Identification of Plant Sterols Using Combined
GLC/Mass Spectrometry
8. A. Knights, University of Glasgow, Botany Department Research Laboratory, Glasgow, Scotland
Abstract
Usingcombined GlC/mass-spectrome-
try, massspectra for fifteen plant sterols
and closely related compounds have
been determined. Free sterols, acetate,
trifluoroacetate, and trimethylsilyl ether
derivatives were investigated, and the
latter two derivatives were found to be
particularly suitable for the method of
analysis. Characteristic features of t:J.5-
and t:J.7·monounsaturated sterols have
been outlined as having features char-
acteristic of sterols possessing double
bonds in the side chain.
Introduction
Sterols occur widely in plants as
well as in animals, and the role of
such compounds in living organisms
is being increasingly studied. Thus,
a sterol requirement for reproduc-
tion of several species of fungi has
been established (1), the metab-
olisms of plant sterols in the intestine
has been studied in several labora-
tories (2,3,4,5), and absorption of
sterols into the dog lymph from the
intestine has been reported (6). In
addition, such compounds are being
studied for use in taxonomic classi-
fication (7).
Plant sterols differ from animal
sterols (e.g., cholesterol IV, desmo-
sterol XV) in having either one or
two extra carbon atoms at C-24.
These two extra carbon atoms are
successively introduced as C-1 units,
arising initially as a methylene
group (e.g., X, XI, XVII) and then
as an ethylidene group (e.g., XII,
XIII, XVIII) (8,9). Further modi-
fication gives rise to sterols with a
methyl group (campesterol V) or
an ethyl group (,B-sitosterol VI) at
C-24, and there may also be a
double bond present at C-22 as in
stigmasterol (XN, R=C 2H5 ) .
The steps involved in the biosyn-
thesis of plant sterols, reviewed by
Clayton (10), apparently overlap
to give rise to sometimes complex
mixtures difficult to separate. Appli-
cation of GLC has revealed the
heterogeneous nature of many plant
sterol fractions (11), and reports
(12, 13) of the heterogeneous na-
ture of previously mentioned plant
sterols (14) have appeared. The
difficulty of separating these com-
pounds has led to the development
of complex chromatographic pro-
cedures (15) and to the application
of combined GLC/mass spectrom-
etry (4, 5, 16) for their analysis
and identification.
The additional carbon atoms
make plant sterols easy to separate
by GLC, particularly if a partial
separation into types of sterol (4,
5, 16) is utilised prior to GLC. The
application of mass spectrometry
for their analysis is also facilitated
since, while the nucleus has a skele-
ton common with that of other ster-
ols, the side chain may be regarded
as constituting successive members
of a highly branched homologous
series (e.g., Figure 1: I - III, IV-
VI, VII - IX) . Thus, ions in a mass
spectrum which retain the nucleus
will be common to each member of
a series, whereas those ions retain-
ing the side chain will show suc-
cessive increases of fourteen mass
units between members of the series.
The validity of this concept for
mass spectrometry of plant sterols
is demonstrated by published spec-
tra (4) for 5,B-cholestan-3-one and
its two C-24 homologues. Ions for
M (molecular ion), M-15, M-
(15+18) and M-70 all show an in-
crease of 14 mass units between
successive members of the series.
Ions at tule 271, 255, 246, 231 and
213, where the side chain has been
lost from the charged fragment, are
common to all three spectra. The
relative intensities of corresponding
ions in all three spectra is similar.
A similar result has been observed
for O-methyloxime derivatives of
ketonic steroids obtained by oxida-
tion of the sterol fraction of oat
seed (16). The latter results have
served to demonstrate the presence
of 14 compounds (I-XIV) in oat
seed.
This concept has been applied
previously to pollen sterols (17). A
complex mixture of C 2 7-C2 9 sterol
acetates was hydrogenated, and
mass spectrometry of the mixture
gave a spectrum showing ions for
M-AcOH and M-(AcOH+ring A)
corresponding to c., C 28 , and C 2 9
sterols. Only single peaks, at mle
257 and 215, were observed where
the side chain had been lost. A
similar method of analysis has been
applied to tobacco sterols (18).
Use of this concept has been made
in studies of the biosynthesis of
ergosterol (9). Ergosterol and ergo-
sterol biosynthesised in the pres-
ence of CDs labelled methioriine
were subjected to mass spectrom-
etry, and it was demonstrated that
the extra methyl group at C 2 4 in
labelled ergosterol contained only
two deuterium atoms. Thus, the
ions M, M-15, M-18, and M-
(15+18) were elevated to mass
units in the labelled substance,
whereas the ions at mle 271 and
253 were common to both spectra.
An ion involving loss of 72 mass
units from cholestanol is matched
by an ion involving loss of. 100
mass units from the corresponding
4, 4-dimethylsterol demonstrating
that this fragmentation involves
ring A (19). That the ion at tale
255 in spectra from ,B-sitosterol
J. of G. C.-June, 1967 273
(VI) and stigmasterol (XIV) is at m/e 299 in the trimethyl sterol been the subject of extensive study
derived by loss of the side chain is tetrahydrodammaradienol (20) . (21) and has included labelling of
confirmed by a corresponding peak Mass spectrometry of steroids has various positions of the nucleus with
deuterium in order to investigate
R R R the fragmentations and hydride
shifts involved. Sterols such as cho-
lesterol have also been the subject
of study, although rather lens is
understood of certain of the frag-
R 1 0 R0 RI0 mentations noted (19, 22, 23, 24).
1 R=H IV R=H VII R=H Because of the difficulty in ob-
11 R=CH 3 V R=CH3 V111 R=CH 3 taining pure samples, plant sterols
!1! R=C 2 H 5 V! R=C H 5 IX R=CZHS have not been exhaustively studied
using mass spectrometry. However,
spectra have been recorded for /3-
sitosterol (VI) (22) , ergosterol (9,
25), stigasterol (XIV) (25), citro-
stadienol (XVIII) (18, 26), and
24-methylenelophenol acetate XVII
(18).
Using GLC/mass spectrometry
Eneroth et al. have recorded spectra
R 0/^/^" Zip R10
for trimethylsilyl ether derivatives
x xl XII
of 24-ethyl-5a-cholestan-38-ol (III),
cholesterol (IV) , campesterol (V) ,
f3-sitosterol (VI) (4), and stigma-
sterol (XIV, R= 2 H 5 ) (5).
In addition to cases referred to
previously, GLC/mass spectrometry
R
of steroids has been used in the
analysis of bile acids in feces (27,
28) and to the analysis of 0 5 -andro-
stenediols and p 5 -pregnenediols in
R 0 R1 R^0
plasma (29) . Mention has been
made of use of the technique for
x111 XIV xv
determining the fate of steroid tol-
uene-p-sulphonate and methane sul-
phonate esters upon GLC (30) .

Experimental

xvttt
Sterols examined in this work are
XVI xvtt
listed in Table 1 together with
Figure 1. details of origin, principal ions ob-
served in mass spectra, and details
1 R=H 5a-cholestan-3p-ol
of mass spectrometers used.
II R=CH3 24-methyl-5a-cholestan-3p-ol
Sterol derivatives were obtained
III R =C:HS 24-ethyl-5a-cholestan-3p-ol
Cholesterol from the "free" sterol (R 1 =H) by
IV R = H
V R =CH3 24-methylcholesterol (campesterol) reaction with the usual reagents
VI R =C2HS 24-ethylcholesterol (p-sitosterol) [i.e. acetic anhydride and pyridine
VII R=H p'-cholesten-3p-ol at room temperature overnight for
VIII R=CH3 24-methyl-p7-cholesten-3p-ol acetates (R 1 =Ac) ; trifluoroacetic
IX R =C:H5 24-ethyl-p'-cholesten-3p-ol anhydride in chloroform for ten
X R= CH2 24-methylene-ps-cholesten-3p-ol minutes (R 1 =T.F.Ac); and hexa-
XI R= CH2 24-methylene-p7-cholesten-3p-ol methyldisilazane and trimethylchlo-
XII R= CHCH3 24-ethylidene-ps-cholesten-3p-ol (fucosterol, 29-isofucosterol) rosilane in chloroform (R 1 =TMS) ].
XIII R CHCH3 24-ethylidene-p 7 -cholesten-3p-ol
Reagents were removed using a
XIX R =CH3 24-methyl-,15.22-cholestadien-3p-ol (brassicasterol)
24-ethyl-,15'22-cholestadien-3p-ol (stigmasterol)
stream of nitrogen and the product
XIV R =C2HS
XV R =H O5'2&cholestadien-3p-ol (desmosterol) dissolved in ether or chloroform for
XVI R=H 4«-methyl-©'-cholesten-3p-ol (Iophenol) GLC. All sterols and products were
XVII R= CH2 24-methylenelophenol analysed by GLC for impurities.
XVIII R= CHCH3 24-ethylidenelophenol A Pye 104 gas chromatograph was

274 J. of G. C:—June,1967-
used for analytical runs using a 9-ft.
column packed with 1% SE-30
coated on 100-120 mesh acid washed
and silanised Gas Chrom P. The
instrument was operated at 250·
using nitrogen as carrier gas.
Isolation of plant sterol fractions
has been described elsewhere for oat
seed (Avena sativa) (16, 33), cab-
bage seed (Brassica oleracea f capi-
tata) (11), and potato (Solanum
tuberosum) (35). The oat seed
sterol was divided into two frac-
tions. Fraction A consisted mainly
of monounsaturated sterols IV-IX
inclusive, and fraction B of com-
pounds X-XIII. These fractions
afforded mass spectra denoted by
source e (Table 1). Cabbage seed
sterol, consisting mainly of com-
pounds V and VI with approxi-
mately 15% of brassica-sterol (XIV,
R=CHa) , was used as source mate-
rial c and d (Table 1).

Table I.
Base Most abundant
Mass Peak ion above mle
Sterol Fig. 1 Hl Source M.W. Spectrometer (100) 210

Cholesterol IV H a 386 LKB 9000 43 275 (50)

Ac b 428 M.S. 9 368 368 (100)
TFA b 482 LKB 9000 368 368 (100)
TMS e 458 Karolinska 129 329 (88)
Campesterol V H c 400 LKB9000 43 400 (35)
Ac d 442 LKB9000 382 382 (100)
TFA d 496 LKB9000 43 382 (78)
TMS e 472 Karolinska 129 343 (61)
,B-sitosterol VI H c 414 LKB9000 43 414 (37)
Ac d 456 LKB9000 81 396 (100)
TFA d 510 LKB 9000 396 396 (100)
TMS e 486 Karolinska 129 357 (78)
A7-cholesten 3p-ol VII H a 386 LKB 9000 57 255 (70)
Ac b 428 LKB 9000 43 255 (100)
TFA b 482 LKB9000 43 255 (41)
TMS b 458 LKB9000 75 255 (48)
24-ethyl-A7-cholesten-3,B-01 IX TFA e 510 LKB9000 43 255 (48)
24-methylenecholesterol X TMS e 470 Karolinska 79 341 (5)
24-methylene-A7-cholesten-3,B-ol XI TMS e 470 Karolinska 79 343 (7)
24-ethylidenecholesterol XII Ac e,f 454 M.S. 9 296 296 (100)
TMS e 484 Karolinska 129 386 (86)
24-ethylidene-A 1-cholesten-3,B-ol XIII TMS e 484 Karolinska 79 343 (96)
Stigmasterol XIV H a 412 LKB 9000 69 255
Ac b 454 M.S. 9 394 394 (100)
TFA b 508 LKB 9000 55 255 (36)
Brassicasterol XIV Ac d 440 LKB 9000 69 380 (57)
4a-methyl-A 7-cholesten-Ss-ol
(Lophenol) XVI Ac g 442 LKB 9000 442
24-methylenelophenol XVII Ac g 454 LKB 9000 327 327 (100)
24-ethylidenelophenol XVIII Ac g 468 LKB 9000 59 327 (100)
Desmosterol XV H h 384 M.S. 9 271 271 (100)

Sources
a Commercial sample
b Prepared from commercial sample
c Brassica oleracea f. capitata seed sterol fraction
d Derived from source c
e Derived from fractionated sterol fraction of Avena sativa seed (16, 33)

J. of G. C.-June, 1967 275

 Table I.-Continued
f Authentic sample from J. Dusza
g Isolated from Solanum tuberosum by K. Schreiber (35)
h Authentic sample from C. J. W. Brooks
Mass spectrometers
M. S. 9: probe inlet system, ion source operated at 250°
scan: 70 eV, ion current: loopA.
LKB 9000: 10 ft. 1% SE-30 on 100-120 mesh acid washed and silanised Gas Chrom P. Operating conditions:
column 2500, helium carrier gas. Molecular separator 250°, ion source 2750, seam: 70 eV, ion
current 60 pA.
Karolinska: Atlas CH 4 instrument installed in Karolinska Institutet, Stockholm, Laboratory of Mass Spectrom-
etry (ref. 4). Inlet system 1% SE-30 1232°, molecular separator 23r, ion source 238°. Scan:
70 eV, ion current 20-25 pA.

Results
In Table 1 are listed details of
the sterols and their derivatives
analysed in the current work to-
gether with the observed base peak
of the obtained mass spectra and
the most abundant ion present
above mle 210. The principal ions
above mle 210 in the resultant mass
spectra are listed in Table 2 (for
ils-sterols), Table 3 (for il 7-sterols)
and Table 4 (for diunsaturated
sterols). In Tables 2-4 the letters in
the left hand column refer to the
ions as illustrated in line diagrams
(Figures 2-8) representing the mass
spectra obtained. In all tables, R
denotes the substituent at C-24
(i.e., H, CHa, C 2Hs, methylene, or
ethylidene) and RI denotes the
derivative attached to the oxygen
at C-3 [i.e., H, acetyl (Ac) , tri-
fluoroacetyl (TFA) or trimethyl-
silyl (TMS)].
In Tables 2-4 figures in paren-
theses indicate relative abundances
for ions measured in the range mle
210 to the molecular ion only, and
a dash indicates that the indicated
ion was not observed or that it was
not an observable ion.

Table II. il 5-Sterols and Derivatives

R= H CH a C 2Hs H H H
Ion Rl= H H H Ac TFA TMS
Fragmentation

M Molecular ion 386 (98) 400 (100) 414 (100) 428 482 (2) 458 (22)
a M-15 371 (46) 385 (50) 399 (45) 413 367 (2) 443 (20)
b M-RIOH 368 (64) 382 (62) 396 (54) 368 (100) 368 (100) 368 (52)
c M-15+RIOH 353 (52) 367 (50) 381 (42) 353 (28) 353 (20) 353 (30)
j M-R10H+67 (CsH r) 301 (56) 315 (60) 329 (50) 301 301 301 (4)
k M-R10H +93 (Cr!Ig) 275 (100) 289 (65) 303 (70) 275 (4) 275 (6) 275 (8)
1 M-R10H + 121 (CeRIS) 247 (40) 261 (20) 275 (18) 247 (26) 247 (20) 247 (16)
n M-I29 (CcCa+RIO) * 329 (100)
0 M-RIOH+ 108 (CsH 12 ) 260 (12) 274 (14) 288 260 (13) 260 (14) 260

d M-side chain 273 (36) 273 (50) 273 (54) 315 369 (40) 345
e M-side chain-l-R'Off 255 (50) 255 (82) 255 (60) 255 (22) 255 (18) 255 (16)
f M-side chain+42 231 (42) 231 (44) 231 (58) 273 327 (8) 303
g M-side chain+42+RIOH 213 (68) 213 (74) 213 (78) 213 (18) 213 (22) 213 (10)
h M -side chain+ 27 246 (12) 246 (10) 246 (12) 288 342 318
M-side chain+27+RIO 229 (36) 229 (25) 229 (25) 229**(3) 229** (6) 229 (4)

*specific for il 5-3,8-0.T.M.Si derivatives. **peak at 228 (loss of RtOH?) more prominent.

276 J.of G. C.-June, 1967



Discussion oxygen was ever observed for this Monounsaturated as-sterols: Ions a,
group of compounds. By contrast b, c, j,-k, 1, n, o
General: With the exception of most 0 7 -sterol derivatives showed On the assumption that ions re-
3 f3-acetoxy-d 5-sterols, all substances large molecular ions, the exceptions taining the side chain will show
analysed e thibited an ion corre- being compounds with 24-methylene increases of 14 mass units between
sponding to the molecular ion. For or 24-ethylidene groups (XI, XIII, successive members of the series
3,Q-acetbxy-cl 5-sterols the ion of XVII, XVIII) where the fragmen- R=H, CH 3 and C 2H 5f it is apparent
highest mass corresponded to that tation pattern is strongly orientated from Table 2 that the above ions all
for loss of acetic acid from the mol- by these groups. The only 0 6 -sterol retain the side chain. The ions a
ecule (peak b : M—R 1 OH), and in derivatives to show large molecular (M-15), b (M-RbOH), and c (M-
general no ion retaining the C-3 ions were the free sterols (R 1 =H). 15 and R 1 OH) are common features

Trimethtsilyt ether.
e 255
9 213 e d k p-Sitosterol R=CZHS M
9
231 255 273 1 j b 213 i
c 229 c o M458
t f h d 353
bi LL IEA,
— d M482
327
9 e Campesterol R=CH 3 M

d
k 1
c
b
a I
9

L
e
h
342
ktl
h

M 428
a

1
1
h

g e
jf d
1
Cholesterol R=H
t b
a
IM
i i
e M
t
273 h
d
315 C Df

386
a

A7-Chotestenct

h a
9 ,231 d 371

200 250 300 350 400 ht e b

200 250 300 n* 350 400 450 500
Figure 2. Mass spectra of p 5 -3p-hydroxysterols (R 7 =H).
Figure 5. Mass spectra of 1 7-cholesten-3p-ol (VII) derivatives.

8-Sitosterot T.MSE . r Desmosterot

D

9 i e t k j c a M
a M
n Carnpesterol TJ{$E. 9 i f se d u q c
b
e c M khotesterot M
9 i t k j a 9 b
Cholesterot iM.SE.
J Ie d c la

200 -
i L e

250
k

300
c a

350 400
M

450 5 00
- _diiI ....lL. 'ii
e A7-Cholestenot M

Figure 3. Mass spectra of p 5-3p-trimethylsilyloxysterols

(R 1 =T.M.Si.). 9 i f a
h
c b

200 250 300 350 400

n 329 TrimethytsiLyt ether
b Figure 6. Mass spectra of cholesterol (IV) p7-cholesten-3p-ol
c ^ (VII) desmosterol (XV) (Rt=H).
353
9 t a
i k a M458
1
b T.
e b ]FA
d
369 n 367 M
fl te t
f s d w 508
k 3271 a M 482
f c P a
b AGClili
e b Acetatt

9 0 M42& n
i a 260 k al s

9 ,. - k275 M386 StiraSterol

213 1 e r M 412

r
f 301 cD a
l 255 d
231 247. ^ x
273 d 351
h
^ t s e ba
200 250 300 350 400 450 500 ioo ... 250 . 900 750. 400 450 500

Figure 4. Masssspectra of cholesterol (IV) derivatives. Figure 7. Mass spectra of stigmasterol (XIV) derivatives.

J. of G. C.—tune, 1967 277

 of sterol spectra and were observed
r 24-Ethylidene-4¢-methyl-p7- for all compounds examined in this
p A ^^
c work, other than 3/3-acetoxy-i 5
1 =Ac) which did not-sterol(R

9 s° • f h q c b a M
show ion a.
The ions j, k and 1 appear to
24-Methytene-4¢-methy1-&-
327 oe=Ur malnly in O S -monounsaturated
370^ sterols and are only intense when
p
9 e 1 a M R'= H (Figure 2) . They are present
i s f h q c b
in O 5 -sterol derivatives (Figure 4)
r p 24-Ethytidene-p7 - TMS.E. also and may serve to distinguish
343 386 this class of sterol from the corre-
u q sponding p'-compounds (Figure 5) .
s e 281 296 h t c b a M All three ions are observed at the
,Jt..__. same mass numbers in cholesterol
r 24-Methylidene-&- T.M.S.E. and in 6-methylcholesterol (24),
e
9 255 p suggesting elimination of C-6 from
i s d a M the charged fragment. It has been
v u q h ct b suggested (22) that the ion 1 con-
P 24-Ethylidene-- T.M.S.E .
sists of rings C and D together with
Y the side chain after loss of a hydro-
257 u gen atom, but the origin of the
9 i se n t c b other two ions in this group is not
J1 h r a M
so readily explained. The ion j
q b 24-Methy4ere-t5 T.M.S.E . (M—RbOH+C,H,) may arise from
Y c loss of C-2 to C-6, or C-3 to C-7, as
9 s u r
M
a unit (to include (J-6) but would
h t
need to lose two additional hydro-
gen atoms. Similarly, elimination of
200 250 320 350 400 450 500 C-1 to C-7 as a unit could furnish
Figure 8. Mass spectra of diunsaturated sterols derivatives. the ion k directly. The ion 1 is not

Table III. o 7-Sterols and Derivatives

4a-methyl
Sterol: R= H H H H CZH, H
Ion R1= H Ac TFA TMS TFA Ac
Frag mentation

M Molecular ion 386 (98) 428 (74) 482 (72) 458 (64) 510 (67) 442 (100)

a M-15 371 (32) 413 (18) 467 (34) 443 (16) 495 (23) 427 (16)

b M—R 1 OH 368 (4) 368 (6) 368 (18) 368 (8) 396 (68) 382 (14)

c M-15+R 1 OH 353 (6) 353 (12) 353 (10) 353 (16) 381 (14) 367 (14)

d M—side chain 273 (25) 315 (10) 369 (62) 345 (6) 369 (56) 329 (7)

e M—side chain+R 1 OH 255 (100) 255 (100) 255 (100) 255 (100) 255 (100) 269 (70)

f M—side chain+42 231 (38) 273 (14) 327 (58) 303 (6) 327 (50) 287 (9)

g M—side chain+42+R'OH 213 (40) 213 (52) 213 (44) 213 (40) 213 (32) 227 (30)

h M—side chain+27 246 (13) 288 (6) 342 (20) 318 (2) 342 (23) 302 —

i M—side chain+27+R 1 O 229 (33) 229 (32) 229 (28) 229 (28) 229 (23) 243 (16)

278 J. of G. C.—June.1967
 Table IV. Diunsaturated Sterols
~5, 24 (25) (XV) ~5, 22 (XIV) ~5, 24 (28) ~7, 24 (28) 4a-methyl-~7, 24 (28)
C-28 C-29 C-28 C-29 C-28 C-29
Ion Sterol C~27 C-29 C-29 C-29 (X) (XII) (XI) (XIII) (XVII) (XVIII)
Rl H H Ac TFA TMS TMS TMS TMS Ac Ac
Fragmentation

M Molecular ion 384 (30) 412 (78) 454 508 (23) 470 (30) 484 (6) 470 (24) 484 (6) 454 (20) 468 (3)
a M-15 369 (30) 397 (8) 439 493 (3) 455 (23) 469 (7) 455 (26) 469 (10) 439 (20) 453 (3)
b M-R1OH 366 (14) 394 (6) 394 (100) 394 (80) 380 (78) 394 (8) 380 (12) 394 (3) 394 (7) 408 (2)
c M-15+R1OH 351 (14) 379 (7) 379 (8) 379 (8) 365 (52) 379 (10) 365 (14) 379 (6) 379 (10) 393 (2)
n M-l29 (CcC 3+R1O) 341 (100) 355 (16)
y M-l29 + (C 23-C27+ 1 x H) 257 (60) 257 (45)
w M-43 (C 25-C27) 369 (24) 411 465 (24)
x M-43+R 1OH 351 (31) 351 (21) 351 (48)
d M-side chain 273** (14) 273**(26)315 369 (20) 345 (30) 345 345 (33) 345 (8) 329 (25) 329 (2)
e M-side chain+R1OH 255 (12) 255 (100) 255 (94) 255 (100) 255 (48) 255 (15) 255 (47) 255 (18) 269 (28) 269 (13)
f M-side chain+42 231 (10) 231 (18) 273 327** (4) 303 - 303 (2) 303 - 303 - 287 (8) 287 (4)
g M-side chain+42+R1OH 213 (21) 213 (42) 213 (26) 213 (26) 213 (42) 213 (18) 213 (42) 213 (22) 227 (29) 227 (15)
h M-side chain + 27 246 246 (4) 288 342 318 (3) 318 (4) 318 (4) 318 (7) 302 (7) 302 (7)
i M-side chain+27+R1O 229 (6) 229 (20) 229* (7) 229* (6) 229 (30) 229* (7) 229* (22) 229* (12) 243* (10) 243* (5)
p M- (C23-C~7+1xH) 314 314 (3) 356 410 (6) 386 (67) 386 (100) 386 (45) 386 (62) 370 (40) 370 (52)
q M-(C 23-C27+1 xH) +R1OH 296 296 - 296 (4) 296 296 (67) 296 (94) 296 (10) 296 (15) 310 (8) 310 (7)
r M-side chain+2xH(p-43) 271 (100) 271 (73) 313 367 (46) 343 (42) 343 (6) 343 (100) 343 (100) 327 (100) 327 (100)
s M-side chain+2xH+R1OH 253 (14) 253 (22) 253 (22) 253 (16) 253 (52) 253 (15) 253 (24) 253 (15) 267 (13) 267 (13)
t M-15+ (C 23-C27+1 xH) 299** (16) 299 (54) 341 395***(38) 371 (14) 371 (14) 371 (6) 371 (6) 355 (t) 355 (t)
u M-15+ (C 23-C27+1 xH) +R1OH 281 (4) 281 (t) 281 281 (t) 281 (45) 281 (47) 281 (18) 281 (12) 295 (t) 295 (t)
v M ......side chain+2xH+42+R1OHt 211 (4) 211 (12) 211 (10) 211 (8) 211 (25) 211 (15) 211 (12) 211 (4) 225 (4) 225 (2.5)
~
0
y M-(C 23-C27+1xH) + (C1 - C3+R1O 257 (60) 257 (45)
p
- j M-R1OH+67 299** (16) 327 (4) 327 327** (4) 313 (7) 327 (0.5)
o (2) 301 (8) 301 (5)
k M-R1OH+93 273** (14) 301 301 287
L
c: I M-R1OH+121 245 (5) 273* * (26) 273 273 (2) 259 (24) 273 (4)
:J
.so 0 M-R1OH+108 258 (4) 286 - 286 (4) 286 272 - 286
....
<D
O'l
......
*228 more abundant **indicates more than one possible ***partly 394+1 (isotope tpeaks coincide with stationary
N
....
CD
fragmentation giving the same ion. abundance effect) phase background spectrum.
specific for i -sterols since Djerassi more abundant ion is observed one
has shown (31) that 0 4-cholesten- mass unit lower. This pair of ions
3, 6-dione and the corresponding appears to be much more aboundant
androstane and pregnane derivative for p 7 -sterols than for other groups,
give an ion of equivalent mass con- and it has been suggested that
taining the side chain. double bond migration may occur
The ion n is specific, in this class to give a charged species such as HO
of sterol, for 3j3-hydroxy-z 5 -sterol shown in Figure 9 (32). Figure 11. Labelling pattern for cho-
trimethylsilyl ether derivatives lesterol biosynthesised from 2-C"-meva-
(R 1 =TMS). From Figure 3 it is lonic acid.
apparent that this ion is very in-
tense and serves to distinguish
monounsaturated A 5 -sterols from
corresponding diunsaturated sterols
(Figure 8) and OT- sterols (Figure
5) . A corresponding abundant ion,
usually the base peak of the spec- HO
trum, is observed at m/e 129 for Figure 12. Labelling pattern for cho-
all 0 5 -sterol TMSi. derivatives and lesterol biosynthesised from 4-C 13 -meva-
is thought to contain C-1 to C-3 lonic acid.
with the C-3 oxygen function (36).
The ion o appears to require a Figure 10. Postulated 1, 6-hydride shift
From the known sequence of re-
C-3 acyl group (R 1 = Ac or TFA) involved in formation of ion p.
actions leading to biosyntheses of
and is only appreciably observed for cholesterol, one may expect that use
i 5 -sterols.
Other characteristic features of of 2-C13 -labelled mevalonic acid
maas spectra from 0 7 -sterol deriv- would give rise to cholesterol with
Tons involving loss of the side chain atives include large parent molec- "labelled" carbon atoms at C-1, C-7,
This group of ions may be divided ular ions. Also, the ion e is usually C-15, C-22, and C-26 of M.W. 391
into those which have retained the the base peak of the spectrum. The (Figure 11) . From this substance
C-3 oxygen (d, f, h) and those ions arising from simple loss of the one may expect that ions a, b, and c
which have lost this group as R 1 OH C-3 oxygen as R 1 —OH (i.e., b and would all be elevated five mass
(e, g), or apparently as R1O (i). c) are very much weaker than for units, compared with normal chole-
The Jatter group were observed for A 5 -sterols. sterol (Table 5) . Similarly, ions d
all compounds examined, but the
former were not detected in spectra
of 0 5 -sterol acetate or trimethylsilyl
derivatives. Table V. Expected lons in Mass Spectra of Normal Cholesterol
The origin of the ions d and e and Biosynthetically Labelled Material
(loss of the side chain) and f and g 2_C13 4-C13
(loss of side chain+42 mass units) Ion Normal mevalonic mevalonic
have been discussed in detail else- acid acid
where (21).
The ion h effectively involves loss Molecular ion 386 391 392
of the side chain and 27 extra mass
units which may involve C-16 and a M-15 371 376 377
C-17 (32). The ion is observed for
b M-18 368 373 374
0 5 -sterol alcohols (R 1 =H) and for
all derivatives of z 7 -sterols (Fig- c M-15+18 353 358 359
ures 5, 6, and 8) . The ion i appar-
ently arises as a net loss of RiO
d M—side chain 273 276 278
from h, except for 0 5 -sterol deriva- e M—side chain+18 255 258 260
tives (other than R'=H) when a
f M—side chain+Ring D? 231 233 235
g M—side chain+Ring D+18 213 215 217
h M—side chain+C-16, 0-17? 246 249 250
i M—side chain+C-16, C-17+18 229 232 233
j (a) M—[C-2—C-6] 301 306 305
(b) M—[C-3—C-7] 301 305 305

R01 k M—[C-1—C-7] 275 278 279

Figure 9. Postulated partial structure 1 M—[Rings A and B] 247 250 251
for the ions h and i (32).

280 J. of G. C.—June, 1967

and e (involving loss of side chain)
will be elevated three mass units,
and ions f and g will be elevated
two mass units providing C-15 is
lost in the fragmentation. The reten-
tion of C-15 in ions h and i would
be confirmed by the presence of this
ion three mass units higher in la-
belled cholesterol than in normal
material.
Similar use of 4-C 13 -labelled mev-
alonic acid would produce the label-
ing pattern illustrated in Figure 12
for cholesterol (M.W. 392) . By the
same argument, loss of the side
chain (d and e) would produce ions
five mass units higher and addi-
tional loss of ring D (f and g) ions
four mass units higher than usual.
Similarly, if the structure of ions h
and i is correct then these ions will
be four mass units higher than in
normal cholesterol.
Fiom the data for 6-methylchole-
sterol (24), it seems likely that C-6
is eliminated from ions j, k, and 1.
If the ion j is formed by removal of
a single unit involving C-6, the two
most likely groups (not involving
carbon rearrangement) appear to
involve either C-2 to C-6 as a body
or C-3 to C-7. The former mode
would produce an ion at m/e 306
in "labelled" cholesterol and the
latter at m/e 305 using 2-C 14 -mev-
alonic acid, and both alternatives
would appear at m/e 305 from 4-
C 14 -mevalonic acid.
A possible origin for the ion k
at m/e 275 in maas spectra of cho-
lesterol is for a unit consisting of
C-1 to C-7 to be eliminated, and
cholesterol from 2-C 14 -mevalonic
acid would show this ion at m/e
278. The presence of C-7 in the
charged fragment would be indi-
cated if this ion appeared at m/e
279.
From the suggested structure
(22) for ion 1, consisting of rings C
and D plus the side chain, it would
be expected that this ion would
occur at m/e 250 for material from
2-C 14 -mevalonic acid and at m/e 251
for 4-C 14 -mevalonic acid derived
material.
In order to eliminate more com-
plex fragmentation schemes, it may
be necessary to label the other posi-
tions of mevalonic acid also. The
effects of dilution with material syn-
thesised by endogenous mevalonic
acid and relative isotope abundance
effects suggest that this application
may need high resolution maas spec-
trometry.
Diunsaturated sterols
Except for the acetates of 29-
isofucosterol (XII) and stigma-
sterol (XIV, R= C 3H5 ) all diun-
saturated sterols investigated ex-
hibited a molecular ion and the ion
a in their maas spectra. Other ions
common to all spectra include b
and c where the side chain was pres-
ent intact and ions d to i where it
had been eliminated.
The presence of a double bond in
the side chain markedly reduced
ions characteristic of 0 5 -sterols, al-
though weak Tons corresponding to
j, k, and l were sometimes observed
when R 1 = H or TMS.
Ions which were noted for all such
compounds examined are denoted r
and s (Table IV) . The former ion
involves elimination of the side
chain with two additional hydrogen
atoms, and s is derived from it by
further elimination of R 1 OH.
As s$-sterols (stigmasterol XIV
,
R=C H S ) (Figure 7)
Q
In addition to the two ions char-
acteristic of side chain double bonds,
stigmasterol and its derivatives ex-
hibit the Tons w, involving loss of
43 mass units from the molecular
ion, and x derived from w by loss of
R 1 OH. This fragmentation is appar-
ent in the spectrum of the trimethyl-
silyl ether derivatives (5) and has
been suggested to involve the iso-
propyl group at the end of the side
chain. Confirmation of this is af-
forded by spectra of derivatives of
the corresponding C-28 sterol (XIV,
R = CH 3 ) brassicasterol. Ions in-
volving Toss of 43 mass units are
observed in this example also, sug-
gesting retention of C -24 and the
group R. This fragmentation ap-
pears to be characteristic of 022-
sterols among those examined in
this work.
As, 24 r 28 )-sterols (compounds
X, XII) (Figure 8)
The ions most characteristic of
this group of compounds are p and
q. Since these Tons occur at m/e 386
and m/e 296 respectively for both
the C-28 and C-29 sterols when
R 1 =TMS, it is evident that C-24
and the group R must be eliminated.
It has been proposed that this frag-
mentation, observed for fucosterol
(26) , its C-29 isomer (33) , and
derivatives of 24-methylenelophenol
(XVII) (18) and citostadienol
(XVIII) (18, 26), involves a six-
membered transition state with the
hydrogen atom at C-20 (Figure 10)
(18, 33). However, cycloeucalenol,
a 14 a -methyl-substituted 24-methyl-
enesterol, shows no sign of the ion p
in its maas spectrum, although a
small ion one maas unit higher is
observed. This suggests that the
fragmentation may be more complex
than originally thought.
Being 0 5 -3(3-hydroxysterols, TMS
derivatives of compounds X and
XII exhibit the ion n (M-129) in
their maas spectra and also a highly
characteristic ion y at m/e 257
combining the fragmentations p and
n.
A 7, 22 ( 2 R) Sterols (compounds
XI, XIII, XVII, XVIII)
Sterols of this group are readily
characterised by the strength of the
ions p and r compared with other
Tons in the spectrum (Figure 8) . It
has been suggested that 'the ion r is
dei'ived from ion p by loss of an
isopropyl group, the presence of a
metastable ion in the spectrum of
citrostadienol derivatives being
cited as evidence (26) .
Summary
p 5 -sterols may be distinguished
from all other groups of sterol. ex-
amined by analysis of the TMS
derivative, where ions involving loss
of 129 maas units from the molecule
were characteristic. Other charac-
teristic ions are j, k, and 1. á 7 -sterols
did not appear to show any unique
Tons but may be characterised most
satisfactorily by the ions h and i
and by having longer retention
times upon GLC that the corre-
sponding OS-sterol.
p 22 -sterols were characterised by
loss of 43 maas units, considered to
be the terminal isopropyl group of
the side chain (5) . Other áó-diun-
saturated sterols examined were
easily distinguished from the corre-
sponding 0 7-compound as trimethyl-
silyl ethers. Apart from ions arising
from loss of 129 maas units, i 5-
J. of G. C.—June, 1967 281
sterols showed a strong ion at m/e
296 (q) due to loss of R1OH from
the ion p and only a weak ion r. In
the p 7 -sterol the ion q was weak,
whereas the ion r was very strong.
In all spectra ions M, a, b, and c
retained the side chain intact. In
spectra of p 5 -sterols the side chain
was retained in ions j, k, 1, n, and o.
The group R and C -24 were re-
tained in ions w and x in mals spec-
tra of 0 22-sterols. In diunsaturated
sterols the ions p, q, t, u, and y
retained part of the side chain but
had lost C-24 and the group R. All
other ions in the range from m/e
210 to the molecular ion M had lost
the side chain. Of these ions r and s
had lost an additional two hydrogen
atoms, ions f and g an extra 42 mass
units, and /& and i an extra 27 mass
units respectively in addition to the
side chain.
Acknowledgments
A generous grant from the S.R.C.
to Dr. C. J. W. Brooks and Di'. G.
Eglinton enabling the purchase of
an L.K.B. 9000 GLC/Mass Spec-
trometer must be acknowledged,
and the author is greatly indebted
for permission for samples to be run
on this instrument. The technical
assistance of Miss H. Humphrys
and Miss J. Malcolm is gratefully
acknowledged. Thanks are due to
Dr. P. Eneroth for analysis of oat
seed sterol extracts on a prototype
GLC/mals spectrometer at the
Karolinska Institutet, Stockholm,
and to Miss J. Wilkie for samples
run on the M.S. 9 instrument. n
282 J. of G. C.—June, 1967
Literature Cited
1. Elliott, C. G., Hendrie, M., and
Knights, B. A., J. Gen. Microbiol. 42,
425 (1966).
2. Wells, W. W., and Makita, M., Anal.
Biochem. 4, 204 (1962).
3. Miettinen, T. A., Ahrens, E. H., and
Grundy, S. M., J. Lipid Res. 6, 411
(1965).
4. Eneroth, P., Heilstrom, K., and Ry-
hage, R., J. Lipid. Res. 5, 245 (1964).
5. Eneroth, P., Hellstrom, K., and Ry-
hage, R., Steroids 6, 707 (1965).
6. Kuksis, A., Can. J. Biochem. 42, 407,
419 (1964).
7. Knights, B. A., The Chemistry of Nat-
ural Products Symposium, I.U.P.A.C.,
Stockholm., Abstr. No. 4-17, (1966).
8. Goad, L. J., Hammam, A. S. A., Dennis,
A., and Goodwin, T. W., Nature 210,
1322 (1966).
9. Lederer, E., Biochem. J. 93,449 (1964).
10. Clayton, R. B., Quart. Rev. 19, 168, 201
(1965).
11. Knights, B. A., Mem. Soc. Endocrinol
1966 No. 16, 211.
12. Thompson, M. J., Robbins, W. E., and
Baker, G. L., Steroids 2, 505 (1963).
13. Linde, H., Ergenc, N., and Meyer, K.,
Hely. Chim. Acta. 49,1246 (1966).
14. Fieser, L. F., and Fieser, M., Steroids,
New York: Reinhold Publishing Corp.,
1959, p. 341.
15. Copius-Peerboom, J., J. Gas Chro-
matog. 3, 325 (1965).
16. Knights, B. A., Phytochemistry, In
Press.
17. Hugel, M. F., Vetter, W., Audier, H.,
Barbier, M., and Lederer, E., Phyto-
chemistry 3, 7 (1964).
18. Benveniste, P., Hirth, L., and Ouris-
son, G., Phytochemistry 5, 31 (1966).
19. Ryhage, R., and Stenhagen, E., J.
Lipid. Res. 1, 361 (1960).
20. Bergstrom, S., Ryhage, R., and Sten-
hagen, E., Acta Chem. Scand. 12, 1349
(1958).
21. Budzikiewicz, H., Djerassi, C., and
Williams, D. H., Structure Elucida-
tion of Natura) Products by Mass
Spectrometry, Vol. 2, San Francisco:
Holden Day (1964).
22. Friedland, S. S., Lane, G. H., Long-
man, R. T., Train, K. E. and O'Neal,
M. J., Anal. Chem. 31, 169 (1959).
23. Gohike, R. S., Chem. Ind. 1963, 946.
24. Wulfson, N. S., Zaretskii, V. I., Zaikin,
V. G., Segal, G. M., Torgov, I. V., and
Fradkin, T. P., Tetrahedron Letters
1964, 3015.
25. Fitches, H. J. M., Advances in Mass
Spectrometry 1I, M. Elliott, ed., Ox-
ford: Pergamon Press (1962), p. 428.
26. Bergman, J., Lindgren, B. 0., and
Svan, C. M., Acta. Chem. Scand. 19,
1661 (1965).
27. Eneroth, P., Gordon, B., Ryhage, R.,
and Sjovall, J., J. Lipid Res. 7, 511
(1966).
28. Eneroth, P., Gordon, B., and Sjo-
vall, J., J. Lipid Res. 7, 624 (1966).
29. Sjovall, J., and Vihko, R., Steroids 7,
447 (1966).
30. Vanden-Heuvel, W. J. A., Stillwell,
R. W., Gardiner, W. L., Wikstrom, S.,
and Horning, E. C., J. Chromatog. 19,
22 (1965).
31. Djerassi, C., Karliner, J., and Alpin,
R. T., Steroids 6, 1 (1965).
32. Clayton, E., Ph.D. Thesis, University
of Glasgow, p. 137, (1964).
33. Knights, B. A., Phytochemistry 4, 857
(1965).
34. Cornforth, J. W., Cornforth, R. H., Pel-
ter, A., Horning, M. G., and Popjak, G.,
Proc. Chem. Soc. 1958, 112.
35. Osske, G., and Schreiber, K., Tetra-
hedron 21, 1959 (1965).
36. Brooks, C. J. W., Personal Communi-
cation.
Manuscript received January 30, 1967