trends in analytical chemistry, vol. 9, no.
IO, 1990
*
Stable carbon isotope ratio analysis on
single components in crude oils by direct
gas chromatography-isotope analysis
Malvin Bjoray and Keith Hall
Trondheim,Norway
and
Jar&e Jumeau
Cheshire,U.K.
A new instrument for direct stable carbon isotope analysis of
effluent from a capillary gas chromatography column has
been developed and tested out on various hydrocarbon sam-
ples.
This article describes technical details of the instrument
and its use in direct analyses of a hydrocarbon standard mix-
ture, natural gases, crude oils and saturated hydrocarbon
fractions. Good reproducibility is found for the different
types of hydrocarbon samples, and analyses of whole oils
give the same genetic trends as isotopic analysis of distilla-
tion cuts from crude oil samples.
Introduction
Isotope Ratio Mass Spectrometry (IRMS) is a
technique that has been widely used in the field of or-
ganic chemistry for the past decade. It has yielded
valuable information in studies of oil-oil and oil-
source rock correlationsi. In particular, the bulk
r3C/‘*C compositions of whole oils, separate frac-
tions and distillate fractions can be routinely mea-
sured. Whilst these bulk measurements give useful
information, the 13C/‘2Cisotopic compositions of in-
dividual components within an oil represent a unique
signature of its origin and maturation. In order to ob-
tain such information using conventional IRMS, it is
first necessary to separate the individual compo-
nents to a very high state of purity, and then to com-
pletely combust each one to CO2 prior to introduc-
tion into the inlet of the mass spectrometer. The
whole process may lead to isotopic fractionation but,
more importantly, it is prohibitively labour inten-
sive. As a result very few such studies have been re-
ported in the literaturez-5.
In order to make valid oil-oil correlations with
carbon isotopes identical fractionation of the oil
must, in theory, be obtained; samples thus require
standardisation prior to analysis. In the simplest case
this involves selection of a particular boiling range6.
In other studies the oil is separated into various com-
01659936/90/$03.00.
331
pound classes (e.g. saturated hydrocarbons, aromat-
ic hydrocarbons, asphaltenes, etc.)7. All such work-
up processes are operator dependent and the result-
ing fractions can become isotopically fractionated to
different extents.
Sano et al.’ first recognized the importance of cou-
pling a combustion furnace to a gas chromatograph
with subsequent analysis on a mass spectrometer in
order to determine the isotopic composition of the
individual components of a complex mixture. The
technique, called gas chromatography (GC)-com-
bustion-IRMS, was pursued by others who repo&ed
improved sensitivity and measurement precision .
The advent of commercially available GC-com-
bustion-IRMS instruments has made it possible to
obtain the i3C/ I2C composition of the individual
components of an oil. Such systems offer unprece-
dented levels of sensitivity and measurement preci-
sion, combined with a simple user interface which
enables the analysis of a whole oil or fraction (satu-
rated/aromatic hydrocarbons) to be obtained in less
than two hours. Previously, many man days of prep-
aration were required.
Instrumentation
The Isochrom GC-combustion-IRMS instru-
ment (Fig. 1) consists of a gas chromatograph, a
combustion unit and a parallel reference gas injec-
tion system, all on line with an isotope ratio mass
spectrometer. The mass spectrometer is fitted with a
continuous flow dual inlet which permits the alter-
nate admission of the sample and reference gases di-
rectly into the ion source.
The mass spectrometer
Electrons emitted from a hot, thoria-coated iridi-
um filament produce positive ions by impact with ga-
seous molecules within the source enclosure. The
electrons are accelerated through a 100-V potential
gradient towards a trap plate and are constrained to
follow a helical path by a small magnetic field gener-
ated by a pair of small magnets mounted externally
to the source block. This increases the electron path
length, thereby increasing the probability of an en-
counter with a gas molecule within the source. From
0 Elsevier Science Publishers B .V.
332
GAS CHROMATOGRAPH
FURNACE TUBE
INJECTOR DETECTOR
CO2
Fig. 1. Schematic drawing of the GC-IRMSsystem.
ence line; 5 = splitter union; VI = heart split valve; V2 = reference injection valve.
CO2 gas, ions at masses 44 (12C160160), 45
(1sc160160 ) 12C160170) and 46 (12C’60180,
13C’70160) are produced.
These ions are focussed by a stack of electrostatic
lenses into an ion beam and accelerated down a
4000-V potential gradient into a 7.5 KGauss magnet-
ic field provided by a permanent magnet. Ions of
masses 44, 45 and 46 are separated in the magnetic
analyser, deflected through 90” along separate circu-
lar paths with radii of curvature of approximately 9
cm. They are collected simultaneously within 3 Fara-
day cups where the currents produced are proportio-
nal to the number of ions collected.
The interface
The interface consists of a continuous flow dual in-
let. High purity helium gas is drawn continuously
through the inlet into the high vacuum enclosure of
the mass spectrometer, from the sample line open
split (OS,_&) and the reference line open split (OS&.
The open splits prevent any disturbance within the
ion source from pressure transients which may occur
during the mechanical switching of valves in both the
reference and sample lines.
The sample to be analysed is injected onto the col-
umn of the gas chromatograph and its components
are chromatographically separated. At the exit of
the column the components may be directed towards
the GC detector or towards the MS sample inlet line.
trends in analytical chemistry, vol. 9, no. IO, 1990
.
MASS SPECTROMETER
ION SOURCE
I = Sample inlet capillary; 2 = reference inlet capillary; 3 = sample line; 4 = refer-
It is possible to select a whole section of the chroma-
togram, or just isolated components from the origi-
nal mixture, for isotopic analysis in the mass spec-
trometer .
The heart cutting process involves the combina-
tion of a mechanical and pressure switching system.
When the heart split valve (VI) is in the closed posi-
tion the effluent of the GC column is directed wholly
down the sample line (3). When the heart split valve
is in the open position, the effluent at the GC column
is forced wholly towards the GC detector by the back
pressure, which is applied at the exit of the sample
line capillary by the helium make up gas [He(l)].
The helium make up gas [He(2)] ensures efficient
flushing of the heart split valve.
Material eluting from the sample line capillary is
completely combusted to CO2 and water on passage
through a furnace tube filled with CuO granules and
maintained at 900 “C. The helium make up gas He-
(1) gives a high linear velocity of the chromatograph-
ic components through the furnace tube, in order to
maintain chromatographic resolution.
A cryogenic trap, maintained at -100 “C, is fitted
along the sample inlet capillary to remove water
from the sample gas. Pulses of CO2 reference gas are
injected via the on/off valve V2. The reference gas
pulses are injected such that they do not coincide
with any sample peaks. The reference gas is precali-
brated and its 13C and “0 values are calculated on
trendsin analyticalchemistry, vol. 9, no. IO, 1990 333

the international PDB standard scale. As in conven-
tional IRMS, the isotopic ratios 45/44 and 46144 for
both the sample and reference CO gases are mea-
sured by the mass spectrometer; Q452CO, and d4%02
are calculated from:
(45/44)sample-(4Y44)reference
d45co 2 =
(45W)reference
(46/44)sample-(46/44)reference
6%0, =
(46/44)reference
The contribution from 170 is corrected for, ac-
cordin to formulations established by Craig”, and
the 6 ,BC is thus obtained. System control, data ac-
quisition and data processing are provided by an
IBM PS/2 Model 50 which communicates with the in-
strument via the RS232C interface.
Experimental conditions
The GC-IRMS analysis was performed on a VG
Isochrom II system interfaced to a Dani 8510 gas
chromatograph. For standard samples, oils and satu-
rated fractions, the GC was fitted with a fused silica
OV-1 column (25 mm x 0.22 mm I.D.). Helium (0.8
bar) is used as a carrier gas and the injections were
performed in split mode. For whole oil analysis, the
GC system was programmed from -10 “C to 300 “C
at 4 “Urnin and held isothermally at 300 “C for 20
min. For analysis of the saturated hydrocarbon frac-
tion, the temperature programme was from 60 “C to
300 “C at 4 “Urnin and held isothermally at 300 “C
for 20 min.
For gas analysis, the gas chromatograph was fitted
with a 10 m X 0.32 mm I.D. Poraplot Q capillary col-
umn from Chrompak. Helium (3 bar) is used as a
carrier gas and injections were performed in split
mode. The temperature programme was from 35 “C
to 150 “C at 4 “Clmin. Due to the large concentra-
tion of methane in the natural gas samples, 1~1 gas
was injected at a split ratio of 3O:l for the analysis of
methane while a splitless injection of 4 ,ul was used
x looo for the analysis of the remaining components.
The MS conditions for these analyses were 200,~A
trap current, 100 eV ionisation energy.
Data collection is on an IBM PS2 model 50 com-
x looo puter system.
For calculation purposes a CO, reference gas was
automatically introduced into the IRMS system in a
series of pulses before and after the array of chroma-
tographic peaks of interest.
Results and discussion
Reproducibility of standard mixture
The reproducibility of the instrument was tested
by analysing a standard mixture made up of C,,-C,,
n-alkanes together with pristane and phytane, dis-
solved in hexane. A volume of 1 ,ul of the standard
mixture was injected at 1:50 split ratio, allowing 50
nanograms of each component on the column. The
standard mixture was analysed using both the ‘heart
cutting’ process and allowing the whole run through
the mass spectrometer. There were no significant
differences found between the two techniques. The
results from five parallel analyses are shown in Table I.
These analyses show good reproducibility with a
standard deviation of 0.08-0.17, based on five runs
(Table I). The same compounds were also analysed
on a conventional dual inlet instrument after sealed
tube combustion (Table I). These analyses give a
standard deviation on five runs of 0.05-0.30 on the
different alkane standards. Comparison between the
results for single components on the dual inlet instru-

TABLE I. Stable carbon isotope ratios of standard alkane mixture by GC-IRMS and dual inlet IRMS

Compound 6Wpmj (%0) Standard deviation (5 parallels)

Dual inlet-IRMS GC-IRMS Dual inlet-IRMS GC-IRMS Deviation IRMS
and GC-IRMS

nCi* -28.60 -28.55 0.08 0.12 0.05

G4 -28.55 -28.61 0.05 0.14 0.06
nCi6 -27.64 -27.72 0.07 0.16 0.08
nCi7 -22.32 -22.16 0.06 0.14 0.16
Pristane -26.25 -26.39 0.12 0.10 0.14
nCi8 -32.15 -32.14 0.02 0.17 0.01
Phytane -29.29 -29.15 0.30 0.13 0.14
nCz0 -27.98 -28.17 0.15 0.16 0.19
nC,z -33.84 -33.93 0.20 0.12 0.09
nC24 -26.33 -26.58 0.06 0.15 0.25
nC26 -29.83 -30.06 0.20 0.11 0.23
nC,0 -32.38 -32.41 0.06 0.08 0.03
nC32 -27.29 -27.74 0.09 0.13 0.45
 334 trends in analytical chembtry, vol. 9, no. JO, 1990

ment and a mixture of these components on the TABLE III. Stable carbon isotope ratios of n-alkanes in North
GC-IRMS instrument shows only minor variation. Sea crude oils analysed by GC-IRMS
Most of the components have mean 613C values
Compound 613C,,, (X0) Standard deviation
which show less than 0.3%0 variation between the (5 parallels)
two techniques (Table I).
Ekofisk Statfjord Ekofisk Statfjord
oil oil oil oil
Analysis of natural gas samples
Natural gas samples from an onshore U.K. field nC6 -28.08 -29.59 0.37 0.18
were analysed and isotopic composition of C, to C, nc7 -27.93 -29.18 0.18 0.38

components measured. Five separate analyses were

G -28.21 -29.09 0.19 0.29
G -27.93 -28.95 0.13 0.17
performed and good reproducibility with a standard 60 -28.34 -29.19 0.20 0.22
deviation of 0.03 to 0.21 was found (Table II). Gl -28.08 -28.83 0.11 0.27
nC12 -28.00 -29.01 0.09 0.24
-28.11 -29.14 0.18 0.24
Analysis of whole oil samples G3

nG -27.95 -29.03 0.04 0.25

The GC-isotope technique was also used to ana- G5 -28.04 -29.14 0.17 0.16
lyse whole crude oil samples. Two oils, both from the nC16 -28.33 -29.37 0.12 0.19
Kimmeridge Clay source rock in the North Sea, G7 -28.16 -29.29 0.18 0.28
were used. Oil A is from the Ekofisk reservoir, a nG3 -28.19 -29.44 0.14 0.19
-27.76 -29.44 0.21 0.20
chalk formation in the Central Graben, while oil B is G

flC20 -27.93 -29.34 0.21 0.20

from the Statfjord reservoir which is a sandstone for- -27.93 -29.48 0.23 0.24
Gl
mation in the Viking Graben. In these analyses each nC22 -28.08 -29.54 0.20 0.21
n-alkane from nC, to nCsO and acyclic isoprenoid al- nC2, -27.65 -29.45 0.24 0.17
kanes from Ci, to C,, were analysed by the GC-iso- 44 -28.11 -29.41 0.20 0.16
-28.04 -29.35 0.21 0.25
tope method by allowing the whole of the sample to G5

nC2, -28.22 -29.15 0.26 0.34

go through the mass spectrometer. The results from -28.10 -29.40 0.19 0.24
nC27
these analyses are shown in Tables III and IV. A nC2, -27.98 -29.52 0.01 0.23
typical GC-isotope trace of a whole oil is presented nC29 -27.97 -29.41 0.01 0.25
in Fig. 2, showing baseline separation between nC,s nC30 -29.42 0.18
-29.59 0.18
and phytane, an acyclic isoprenoid alkane. nC3,

The isotopic ratios are plotted against the number

of C atoms in the molecules in Fig. 3. These analyses
show good reproducibility between the different par-
allel runs. The standard deviation generally shows a
variation of 0.01-0.38 for both n-alkanes and acyclic
isoprenoids. Samples were tested both on narrow
bore (0.25 mm I.D.) and wide bore (0.32 mm I.D.)
capillary columns. The narrow bore columns give a
lower standard deviation (mainly <0.2) than was
found for the wide bore columns, due to a better re-
solution between the different compounds. This was
especially true for the lower molecular weight com-
pounds, up to nC,s.
Saturated hydrocarbon fractions of the two oils
were also analysed and these give similar results as
those for the whole oil analyses (Tables V and VI),
but with a lower standard deviation. This is due to
background material, such as aromatic compounds,
TABLE IV. Stable carbon isotope ratios of acyclic isoprenoid
alkanes in North Sea crude oils analysed by GC-IRMS

Compound 613Cpr,a (%0) Standard deviation

(5 parallels)
TABLE II. Stable carbon isotope ratios of components in natu-
ral gas samples Ekofisk Statfjord Ekofisk Statfjord
oil oil oil oil
Compound 613Cp,,a (%o) Standard deviation
G3 -25.08 -29.39 0.04 0.25
(5 parallels)
if-% -27.11 -28.09 0.18 0.25
Methane -51.00 0.18 iCl5 -25.48 -29.24 0.23 0.30
Ethane -31.42 0.10 ic16 -24.05 -29.80 0.17 0.21
Propane -29.08 0.07 G8 -25.93 -29.64 0.06 0.23
Isobutane -31.47 0.11 G9
Butane -29.27 0.03 (Pristane) -27.94 -30.51 0.27 0.17
Isopentane -28.34 0.21 iC20
Pentane -28.78 0.07 (Phytane) -27.41 -30.21 0.24 0.21
trends in.analytical chemistry, vol. 9, no. lo,1990 335

EKOFISK OIL

Fig. 3. Plot of 6°C versus carbon number in analysed compounds

for Ekofisk and Statford oils. 0 = Isoprenoids, Ekofisk oil;
l = n-alkanes, Ekofisk oil; 0 = isoprenoids, Statford oil;
n = n-alkanes, Statfiord oil.

The Statfjord oil changes from -29.6%0 for nC, to

-28.8%0 for Krr, then becomes slightly more neg-
ative again with increasing alkane number to -29.4%0
for nCIg. From this point the isotopic ratios level out
at approximately -29.5%0 for the remaining alkanes.
The Ekofisk oil has a uniform value of approximate-
ly -28%0 for the whole range of n-alkanes. This
shows that a single GC-isotope analysis and mea-

ot.\.. I~.,I~~,,~..,,..,,..,...,..,,,,.J
42 ‘43 44 45 46 47 48 49 50 51
Time (minutes) TABLE V. Stable carbon isotope ratios of n-alkanes in satu-
rated hydrocarbon fractions of North Sea crude oils analysed by
Fig. 2. Isotope gas chromatogram of Ekofisk oil using Mass 44 GC-IRMS
from the MS on Isochrom II.
Compound 613CPna (X0) Standard deviation
(5 parallels)
Ekofisk Statfjord Ekofisk Statfjord
oil oil oil oil
which will have a greater effect for the whole oil
analysis than the background material in the satu- G3 -28.38 -29.58 0.10 0.03
rated fractions. Determinations of the stable carbon 44 -28.24 -29.63 0.05 0.11

isotope values of single compounds could therefore

n% -2844 -29.62 0.06 0.04
G6 -28.13 -29.71 0.15 0.07
be sensitive to background substraction, especially G -28.53 -29.81 0.08 0.09
where there is no baseline separation. G -28.39 -29.92 0.14 0.05
Both Ekofisk and Statfjord oil have been analysed nC19 -27.82 -29.85 0.18 0.01
nC20 -28.12 -29.83 0.12 0.05
by determining isotopic ratios on narrow distillation
cuts’*. These analyses show that the isotopic ratio of
n% -27.88 -29.84 0.16 0.06
nC22 -28.23 -29.85 0.17 0.02
the Statfjord oil became heavier with increasing boil- n% -27.74 -29.74 0.16 0.03
ing ranges from -29.4%0 for 50 “C to -28.3%0 for nC2, -28.27 -29.53 0.18 0.01
G -28.09 -29.32 0.22 0.02
100 “C, and then lighter (-29.4%0) at 150 “C. The re-
nC, -28.29 -29.50 0.26 0.03
maining distillation cuts show approximately the -28.38 -29.45 0.14 0.01
n%
same isotopic ratios. The Ekofisk oil has a rather G -28.36 -29.51 0.20 0.06
uniform value of approximately -27.8%0 for the nC29 -28.49 -29.40 0.30 0.06
whole range of distillation cuts, This is similar to nC30 -28.27 -29.31 0.19 0.08
what is found for the measurement on single n-al- nC3, -28.31 -29.27 0.24 0.06
nc32 -29.34 0.09
kanes shown in Fig. 3.
336
TABLE VI. Stable carbon isotope ratios of acyclic isoprenoid
alkanes in saturated hydrocarbon fractions of North Sea crude
oils analysed by GC-IRMS
Compound 6t3C,oa (%o) Standard deviation
(5 parallels)
Ekofisk Statfjord Ekofisk
oil oil oil
iC,s -24.27 -30.02 0.17
iC,, -24.48 -30.20 0.29
-25.95 -29.91 0.15
iCis
iCi,
(Pristane) -27.79 -30.73 0.17
iC2a
(Phytane) -27.28 -30.68 0.24
surement of n-alkanes in whole oil gives a similar
pattern to that found by laborious distillation and
analysis of separate cuts.
The most striking difference between the two oils
is the variation in the isotopic composition of the acy-
clic isoprenoid alkanes. The Ekofisk oil contains iso-
prenoids which are isotopically heavier than the cor-
responding n-alkanes, while the Statfjord oil con-
tains isoprenoids which are isotopically lighter than
the corresponding n-alkanes (Fig. 3). It is presently
not known what causes this large variation between
two oils that have a similar thermal maturity and are
generated from the same source rock, albeit in two
different basins. A possible explanation may be re-
lated to a variation in the oxicity of the water column
during deposition of the organic material in the two
basins. The Kimmeridge Clay Fm. sediments in the
Central Graben were deposited in less oxic bottom
waters than in the Viking Graben. Investigation of
this is presently in process.
Another surprising effect is the large variation in
the isotopic composition found between individual
isoprenoids, especially for iC,,-iC,, isoprenoids in
the Ekofisk oil. Similar variation was not recorded
for those isoprenoids which are present in the satu-
rated hydrocarbon fraction. A possible explanation
is therefore that some of these components coelute,
or elute close to, aromatic compounds. Aromatic
components in this boiling range give different isoto-
pic ratios than are found for these isoprenoids.
Words of caution
In this article, the discussion has mainly concen-
trated on simple mixtures such as n-alkanes and iso-
prenoids, where there is normally good separation
and minimal overlapping with other compounds.
The technique has also been used on far more com-
plex samples such as those rich in biomarkers, i.e.
steranes and triterpanes and complex mixtures of
trendsin analyticalchemistry,vol. 9, n0.~10,1990
iso-alkanes. It is too early to give an exact definition
of how complex a sample can be and yet still give
good results. A rule of thumb is that there should be
good separation between two peaks to give good re-
sults. The case where one compound is only repre-
sented as a shoulder on the peak of another com-
Statfjord
oil pound will not give reliable results.
Another problem is the quantity of material
0.03
needed. Small peaks will yield carbon isotope data,
0.13
0.02 but these may be quite erroneous. The amount of
material will vary from compound to compound and
0.08 depends on the amount of CO, generated. If the
peaks are small, extra caution should be exercised in
0.11
interpreting the isotopic values given by the instru-
ment.
Other applications
This article has dealt with petroleum geochemical
problems, and the use of the GC-isotope technique
in solving such problems. Other fields where the
technique has been successfully applied are:
flavour industry, i.e. to distinguish whether a
product is synthetic or a natural product extracted
from plants,
perfume industry,
pharmaceutical industry,
medical problems (glucose metabolism),
pollution problems,
recent sediment geochemistry,
natural product chemistry.
Conclusions
Direct stable carbon isotope ratio analysis of sin-
gle compounds in crude oils can now be undertaken
using capillary column GC analysis interfaced on-
line to an isotope ratio mass spectrometer. These
analyses give the same genetic trends as have earlier
been described in the literature by analysing sepa-
rate distillation cuts. Direct GC-isotope analysis
significantly reduces the time and complexity of such
analyses.
Determination of the isotopic composition of sin-
gle compounds in whole oils will be a significant ad-
ditional tool for oil-oil and oil-source rock correla-
tion studies in petroleum geochemistry.
References
M. Schoell, in J. Brooks and D. Welte (Editors), Advances
in Petroleum Geochemistry, Vol. I, Academic Press, New
York, 1984, pp. 215-245.
D. H. Welte, in P. A. Schenck and I. Havenaar (Editors),
Advances in Organic Geochemistry, Pergamon Press, OX-
ford, 1969, pp. 269-277.
E. A. Vogler, P. A. Meyers and W. A. Moore, Geochim.
Cosmochim. Actu, 45 (1981) 2287-2293.
trendsinanalytical
chemistry,vol.9, no. lo,1990
4 J. M. Hayes, R. Takigiku, R. Ocampo, H. J. Callot and P.
Albrecht, Nature, 329 (1987) 48-51.
5 I. Gilmour, P. K. Swart and C. T. Pillinger, Org. Geochem.,
5 (1984) 665-670.
6 H. H. Chung, W. B. Sheri and P. L. Grizzle, Geochim. Cos-
mochim. Acta, 45 (1981) 1803-1815.
7 W. H. Stahl, Geochim. Cosmochim. Acta, 42 (1978)
1573-1577.
8 M. Sano, Y. Yotsui, H. Abe and S. Sasaki, Biomed. Mass
Spectrom., (1976) l-3.
9 D. E. Matthews and J. M. Hayes, Anal. Chem., 50 (1978)
1465-1473.
The analysis of brassinosteroids -
growth-promoting substances
Nubuo lkekawa
Fukushima,Japan
Brassinosteroid is the generic name for a new group of ste-
roidal plant growth substances which possess B-ring lactone
and two vicinai dials. Microamounts of it are distributed
widely in the phtnt kingdom as a mixture of several ana-
logues. Analytical methods for these special steroids by gas
chromatography (GC), GC-mass spectrometry and high-
performance liquid chromatography as the boronate deriva-
tive and their applications are discussed.
Introduction
Brassinolide (Fig. l), which was first isolated from
rape (Brmsica nupus L.) pollen by USDA scientists
in 1979, is a newly discovered plant growth-promot-
ing substance’. It is a steroidal compound with a sev-
en-membered lactonic B-ring and two pairs of vici-
nal diol groups in the A-ring and the side chain. Since
its discovery, extensive studies have been devoted to
its chemical synthesis, the screening of brassinolide
and related substances in plants, and the possible ap-
plication of these steroids in agriculture.
To date, more than 20 brassinolide analogues, or
brassinosteroids as they are now called, have been
isolated and characterized in plants, mainly by Japa-
nese scientists.
Brassinosteroids generally show plant growth-
promoting activities such as cell elongation and cell
division, resulting in curvature, swelling and splitting
of the internode in the bean second-internode as-
say2, and inclination of rice lamina3. In particular,
the rice-lamina inclination assay, originally devel-
oped by Maeda4, is specific and highly sensitive for
brassinosteroids. Brassinosteroids also exhibit a
0165-9936/90/$03.00.
337
10 D. E. Matthews, E. Ben-Galin and D. M. Bier, Anal.
Chem., 51(1979) 80-84.
11 H. Craig, Geochim. Cosmochim. Acta, 12 (1957) 133-149.
12 M. A. Northam, in B. M. Thomas et al., Petroleum Geo-
chemistry in Exploration of the Norwegian Shelf, Graham &
Trotman, 1985, pp. 93-100.
Dr. Malvin Bjoray is managing director of Geolab Nor AIS, Hor-
nebergveien 5, 7038 Trondheim, Norway since 1984; Keith Hall
does chromatographic consultancy work in geochemical, medical
and chemical R&D; Janine Jumeau is Product Manager for VG
Isotech Ltd., Aston Way, Cheshire CWIOOHT, U.K.
plant
broad range of biological activities commonly ob-
served in many of the known plant hormones includ-
ing auxin, gibberellin and cytokinin. Recently we
have synthesized a number of brassinosteroids and
assayed them for their biological activity in order to
investigate structure-activity relationships. We
have also developed analytical methods for brassi-
nosteroids based on gas chromatography-mass
spectrometry (GC-MS) and high-performance
liquid chromatography (HPLC) techniques, which
are very suitable for the analysis of such mixtures.
Application of our methods has resulted in the iden-
tification of new brassinosteroids in plants, and as a
consequence, through the studies of our group and
others, it is now apparent that brassinosteroids are
ubiquitously distributed in the plant kingdom.
In this article I would like to describe these analyt-
ical methods and their application to the screening of
brassinosteroids.
Structure and occurrence of brassinosteroids
Fig. 2 summarizes the structures of typical brassi-
nosteroids. In 1982, Yokota and co-workers, on the
basis of the rice lamina inclination assay, isolated
castasterone (2)5 from the insect galls of Casanea
crenatu and dolicholide (8)6 from the immature seeds
of Dolichos lablab.
In the same year, we identified brassinolide and
castasterone in three plants, i.e., the immature seeds
and sheaths of the Chinese cabbage, Brassicu cam-
pestris L. var. pekinensis, the leaves of green tea,
Thea sinensis, and the insect galls of the chestnut
tree, Cusanea crenatu L. Sieb. et SUCC.~,*.Further
analysis of these plant sources revealed the occur-
rence of brassinone (5), 28-norbrassinolide (4) and
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