A direct gas chromatography-mass spectrometry (GC-MS) method for the detection of orellanine present in stomach content (Part I)

doi:10.4236/jbpc.2012.31003

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Vol.3, No.1, 29-34 (2012) Journ al of Biophysical Chemistry

http://dx.doi.org/10.4236/jbpc.2012.31003

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: [email protected]

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

ABSTRACT

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

1. INTRODUCTION

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-

tioned.

There could be a delay (latent period) of up to three

weeks from the time of ingestion of food containing C.

I. Brondz et al. / Journal of Biophysical Chemistry 3 (2012) 29-34

(a)

(b)

Figure 1. (a) The chemical structures for

orellanine; (b) The chemical structures for

orelline.

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.

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. MATERIALS AND METHODS

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

30˚C/min.

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.

orellanus.

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.

3. RESULTS

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

[14,15].

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

I. Brondz et al. / Journal of Biophysical Chemistry 3 (2012) 29-34

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.

OPEN ACCESS

Figure 4. Mass spectrum recorded with

negative detection [8].

in biological samples and comparable to previous pub-

lished in literature [8,14,15].

4. DISCUSSION

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].

5. CONCLUSIONS

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.

6. ACKNOWLEDGEMENTS

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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