Food Control 17 (2006) 271285

www.elsevier.com/locate/foodcont

Review on the qualitative and quantitative analysis

of the mycotoxin citrinin
Bao-jun Xu
a

a,b

, Xiao-qin Jia a, Li-juan Gu a, Chang-keun Sung

a,*

Department of Food Science and Technology, College of Agriculture and Biotechnology, Chungnam National University, 220 Gung-Dong,
Yusung-Gu, Taejon 305-764, South Korea
b
The Pharmaceutical Institute, Dalian University, Dalian 116622, China
Received 9 March 2003; received in revised form 10 October 2004; accepted 11 October 2004

Abstract
Citrinin is a toxic metabolite produced by several lamentous fungi of the genera Penicillium, Aspergillus and Monascus, which
has been encountered as a natural contaminant in grains, foods, feedstus, as well as biological uids. This mycotoxin is hepatonephrotoxic and implicated in disease outbreaks in animals and humans. Some analytical systems have been developed for its detection and quantication. The purpose of this paper is to review physicochemical properties, qualitative and quantitative analytical
methods of citrinin, evaluate advantages and disadvantages of various analytical techniques, and bring forward some constructive
suggestions on establishment of international criteria for quality control of products contaminated with citrinin by comparing chromatographic properties, sample pre-treatment, recovery rate and detection limit of citrinin among various analytical methods. This
paper concentrates the most important achievements on the analytical methods of citrinin published from 1980 to early 2004.

2005 Published by Elsevier Ltd.
Keywords: Mycotoxin; Citrinin; TLC; HPLC; Enzyme immunoassay

Contents
1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

272

2.

General characteristics of citrinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2.1. Physicochemical properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.2. Biosynthetic pathway and physiological factors affecting production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2.3. Toxicity and stability of citrinin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

272
272
273
273

3.

Qualitative and quantitative analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

3.1. Colorimetric technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2. Chromatographic technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.1. TLC technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.2.2. High-performance liquidchromatographic technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3. Chromatography and mass spectrum combination technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3.3.1. LCMS technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

274
274
274
274
276
281
281

Corresponding author. Tel.: +82 42 821 6722; fax: +82 42 822 2287.
E-mail address: kchsung@cnu.ac.kr (C. Sung).

0956-7135/$ - see front matter
2005 Published by Elsevier Ltd.

doi:10.1016/j.foodcont.2004.10.012

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B. Xu et al. / Food Control 17 (2006) 271285

3.4.

4.

3.3.2. GCMS technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

Bioassay technique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
3.4.1. Enzyme immunoassaytechnique . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281

Conclusion and perspective. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

282

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

282

1. Introduction
Mycotoxins are a group of structurally diverse secondary metabolites produced by various fungal species.
These toxic compounds can contaminate foodstus,
crops or human foods. The ingestion of these contaminated materials may be pathogenic in animals and humans as they may lead to serious health problems,
such as liver, kidney or nervous system damage, immunosuppression and carcinogenesis (Bennett & Klich,
2003). Due to the widespread nature of fungi in the environment, mycotoxins are considered unavoidable contaminants in foods and feeds; therefore, one of the
most eective measures to protect the public health is
to establish reasonable regulatory levels of these toxins.
It is important to develop rapid, sensitive and reproducible assays to detect the presence of mycotoxins. Numerous studies were performed on the occurrence of
mycotoxins in raw commodities and foodstus. However, due to trace property of mycotoxin and co-occurrence of diverse mycotoxins, their accurate and rapid
qualitative and quantitative analysis remains still a challenging task.
The mycotoxin citrinin is a toxic secondary metabolite, rst isolated from lamentous fungus Penicillium
citrinum (Hetherington & Raistrick, 1931). It is also produced by other species of Penicillium (Ei-Banna, Pitt, &
Leistner, 1987), Aspergillus (Kurata, 1990) and Monascus (Blanc, Loret, & Goma, 1995a, 1995b; Li, Xu, Li,
& Chen, 2003). On account of its antibacterial eects,
citrinin was investigated as an antibiotic (Wong &
Koehler, 1981), but relative toxicity studies showed that
this secondary metabolite acted in animals as a nephrotoxin (Betina, 1989a), damaged the proximal tubules of
the kidney (Phillips, Hayes, Berndt, & Williams, 1980b),
and was implicated as a potential causative agent in human endemica Balkan nephropathy (Frank, 1992;
IARC, 1986).
Contaminations of citrinin were reported in a number
of agricultural commodities, foods, feedstus as well as
biological uids at geographically diverse locations
(Abramson, Hulasare, White, Jayas, & Marquardt,
1999; Bailly, Querin, Le Bars-Bailly, Benard, & Guerre,
2002; CAST, 2003; Comerio, Fernandez Pinto, & Vaamonde, 1998; Gimeno & Martins, 1983; Heber, Lembertas, Lu, Bowerman, & Go, 2001; Kpodo, Sorensen, &

Jakobsen, 1995; Meister, 2004; Phillips, Hayes, &

Berndt, 1980a) (Table 1). However, these relative limited
analytical data were based on diverse analytical methods
with dierent sensitivity and accuracy. It was dicult to
establish widely acceptable limits for citrinin. Presently,
there is no specic legislation for citrinin worldwide. The
main reason was either lack of suitable analytical routine methods (Pohland & Wood, 1987), or its instability
in foodstus. So far, commonly used methods to analyze
citrinin are thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC) with UV or
uorescence detection (FD), and enzyme immunoassays
(EIA). Recently, LCMS and GCMS techniques have
become available for qualitative and quantitative determination of citrinin. In order to protect public health
and promote international trade, more sensitive and
accurate analytical methods for citrinin should be developed, and international criteria should be established for
quality control of products contaminated with citrinin.
Hence, we summarized physicochemical properties and
quantitative analytical methods of citrinin, compared
their chromatographic properties, sample pre-treatment,
recovery rate and detective limit among various analytical methods.

2. General characteristics of citrinin

2.1. Physicochemical properties
Citrinin [C13H14O5, IUPAC: (3R,4S)-4,6-dihydro-8hydroxy-3, 4, 5-trimethyl-6-oxo-3H-2-benzopyran-7carboxylic acid; CAS No.: 518-75-2] (Fig. 1), is an acidic
lemon-yellow crystal with maximal UV absorption at
250 nm and 333 nm (in methanol), melting at 172 C.
It is sparingly soluble in water but soluble in dilute sodium hydroxide, sodium carbonate, or sodium acetate;
in methanol, acetonitrile, ethanol, and most of other
polar organic solvents (Deshpande, 2002). It is capable
of forming chelate complexes, and can be degraded in
acidic or alkaline solution, or by heating. It is a quinone
methide with two intramolecular hydrogen bonds. Citrinin crystallizes in a disordered structure, with the p-quinone and o-quinone two tautomeric forms in a dynamic
equilibrium in the solid state. In methanol or methanol/
methylene chloride mixtures, citrinin undergoes a

B. Xu et al. / Food Control 17 (2006) 271285

273

Table 1
Natural occurrence of citrinin in commodities
Commodity contaminated

Citrinin contents (lg/kg)

Reporting country

References

Fermented maize
Cheese

548 g/kg
600 mg/kg

Ghana
France

Wheat

65 lg/kg at aw 0.810
460 lg/kg at aw 0.825
25,000 lg/kg at aw 0.885
38 g/kg in the 19% moisture
absent in the 15% moisture
12 lg/kg
0.4711.82 lg/capsule
0.2140 mg/kg
4.225.1 mg/kg
2.464.2 lg/kg
280400 lg/kg for un-irradiated grape,
g, pear; absent for all irradiated fruits
320920 lg/kg

Argentina

Kpodo et al. (1995)

Franco et al. (1996), Vazquez et al. (1996)
and Bailly et al. (2002)
Comerio et al. (1998)

Canada

Abramson et al. (1999)

India
USA
China
Taiwan, China
Germany
Egypt

Janardhana et al. (1999)

Heber et al. (2001)
Xu et al. (1999)
Shu and Lin (2002)
Schneweis et al. (2001)
Aziz and Moussa (2002)

Portugal

Gimeno and Martins (1983)

and Martins et al. (2002)
Odhav and Naicker (2002)
Meister (2004)

Barley
Maize
Red yeast rice

Silages
Fruits
Apples
Brewed beers
Cereal products

Not detected
Rye wholemeal: 1.1 lg/kg
Wheat wholemeal: 0.5 lg/kg
Weat bran: 1.92.0 lg/kg
Cocoa shells: 2.4 lg/kg
Red yeast rice: 2.9 lg/kg

OH

OH
O

HO
O

O
O

O
HO

p-quinone

South Africa
Germany

Vaamonde, Resnik, & Buera, 1988), preservation techniques (Santos, Abrunhosa, Venancio, & Lima, 2002),
as well as circulation and storage techniques of commodity (Nelson, Kirby, Beasley, Johnson, & Ciegler,
1985).

o-quinone

Fig. 1. Structural formula of citrinin isomers.

Michael-type nucleophilic addition reaction. This reaction is reversible, and the equilibrium shifts toward the
normal citrinin if temperature is increased in methylene
chloride (Poupko, Luz, & Destro, 1997).
2.2. Biosynthetic pathway and physiological factors
aecting production
Polyketide pathway is a well known route for the formation of secondary metabolites, including various
mycotoxins, in the lamentous fungi. The latest study
on biosynthetic pathway by NMR technique has revealed that the biosynthesis of citrinin originated from
a tetraketide in lamentous fungus Monascus ruber
(Hajjaj et al., 1999b), diering with a pentaketide as
has been shown for Penicillium and Aspergillus species
(Barber & Staunton, 1980; Sankawa et al., 1983). In
addition, citrinin production was inuenced by various
parameters, including nutritional factors such as oxygen
supply (Hajjaj et al., 1999a, 2000a), carbon and nitrogen
sources (Wang, Lee, & Pan, 2003), fatty acids (Hajjaj et
al., 2000b) and environmental factors such as water
activity (Comerio et al., 1998), temperature (Montani,

2.3. Toxicity and stability of citrinin

As one of mycotoxins, citrinin possesses antibiotic,
bacteriostatic, antifungal and antiprotozoal properties.
While it is also known as a hepato-nephrotoxin in a wide
range of species (Berndt, 1990; Bilgrami, Sinha, & Jeswal, 1988; Hanika, Carlton, & Tuite, 1983), in vitro
studies have demonstrated that citrinin produced multiple eects on renal mitochondrial function and macromolecule biosynthesis that ultimately resulted in cell
death (Chagas, Campello, & Kluppel, 1992a, 1992b,
1995). In addition, citrinin occurred frequently together
with another nephrotoxinochratoxin A in foodstus
such as cereals, fruits, meat (Nishijima, 1984) and cheese
(Lepom, 1986; Vazquez et al., 1996; Vrabcheva, Usleber,
Dietrich, & Martlbauer, 2000) and acted synergistically
(Glahn, Shapiro, Vena, Wideman, & Hu, 1989). To
avoid the direct/indirect intake of citrinin, it is important
to develop detoxication methods for citrinin during
food processing. So far, there have been several reports
on the detoxication of citrinin. The investigation on
thermal decomposition and detoxication showed that,
in the presence of a small amount of water, heating citrinin at 130 C caused a signicant decrease in its toxicity
to Hela cells (Kitabatake, Trivedi, & Doi, 1991);
whereas heating at 140 C or 150 C in water caused

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B. Xu et al. / Food Control 17 (2006) 271285

O
O
H
H
O

H
O

OH

H
O

HO

HO

Citrinin H1

Citrinin
H
O

OH O
H

O
OH

O
O

CH

HO
Citrinin H2

In an acidic environment, the weak native uorescence

of citrinin could be strongly enhanced. A standard curve
was, therefore, established where the concentration of
chlorhydric acid needed to show the yellow uorescence
signal of dierent concentrations of citrinin, and thereby
providing a semi-quantitative method to prove the
capacity of toxin production (Vazquez et al., 1997).
Due to diverse inuencing factors of colorimetric technique, low mean recoveries of added citrinin from corn
(56.4%), barley (66.6%), and peanuts (40.0%), and relative low sensitivity levels (1008000 ng/g) restricted citrinin application in commercial analysis. However, this
analysis procedure is rapid and low cost for analyzing
a large number of samples. In addition, this technique
allows the high throughput analysis of a large number
of industrial samples for automation by means of a
microplate system.

Fig. 2. Structures of citrinin H1 and citrinin H2.

3.2. Chromatographic technique

formation of highly toxic compounds (Trivedi, Doi, &
Kitabatake, 1993). Citrinin was also known to form a
novel toxin citrinin H1 (Fig. 2), which is made up of
two citrinin molecules at temperature above 100 C
(Trivedi, Hirota, Doi, & Kitabatake, 1993). Further
examination of the decomposition products from heated
citrinin at 140 C in water led to the isolation of another
compound, citrinin H2 [3-(3,5-dihydroxy-2-methylphenyl)-2-formyloxy-butane] (Fig. 2), which showed much
weaker cytotoxicity than that of citrinin (Hirota, Menta,
Yoneyama, & Kitabatake, 2002). It was found that after
boiling in water, concentration of citrinin in Monascus
was dramatically decreased; 20 minutes of heating could
decrease the concentration of citrinin by 50% (Shu &
Lin, 2002). These facts indicated that citrinin was unstable and thermolabile in aqueous solution. Our previous
study also veried that its stability was aected by temperature, solvent compositions used for sample preparation, HPLC mobile phase and preparation of standard
citrinin solution (Xu, Wang, Lee, Jia, & Sung, 2003).
These facts explained the necessity for a sensitive analytical procedure for determination of citrinin in various
foodstus.

3. Qualitative and quantitative analysis

3.1. Colorimetric technique
Citrinin has a conjugated, planar structure that gives
it natural uorescence ability (Fig. 1), which makes it
feasible for qualitative and quantitative determination
of citrinin by using a uorometer. Rapid semi-quantitative uorimetric assay was used for citrinin testing in
corn, barley, and peanuts (Trantham & Wilson, 1984),
fungal cultures as well as cheese (Vazquez et al., 1997,
Vazquez, Fente, Franco, Vazquez, & Cepeda, 2001).

In the 1980s and the early of 1990s, various reviews

and book chapters on the chromatography of mycotoxins were published (e.g., Betina, 1984, 1985, 1989b,
1993). In current review, novel applications of chromatographic techniques in the eld of citrinin determination are summarized.
3.2.1. TLC technique
TLC was an extremely powerful, rapid and inexpensive separation technique in mycotoxicology before
HPLC techniques became popular. Several TLC methods were proposed for citrinin quantitative (Betina,
1989b; Gimeno, 1984; Gimeno & Martins, 1983; Golinski & Grabarkiewicz, 1984; Pepeljnjak, Segvic, & Ozegovic, 2002) and qualitative determination (Abrunhosa,
Paterson, Kozakiewicz, Lima, & Venancio, 2001; Blanc
et al., 1995a, 1995b; Odhav & Naicker, 2002; Rasheva,
Nedeva, Hallet, & Kujumdzieva, 2003) (Table 1). But
as reported in earlier reviews (Betina, 1984, 1985,
1993), they exhibited relatively low sensitivities in the
range of 1050 ng/g.
In general, citrinin was easily separated by TLC using
several solvents (shown in Table 2). For chemical conrmation of citrinin, two treatment methods were often
used. Firstly, TLC plates were previously impregnated
with acidic-organic solution (Blanc et al., 1995a,
1995b; Gimeno, 1984); secondly, the TLC plates, with
the developed chromatogram, were exposed to vapors
of pyridine or acetic anhydride (Golinski & Grabarkiewicz, 1984) or dipped into aluminium chloride reagent (Rasheva et al., 2003). After these treatments,
citrinin was converted into new uorescent compounds,
and then the TLC plates were observed under 365 nm
light. The major problems with the TLC of citrinin were
its weak uorescence, tailing in normal-phase TLC on
silica gel and instability (Betina, 1989b). In order to

Table 2
TLC conditions of citrinin determination
Mobile phase

Stationary phase

Color reagents

Recovery rate and detectable limit

References

Apples, pears

Toluene-ethyl
acetate-chloroform-90%
formic acid = 70:50:50:20
(a) or ethyl ether-hexane-ethyl
acetate-90% formic
acid = 70:90:40:2 (b)

Silica gel pre-coated

G-25 HR TLC plates

(a) was exposed to NH3

vapour for 1015 s, observed
under 366/254 nm (b) was
sprayed with 20% AlCl3
solution in methanol, heated
5 min at 105 C, observed
under 366/254 nm

Recovery rate: 8796%;

detection limit: 1520 lg/kg

Gimeno and Martins

(1983) and Martins et al.
(2002)

Grain samples,
fungal cultures

Toluene-ethyl acetate-90%
formic acid = 6:3:1

Silica gel TLC plates

Exposed to vapors of pyridine

or acetic anhydride. observed
under 365 nm light

Qualitative analysis

Golinski and Grabarkiewicz

(1984)

Recovery rate: 9198%;

detection limit: 1520 lg/kg

Gimeno (1984)

Corn and barley

TLC plates previously

impregnated with 10%
glycolic acid solution in
ethanol

Decaying apples

Acetone-ethyl
acetate-water = 10:10:4

Silica gel H TLC plates

Observed yellow-green
uorescent spot under 366 nm

Recovery rate: 33.372.2%;

detection limit: 36 lg/L

Pepeljnjak et al. (2002)

Monascus spp.

Two dimensions
development:one
dimension:chloroformacetone = 90:10, Second
dimension:toluene:ethyl
acetate:formic acid = 6:3:1

Silica gel 60 TLC plates,

previously impregnated with
10% oxalic acid in methanol

Observed yellow uorescent

under 366 nm

Qualitative analysis

Blanc et al. (1995a,b)

Fungi isolated from

grapes

Toluene:ethyl acetate:formic
acid = 5:4:1

Silica gel 60 TLC

5 g/l 1 3-methyl-2-benzothiazoline
hydrazone hydrochloride, with
heating for 15 min at 110 C

Qualitative analysis

Abrunhosa et al. (2001)

Traditional brewed
beers
Monascus purpureus

Toluene:ethyl acetate:formic
acid = 6:3:1
Chloroform:ethyl acetate:formic
acid = 4.5:5.5:1

F254 uorescent silica gel TLC

Observed under 254 nm UV light

Qualitative analysis

Odhav and Naicker (2002)

Kiesel gel 60 F254 HPTLC plate

Dip into 1% solution of AlCl3 in

95% ethanol for 3 s

Qualitative analysis

Rasheva et al. (2003).

B. Xu et al. / Food Control 17 (2006) 271285

Samples

275

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B. Xu et al. / Food Control 17 (2006) 271285

overcome these disadvantages, more intense uorescence of citrinin and easier detections were accomplished
by using an aluminium chloride spray followed by heating, which changed the yellow uorescence to blue (Gimeno & Martins, 1983). In order to reduce tailing,
another improvement was the incorporation of an acid
in the silica gel. Oxalic acid was used initially (Betina,
1989b), but subsequently, glycolic acid was found to
be better because of reduced diusion of the citrinin
spots and hence enhanced detectability (Gimeno,
1984). These modied procedures were satisfactory for
conrming the present of citrinin in standards, articially contaminated grain samples, extracts from both
fungal cultures (Rasheva et al., 2003), and naturally contaminated grain samples such as corn and barley (Gimeno, 1984). Citrinin was then quantitated by the limit of
detection method. From corn and barley samples,
spiked at citrinin levels of 50, 80, 150, 300, 500, and
1000 lg/kg, the recoveries were in the range 9198%.
The minimum detectable concentration was 1520 lg/
kg. However, due to low sensitivities and poor recoveries, these TLC methods are suitable for qualitative analysis at best.
3.2.2. High-performance liquid chromatographic
technique
During the last decades, high-performance liquid
chromatographic (HPLC) has been popular and used
increasingly for the analysis of mycotoxins in foods
and fungal cultures. Several reviews on the HPLC of
mycotoxins have been written (Betina, 1984, 1989b,
1993; Scott, 1981; Shepherd, 1986). However, we summarize here the progress of HPLC technique in the eld
of citrinin determination. So far, HPLC techniques have
been successfully applied to the analysis of citrinin in
grains (Lepom, 1986; Meister, 2004; Nakagawa,
Kawamura, Fujimoto, & Tatsuno, 1982; Zimmerli,
Dick, & Baumann, 1989), fungal cultures (Frisvad,
1987; Frisvad & Thrane, 1987), cheese (Franco et al.,
1996; Vazquez et al., 1996), feeds (Schneweis, Meyer,
Hormansdorfer, & Bauer, 2001), dietary supplement
red yeast rice (Meister, 2004; Xu et al., 2003) and biological uids (Orti, Hill, Liddle, Needham, & Vickers, 1986;
Phillips et al., 1980a). These HPLC methods diered signicantly in the choice of normal-phase or reversedphase columns of dierent types, elution mixtures and
gradients, detection methods and sample preparation
and purication procedures (shown in Table 3). Among
those, most chromatography were performed in the
form of reversed-phase based on acidic mobile phase
with orthophosphoric acid and uorescence detection
(FD) (Dietrich, Usleber, Martlbauer, & Gareis, 1999;
Lepom, 1986; Orti et al., 1986; Scudamore & Hetmanski, 1992; Scudamore, Hetmanski, Chan, & Collins,
1997), or UV detection (Phillips et al., 1980a); the ionpair techniques with UV detection (Nakagawa et al.,

1982) or FD (Franco et al., 1996; Vail & Homann,

1990; Vazquez et al., 1996) were applied. HPLC in normal phase mode on a buered silica gel column was also
proposed (Zimmerli et al., 1989).
3.2.2.1. HPLCUV technique. Reversed-phase HPLC
UV technique was an early method used for determination of citrinin in biological uids (Phillips et al., 1980a),
which was established based on an acidic mobile phase
with phosphoric acid. Highly reproducible retention
time and peak area could be acquired, and as little as
25 ng citrinin was detectable. Later, Frisvad developed
a general method for most known mycotoxins including
citrinin based on HPLCUV techniques (Frisvad, 1987).
However, the result showed that even using the same
type of columns, the retention times were highly variable. In succession, a general method (Frisvad &
Thrane, 1987) for mycotoxin analysis was developed,
based on HPLC with an alkylphenone retention index
and photodiode-array (PDA) detection in two dierent
eluents. The application of PDA technique allowed the
simultaneous qualitative detection and identication of
multi-mycotoxins and other fungal secondary metabolites. By analyzing the organic solvent extracts of fungal
cultures, this system was found eective for comparison
of chemotaxonomic data and for precise identication
of fungi. Based on RPHPLCUVPDA techniques,
multi-mycotoxins (including citrinin) estimation was
further developed (Kuronen, 1989) by using linear gradient elution with an acetonitrile/water solvent system.
The toxins were characterized by retention times and
on-line UV spectra produced with a diode array detector
(DAD).
Because the choice of stationary phase material was
crucial for optimizing HPLC separation, RPHPLCUV
chromatographic behavior of citrinin was studied systematically, using ve RP-materials, as a function of
the hydrophobicity and silanophilic activities of the
stationary phase (Reinhard & Zimmerli, 1999). Citrinin
showed a high anity to hydrophobic phase materials,
and its elution order depended strongly on the phase
material chosen. However, there was no recommended
column type for citrinin chromatography, i.e., successful
chromatography was strongly dependent on the interaction of stationary phase material and chosen chromatographic parameters, such as pH, type of acid in the
eluent, eluent composition and temperature, which will
be discussed in the following sections.
3.2.2.2. HPLC-uorescence technique. The natural uorescence property of citrinin under long wave ultraviolet
light permitted detection at ng/g concentrations. Recent
years, uorescence detection has almost been universally
adopted for citrinin, which showed much more sensitive
than UV absorbance detection. A comparative analysis
revealed that uorescence detection of citrinin was 100

Table 3
HPLC conditions for citrinin determination
Sample
preparation

Stationary
phase

Mobile
phase

Detection

Recovery (%);
detection limits

References

Urine and bile

None

lBondapak C18, 10 lm,

300 4 mm I.D.

Acetonitrile-isopropanol0.25 N Phosphoric acid/

(35:10:55 v/v); ow rate:
2.0 ml/min
Gradsient solvent system
consist of water-0.05%
TFA in acetonitrile; ow
rate: 2.0 ml/min at 30 C
Gradient solvent system
consist of water-0.05%
TFA in acetonitrile; ow
rate: 2.0 ml/min
Linear gradient elution
with acetonitrile-water
system
Both gradient elution and
isocratic elution was used,
methanol 0.25 M
orthophosphoric acid
(80:20, v/v); ow rate:
0.5 ml/min at 25 C
Acetonitrile-isopropanol0.08 M phosphoric
acid = 35:10:55; ow rate:
1.5 ml/min at 40 C

UV detection at
340 nm

Recovery: 95.5100;
detection limits:
25 ng

Phillips et al. (1980a)

UV detection at
254 nm

Qualitative analysis

Frisvad (1987)

UV-photodiode-array
(PDA) detection

Qualitative analysis

Frisvad and Thrane

(1987)

UV-diode-array
detection (DAD)

Qualitative analysis

Kuronen (1989)

UV DAD detection at
321nm; DAD range
was set from 200 to
400 nm; uorescence
detection kex = 331 nm,
kem = 500 nm
Fluorescence detection
kex = 330 nm

Detection limits:
100 pg

Reinhard and
Zimmerli (1999)

Recovery: 62.577.0%;
detection limits:
10 ng/g

Lepom (1986)

Fluorescence detection

Detection limits
10 ppb

Orti et al. (1986)

n-Hexane-chloroform = 60:40,
v/v; ow rate: 1.0 ml/min

Fluorescence detection

Recovery: 85.2%;
detection limits:
0.1 ng/g

Zimmerli et al.
(1989)

Acetonitrilewateracetic
acid (40:59:1) containing
tetrabutylammonium
phosphate (0.025 M)
(pH 3.8); ow rate: 2.5 ml/min
Acetonitrile-isopropanol0.75 M phosphoric
acid = 35:10:55 at 40 C

Fluorescence detection
kex = 330 nm,
kem = 500 nm

Recovery: 91102%;
detection limits:
0.1 ng

Vail and Homann

(1990)

Fluorescence detection
kex = 360 nm,
kem = 500 nm

Detection limits:
0.1 ng/g

Kpodo et al. (1995)

Fungal extracts

Nucleosil, C18, 5 lm,

Fungal extracts

Nucleosil, C18, 5 lm

Multiple
mycotoxins

C18

Citrinin

Hypersil ODS, Inertsil

ODS2, Kromasil,
Nucleosil and
Spherisorb ODS1

Wheat and barley

Sample was extract

with a acidbase
partition clean-up.

Human urine

Sample clean-up by
Solid-phase extraction
(SPE)

Cereals

Microbial
fermentations

Without sample
extraction

Fermented maize

Mycotoxins was
extracted into aqueous
acidied acetonitrile
solution then defatted
with isooctane and
puried by several
partition steps

Separon SI, C18, 10 lm

Normal-phase
LiChrospher Si
100 column, acidbuered silica gel
radial-compression
C18, 5 lm

Nova-Pak, C18,
150 3.9 mm I.D.

277

(continued on next page)

B. Xu et al. / Food Control 17 (2006) 271285

Samples

278

Table 3 (continued)
Samples

Sample
preparation

Mobile
phase

Detection

Recovery (%);
detection limits

References

Soft cheese
cheese extracts

C18 Nucleosil, 5 lm,

2504.6 mm I.D.

Fluorescence detection
kex = 331 nm,
kem = 500 nm

Detection limits:
0.9 10 7 M

Franco et al. (1996)

Soft cheese

Intertsyl ODS-2,
250 4.6 mm I.D.

Tetrabutylammonium
hydroxide in methanol,
adjusted to pH to 5.5 by
addition of aqueous
hydrochloric acid (1 M)
1 M hydrochloric acid
as an acidic post-column
reagent
An isocratic eluent
consisting of methanol
water (70:30, v/v)
containing
tetrabutylammonium
hydroxide (10 3 M),
acidied to pH 5.5 with
HCl; ow rate:
0.8 ml/min at 21 C
0.25 N Phosphoric acidacetonitrile

Time-resolved
luminescence (TRL)
detection kex = 331 nm,
kem = 545 nm

Detection limits:
2.0 10 6 M

Vazquez et al. (1996)

Fluorescence detection

Recovery: 3050%;
detection limits:
1 ng/g

Fluorescence detection

Recovery: 90%,
detection limits:
2002000 ng/g
Recovery: 71.286.3%

Scudamore and
Hetmanski (1992),
Scudamore et al.
(1997), Scudamore
et al. (1998) and
Scudamore et al.
(1998)
Kycko et al. (1998)

Raw ingredients
used for feedstus

Gel permeation (GPC)

clean-up

Monascus colour

HP20 column

Corn

LLE

LiChrospher, C18, 10 lm,

250 4.0 mm I.D.

Silages

Samples treatment by
solvent extraction
procedures, column
chromatograpy clean up.

LiChrospher, C18, 5 lm,

250 4.0 mm I.D.

Acetonitrile-water
(containing 2% formic
acid) = 2:3; ow rate:
1.5 ml/min at 30 C
Acetonitrile-0.25 N
orthophosporic
acid = 50:50, v/v; ow
rate: 1.0 ml/min

Fluorescence detection
kex = 333 nm

Fluorescence detection
Kex = 325 nm,
kem = 512 nm

Recovery: 55%;
detection limits:
2.4 ng/g

Abramson et al.
(1999)

Schneweis et al.
(2001)

B. Xu et al. / Food Control 17 (2006) 271285

Stationary
phase

Meister (2004)
Recovery: 7490%,
detection limits:
12 ng/g
Fluorescence detection
Kex = 340 nm,
kem = 495 nm

Nielsen and
Semedsgaard (2003)
Qualitative analysis
Mass system was operated
in the positive ESI mode

Polyamide column
clean up
Cereals and cereal
products

LiChrospher 100,
C18, 5 lm,
250 4.0 mm I.D,
with pre-column

Solid phase microextraction method

Fungal cultures on
agar substrates

Agilent Hypersil
BDS-C18, 3 lm,
125 2 mm I.D.

Samples were saturated

with nitrogen, then
clean-up by C8 SPE
cartridges
Samples
contaminated by
microbes

LichroCart 250-3
Purospher, RP-18

Programmed gradient
eluent consist of
methanol-aqueous buer
(10 Mm ammonium
acetate); ow rate:
0.4 ml/min at 30 C
Wateracetonitrile
gradient (TFA,
50 ll/l, in water); ow
rate: 0.3 ml/min
Programmed gradient
eluent consist of methanol,
ethylacetate, phosphoric
acid solution; ow rate:
0.4 ml/min at 30 C

Mass detector tted with

an electrospray ionization
(ESI) probe operated in
the positive ion mode

Recovery: 28%
(without adding HCl);
2% (added HCl);
detection limits:
100 ng

Tuomi et al. (2001)

B. Xu et al. / Food Control 17 (2006) 271285

279

times more sensitive than conventional ultraviolet detection (Vail & Homann, 1990). An early RPHPLCFD
coupled with solid-phase extraction (SPE) clean-up
and concentration procedure was developed for the
analysis of citrinin from hydrolyzed human urine (Orti
et al., 1986). By this method, the detection limit for
citrinin was achieved to 10 ng/g. In succession, liquid
chromatography using reversed-phase columns and
uorescence detection was successfully used to quantify
citrinin in grains (Dick, Baumann, & Zimmerli, 1988;
Lepom, 1986; Scudamore, Nawaz, & Hetmanski, 1998;
Scudamore, Nawaz, Hetmanski, & Rainbird, 1998;
Zimmerli et al., 1989), feeds (Schneweis et al., 2001;
Scudamore & Hetmanski, 1992; Scudamore et al.,
1997), cheese (Franco et al., 1996; Vazquez et al.,
1996), and fungal cultures (Franco et al., 1996; Vail &
Homann, 1990).
Selective separations were generally optimized by
using ternary or even quaternary eluent systems. Water,
methanol and acetonitrile were mostly used as a ternary
system (Table 3). The retention of citrinin depended on
the content of water, whereas the composition and ratio
of methanol and acetonitrile determined the elution
order and resolution of citrinin with other analytes.
Since citrinin was solvated much more in methanol than
in acetonitrile and the anion was non-uorescent, noted
increase in detection sensitivity of citrinin with increasing acetonitrile content of the organic modier was
achieved (Reinhard & Bernhard, 1999). Acidication
of the eluent was mandatory for chromatography because of the acidic nature of citrinin. This acidication
reduced the interactions between analytes and free
silanol groups of stationary phase. A latest testing for
evaluating of dierent HPLC mobile phases as described
in the literature resulted in citrinin peaks that were too
low with strong tailing (Meister, 2004). The eluents of
Dietrich (Dietrich et al., 1999) resulted in very small
peaks with relatively strong tailing. After injection of 1
ng citrinin (absolute amount), HPLC conditions specied by Abramson (Abramson, Usleber, & Martlbauer,
1999) did not result in any peak. The eluent of Valenta
(Valenta, 1993) (methanol/ethyl acetate/0.6 M phosphoric acid = 65/3/32, v/v) was used for optimization, which
revealed that the peak form was inuenced by the
molarity of phosphoric acid as well as by the mixing
proportion of organic and aqueous components. This
was improved by decreasing pH (Meister, 2004).
From the chemical-analytical point of view, it was recommended that the chelating ability, the eects of pH
and temperature on citrinin properties should be considered (Stolo, 1983). The chromatographic behavior of
citrinin based on RPHPLCFD technique was investigated in details, as a function of stationary phase, pH,
type of acid in the eluent, its composition as well as of
the column temperature (Reinhard & Zimmerli, 1999).
As a conclusion, like foregoing discussion, a successful

280

B. Xu et al. / Food Control 17 (2006) 271285

chromatography strongly depends on the interaction of

stationary phase material and chosen chromatographic
parameters. The higher acetonitrile content increased
sensitivity in ternary system but decreased resolution.
From the selected acids, orthophosphoric acid appeared
to be of universal applicability since it allowed detection
of citrinin and ochratoxin A at about comparable sensitivities independent of chosen RP materials.
In order to obtain ne peak forms and resolution,
RP-Ion-pair HPLC techniques were also applied for
determination of citrinin. So far, several sensitive assays
based on HPLCFD technique have been developed for
determination of citrinin in fungal cultures and cheese
extracts (Vail & Homann, 1990; Franco et al., 1996;
Vazquez et al., 1996). Owing to the acidic character of
citrinin and its partial ionization at the pH of the mobile
phase, several ion-pairing reagents, e.g., tetrabutylammonium phosphate and tetrabutylammonium hydroxide
were commonly used (shown in Table 3). These ionpairing reagents were added to the mobile phase to
make chromatography reproducible and ensure a convenient resolution (RS = 1.4) between citrinin and ochratoxin A (Vazquez et al., 1996). A rapid and sensitive
assay, based on ion-pair HPLC technique for citrinin
determination, was developed (Vail & Homann, 1990).
This assay was linear over the concentration range between 0.01 and 100 lg/ml. Recovery experiments, conducted by adding citrinin to fermentation samples,
revealed the quantitation eciency to be 91102%. Unlike previously reported methods, this procedure had the
advantage of enabling the direct quantitative analysis of
citrinin in crude microbial fermentations without extensive sample extraction protocols or pre-column concentration. Later, an improved ion pair RPHPLC coupled
with post-column uorimetric detection technique for
citrinin determination was developed (Franco et al.,
1996). Based on this technique, a highly sensitive analysis was successfully applied for determination of citrinin
in fungal cultures and cheese extracts. Spectroscopic
studies demonstrated that the uorescence of this
metabolite was inuenced by the pH of the environment.
The proposed method, based on the addition of 1 M
hydrochloric acid as an acidic post-column reagent,
had a limit of detection of 0.9 10 7 M. The repeatability and reproducibility were satisfactory, with
R.S.D. = 5.1% (n = 9, c = 10 5 M). In the meantime, another ion-pair RPHPLC procedures coupled with postcolumn technique and time-resolved luminescence
(TRL) detection were developed (Vazquez et al., 1996)
for simultaneous determination of citrinin and ochratoxin A in soft cheese. Prior to detection, a butanolic
solution of 5 10 3 M terbium 5 10 4 M trioctylphosphine oxide (TOPO) 2.5 10 2 M triethylamine
(TEA) was pumped in a post-column mode at a owrate of 0.2 ml/min to perform TRL detection of the corresponding terbium chelates. The method was linear

from 3.5 10 6 to 2 10 5 M of citrinin. The repeatability and reproducibility of citrinin were 1.9 and
2.4% (n = 10). By the proposed method, citrinin was
easily determined in soft cheeses, with a signicative increase in selectivity in comparison with direct uorescence detection.
Reversed-phase ion-pair HPLC provided good peak
forms, while the native uorescence of citrinin was
partly lost. Thereby the sensitivity and selectivity of this
detection method was diminished. This drawback had
been overcome by acidifying the eluate of HPLC column
before entering the uorescence detector. RPHPLC is
widely used because of its advantages over conventional
normal-phase HPLC. However, a normal-phase HPLC
procedure, based on an acid-buered silica gel column
chromatographic separation and uorescence detection
(Zimmerli et al., 1989), was successfully applied for
determination of citrinin in soft wheat, wheat bran, rice,
barley, corn and so on. A limit of quantication of
about 0.1 ng/g was achievable. The modied silica gel
was a remarkable alternative, especially in trace analysis
of very polar compounds. However, the disadvantage of
this technique was low reproducibility of chromatographic behavior on retention time. Because of prolonged use of the column, the retention time of citrinin
became shorter and shorter, the number of theoretical
plates decreased.
Some attempts were done for improving chromatographic behavior of citrinin on HPLC by using various
extraction methods and clean-up procedures, however,
those methods did not provide satisfying results. Generally, satisfying sensitivity was counteracted poor or variable recovery. While, the method of Abramson et al.
(1999) of extract clean-up with liquidliquid extraction
(LLE) cartridges (lled with an diatomaceous-earth
adsorbent) resulted in signicantly better recovery.
Recovery to the extent of 78% was achieved for standard
solution and 7585% for spiked wheat extract. However,
the chromatograms of rye could not be quantied, due
to interfering peaks. TLC method was not found useful
as the pretreatment procedure of HPLCFD analysis,
due to low recovery rate. When a powdered sample
was subjected, the Rf value of citrinin was inuenced
by co-existing dextrin. Using eight kinds of solid-extraction cartridges for citrinin extraction was also not eective. However, pretreatment with the Daiaion HP20
column was satisfactory for citrinin extraction. The
recovery of citrinin was more than 90%. Moreover, the
detection limits of HP20 column pretreatment-HPLC
method were 0.2 lg/g for liquid samples and 2 lg/g
for powdered samples (Kycko, Shiho, Yoko, & Tamio,
1998). More recently, a novel HPLC method was established with sensitive uorescence detection, based on
solid phase extraction in combination with a gradient
eluent (Meister, 2004). The samples were extracted with
dichloromethane with addition of phosphoric acid, and

B. Xu et al. / Food Control 17 (2006) 271285

the extract was cleaned up on polyamide columns. Following this method, citrinin was detected to the extend
of 12 ng/g in the cereals and milling products. The
mean recovery rates were 7490%. Suitable for determination of citrinin in cereal products, this method was
highly applicable to red yeast rice, which was regarded
as a dicult matrix because of very high content of pigment compositions.
Although these RPHPLCFluorescence detection
methods aorded relative good sensitivity and recovery,
in practice, application of all these methods to various
complex matrices was tedious and time-consuming.
Lengthy clean-up procedures were generally necessary,
and sometimes decient in specicity. Some problems
still existed, such as low reproducible LC retention times
when normal-phase columns were used (Dick et al.,
1988; Zimmerli et al., 1989), decreased sensitivity and
accuracy resulting from stability of citrinin in organic
eluents, ion-pair reagents and acid environment.
3.3. Chromatography and mass spectrum combination
technique
3.3.1. LCMS technique
A HPLCMS analysis method for determining citrinin was established (Tuomi, Johnsson, Hintikka, &
Reijula, 2001), which included extraction, sample pretreatment and reversed-phase HPLC separation with
MS identication and quantication using electrospray
ionization on a quadrupole ion trap mass analyser
(ESIMSMS). Mass spectral analysis was performed
on a Finnigan LCQ tted with an electrospray ionisation (ESI) probe in the positive ion mode. Aqueous
methanol was used in the initial extraction, solvent partition and solid phase extraction in the purication of
samples. The HPLC separation was run on-line with
the ESI-MS-MS detection. The limit of quantication
of the procedure was 200 ng for all compounds. Recoveries of the sample pre-treatment varied from 28 to 99%.
The average compound- and concentration-dependent
accuracy and precision (RSD) were 21 and 113%,
respectively. In addition, a standardized LCUVMS
micro-scale method for screening of fungal metabolites
and mycotoxins in culture extracts was presented, the
database of 474 mycotoxins including citrinin was established (Nielsen & Semedsgaard, 2003).
3.3.2. GCMS technique
Historically, gas chromatography (GC) was introduced in the eld of mycotoxins in the early 1970s. If
mycotoxins are suciently volatile at the column temperature, or can be converted into volatile derivates,
GC technique can be used for their determination. Recently, a new method for the qualitative and quantitative
analysis of citrinin in Monascus by gas-chromatographyselected ion monitoring (SIM) mass spectrometry was

281

developed (Shu & Lin, 2002). GC separation of citrinin

in Monascus extract was achieved without the need for
chemical derivatization, and could be detected as a single
peak when the SIM mode selected ve prominent fragmentations (m/z of 220, 205, 177, 105 and 91). The quantitative detection limit for citrinin was approximately 1
ppb. Finally, the GC-separated analyte from Monascus
extract, at a retention time of 10.89 min, was studied
by the method of pattern recognition by comparison
with a citrinin standard. Citrinin could be detected in
all the six commercial Monascus samples at concentration varying between 4.2 and 25.1 mg/kg.
3.4. Bioassay technique
In general, the above-mentioned physicochemical
detection methods required a tedious sample extract
and clean-up with large volumes of halogenated solvents. Besides, there is loss of citrinin during sample
treatment, instable chromatographic behavior of citrinin, or relative low sensitivity and recovery. Therefore,
bioassays have become increasingly useful for mycotoxin detection (Watson & Lindsay, 1982; Yates, 1986)
as a precursor of chemical analysis. Bioassay provided
a rapid means for screening samples and allowed the
analyst to make an informed decision. Immunochemical
methods provided a convenient and sensitive alternative
for detecting many mycotoxins (Chu, 1991). So far,
immunoassay techniques have been devised for many
mycotoxins as a rapid alternative to chromatography.
3.4.1. Enzyme immunoassay technique
Although enzyme immunoassay (EIA) methods for
major mycotoxins have been known for many years,
only recently, EIAs for citrinin determination has been
reported in wheat (Abramson, Usleber, & Martlbauer,
1995), barley (Abramson, Usleber, & Martlbauer,
1996), corn (Abramson et al., 1999), red-fermented rice
(Dietrich et al., 1999; Heber et al., 2001), and other cereals (Vrabcheva et al., 2000).
An indirect competitive EIA (Abramson et al., 1995)
using rabbit antisera was rstly performed to detect
citrinin in buer solutions at 113 ng/ml (0.050.65 ng
per assay). By using an indirect assay format, a simple
and reliable procedure with a low level of intraplate color intensity variation was established. Recovery of citrinin added to wheat our at 2002000 ng/g was 89104%,
with a coecient of variation of 6.913%. In succession,
an indirect and a direct EIA were simultaneously applied to determination of citrinin in barley (Abramson
et al., 1996). Polyclonal antibodies against the mycotoxin citrinin were raised in rabbits after immunization
with citrinin conjugated to keyhole limpet hemocyanin.
The antibodies were used in a competitive indirect EIA
with citrinin coupled to glucose oxidase as the solidphase antigen for coating microtiter plates. Detection

282

B. Xu et al. / Food Control 17 (2006) 271285

limits of this indirect EIA for citrinin ranged from 0.4 to

0.8 ng/ml for buer solutions. Recoveries of citrinin
added to ground barley at 1002000 ng/g ranged from
105 to 112%, with coecients of variation between 4.5
and 12%. A direct competitive EIA also was established,
with citrinin coupled to horseradish peroxidase as the
labeled antigen. Detection limits of this direct EIA for
citrinin ranged from 2 to 4 ng/mL for buer solutions.
Recoveries of citrinin added to ground barley at 500
2000 ng/g ranged from 108% to 111%, with coecients
of variation between 8.4% and 26.9%. Later, a rapid
assay procedure for mycotoxin citrinin in corn using
liquidliquid extraction (LLE) cartridges for samples
clean-up was developed (Abramson et al., 1999). An
indirect EIA was evaluated, using sodium carbonate
solution for extraction. Recoveries of citrinin added to
ground corn at 2002000 ng/g ranged from 53.2 to
67.2%, but the coecients of variation varied between
18.4 and 51.5%. The indirect EIA produced much lower
recoveries and much higher coecients of variation
(CV) gures at all spiked levels except 2000 ng/g. The
EIA method underwent some unsuccessful trail modication in search of higher recoveries at the 1000 and
2000 ng/g. These indicated that the sensitivity of the
indirect EIA, using polyclonal antibodies, would probably not be sucient to assay citrinin in corn at levels below 2000 ng/g. More recently, a competitive EIA for
determination of citrinin concentration in dietary supplements was developed (Heber et al., 2001). Detection
limit of 15 lg citrinin per gram solid fermentation preparation was achieved. The results suggested that citrinin
(with concentration range at 0.4711.82 lg/capsule) was
found at measurable concentrations in 7 of the 9 dierent commercially available dietary supplements.
Although EIAs in the ppb-range were developed for
citrinin determination, due to cross reactions, EIA frequently gave analytical results that were too high. So
the results of EIA must be conrmed by other analytical
methods such as sensitive HPLC. The antibodies developed for EIA were also used for self-made immuno
anity columns for HPLC extract clean-up (Dietrich
et al., 1999; Vrabcheva, Usleber, Dietrich, & Martlbauer, 1999).

4. Conclusion and perspective

This review was written with the aim of demonstrating the scope of various analytical techniques in citrinin
detection and estimation. In most instances, citrinin to
be analysed was present in contaminated samples.
Hence, citrinin must be extracted and cleaned-up prior
to TLC, HPLC, GC or immunoassay if reliable results
were to be obtained. Extraction procedures included
extractions of mycotoxins from feeds, foodstus, cultivation media or mycelia of toxigenic fungi, and body

uids or tissues. In the early 1980s, TLC was the most

widely used chromatographic technique applied to citrinin owing to its relative simple, fast and inexpensive
properties. The several disadvantages of TLC analysis
included its lack of potential for automation, low sensitivity and the subjective nature of the quantitation steps.
Using densitometer overcame the latter limitation but at
a cost equivalent to that of a single HPLC system.
The rapidly increasing applications of the HPLC techniques showed their inherent advantages over other chromatographic techniques and it seemed destined to
supplant the other methods in the routine analysis of
citrinin. Among the available detectors, the one most frequently used was the variable-wavelength detector (e.g.
PDA or DAD), which had a particular application in
the eld of co-occurrence of citrinin with other mycotoxins. The main alternative to UV detector is the uorescence detector, which is more sensitive than UV
detectors and has been used in the detection of citrinin.
HPLCMS could exert its advantages for trace level
detection and conrmation, especially for complex matrices. In addition, HPLC with evaporative light scattering
detection (HPLCELSD) and with thermospray interfacing chromatography (HPLCTSP) were commercially
available systems. The ELSD for HPLC is relatively new
technique, but the ELSD was not a universal detector,
and its application for citrinin analysis needs to be researched. While TSP technique has been successfully applied to determination of other mycotoxins in grain
(Rajakyla, Laasasenaho, & Sakkers, 1987), the utility
of this technique for citrinin determination needs to be
explored. Till now, it is not reported that countercurrent
chromatography (CCC), supercritical uid chromatography (SFC), and capillary electrophoresis (CE) techniques
were applied in the elds of citrinin determination,
although these have been successfully applied to determine natural compounds. There remains some bottle
necks for analyst to develop more sensitive methods.
Comparing to HPLC, GC owns higher sensitivity and
could be eectively coupled with mass spectrometry to
obtain qualitative data concerning the identity of citrinin
and its derivates. Immunoanity clean-up techniques
with high-resolution chromatography showed particular
promise for mycotoxin. Recently, advances using tandem
or mixed selectivity immunoanity cartridges demonstrated the feasibility of multi-target mycotoxin assays.
In a nutshell, the above-mentioned methods have
their advantages and disadvantages, and selection of
qualitative or quantitative analytical methods depends
on analytical aim, sample properties, and environmental
conditions. The properties of dierent available methods
are summarized in Table 4, which oers references for
the appropriate choice of analytical method depending
on the analytical task.
Although some work on qualitative and quantitative
analysis of citrinin has been done, rapid and sensitive

B. Xu et al. / Food Control 17 (2006) 271285

283

Table 4
Summary of analytical methodology of mycotoxin citrinin
Quantitation

Detection limit

Recovery (%)

Fluorimetric assay

1008000 lg/kg

4066

TLC
RPHPLCUV
RPHPLCFD

1520 lg/kg
25 ng
12 lg/kg
2002000 lg/kg
10 lg/kg
1 lg/kg
2.4 lg/kg
10 lg/kg
0.1 ng
2.0 10 6 M
0.1 g/kg
100 ng
1 lg/kg
24 ng/ml
0.413 ng/ml
200 lg/kg

33.396
95.5100
7490
90%

Normal-phase-HPLCFD
HPLCMS
GCMS
EIA

Technical characteristic

Clean-up

Polyamide column clean-up

Daiaion HP20 column
C18-SPE
GPC
Silica column chromatograpy
Acidbase partition
Ion-pair HPLCFD
Ion-pair HPLCTRL
Acid-buered silica gel
ESI-MS/MS
SIMMS
Direct EIA
Indirect EIA

C8-SPE
Without derivatization

LLE cartrige

quantitative analysis methods needed to be established.

On account of the instability of citrinin and trace properties in foodstu, its analysis remains a challenge. In
order to increase the security of food and promote circulation of foodstu in the international market, suitable
analytic criteria need to be established.

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