Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol32.Issue1.2001:

Organic Geochemistry 32 (2001) 115±126
www.elsevier.nl/locate/orggeochem

Complex patterns of steroidal biomarkers in Tertiary
lacustrine sediments of the Biyang Basin, China
Junhong Chen 1, Roger E. Summons *
Australian Geological Survey Organisation, GPO Box 378, Canberra ACT 2601, Australia
Received 21 February 2000; accepted 20 September 2000
(returned to author for revision 25 May 2000)

Abstract
Gas chromatography (GC), gas chromatography±mass spectrometry (GC±MS) and gas chromatography±mass
spectrometry±mass spectrometry (GC±MS±MS) with co-injected synthetic standards were used to analyse the biomarker patterns of some Tertiary lacustrine clayey dolomites from the Biyang Basin, China. The lithology, low Pr/Ph
ratio and high gammacerane content of these sediments indicated that high salinity prevailed during their deposition.
The distributions of steroidal hydrocarbons were particularly unusual and several pseudohomologous series, including
regular steranes (C27±C29), 4-methyl steranes (C28±C30 including dinosteranes), 3b-ethyl steranes (C29±C31), lanostanes
(C30±C32), and a variety of other 3b-alkylated steranes were identi®ed. 3b-n-Propylcholestane, 3b-n-propylstigmastane
and 4,4-dimethylcholestane were identi®ed using authentic standards and this is the ®rst time these compounds have
been unambiguously characterised in sediments. Crown Copyright # 2001 Published by Elsevier Science Ltd. All
rights reserved.
Keywords: Biomarkers; Steroids; Hydrocarbons; Dinosterane; 3b-Alkylated steranes; Lanostanes; 4,4-Dimethyl steranes; Lacustrine

sediments; Tertiary; Hypersaline; Biyang Basin; China

1. Introduction
Lipid biomarker compounds have been widely used
to assess depositional environments, types of organic
input, thermal maturity of organic matter and to
demonstrate the relationship between oils and their
sources (e.g. Mackenzie et al., 1980, 1981; Brassell et al.,
1986, 1987; ten Haven et al., 1987; Volkman, 1988;
Peters and Moldowan, 1993; Ritts et al., 1999). Steroids
are an important class of biomarker and unambiguous
determination of their chemical structures is fundamental to understanding their sources and application
in paleoenvironmental reconstruction. With continuing
improvements in analytical techniques numerous ster-

* Corresponding author. Fax: +61-6-249-9956.
E-mail address: rsummons@agso.gov.au (R.E. Summons).
1
Present address: Department of Earth and Planetary Sciences, Macquarie University, NSW 2109, Australia.

oids, many with only subtly di€erent molecular structures, have been reported (e.g. Maxwell et al., 1980;
Brassell and Eglinton, 1981; Robinson et al., 1984;
Summons and Capon, 1988, 1991; Chen et al., 1989;
Moldowan et al., 1990; Nichols et al., 1990; Volkman et
al, 1990; Dahl et al., 1992, 1995).
Lacustrine sediments often show complex and distinctive biomarker compositions, since these settings
can, through time, receive a wide spectrum of organic
inputs. Lakes also show a wide variety of water column
chemistries with consequent variability in diagenetic
conditions. This provides organic geochemists with
opportunities for detailed study of the controls on biomarker distribution. The Eocene Biyang Basin located
in central China has previously been investigated for its
petroleum geology and geochemical characteristics of
the oils and sediments (Zhu et al., 1981; Jiang and Jia,
1986; Chen et al., 1988; Philp et al., 1992). A pseudohomologous series of C30±C32 lanostanes was identi®ed
there for the ®rst time (Chen et al., 1989). In this paper,

0146-6380/01/$ - see front matter Crown Copyright # 2001 Published by Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00145-5

116

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

we report further aspects of the Biyang biomarker distributions and the use of synthetic standards to aid
characterisation of conventionally and unconventionally
alkylated steroids.

2. Geological setting and samples
2.1. Geological setting
The Biyang Basin is an Eocene faulted lake basin
located in the southern part of Henan Province in central
China. Although it has an area of only 1000 km2, it has
been a prospective target for oil exploration (Zhu et al.,
1981; Chen et al., 1988). The basin developed on the
background of the Qinling mountains and gradually
evolved from a freshwater environment to a much
smaller and increasingly saline lake. The development of
the basin was controlled by the presence of two large
faults on the southern border, trending NNE and

NWW. The sediments were deepest in the south and the
burial depth decreased from south to north in the basin.
In the early stages of its evolution 3000±4000 m of red
clastic sediments were deposited relatively rapidly during periods of river ¯ooding to form the Yuhuangding
and Dachangfang formations which are not oil prone.
The latter stage was one of steady sediment deposition
to produce the Oligocene Hetaoyuan formation, which
is divided into three sections: Eh1, Eh2 and Eh3. Eh3 is
the oldest and was deposited as a suite of dark grey or
grey claystones interbedded with oil shales and sandstones. This unit is the major source of the oils in the
Biyang Basin. The second section of the Hetaoyuan
formation (Eh2) represented a generally more saline
environment, rich in organic matter and with a lower
thermal maturity than Eh3. The youngest section is Eh1,
which is immature and has little contribution to oil
accumulation. In the ®nal stage of lake evolution
(Hetaoyuan to Niaozhuang formation), uplift occurred,
the water volume of the lake decreased and the salinity
increased. Clastic sediments with little petroleum
potential were deposited from rivers onto the ¯oodplains.

2.2. Samples
The core samples employed in this study were collected from a clayey dolomite formation belonging to
the second section of the Hetaoyuan formation (Eh2) in
well Y2 located in the central area of the Biyang Basin.
Previous studies have shown that sample nos. 1 (1992
m) and 3 (2036 m) contained C30±C32 lanostanes (Chen
et al., 1989). In our present study, these two samples and
another two core samples (nos. 2 and 4), also from well
Y2 (1994 and 2085.5 m, respectively), were analysed by
GC, GC±MS and GC±MS±MS.

3. Experimental
3.1. Bitumen isolation
Sediment samples were ground to a ®ne powder and
extracted by Soxhlet using dichloromethane:methanol
(87:13). After removal of elemental sulfur, the extracts
were further separated into saturated, aromatic and
polar-asphaltene fractions using column chromatography on silica gel.
3.2. GC analysis
GC analyses of saturated hydrocarbons were carried

out to examine the distributions of n-alkanes, isoprenoids and the relative contents of steranes, hopanes
and other compounds. These experiments were conducted with a HP 6890 GC using a 25 m  0.25 mm i.d.
DB-1 capillary column, coupled to an auto sampler with
on-column injection and hydrogen carrier gas. The oven
temperature was programmed from 60 C (held 2 min)
to 310 C at 4 C/min, followed by an isothermal period
of 15 min.
3.3. GC±MS and GC±MS±MS analysis
The full-scan and selected ion monitoring (SIM) of
GC±MS and metastable reaction monitoring (MRM) of
GC±MS±MS were carried out with a VG Autospec
Ultima-Q coupled to a CarloErba GC (8000 series), to
further evaluate the distribution of sterane biomarkers
and to compare with unambiguously identi®ed synthetic
standards. Chromatography was conducted using a 60
m  0.25 i.d. mm DB-5 capillary column with H2 carrier
gas. For the identi®cation of 4,4-dimethylcholestanes, a
polar column (SGE, type: BP-10; length: 50 m; i.d. 0.22
mm; ®lm: 0.25 mm) was also used. Samples were injected
using a vaporising injector at 300 C in the splitless

mode. The oven temperature was programmed from 70
to 210 C at 10 C/min and then to 310 C at 2 C/min,
then held at the ®nal temperature for 20 min. The mass
spectrometer was operated at 70 eV with a source temperature of 240 C. During the fullscan acquisition mode
the mass spectrometer was scanning from m/z 600±50,
with a scan time of 1.0 s. For GC±MS±MS metastable
analysis, we analysed 17 di€erent parent ion to daughter
ion transitions, each with 30 ms scanning acquisition
and 50 ms delay periods.
3.4. Synthesis of standards
Standards, including 3b-ethylcholestane, 3b-ethylstigmastane plus their 3b-propyl, 3b-n-pentyl, 3b-i-pentyl and 4,4-dimethyl analogues, were prepared by
variations of methods reported before (Summons and
Capon, 1988, 1991). Isomerisation of the aaa 20R iso-

117

+f
+
++
++

0.61
0.15
0.73
0.23
3.57
4.60
4.70
9.09
32.9
46.1
45.5
50.5
30.0
29.2
32.2
31.9
37.1
24.7
22.2
17.6

g

f

e

d

c

b

a

Ratio of pristane to phytane.
OEP=(nC23+6*nC25+nC27)/(4*nC24+4*nC26).
Regular sterane relative contents calculated from the areas of aaa 20R isomers in SIM data.
Ratio of C30 ab-hopane to C30 moretane.
Ratio of gammacerane to C30 ab-hopane.

Indicates relative abundance.
nm, not measured.

0.20
0.24
0.25
0.22
1.16
1.38
1.39
1.19
0.19
0.48
0.32
0.73
Phytane
n-C17
n-C17
C30 abH
14±38

11±31
11±31
11±32
nm
3.9
3.0
4.9
nm
14.6
16.3
2.2
nm
17.5
16.3
52.2
nmg
3.1
0.9
3.6

Lithological and organic parameters for the Biyang
Basin sediment samples are summarised in Table 1.
Saturated hydrocarbon fractions isolated from the bitumens had low Pr/Ph ratios (0.19±0.73) and relatively
high gammacerane/C30 hopane ratios (0.15±0.73) and
both features are associated with sedimentation from
saline waters. This conclusion is supported by the presence of relatively high contents of b-carotane, a noted
component of oils and bitumens from saline lacustrine
environments (Fu et al., 1985; Volkman, 1988; Peters
and Moldowan, 1993). The saturated hydrocarbon
fractions of sample nos. 1±3 were dominated by n-C17 or
phytane, hydrocarbons considered to signal input from
cyanobacteria and/or algae. Sample no. 4 was unusual
in that C30 hopane was the most abundant hydrocarbon
(Fig. 1). Waxy n-alkanes had low odd over even preference (OEP=1.16±1.39), which indicates that there
may have been a minor input from vascular plant
waxes, an observation supported by the presence of
oleanane in sample nos. 1 and 3 (Fig. 3). Stigmastane
was the predominant desmethyl sterane in all samples
except no. 1 and 24-n-propylcholestane was notable for
its absence from all samples.
All samples are immature as shown by the predominance of steranes with biological stereochemistry
(i.e. 20R>>20S and aaa>>abb) (Fig. 2) and high
contents of C30 moretane (Fig. 3). Values of the ratio of
C29 sterane-20S/(S+R) range from 0.22 to 0.25 and the
ratio of C30 hopane/moretane from 1.59 to 4.27 (Table
1). 17a(H)-22, 29, 30-Trisnorhopane (C27Tm) was the
most abundant C27 hopane with more thermodynamically stable 18a(H)-22, 29, 30-trisnorneohopane
(C27Ts) almost absent, which was consistent with the
low maturity of these samples. The C31 methyl hopanes
detected in the Biyang Basin samples included a mixture
of isomers of 2a-, 2b- and 3b-methylhopanes, with the
less thermodynamically stable 2b- and 3b-methyl isomers
in approximately equal abundance. These A-ring methylated hopanes have been detected in a variety of other
sediments and oils (Summons and Walter, 1990; Collister et al., 1992; Summons and Jahnke, 1992). Possible
precursors for C31±C36 methyl hopanes include a range
of organisms including acetic bacteria, methylotrophs,
methanotrophs and cyanobacteria (e.g. Rohmer et al.,
1984; Bisseret et al., 1985; Zundel and Rohmer, 1985a,b;
Summons et al., 1999). Isotopic evidence supports the

Table 1
Geochemical parameters of the Tertiary lacustrine sediment samples from Well Y2, Biyang Basin

4.1. General geochemical characteristics

1992.0
1994.0
2036.0
2085.5

4. Results and discussion

1
2
3
4

mers was carried out as previously described (Abbott et
al., 1984; Summons and Capon, 1988; Bisseret and
Rohmer, 1990). Lanostane and 18a(H)-oleanane were
obtained from Chiron Laboratory (Norway).

Sample Depth %TOC % Illite % Fe
% Calcite Range of Largest peak Pr/Pha OEPb C29 sterane
no.
(m)
dolomite
n-alkanes in the GC
20S/R+S

% C27 % C28 % C29 C30H Gammac/C30He b-Carotane
steranec sterane sterane ab/bad

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

118

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

Fig. 1. GC traces for saturated hydrocarbon fractions of the
Tertiary lacustrine sediment samples from Well Y2, Biyang
Basin. The peak marked with an asterisk is 3-methylhenicosane
(a-C22), the internal standard.

idea that sedimentary 3-methylhopanes are derived from
methanotrophs (Freeman et al., 1990; Collister et al.,
1992) while a recent survey of cultures and microbial
mats indicates that their 2-methyl counterparts probably re¯ect inputs from cyanobacteria, especially in
lacustrine sedimentary environments (Summons et al.,
1999).
4.2. C30 A-ring methylated steranes with base peak at
m/z 231
The A-ring methylated C30 steranes detected in the
Biyang sediment samples comprise dinosteranes, 4amethylstigmastane, 2a-methylstigmastane and 3bmethylstigmastane (Fig. 4). Dinosteranes were predominant only in Biyang Basin sample no. 1 but were present
as minor components in the other three samples that
were dominated by 3b-methylstigmastane and 4amethylstigmastane (Fig. 4). Lower homologues,

Fig. 2. C27 to C29 sterane patterns evident in GC±MS m/z 217
selected ion chromatograms for four samples from the Biyang
Basin.

that is methylated cholestanes and ergostanes, also
comprise mixtures of 2-, 3- and 4-methyl analogues in
the Biyang samples.
Mixtures of C30 4-methyl steranes, including dinosteranes (i.e. 4,23,24-trimethylcholestanes) and 4-methylstigmastane, have long been regarded as indicating
dino¯agellate input to geological samples (e.g. Boon et
al., 1979; de Leeuw et al., 1983). However, Edmunds
and Eglinton (1984) suggested that dinosterol and related sterols were not exclusive markers for dino¯agellates, since they may be produced by other
organisms. Nichols and co-workers (1990) detected
dinosterol as a minor component (0.1±3.2% of total
sterols), with two other novel 4-methyl-C30 sterols, in
sea-ice diatom communities. The predominance of
dinosteranes over 4-methyl stigmastanes in lacustrine
sediments is quite unusual (Goodwin et al., 1988; Summons et al., 1992) and, in the present case, may be a
re¯ection of water column chemical conditions. Diagenesis in sediments subject to high pH, low Eh and/or
sulfate reduction, as compared to those deposited under

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

Fig. 3. Hopanes, gammacerane and oleanane distribution patterns evident in GC±MS m/z 191 selected ion chromatograms
for four samples from the Biyang Basin. Tm=17a(H)-22,29,30trisnorhopane.

freshwater, may lead to enhanced preservation of
dinosterol, a side-chain unsaturated steroid, through
formation of sul®de adducts. The main precursor of 4methylstigmastane, on the other hand, is likely to be the
4-methyl-24-ethylcholestanol and diagenetic conditions
(Eh or pH) should not preferentially in¯uence its preservation. In other words, the high abundance of 23,24dimethyl isomers of the 4-methyl steranes compared to
the 24-ethyl analogues, generally observed in marine
settings, is possibly due to protection of the unsaturated
dinosterol side-chain feature from oxidation. Such a
mechanism has been proposed to explain the relative
abundance of oleanane in marine sediments through
preferential preservation of oleanoid triterpene precursors. In contrast, the same precursors in non-marine
settings appear to follow diagenetic pathways toward
partial or complete aromatisation (Murray et al., 1997).
4.3. C29-C31 steranes with a base peak at m/z 245
A pseudohomologous series of C29±C31 steranes with
base peak m/z 245 is prominent in the Biyang Basin

119

Fig. 4. m/z 414 ! 231 GC±MS±MS chromatograms for the
Tertiary lacustrine sediment sample no. 1 from the Biyang
Basin, displaying the natural distributions of C30 A-ring methyl
alkylated steranes.

samples. They could have either one C2 or two C1 substituents in ring-A+B+C. Compounds identi®ed by coinjection with the 20S and 20R isomers of C29 and C31
synthetic standards comprised the 3b-ethylcholestanes
and 3b-ethylstigmastanes with 3b-ethylergostanes identi®ed, by analogy as the C30 components. As observed
with the desmethyl steranes, all compound series were
dominated by 5a, 14a, 17a(H)-20R isomers (Fig. 5) and
indicated a low maturity for the samples.
Full scan mass spectral analyses of synthetic 3b-ethyl
steranes and 4,4-dimethyl analogues showed that their
mass spectra are virtually identical. Chromatographic
behaviour, therefore, o€ers the best means to distinguish them in complex mixtures and we observed that
the 4,4-dimethyl sterane aaa-20R isomer eluted earlier
than its 3b-ethyl counterpart on both DB-5 and Ultra-1
columns. Furthermore, under the GC condition discussed above, the 3b-ethylcholestanes could be completely separated from their 4,4-dimethylcholestane
counterparts (Fig. 5). While the 20S isomer of 4,4dimethylstigmastane was totally separated from the 20S

120

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

were based on the appearance of mass spectra, without
appreciating the possible interference of 3b-ethyl steranes that were not known at the time.
In our present study, a further attempt to identify 4,4dimethyl steranes from the geological record was made
by GC±MS±MS analysis. Only C29 4,4-dimethylcholestane with a con®guration of 20R was found to occur in
Biyang sediments, and it was in very low concentration
(Fig. 5). Furthermore, 4,4-dimethylergostane (C30) and
4,4-dimethylstigmastane (C31), compounds having alkylation at C-24, were not detected in these samples (Fig.
6). This is consistent with the observed order of biosynthetic reactions whereby alkylation at C-24 follows loss
of the methyl at C-14 and at least one of those at C-4
(Nes and McKean, 1977). In contrast to the 4,4-dimethylsteroids, 3b-ethyl steroids (and other 3b-alkylated
steroids) have not been reported in any natural living
system while occurring ubiquitously in geological samples (Summons and Capon, 1988, 1991; Dahl et al.,
1992, 1995).
4.4. C30±C32 steranes with a base peak at m/z 259

Fig. 5. m/z 400 ! 245 GC±MS±MS chromatograms of sample
no. 1 from the Biyang Basin showing co-injection with standards of 4,4-dimethylcholestane (20R) and isomerised 3bethylcholestanes (20S+20R).

isomer of 3b-ethylstigmastane, the 20R epimers could
not be fully separated (Fig. 6) under routine conditions.
For unambiguous identi®cation we conducted GC±MS
(MRM) analyses on a polar (SGE BP-10) column and
con®rmed the target 20R isomer of 4,4-dimethylcholestane also eluted with the standard. We also observed
that GC±MS analyses using a delayed GC program
enabled a complete separation of all 3b-ethyl and 4,4dimethyl isomers to be achieved (Chen et al., 1993).
4,4-Dimethylsterols such as 4,4-dimethyl-5a-cholesta8(14),24-dien-3b-ol and 4,4-dimethyl-5a-cholesta-8(14)en-3b-ol may be regarded as precursors of 4,4-dimethylcholestane. They have been reported in living
organisms, being intermediates in the biosynthetic
pathway from lanosterol to desmethyl sterols through
the loss of the C-14 methyl group (e.g. Bouvier et al.,
1976; Seher, 1976; Nes and McKean, 1977; Weete, 1980,
and references therein). 4,4-Dimethyl steranes, to our
knowledge, have not been unambiguously identi®ed in
geological samples. A report of the identi®cation of 4,4dimethylpregnanes and 4,4-dimethylhomopregnanes has
been made (ten Haven et al., 1985), but these results

A series of C30±C32 lanostanes with base peak m/z 259
had been detected in previous studies of the Biyang
Basin samples using GC±MS analysis. These compounds were characterised by comparisons of mass
spectra and co-injection with a synthetic standard for
the C30 analogue (Chen et al., 1989,1993). In the present
study, a second series of C30±C32 compounds with base
peak m/z 259 was observed and characterised. They
eluted after the lanostane series and the major series
members were hypothesised to be the 5a, 14a, 17a(H)20R isomers of 3b-n-propylcholestane, 3b-n-propylergostane and 3b-n-propylstigmastane, respectively.
This was con®rmed (Fig. 7) by co-injection with
authentic standards. 3b-Propyl steranes have been
reported to occur in crude oils and rock extracts (Dahl
et al., 1992, 1995) with the assignment being made on
the basis of their being part of homologous series of 3balkyl sterane isomers. Given the complexity of the M+
! 259 reaction chromatograms of some petroleum and
bitumen samples, our characterisation here provides
necessary rigour to the compound class assignments.
Both lanostanes and 3b-n-propyl steranes show m/z
259 (rings-A+B+C) as the base peak in their main
beam mass spectra. The 3b-n-propyl steranes also have
a strong ion at m/z 191, the latter being analogous to the
rings-A+B fragment at m/z 149 in regular desmethyl
steranes. On the other hand, lanostanes have a very
strong ion at m/z 190 instead of m/z 191 and consequently high m/z 190 vs 191 ratios in lanostanes serve to
distinguish the two carbon skeletons. These two series of
compounds can also be readily di€erentiated based on
their relative elution times from a DB-5 capillary column. Recently, a third compound class has been identi-

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

Fig. 6. m/z 428 ! 245 GC±MS±MS chromatograms showing
the elution order and co-injections of standards of isomerised
3b-ethylstigmastane (20S+20R) and isomerised 4,4-dimethylstigmastane (20S+20R). Under the analytical conditions there
is only partial separation of 20R isomers while 20S isomers are
completely separated. No 4,4-dimethylstigmastane is evident in
contrast to the equivalent experiment for cholestanes shown in
Fig. 5.

®ed using m/z 414!259 reaction chromatograms. The
so-called TPP or `C30 tetracyclic polyprenoids' (Schaeffer et al. 1994; Holba et al., 2000) have been identi®ed in
lacustrine sediments and in their derived oils and
attributed to a freshwater algal source. Comparison of
the m/z 414!259 reaction chromatograms for the
Biyang Basin sediment sample no. 1 with those of
freshwater lacustrine oils, from Indonesia (LAC oils of
Murray et al., 1994) enabled the TPP doublet to be
assigned (Fig. 7) on the basis of relative retention times.
The m/z 259 selected ion chromatograms and m/z
414!259 reaction chromatograms of the latter oils
presented an intense doublet of peaks, with higher
homologues apparently absent, and we presumed these
to be the C30 TPP of Holba et al. (2000).
In their recent study, Peng et al. (1998) identi®ed C30
and C31 lanostane sul®des occurring in an immature oil

121

Fig. 7. M+ ! 259 GC±MS±MS chromatograms showing
sample no. 1 co-injected with standards of lanostane, isomerised 3b-n-propylcholestane (20S+20R). The results display
the occurrence of lanostane and 3b-n-propylcholestane
(20S+20R) and their C31 and C32 pseudohomologous ergostane (20S+20R) and stigmastane (20S+20R) isomers. The 20R
isomers of 3b-n-propyl steranes are predominant over 20S isomers. Peaks marked a and b are presumed to be isomers of the
tetracyclic polyprenoid (TPP) reported by Holba et al. (2000)
and based on their elution positions relative to intense doublets
in the Minas and Duri oils of Central Sumatra.

from Paleocene evaporite source rocks in Jianghan
Basin, China. No saturate counterparts were found in
this sample, indicating that the lanosterol precursors
were completely converted to sul®des under an environment with ready availability of H2S or polysul®de.
Accordingly, occurrence of lanostanes in sedimentary
bitumens and oils might be controlled by a combination
of source and diagenetic factors such as sul®de availability.
4.5. Other A-ring alkylated steranes
Additional A-ring alkylated steranes were present in
the Biyang samples and identi®ed as A-ring-C4 steranes
(C31±C33) (Fig. 8), and 3b-n-pentyl steranes and 3b-ipentyl steranes (C32±C34) (Fig. 9). The 3b-n-pentyl steranes and 3b-i-pentyl steranes (C32±C34) were con®rmed

122

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

Fig. 8. M+ ! 273 GC±MS±MS chromatograms of C31±C33
A-ring-C4 sterane compounds in sample no. 1.

with co-injected isomerised 3b-n-pentylcholestane and
3b-i-pentylcholestane. The relative content of 3b-n-pentyl steranes is much higher than that of 3b-i-pentyl steranes and supports the hypothesis of Dahl et al. (1995)
that the ratio of 3b-n-pentyl steranes to 3b-i-pentyl
steranes is related to source environments. In their earlier study, Dahl et al. (1995) found that the Green River
Formation and Rozel Point oil (both lacustrine samples)
showed a preference for 3b-n-pentyl steranes while most
other samples (e.g. marine Monterey oil) were dominated by 3b-i-pentyl steranes.
Although no standards were employed in the identi®cation of the A-ring-C4 compound series, they appear to
be dominated by 20R isomers of the 3b-n-butyl analogues based on the normal structure and 20R stereochemistry of the co-occurring series and the low
maturity of the sediments.
4.6. Signi®cance and implications
Except for 4,4-dimethylcholestane (20R), the steroids
discussed above were found occurring in all four Biyang

Fig. 9. M+ ! 287 GC±MS±MS chromatograms showing distribution of C32±C34 3b-n-pentyl steranes and 3b-i-pentyl steranes with the predominance of 3b-n-pentyl isomers. These
compounds were con®rmed with co-injected isomerised 3b-npentyl cholestanes and isomerised 3b-i-pentyl cholestanes.

Basin samples. Based on the biomarker ratios and particularly relative abundances of gammacerane, Pr, Ph
and b-carotane (Table 1), these lacustrine sediments
were immature and deposited from highly saline waters
carrying a biota dominated by algae and bacteria with
only minor inputs from vascular plants. Samples nos. 2
and 3 had very similar geochemical characteristics and
biomarker distributions despite a large di€erence in
TOC values. Sample no. 1 was di€erentiated by higher
relative abundance of phytane and C27 steranes (37%)
and predominance of dinosteranes over 4-methylstigmastanes, while no. 4 was characterised by very high
concentration of C30 hopane and the dominance of C29
steranes (50%). This is probably a re¯ection of changes
of depositional conditions, since sample no. 4 has a
much higher content of clay minerals than others (Table
1), which may indicate a depositional environment with
in¯ux of fresher water and more terrigenous debris. This
is also consistent with the high Pr/Ph ratio and low
gammacerane content for sample no. 4.
The biological origins of lanostanes and 3b-alkyl
steranes (C1±C5) remain to be determined. Lanosterol,

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

the most likely precursor of lanostane, is normally
associated with sterol biosynthesis in animals and fungi
and has been identi®ed as a precursor to the methyl and
dimethyl sterols of the methylotrophic bacterium
Methylococcus capsulatus (Bouvier et al., 1976). Triterpenes with a lanostane carbon skeleton have been
identi®ed in the fern Adiantum venustum (Alam et al.,
2000) but these compounds did not have alkylation at
C-24. Although there has been a report of 24-methyllanosterol (C31) occurring in fungi (e.g. Aspergillus fumigatus and Penicillium expansum etc., Weete, 1980 and
references therein), 24-ethyllanostane has no known
natural precursor identi®ed in any organisms. Although
3b-alkylated steroids have not yet been found to occur
as natural products in living organisms, 3b-carboxyl
steroids have been found in sediments (Dany et al.,
1990) and kerogen hydrolysates (Barakat and RullkoÈtter, 1994). The evidence from their carbon number
and isomer distributions suggests they are possibly
diagenetic products of sedimentary
2-sterenes (Summons and Capon, 1991; Dahl et al., 1992, 1995).
4,4-Dimethylcholestane (20R) was unambiguously
characterised in Biyang Tertiary lacustrine sediments.
The very low concentration of 4,4-dimethylcholestane is
most probably a re¯ection of the low concentration of its
precursors in algae or bacteria (e.g. 4,4-dimethyl-5acholesta-8(14),24-dien-3b-ol and 4,4-dimethyl-5a-cholesta-8(14)-en-3b-ol), which are intermediates in the biosynthetic pathway from lanosterol to desmethyl sterols.
Our failure to detect 4,4-dimethylergostane (C30) and
4,4-dimethylstigmastane (C31) is possibly a consequence
of the ordering of reactions in sterol biosynthesis,
whereby alkylation at C-24 generally takes place after
removal of the C-14 methyl group and at least one C-4
methyl group (Nes and Mckean, 1977; Weete, 1980).

5. Summary
In this study we employed GC as well as GC±MS in
full scan, SIM and MRM modes to investigate the detail
of steroidal hydrocarbons in lacustrine clayey dolomites
from the Eocene of the Biyang Basin. Isomerised standards of A-ring alkylated cholestanes and stigmastanes
were critical to the unambiguous characterisation of

123

several groups of compounds. In our experience, these
sediments are unusual for showing such a broad range of
steroids in a single sample. Sample no. 1, for example,
contained isomeric mixtures of lanostane, 24methyllanostane, 24-ethyllanostane, 4,4-dimethylcholestane, 4-methylcholestane, 4-methylergostane, 4-methylstigmastane and 4,23,24-trimethylcholestane (dinosterane).
All of these compounds, with the exception of 24-ethyllanostane can be traced to known sterol precursors
which have been identi®ed in algae, fungi, methylotrophic bacteria and plants. In addition, the samples
contained series of 2a- and 3b-alkyl steroids without
known precursors, the most prominent being the latter
with chain lengths of C1±C5. Because of the complexity
of co-eluting and closely eluting peaks, metastable
analysis with coinjections of 20S+20R sterane standards
was required to distinguish between 3b-ethylcholestane
and 4,4-dimethylcholestane on the one hand, and the
3b-n-propylcholestane (C30) to 3b-n-propylstigmastane
(C32) and lanostane (C30±C32) series on the other.
GC analyses showing low Pr/Ph ratios and prominent
peaks for gammacerane and b-carotane indicate sedimentation in highly saline waters. These conditions were
evidently hospitable to an unusual microbial ¯ora and
the probable source of the diverse array of steroids.
Minor plant inputs were also evident through the presence of waxy hydrocarbons with a low odd/even preference and oleanane in some samples.

Acknowledgements
We would like to thank Zarko Roksandic, Graham
Logan and Paul Greenwood for their assistance with
GC±MS analysis and Peng Ping'an for collecting some
reference material. Rob Capon provided the synthetic
standards for 4,4-dimethylcholestane and 4,4-dimethylstigmastane while Janet Hope synthesised the 3b-alkyl
steranes. Graham Logan and Jochen Brocks provided
comments that improved this manuscript. We also
thank Michael Moldowan and an anonymous reviewer
for their constructive reviews. Roger Summons publishes with the approval of the CEO of AGSO.
Associate EditorÐB.R.T. Simoneit

Appendix on next page

124

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

Appendix. Chemical structures of tetracyclic biomarkers discussed in text

References
Abbott, G.D., Lewis, C.A., Maxwell, J.R., 1984. Laboratory
simulation studies of steroid aromatisation and alkane isomerisation. Organic Geochemistry 6, 31±38.
Alam, M.S., Chopra, N., Ali, M., Niwa, M., 2000. Normethyl
pentacyclic and lanostane-type triterpenes from Adiantum
venustum. Phytochemistry 54, 215±220.
Barakat, A.O., RullkoÈtter, J., 1994. Occurrence of bound 3bcarboxylsteroids in geological samples. Energy & Fuels 8,
481±486.

Bisseret, P., Rohmer, M., 1990. Bromine, n-bromosuccinimide
and sulfur induced isomerisations in the hopane series. Tetrahedron Letters 31, 7445±7448.
Bisseret, P., Zundel, M., Rohmer, M., 1985. Prokarytic triterpenoids. 2. 2b-Methylhopanoids from Methylobacterium
organophilum and Nostoc muscorum, a new series of prokarytic triterpenoids. European Journal of Biochemistry 150,
29±34.
Boon, J.J., Rijpstra, W.I.C., de Lange, F., de Leeuw, J.W.,
Yoshioka, M., Shimizu, Y., 1979. Black Sea sterol-a molecular fossil for dino¯agellate bloom. Nature 277, 125±127.
Bouvier, P., Rohmer, M., Benveniste, P., Ourisson, G., 1976.

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

8(14)-steroids in the bacterium Methylococcus capsulatus.
Biochemistry Journal 159, 267±271.
Brassell, S.C., Eglinton, G., 1981. Biogeochemical signi®cance
of a novel sedimentary C27 stanol. Nature 290, 579±582.
Brassell, S.C., Eglinton, G., Fu, J.M., 1986. Biological marker
compounds as indicators of the depositional history of the
Maoming oil shale. In: Leythaeuser, D., RullkoÈtter, J. (Eds.),
Advances in Organic Geochemistry 1985, Organic Geochemistry, Vol. 10. Pergamon, Oxford, pp. 927±941.
Brassell, S.C., Eglinton, G., Howell, V.J., 1987. Palaeoenvironmental assessment of marine organic-rich sediments using
molecular organic geochemistry. In: Brooks, J., Fleet, A.J.
(Eds.) Lacustrine Petroleum Source Rocks Geological
Society Special Publication No. 26, pp. 179-198.
Chen, J.Y., Wang, Q.J., Ma, W.Y., 1988. Geochemical features
of source rocks in an alkaline lake basin. In: 2nd Conference
on Petroleum Geochemistry and Exploration in the AfroAsian Region (abstract), Beijing, p. 145.
Chen, J.H., Philp, R.P., Fu, J.M., Sheng, G.Y., 1989. The
occurrence and identi®cation of C30±C32 lanostanes: a novel
series of tetracyclic triterpenoid hydrocarbons. Geochimica
et Cosmochimica Acta 53, 2775±2779.
Chen, J.H., Summons, R.E., Fu, J.M., Sheng, G.Y., 1993. The
distribution and implications of biomarkers from some Chinese Tertiary lacustrine sediments, Biyang Basin, In: éygard
K. (Ed.), Organic Geochemistry: Poster Sessions from the
16th International Meeting on Organic Geochemistry, Stavanger, Norway, 20±24 September 1993. Falch Hurtigtrykk,
pp. 313±316.
Collister, J.W., Summons, R.E., Lichtfouse, E., Hayes, J.M.,
1992. An isotopic biochemical study of the Green River Oil
Shale. Organic Geochemistry 19, 265±276.
Dahl, J., Moldowan, J.M., McCa€rey, M.A., Lipton, P.A.,
1992. A new class of natural products revealed by 3b-alkyl
steranes in petroleum. Nature 355, 154±157.
Dahl, J., Moldowan, J.M., Summons, R.E., McCa€rey, M.A.,
Lipton, P.A., Watt, D.S., Hope, J.M., 1995. Extended 3balkyl steranes and 3b-alkyl triaromatic steroids in crude oils
and rock extracts. Geochimica et Cosmochimica Acta 59,
3717±3729.
Dany, F., Riolo, J.-M., Albrecht, P., 1990. 3-Carboxylsteranes,
a novel family of fossil steroids. Journal of the Chemical
Society, Chemical Communications, 1228-1230.
Edmunds, K.L.H., Eglinton, G., 1984. Microbial lipids and
carotenoids and their early diagenesis in the Solar Lake
laminated microbial mat sequence. In: Cohen, Y., Castlenholtz, R.W., Halvorson, H.O. (Eds.), Microbial Mats:
Stromatolites. A.R. Liss, New York, pp. 343±389.
Freeman, K.H., Hayes, J.M., Trendel, J.-M., Albrecht, P.,
1990. Evidence from carbon isotope measurements for
diverse origins of sedimentary hydrocarbons. Nature 343,
254±256.
Fu, J., Sheng, G., Peng, P., Brassell, S.C., Eglinton, G., Jiang,
J., 1985. Peculiarities of salt lake sediments as potential
source rocks in China. In: Leythaeuser, D., RullkoÈtter, J.
(Eds.), Advances in Organic Geochemistry 1985. Organic
Geochemistry, Vol. 10. Pergamon, Oxford, pp. 119±126.
Goodwin, N.S., Mann, A.L., Patience, R.L., 1988. Structure
and signi®cance of C30 4-methylsteranes in lacustrine shales
and oils. Organic Geochemistry 12, 495±506.
ten Haven, H.L., Baas, M., Kroot, M., de Leeuw, J.W.,

125

Schenck, P.A., Ebbing, J., 1987. Late Quaternary Mediterranean sapropels. III: Assessment of source of input and
palaeotemperature as derived from biological markers. Geochimica et Cosmochimica Acta 51, 803±810.
ten Haven, H.L., de Leeuw, J.W., Schenck, P.A., 1985. Organic
geochemical studies of a Messian evaporitic basin, northern
Appenines (Italy) 1: hydrocarbon biological markers for a
hypersaline environment. Geochimica et Cosmochimica Acta
49, 2181±2191.
Holba, A.G., Ellis, L., Tegelaar, E., Singletary, M.S., Albrecht,
P., 2000. Tetracyclic polyprenoids: indicators of fresh water
(lacustrine) algal input. Geology 28, 251±254.
Jiang, N.H., Jia, F.Y., 1986. Source and migration of crude oils
of Biyang Depression. Oil and Gas Geology 7, 51±58 (in
Chinese).
de Leeuw, J.W., Rijpstra, W.I., Schenck, P.A., Volkman, J.K.,
1983. Free, esteri®ed and bound sterols in Black Sea Unit I
sediments. Geochimica et Cosmochimica Acta 47, 455±465.
Mackenzie, A.S., Ho€mann, C.F., Maxwell, J.R., 1981. Molecular parameters of maturation in the Toarcian shales,
Paris Basin, France III. Changes in aromatic steroid
hydrocarbons. Geochimica et Cosmochimica Acta 45,
1345±1355.
Mackenzie, A.S., Patience, R.L., Maxwell, J.R., Vandenbroucke, M., Durand, B., 1980. Molecular parameters of
maturation in the Toarcian shales, Paris Basin, France I.
Changes in the con®guration of the acyclic isoprenoid
alkanes, steranes and triterpanes. Geochimica et Cosmochimica Acta 44, 1709±1721.
Maxwell, J.R., Mackenzie, A.S., Volkman, J.K., 1980. Con®guration at C-24 in steranes and sterols. Nature 286, 694±697.
Moldowan, J.M., Fago, F.J., Lee, C.Y., Jacobson, S.R., Watt,
D.S., Slougui, N.-E., Jeganathan, A., Young, D.C., 1990.
Sedimentary 24-n-propylcholestanes, molecular fossils diagnostic of marine algae. Science 247, 309±312.
Murray, A.P., Summons, R.E., Boreham, C.J., Dowling, L.M.,
1994. Biomarker and n-alkane isotope pro®les for Tertiary
oils: relationship to source rock depositional setting. Organic
Geochemistry 22, 521±542.
Murray, A.P., Sosrowidjojo, I.B., Alexander, R., Kagi, R.I.,
Norgate, C.M., Summons, R.E., 1997. Oleananes in oils and
sediments: evidence of marine in¯uence during early diagenesis? Geochimica et Cosmochimica Acta 61, 1261±1276.
Nes, W.R., McKean, M.L., 1977. Biochemistry of Steroids and
Other Isopentenoids. University Park Press, Baltimore.
Nichols, P.D., Palmisano, A.C., Rayner, M.S., Smith, G.A.,
White, D.C., 1990. Occurrence of novel C30 sterols in Antarctic sea-ice diatom communities during a spring bloom.
Organic Geochemistry 15, 503±508.
Peng, P., Morales-Izquierdo, A., Fu, J., Sheng, G., Jiang, J.,
Hogg, A., Strausz, O.P., 1998. Lanostane sul®des in an
immature crude oil. Organic Geochemistry 28, 125±134.
Peters, K., Moldowan, J.M., 1993. The Biomarker Guide:
Interpreting Molecular Fossils in Petroleum and Ancient
Sediments. Pretince Hall, Englewood Cli€s, NJ.
Philp, R.P., Chen, J.H., Fu, J.M., Sheng, G.Y., 1992. A geochemical investigation of crude oils and source rocks from
Biyang Basin, China. Organic Geochemistry 18, 933±945.
Ritts, B.D., Hanson, A.D., Zinniker, D., Moldowan, J.M.,
1999. Lower-middle Jurassic nonmarine source rocks and
petroleum systems of the northern Qaidam basin, NW

126

J. Chen, R.E. Summons / Organic Geochemistry 32 (2001) 115±126

China. American Association of Petroleum Geologists Bulletin 83, 1980±2005.
Robinson, N., Eglinton, G., Brassell, S.C., Cranwell, P.A.,
1984. Dino¯agellate origin for sedimentary 4a-methylsteroids and 5a(H)-stanols. Nature 308, 439±442.
Rohmer, M., Bouvier-Nave, P., Ourisson, G., 1984. Distribution of hopanoid triterpenes in prokaryotes. Journal of
General Microbiology 130, 1137±1150.
Schae€er, P., Poinsot, J., Hauke, V., Adam, P., Wehrung, P.,
Trendel, J.-M., Albrecht, P., Dessort, D., Connan, J., 1994.
Novel optically active hydrocarbons in sediments: evidence for
an extensive biological cyclisation of higher regular polyprenols.
Angewandte Chemie International Edition 33, 1166±1169.
Seher, A., 1976. Sterols in the chemistry and technology of
edible fats and oils. In: Paoletti, R., Jacini, G., Porcellati, G.
(Eds.), Lipids, Vol. 2. Technology Raven Press, New York,
pp. 293±313.
Summons, R.E., Boreham, C.J., Thomas, J., Maxwell, J.R.,
1992. Secular and environment constraint on the distribution
of dinosterane in sediments. Geochimica et Cosmochimica
Acta 56, 2437±2444.
Summons, R.E., Capon, R.J., 1988. Fossil steranes with
unprecedented methylation in ring-A. Geochimica et Cosmochimica Acta 52, 2733±2736.
Summons, R.E., Capon, R.J., 1991. Identi®cation of signi®cance of 3b-ethylsteranes in sediments and petroleum.
Geochimica et Cosmochimica Acta 55, 2391±2395.
Summons, R.E., Jahnke, L.L., 1992. Hopenes and hopanes
methylated in ring A: correlation of the hopanoids from
extant methylotrophic bacteria with their fossil analogues. In:
Moldowan, J.K., Albrecht, P., Philp, R.P. (Eds.), Biological

Markers in Sediments and Petroleum. Prentice Hall, Englewood Cli€s, NJ, pp. 182±194.
Summons, R.E., Walter, M.R., 1990. Molecular fossils and
microfossils of prokaryotes and protists from Proterozoic
sediments. American Journal of Science 51, 557±566.
Summons, R.E., Jahnke, L.L., Logan, G.A., Hope, J.M., 1999.
2-Methylhopanoids as biomarkers for cyanobacterial oxygenic photosynthesis. Nature 400, 554±557.
Volkman, J.K., 1988. Biological marker compounds as indicators of the depositional environments of petroleum source
rocks. In: Fleet, A.J., Kelts, K., Talbot, M.R. (Eds.), Lacustrine Petroleum Source Rocks. Geological Society Special
Publication No. 40, pp. 103-122.
Volkman, J.K., Kearney, P., Je€rey, S.W., 1990. A new source
of 4-methyl sterols and 5a(H)-stanols in sediments: prymnesiophyte microalgae of the genus Pavlova. Organic Geochemistry 15, 489±497.
Weete, J.D., 1980. Lipid Biochemistry of Fungi and Other
Organisms. Plenum Press, New York.
Zhu, S.A., Xu, S.R., Zhu, S.B., Wang, Y.X., 1981. Petroleum
geological characterisics of the Biyang depression, Henan.
Acta Petroleum Sinica 2 (2), 21±218 (in Chinese).
Zundel, M., Rohmer, M., 1985a. Prokaryotic triterpenoids. 1.
3b-Methylhopanoids from Acetobacter sp. and Methylococcus capsulatus. European Journal of Biochemistry 150,
23±27.
Zundel, M., Rohmer, M., 1985b. Prokaryotic triterpenoids. 3.
The biosythesis of 2b-methylhopanoids and 3b-methylhopanoids of Methylobacterium organophilum and Acetobacter
pasteurianus spp. pasteurianus. European Journal of Biochemistry 150, 35±39.