 80-150°C. High resolution measurements were obtained with a CEC-Model 21-110B double focussing spectrometer using the photoplate technique. All mass spectra were obtained using direct probe inlet systems. In a few cases, as noted in the text, the spectra were recorded at lower ionization energies. Cholestan-5ceol (I)
Cholest-5-en-3/3-ol (2 g) was converted to cholest-5-en-3/3-chloride by reaction with thionyl chloride (4 ml) at room temperature overnight. The mixture was poured over ice, extracted with ether and the organic layer washed well with water, dried, and the solvent was evaporated to give a light yellow solid. Crystallization from acetone gave beautiful hexagonal plates, 1.3 g, mp 92°C (Lit., mp 95°C [5]); purity established by t.l.c. (S-l); i.r. u max (KBr) 800 and 865 cm-1; m.s. 406 and 404 (M, 33% and 97%), 391 and 389 (M-CH 3, 18% and 45%), 368 (M-HC1, 100%), 353 (M-CH3-HC1, 38%), 301 (25%), 291 (50%), 285 (55%), 249 (85%), 239 (85%) and 213 (46%); n.m.r. 3.74 (m, 1 H, C - 3 - H ) and 5.14 (m, 1 H, C - 5 - H ) . The chloride (1 g) was dissolved in dry ether (20 ml) and added to 50 ml of liquid ammonia. Metallic sodium (2 g) was added in small lumps and the resulting deep blue solution was stirred for 40 min. Ethanol (50 ml) was then added slowly with vigorous stirring
34
F.[: Knapp et al., Mass spectra of sterols
R2 I, R I = H
R2 R2=H
X I, R1 = OH, R2= OH
R1
R2
XXl, R1 = H, R2= H
II, R1 = OH, R2= H
XII
R1 = OAc , R2= OAc
XXII, R1 = CH3, R= H
II I, R1 = OAC, R2 = H
XIII
R1 =OH, R2= CH 3
X X I l I , R1 -C2H3 , R2=H
IV, R1 = OH, R2 = ~-CH 3
XIV
R1= OAc, R2=CH 3
XXIV, R1= CH2CH3, R2=H
V, R1 = O A c , R2= I~-CH 3
XV
RI= OH, R2=C2H 3
XVl
R1= OAc, R2=C2H 3
Vl, R1 =OH, R2=c4-CH 3 VII, R1 =OH, R2= ~-C2H 3 VIII, R1 = OAc, R2=I~-C2H 3 IX, R1 = OH, R2=~-CH2CH 3 X, R1 = OAc, R2= ~-CH2CH 3
XVlt - R I = OH, R2=CH2C,4 3 XVlII XIX XX,
XXV, RI=H, R2=CH 3 XXVi, XXVII,
R1=H, R2= C2H 3 RI=H, R2=CH2CH 3
R1 = OAc, R2 = C~-:2CH3 R1 = OH, R2 = CI R1 =OAc, R2 = CI
to give a clear solution. Extraction with ether followed by several water washes and evaporation of the solvent gave cholest-5-ene as a gum. Crystallization from ethanol gave plates, 657 rag, mp 89-90°C (Lit., mp 92°C [6]); purity established by t.l.c. (S-1) and g.l.c, analysis (3% OV-1 and 3% OV-17); i.r. u max (KBr) 960 cm-1; m.s. 370 (M, 100%), 355 (M-CH3, 80%), 301 (20%), 257 (38%), 247 (10%), 215 (47%) and 201 (12%); n.m.r. 5.34 (m, 1 H, C-5-H). The cholest-5-ene (200 rag) was dissolved in chloroform (20 ml) and stirred with m-chloroperbenzoic acid (150 rag) for two hours. The product, cholestan-5a, 6~-oxide, crystallized as plates from methanol, 197 rag, mp 75°C (Lit., mp 74-75°C) [7]); purity established by t.l.c. (S-1) and g.l.c, analysis (3% OV-1 and 3% OV-17); i.r. u max (KBr) 970 and 924 cm-1; m.s. 386 (M, 100%), 371 (M-CH 3, 52%), 368 (M-H20, 68%), 353 (M-CH3-H20, 31%), 331 (52%), 303 (8%), 273 (23%), 261 (14%), 255 (38%), 247 (24%), 213 (22%) and 201 (25%); n.m.r. 2.90 (d, J --- 4 Hz, 1 H, C - 6 - H ; from Dreiding models ¢ 6/3-7/3 = 90 °, J = 0 and ~ 6/3-7/3 = 30 °, J = 6 Hz; normally observed J = 3.7-4.8, [8]). The epoxide (98 rag) was reduced with lithium aluminum hydride in dry ether to give cholestan-5~-ol which was purified by t.l.c.; plates from methanol-water 42 mg, mp 105-106°C (Lit., mp 109°C [9]); purity established by t.l.c. (S-l) and g.l.c, analysis (3% O%17); i.r. u max (KBr) 3460 cm-1; m.s. (see table 1 and figure 1). 4~-Methyl-cholestan-3/3, 5~-diol (IV) and [ 4~-C2H3]-methyl-cholestan-3/3, 5~-diol
(vii) The method of Julia and Lavaux [ 10] was used to prepare 4/3-methyl-cholestan3/3, 5c~-diol (IV) mp 132-134°C (Lit., mp 134-135°C, [ 10]). This synthesis will be described in detail elsewhere [11]. The mass spectrum is presented in fig. 2. The reaction of 4~, 5a-epoxy-cholestan-3/3-yl acetate (200 mg) with [C2H3]-MgI (2.4 g of [C2H3] I reacted with 225 mg of magnesium turnings in ether) gave [4/3-C2H3]-
F.F. Knapp et al., Mass spectra of sterols
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MASS SPECTRAL F R A G M E N T A T I O N OF 5 ~ - H Y D R O X Y S T E R O I D S a,b Furn F. KNAPP, Jr., Martha S. WILSON, and George J. SCHROEPFER, Jr.
Departments of Biochemistry and Chemistry Rice University, Houston, Texas 77001, USA Received September 14, 1975,
accepted November 4, 1975
The mass spectra of a number of C-4a- and C-4fl-alkylatedcholestan-3fl, 5a-diols have been found to contain an intense ion at m/e 332. The corresponding 6fl-alkylated (R) cholestan-3fl, 5a-diols exhibit an abundant ion at role 331 + R. The mass spectra of cholestan-5~-ol and cholestan-3t3,5c~-diolalso show an ion at m/e 332 which, however, is of relatively low abundance. The ion in question appears to arise from fragmentation processes which are characteristic of 5c~-hydroxysteroids. A similar fragmentation has been found to occur in the cases of C-4 and C-6fl-alkylated5a-hydroxy-cholestan-3-ones. The results of isotopic labeling, high resolution and metastable ion defocusing studies are discussed in terms of the origin of several of the ions in the spectra of the various compounds.
I. Introduction A recent report from this laboratory cencerned the mass spectral fragmentation of C-4 alkylated cholesterols [ 1]. The C-4fl alkylated sterols discussed in this study included 4fl-methyl-cholest-5-en-313-ol, [4fl-C2H3]-methyl-cholest-5-en-3fl-ol,and 4fl-ethyl--cholest-5-en-3fl-ol.These sterols were prepared from the analogous 4~3-alkylated cholestan-3fl, 5a-diols. Upon characterization of these 313, 5a-diols by mass spectrometry we noted an abundant ion at role 332 in the spectrum of each compound. Our interest in the origin of this ion resulted in an investigation of the mass spectral fragmentation of a number of 5c~-hydroxysteroids. The presence of an ion at m/e 332 in the mass spectra of cholestan-3fl, 5a-diol and cholestan-4fl, 5a-diol was initially reported by Wyllie [2]. It was suggested that functionalization at C-3 or C-4 is necessary for the formation of this ion since it was not described in the spectrum of cholestan-5a-ol [2]. However, we and others [3,4] have found this ion to be present in the spectrum of the latter compound, suggesting a Presented in part at the 30th Southwest Regional A.C.S. Meeting, Houston, Texas, December 11, 1974. b The configuration of the hydrogen at carbon atom 5 in the various sterols mentioned in this paper is ~. The designation of the configuration as 5a has been omitted throughout the text to conserve space. 31
F.I:: Knapp et al., Massspectra of sterols
32
that the detection and/or formation of this ion is dependent upon the instrument used or the conditions employed in recording the spectra. More recently, the effect of selected 613-substituents on the mass spectral fragmentation of 5a-hydroxysteroids has been studied by Rotman and coworkers [3] who reported that ions analogous to the m/e 332 ion, in the spectrum of cholestan-313, 5a-diol were not found in the spectra of cholestan-5c~, 6/3-diol, 3i3-methoxy-cholestan-5a, 6i3-diol, cholestan-5a, 6a-diol, and 5~3-hydroxy-cholestan-3i3, 6/3-diacetate. It was suggested that substituents at C-6 inhibit the formation of ions analogous to the m/e 332 ion observed for cholestan-313, 5a-diol. In the present study we have noted ions of high abundance corresponding to the ion at m/e 332 (in the spectrum of cholestan-3i3, 5c~-diol) in the spectra of 6i3-alkylated cholestan-3t3, 5a-diols. Others have reported inability to detect metastable transitions for the formation of the ion at m/e 332 in the spectra of cholestan-5a-ol [3] and cholestan-3/3, 5a-diol [2,3]. In addition, no metastable transitions were found for the transition M - ~ M - H 2 0 in the spectrum of cholestan-3/3, 5c~-diol and it was suggested that the loss of water to form an ion at M - 1 8 in the latter compound may result from a thermal process [2]. In the present study we have detected metastable peaks for the formation of the m/e 332 ion (m/e 331 + R in the case of the C-6 alkylated sterol) from both M and M - H 2 0 in the spectra of cholestan-313, 5a-diol, 4a-methyl-cholestan-3i3, 5c~diol, 4~3-methyl-cholestan-313, 5a-diol, and 613-methyl-cholestan-313, 5a-diol. In addition, we have observed the appropriate metastable ions for the transition M -7 M_H20 for these compounds, indicating that the loss of water from the molecular ion is at least partially due to an electron ionization induced fragmentation. Analysis of the mass spectra of [313ASOH]-cholestan-3i3, 5a-diol and [3~-lSOH]-6/3-methyl-cholestan-3i3, 5a-diol indicate that the ions corresponding to M ~ M - H 2 0 contain, within the error limits of the measurements, the oxygen of the 3~-hydroxyl function and none of the oxygen of the 5ahydroxyl group. Detailed studies of the fragmentation of these two 180-labeled diols and of a number of C-4 and C-6 alkylated cholestan-3/3, 5a-diols have permitted the proposal of several mechanisms for the formation of the ion at m/e 332 (331 + R in the cases of the C-6 alkylated sterols). In addition, the mass spectral fragmentations of a number of 5a-hydroxy-cholestan-3-ones have been studied and mechanisms consistent with the formation of characteristic ions in this series of compounds have been proposed.
lI.
Experimental
A. General The C2H31 used in the present experiments was purchased from the ICN Corporation, Irvine, California. The H2180 was 97.42% enriched and was supplied by Miles Laboratories. Thin-layer chromatographic analyses (t.l.c.) were
b:F. Knapp et aL, Mass spectra of sterols
33
performed on 500 micron thick layers of silica gel G using the following solvent systems: S-l, chloroform; S-2, 35% ethyl acetate in chloroform. Spot colors were detected by heating the plates at 80-100°C after spraying the plates with molybdic acid spray. For preparative purposes bands were visualized by ultraviolet irradiation after spraying with a solution of Rhodamine 6G. Bands were then scraped from the plates and the material was eluted from the adsorbent with ether. Gas-liquid chromatography (g.l.c.) was performed using a Hewlett-Packard Model 402 instrument equipped with dual flame ionization detectors. The carrier gas was helium with a flow rate of 66 ml/min. The column temperature was between 240 and 280°C depending upon the column in use. The detector and injector temperatures were normally 20°C higher than the column temperature. Compounds were analyzed on the four stationary phases SE-30 (1%), QF-1 (1%). OV-1 (3%) and OV-17 (3%) all coated on Gas Chrom Q, (100/120 mesh). Nuclear magnetic resonance spectra (n.m.r.) were obtained with a Perkin-Elmer HR-12 instrument at 60 MHz. Samples were run in CDC13 and peaks are reported in p.p.m. (6) downfield from the tetramethylsilane internal standard. Melting points are uncorrected and were determined using a Thomas-Hoover unimelt apparatus. Low resolution mass spectra were routinely obtained using an LKB Model 9000S single focusing mass spectrometer under the following conditions: ionization energy, 70 eV; trap current, 60/JA; source temperature, 230-270CC; probe temperature, 80-120°C. In addition, a few of the low resolution spectra were recorded on a CEC Model 21-110B spectrometer under the following conditions: ionization energy, 70 eV; trap current, 100/~A; source temperature, 200-220°C. Selected spectra were also recorded using a Finnegan Model 3300 spectrometer under the following conditions: ionizing energy, 70 eV; collector current, 500/~A; source temperature, 90°C; probe temperature 80-150°C. High resolution measurements were obtained with a CEC-Model 21-110B double focussing spectrometer using the photoplate technique. All mass spectra were obtained using direct probe inlet systems. In a few cases, as noted in the text, the spectra were recorded at lower ionization energies. Cholestan-5ceol (I)
Cholest-5-en-3/3-ol (2 g) was converted to cholest-5-en-3/3-chloride by reaction with thionyl chloride (4 ml) at room temperature overnight. The mixture was poured over ice, extracted with ether and the organic layer washed well with water, dried, and the solvent was evaporated to give a light yellow solid. Crystallization from acetone gave beautiful hexagonal plates, 1.3 g, mp 92°C (Lit., mp 95°C [5]); purity established by t.l.c. (S-l); i.r. u max (KBr) 800 and 865 cm-1; m.s. 406 and 404 (M, 33% and 97%), 391 and 389 (M-CH 3, 18% and 45%), 368 (M-HC1, 100%), 353 (M-CH3-HC1, 38%), 301 (25%), 291 (50%), 285 (55%), 249 (85%), 239 (85%) and 213 (46%); n.m.r. 3.74 (m, 1 H, C - 3 - H ) and 5.14 (m, 1 H, C - 5 - H ) . The chloride (1 g) was dissolved in dry ether (20 ml) and added to 50 ml of liquid ammonia. Metallic sodium (2 g) was added in small lumps and the resulting deep blue solution was stirred for 40 min. Ethanol (50 ml) was then added slowly with vigorous stirring
34
F.[: Knapp et al., Mass spectra of sterols
R2 I, R I = H
R2 R2=H
X I, R1 = OH, R2= OH
R1
R2
XXl, R1 = H, R2= H
II, R1 = OH, R2= H
XII
R1 = OAc , R2= OAc
XXII, R1 = CH3, R= H
II I, R1 = OAC, R2 = H
XIII
R1 =OH, R2= CH 3
X X I l I , R1 -C2H3 , R2=H
IV, R1 = OH, R2 = ~-CH 3
XIV
R1= OAc, R2=CH 3
XXIV, R1= CH2CH3, R2=H
V, R1 = O A c , R2= I~-CH 3
XV
RI= OH, R2=C2H 3
XVl
R1= OAc, R2=C2H 3
Vl, R1 =OH, R2=c4-CH 3 VII, R1 =OH, R2= ~-C2H 3 VIII, R1 = OAc, R2=I~-C2H 3 IX, R1 = OH, R2=~-CH2CH 3 X, R1 = OAc, R2= ~-CH2CH 3
XVlt - R I = OH, R2=CH2C,4 3 XVlII XIX XX,
XXV, RI=H, R2=CH 3 XXVi, XXVII,
R1=H, R2= C2H 3 RI=H, R2=CH2CH 3
R1 = OAc, R2 = C~-:2CH3 R1 = OH, R2 = CI R1 =OAc, R2 = CI
to give a clear solution. Extraction with ether followed by several water washes and evaporation of the solvent gave cholest-5-ene as a gum. Crystallization from ethanol gave plates, 657 rag, mp 89-90°C (Lit., mp 92°C [6]); purity established by t.l.c. (S-1) and g.l.c, analysis (3% OV-1 and 3% OV-17); i.r. u max (KBr) 960 cm-1; m.s. 370 (M, 100%), 355 (M-CH3, 80%), 301 (20%), 257 (38%), 247 (10%), 215 (47%) and 201 (12%); n.m.r. 5.34 (m, 1 H, C-5-H). The cholest-5-ene (200 rag) was dissolved in chloroform (20 ml) and stirred with m-chloroperbenzoic acid (150 rag) for two hours. The product, cholestan-5a, 6~-oxide, crystallized as plates from methanol, 197 rag, mp 75°C (Lit., mp 74-75°C) [7]); purity established by t.l.c. (S-1) and g.l.c, analysis (3% OV-1 and 3% OV-17); i.r. u max (KBr) 970 and 924 cm-1; m.s. 386 (M, 100%), 371 (M-CH 3, 52%), 368 (M-H20, 68%), 353 (M-CH3-H20, 31%), 331 (52%), 303 (8%), 273 (23%), 261 (14%), 255 (38%), 247 (24%), 213 (22%) and 201 (25%); n.m.r. 2.90 (d, J --- 4 Hz, 1 H, C - 6 - H ; from Dreiding models ¢ 6/3-7/3 = 90 °, J = 0 and ~ 6/3-7/3 = 30 °, J = 6 Hz; normally observed J = 3.7-4.8, [8]). The epoxide (98 rag) was reduced with lithium aluminum hydride in dry ether to give cholestan-5~-ol which was purified by t.l.c.; plates from methanol-water 42 mg, mp 105-106°C (Lit., mp 109°C [9]); purity established by t.l.c. (S-l) and g.l.c, analysis (3% O%17); i.r. u max (KBr) 3460 cm-1; m.s. (see table 1 and figure 1). 4~-Methyl-cholestan-3/3, 5~-diol (IV) and [ 4~-C2H3]-methyl-cholestan-3/3, 5~-diol
(vii) The method of Julia and Lavaux [ 10] was used to prepare 4/3-methyl-cholestan3/3, 5c~-diol (IV) mp 132-134°C (Lit., mp 134-135°C, [ 10]). This synthesis will be described in detail elsewhere [11]. The mass spectrum is presented in fig. 2. The reaction of 4~, 5a-epoxy-cholestan-3/3-yl acetate (200 mg) with [C2H3]-MgI (2.4 g of [C2H3] I reacted with 225 mg of magnesium turnings in ether) gave [4/3-C2H3]-
F.F. Knapp et al., Mass spectra of sterols
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19F. Knapp et aL, Mass spectra o]sterols
36
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