Vol.3, No.1, 29-34 (2012) Journ al of Biophysical Chemistry
A direct gas chromatography-mass spectrometry
(GC-MS) method for the detection of orellanine
present in stomach content (Part I)
Ilia Brondz1*, Eviatar Nevo2, Solomon P. Wasser3, Anton Brondz4
1Department of Biology, University of Oslo, Oslo, Norway; *Corresponding Author: ilia.brondz@bio.uio.no
2International Graduate Center of Evolution University of Haifa, Haifa, Israel
3Institute of Evolution, Haifa University, Haifa, Israel
4Department of Chemistry, Norwegian University of Science and Technology, Trondheim, Norway
Received 29 October 2011; revised 5 December 2011; accepted 20 December 2011
Intoxication with Cortinarius orellanus Fr. is of-
ten lethal or, at the least, disabling for the v ictim.
Orellanine is recognized as the prime toxic sub-
stance in this mushroom. Poisoning by other
toxic mushrooms can be occasionally mistaken
as poisoning by C. orellanus or vice versa. The
C. orellanus toxins have a prolonged latent pe-
riod after ingestion and onset in the appearance
of symptoms. These properties of the toxin to-
gether with the chemo-, thermo- and photolabil-
ity have made it difficult to develop a direct
analytical method for diagnosing poisoning w ith
orellanine, which in turn is needed to administer
the correct medication. The aim of this study
was to dev elop a dir ect a nalytical method for the
detection of orellanine present in stomach con-
tent. Gas chromatography-mass spectrometry
with supersonic molecular beams (GC-MS with
SMB) was used for the direct detection of
orellanine in the stomach fluids of rats after they
were fed with food containing C. orellanus. This
method can be used as a platform for the future
development of analytical procedures for the
direct analytical detection of orellanine in hu-
mans intoxicated by ingestion of toxic mush-
rooms. The standard orellanine was isolated
from C. orellanus following the procedure de-
scribed by Prast et al. [1] and was used as an
authentic comparison.
Keywords: Cortinarius orellanu s Fr.; Orellanine;
GC-MS with SMB; Nephrotoxicity; Nephrotoxin
The Cortinariaceae are a large family of gilled mush-
rooms found worldwide, containing over 2100 species.
The deadly toxin orellanine has been found in at least 34
Cortinariaceae. Cortinarius orellanus Fr. inhabit also
Northern Europe and stretch as far north as Southern
Scandinavia. Poisoning by C. orellanus and other orella-
nine-containing mushrooms is quite common in East-,
North- and Central Europe and North America. Cases of
intoxication from orellanine-containing mushrooms have
also been registered in Asia.
C. orellanus has attracted the attention of mycologists,
chemists, toxicologists and physicians as a mushroom
that causes intoxication of humans after ingestion. After
an incident of mass intoxication in Poland in the 1950s,
the substance orellanine was recognized as the prime
toxic substance in this mushroom. Orellanine is recog-
nized as a potent nephrotoxin. Besides orellanine, C.
orellanus contains several other toxins such as cortinarin
A, B and C and degradation products formed from orel-
lanine such as orelline.
The molecular weight of orellanine is 252.039, the
molecular formula is C10H8O6N2, its systematic name is 3,
3’,4,4’-tetrahydroxy-2,2’-bipyridyl-N,N’-dioxide and the
structure is shown in Figure 1(a). The molecular weight
of orelline is 220.048, the molecular formula is C10H8O4N2,
its systematic name is 3,3’,4,4’-tetrahydroxy-2,2’-bipyri-
dyl and the structure is shown in Figur e 1(b).
The kidneys are the prime target for orellanine intoxi-
cation. Despite orellanine being widely recognized as the
prime nephrotoxin in C. orellanus [1], some scientists
express doubt about the correct correlation between the
molecular structure of orellanine and its toxicity [2]. The
existence of diglucoside with orellanine as the aglycon
has been discussed in the literature and the exact struc-
ture of the nephrotoxin in C. orellanus may also be ques-
There could be a delay (latent period) of up to three
weeks from the time of ingestion of food containing C.
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I. Brondz et al. / Journal of Biophysical Chemistry 3 (2012) 29-34
Figure 1. (a) The chemical structures for
orellanine; (b) The chemical structures for
orellanus until the onset of symptoms of intoxication.
Orellanine intoxication in humans is characterized by a
latent period from three days to several weeks before the
appearance of poisoning symptoms such as acute renal
failure resulting from damage to the tubular epithelium.
The term “paraphalloid syndrome” [3,4] is used to de-
scribe intoxication, which is associated with a prolonged
latent period between ingestion and the appearance of
symptoms. The mushrooms that cause symptoms more
than 6 h after consumption are associated with serious
and potentially lethal toxicity. C. orellanus is one of
these and the precise diagnosis of orellanine as the prime
toxin in C. orellanus is urgently needed.
The precise diagnosis of intoxication with C. orellanus
is needed to administer the correct medication. To con-
firm poisoning with orellanine, the histology of a kidney
biopsy of the victim is currently the diagnostic method
for humans.
Thin layer chromatography (TLC) methods for the
determination of orellanine have been developed [5-7];
however, most of these methods have been applied to
mushroom tissues. Even electron spin resonance spec-
troscopy (ESR) analysis of the oxidation product from
orellanine was described in [6] as a diagnostic tool. The
high performance liquid chromatography (HPLC) method
has also been described [8]; this method was developed
to analyze orellanine directly in the stomach contents of
rats after ingestion of C. o rellanus. The gas chromatog-
raphy (GC) method provides both significantly better
separation of substances in a mixture than TLC or
HPLC and better reproducibility. The gas chromatog-
raphy-mass spectrometry (GC-MS) analyses are quite
sensitive, and extremely small amounts of material can
be analyzed.
The logical step forward was the development of GC-
MS analysis. The use of MS in connection with GC for
detection provides a unique capacity to identify unknown
substances, or to verify the presence of target molecules
in complex mixtures. GC-MS is a core analytical tech-
nique with a broad range of applications, including the
analysis of pharmaceuticals, pesticides, environmental
pollutants, xenobiotics and toxins.
However, conventional GC-MS instrumentation de-
mands the use of elevated temperatures in the injector,
column, detector and transfer line to the MS during the
separation and detection of substances. The temperature
in the injector must be higher than the boiling point of
the main substance in the mixture to be analyzed. The
conventional capillary columns used in these analyses
have a normal column length of 15 - 30 m. The mobile
phase (gas) flow rate is low, at about 1 mL/min or less.
Current GC-MS technology suffers from a major limi-
tation in that a relatively small range of volatile, ther-
mally stable compounds are amenable to analysis. The
electron impact (EI) ionization mass spectra suffer from
the frequent absence of the molecular ion [M]+, and this
drawback reduces confidence in sample identification.
Less volatile compounds tend to be more fragile and
have a higher probability of being thermally labile. Fur-
thermore, to prevent ion-source-induced peak tailing
(and contamination) with less volatile compounds, the
temperature of the ion source must be increased; this
causes a reduction in the relative abundance of the mo-
lecular ion for all sample compounds (due to increased
sample vibrational energy content). Because of the ther-
molability of orellanine, the use of conventional GC-MS
for its analysis is difficult.
In the past decade, a new type of GC-MS has been
developed based on the use of supersonic molecular
beams (SMB) (also referred to as “supersonic GC-MS”)
and its performance capabilities were explored [9,10].
SMB are used to interface the gas chromatograph to a
mass spectrometer and as a medium for ionization of
sample compounds while in the SMB by EI [9,10]. In
contrast to conventional GC-MS, the temperatures used
in GC-MS with SMB are significantly lower, the dura-
tion of a single analysis is shorter, the column gas flow is
higher and the use of a contact-free fly-through EI ion
source technique is applied. The GC-MS with SMB is
less damaging for thermolabile substances in comparison
to conventional GC-MS [9].
The GC-MS with SMB uses capillary columns with a
length of about 4 m or shorter and the mobile phase (gas)
flow can be adjusted up to 8 mL/min or higher [9].
In the present paper, the GC-MS with SMB method
was developed for the direct analysis of orellanine in the
stomach content of rats after ingestion of C. orellanus.
Copyright © 2012 SciRes. OPEN ACCESS
I. Brondz et al. / Journal of Biophysical Chemistry 3 (2012) 29-34 31
This method can be used as a platform for the future de-
velopment of analytical procedures for the analysis of
orellanine in humans who are suspected to be poisoned
by toxic mushrooms. The presence of orellanine in ani-
mal stomach fluids was analytically detected by GC-MS
with SMB and compared with the mass spectrum of
standard orellanine.
The biological samples of stomach content were do-
nated by the R & D Department, Jupiter Ltd., Norway.
The pattern of MS fragmentation for orellanine described
in the literature and the MS data recorded by analysis of
standard orellanine supported the detection of orellanine
in the stomach fluid from laboratory animals.
The use of GC-MS with SMB provided a unique op-
portunity for orellanine analysis in stomach content after
ingestion of meals containing toxic mushrooms because
of the preservation of intact orellanine molecules during
the analytical procedure.
2.1. Instrumentation and Conditions
The experimental GC-MS with SMB based on a Var-
ian 1200 GC-MS system is described in detail in [9-11].
The separation of compounds was done with a VF-5HT
column, 0.25 mm I.D., 0.1 μm film thickness and 4 m
length (Varian, Middleburg, The Netherlands). The re-
duction of column length was done in the laboratory. The
helium column flow rate was 8 mL/min.
Samples were dissolved in methanol p.a. quality (Mer-
ck, Darmstadt, Germany). 1 μL sample at an approximate
concentration of 20 ppm was injected with a split ratio of
10:1 using the Varian 1079 injector operated at 200˚C.
The GC oven was programmed from 120˚C to 300˚C at
Ion source degradation was prevented in view of the
use of a contact-free fly-through EI ion source [9-12],
and degradation of orellanine in the sample was avoided
due to the use of a short column length and a high gas
column flow rate [9,10,13]. Separation and the detection
of stomach content are shown in the reconstructed ion
chromatogram (RIC) in Figure 2 and the mass spectrum
in Figure 3.
2.2. Standard
The mushrooms were collected in vicinity of Oslo,
Norway, and freeze-dried to a constant weight. Toxins
were extracted from C. orellanus as discussed elsewhere.
The procedure of extraction and isolation of toxin was as
described in [1].
2.3. Sample Preparation
Biological samples of stomach content and a short de-
Figure 2. Reconstructed ion chromatogram
(RIC) from the stomach content of Mus rat-
tus L. fed with pellets containing dried C.
scription of the procedure for recovered biological sam-
ples were kindly provided by the R & D Department of
Jupiter Ltd., Norway, and were as follows: animals, Mus-
rattus L. were fed with pellets containing dried C. orella-
nus (1%), glucose (4%), hydrolyzed protein (20%), fat
(10%) and ground cereals (65%), and were given water
in quantum satis. After 6 h, the stomach content of the
animal was drained by a probe without decapitation of
the animals. The obtained stomach content was then ly-
ophilized and samples were kept in liquid nitrogen. Ly-
ophilized stomach content samples (10 mg) were dis-
solved in methanol (Merck) p.a. quality, homogenized in
an ultrasound bath at 0˚C, centrifuged at 10,000 rpm for
15 min, after which the supernatant was separated from
the precipitate and lyophilized. Prior to analysis, the
samples were adjusted to 0.1 mL volume with methanol
(Merck, Darmstadt, Germany) pro analysis quality.
Orellanine was detected in the chromatogram by MS
as [M]+ ions with m/z 252 as a base signal (Figure 2).
The fragmentation of orellanine from the stomach con-
tent of an intoxicated rat (Figure 3) was comparable to
the spectrum shown in Figure 4, which was published
earlier in [8], and to spectra published in the literature
The mass spectrum of orellanine showed intense sig-
nals at m/z 252 [M]+, moderate signals at m/z 235 and
weaker signals at m/z 236 and m/z 220 in accordance
with [14]. The mass spectrum of standard orellanine is
hown in Figure 5 and is identical to that for orellanine s
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I. Brondz et al. / Journal of Biophysical Chemistry 3 (2012) 29-34
Copyright © 2012 SciRes.
Figure 3. Mass spectrum at point 1A in the chromatogram in Figure 2 from
the stomach content of Mus rattus L. fed with pellets containing dried C.
orellanus. The conditions for GC-MS with SMB analyses are described in the
text above.
Figure 4. Mass spectrum recorded with
negative detection [8].
in biological samples and comparable to previous pub-
lished in literature [8,14,15].
Several authors in the past and up to present time have
studied the toxic properties of C. orellanus and the toxin
orellanine [1,4,8,14-20]. The precise evaluation of cases
of human poisoning with orellanine from C. orellanus or
other Cortinariaceae requires a kidney biopsy followed
by a histological evaluation.
The other possibility is to obtain the stomach content
of a victim and to study it by morphological and histo-
logical means. However, this procedure often has poor
results because the patient usually prepares the meal
from several different species of mushroom, which is
mixed with several other ingredients and finely cut,
cooked or staked, chewed, and until the appearance of
the first symptoms, is partly dejected. Because of this,
the fine structure of biological material that is mixed
together partly disappears.
By the time the initial symptoms appear, severe im-
pairment in renal function is often observed. In a review
of Cortinarius species mushroom poisoning, Danel et al.
[21] highlighted that from 90 described cases, about 69%
developed symptoms of renal toxicity, more than 51% of
patients required hemodialysis, more than 11% devel-
oped end stage renal failure and more than 13% required
kidney transplantation.
The correct diagnosis and medical treatment is very
important in the case of intoxication with C. orellanus
and other species containing orellanine. Generally, sup-
portive care for acute kidney injury with close monitor-
ing of serum creatinine is needed [22,23].
The TLC analyses of orellanine in C. orellanus and
other toxic mushrooms are described in [24]. TLC is also
a useful method for the detection of orellanine in kidney
biopsy samples [25]; however, as the authors of this pa-
per concluded: “Orellanine can be detected after a rela-
tively long period following poisoning by performing a
simple thin layer chromatography technique using small
quantities of renal biopsy material. No toxin was found
in urine or blood samples”.
Kidney biopsy is an invasive diagnostic method and
can be damaging for the patient. In publication [26], the
analytical results are presented as a statement: “This re-
port was confirmed by assaying orellanine in the plasma
and two renal biopsies of patient after specific photode-
omposition into a non-toxic metabolite called orelline”. c
I. Brondz et al. / Journal of Biophysical Chemistry 3 (2012) 29-34 33
Figure 5. Mass spectrum of the isolated toxin following the method de-
scribed in [1]. The conditions for GC-MS with SMB analyses are de-
scribed in the text above.
Orelline is a substance that C. or ell anus and other toxic
Cortinarius species contain as an oxidation/degradation
product of orellanine even before ingestion of the mush-
room by the victim. The substance can be accumulated in
the mushroom under preservation, drying, cooking and
staking. It can be accumulated also after ingestion or as a
result of sample preparation. Improper sample prepara-
tion results in the production of molecules that can mask
the nature of the original molecules in the mushroom
tissue as described in [27].
The effectiveness of transformation of orellanine to
orelline by specific photodecomposition is also not evi-
dent [26]. The presence of orelline in mushroom tissues
can be detrimental to the accuracy of results. Therefore,
the direct determination of orellanine in samples is pref-
erable and needed. In GC-MS with SMB, orellanine was
directly detected. The presence of orellanine in samples
is supported by the appearance of a well recognizable
and distinct base signal of [M]+ ions with m/z 252 in the
mass spectrum. Several characteristic fragment ions are
also visible in the spectrum. The spectrum is well corre-
lated with earlier published spectra as in Figure 4 [8].
This spectrum is produced by HPLC-MS with negative
detection and has a base ion [M-H] with m/z 251. The
MS spectrum obtained in the present study is also in
good agreement with spectra published in [1,2,14,15].
1) GC-MS with SMB gives unequivocal determination
of orellanine in biological samples by [M]+ ions with m/z
252 in the mass spectrum and characteristic fragmenta-
tion ions.
2) The conditions used during analysis by GC-MS
with SMB allow the orellanine molecules to stay intact
and gives a visible mass ion signal.
3) The presented method is a basis for direct orellanine
analysis. This method can be a useful tool for the deter-
mination of the exact cause of intoxication by toxic
mushrooms in clinical and forensic medicine.
The authors are grateful to Jupiter Ltd., Norway, for donation of bio-
logical samples, permission to reproduce mass spectrum recorded with
negative detection at HPLC–MS from the stomach content of Mus
rattus L. fed with pellets containing dried C. orellanus [8] and financial
support. The authors are grateful to Prof. Aviv Amirav, School of
Chemistry, Sackler Faculty of Exact Sciences, Tel Aviv University, Tel
Aviv, Israel, for technical support and scientific cooperation within MS
and to Jon Reierstad, Technical Department, University of Oslo, Nor-
way, for technical assistance in preparation of figures.
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