Ochratoxin A

O OH
O OH O

N O
H

Cl

N-{ [(3R)-5-chloro-8-hydroxy-3-methyl-1-oxo-3,4-dihydro-1H-isochromen-7-yl]carbon
yl}-
L-phenylalanine
C20H18ClNO6 MW: 403.81 CAS No.: 303-47-9

[Summary of ochratoxin A]
Ochratoxin is a kind of mycotoxin discovered by a research group in South Africa
and is a carcinogenic substance.
Reported ochratoxin producers include genus Aspergillus, Aspergillus ochraceus in
particular, and Penicillium such as Penicillium viridicatum. These ochratoxin
producers are widely distributed in foods such as cereals.
Six metabolites as shown in Figure 5.1.18-1 are known to be the toxic components of
ochratoxin, and usually ochratoxin A is viewed as the problem because its toxicity in
animals is more potent than the other metabolites.

O OR2
O OH O

N O
H

R1
 R1 R2 R1 R2
Ochratoxin A Cl H Ochratoxin A methyl ester Cl CH3
Ochratoxin B H H Ochratoxin B methyl ester H CH3
Ochratoxin C Cl C2H5 Ochratoxin B ethyl ester H C2H5

Figure 5.1.18-1 Structural formulas of ochratoxins

[Methods listed in the Feed Analysis Standards]

1 Liquid chromatography (1) [Feed Analysis Standards, Chapter 5, Section 1
18.1]
Scope of application: Grains

A. Reagent preparation
Ochratoxin A standard solution. Weigh accurately 5 mg of ochratoxin A
[C20H18NO6Cl], transfer to a 25-mL amber volumetric flask, dissolve by the
addition of methanol, and add the same solvent up to the graduation line to prepare
the ochratoxin A standard stock solution (1 mL of this solution contains 0.2 mg as
ochratoxin A).
Before use, dilute accurately a certain amount of the standard stock solution with
acetonitrile/ water/ acetic acid (300:700:1) to prepare several ochratoxin A
standard solutions that contain 0.5-20 ng of ochratoxin A in 1 mL.

B. Quantification
Extraction. Weigh 25.0 g of an analysis sample, transfer it to a 200-mL stoppered
Erlenmeyer flask, add 100 mL of acetonitrile/ water/ acetic acid (84:16:1), and
extract by shaking for 30 minutes. Transfer the extract to a stoppered centrifuge
tube, centrifuge at 1,000×g for 5 minutes, to obtain supernatant as a sample
solution to be subjected to column treatment.
Column treatment. Load the sample solution on a multifunctional column (for
ochratoxin pretreatment), Note 1 and discard the first 1 mL of eluate, then collect the
following 3 mL of eluate into a 10-mL test tube. Transfer accurately 2 mL of this
solution to another 10-mL test tube, evaporate under vacuum in a water bath at
50°C or less to be almost dried up, and then dry up by nitrogen gas flow.
Add accurately 1 mL of acetonitrile/ water/ acetic acid (300:700:1) to dissolve the
 residue. Transfer this solution to a plastic centrifuge tube (volume: 1.5 mL),
centrifuge at 5,000×g for 5 minutes, to obtain supernatant as a sample solution to
be subjected to liquid chromatography.
Liquid chromatography. Inject 50 µL each of the sample solution and the respective
standard solutions to a liquid chromatograph to obtain chromatograms.
Example of measurement conditions
Detector: Fluorescence detector (excitation wavelength 385 nm;
fluorescence wavelength 444 nm)
Column: Octadecylsilyl silica gel column (4.6 mm in inner diameter, 250
mm in length, particle size 5 µm) Note 2
Eluent: Acetonitrile/ water/ acetic acid (500:500:1)
Alkalizing solution Note 3: Sodium hydroxide solution (0.1 mol/L)
Flow rate: Eluent, 1.0 mL/min; alkalizing solution, 0.3 mL/min
Column oven temperature: 40°C
Calculation. Obtain peak heights or areas from the resulting chromatograms[1] to
prepare a calibration curve, and calculate the amount of ochratoxin A in a sample.
Note 1 MultiSep 229 Ochra (Romer Labs) or equivalents
2 Inertsil ODS-2 (GL Sciences) or equivalents
3 Using a PEEK mixing tee etc., mix the eluate eluted from the column and
the alkalizing solution, and then send to a fluorescence detector.

<<Summary of analysis method>>

In this method, ochratoxin A in grains is extracted with acetonitrile/ water/ acetic
acid (84:16:1), purified with a multifunctional cleanup (MFC) column, and
quantitated by a liquid chromatograph with post-column alkalization and a
fluorescence detector.
Ochratoxin A can be detected sensitively because its fluorescence intensity increases
under alkaline conditions.
The flow sheet of the analysis method is shown in Figure 5.1.18-2.
 25.0 g sample
100 mL Acetonitrile/ water/ acetic acid (84:16:1)
Shake for 30 minutes
Centrifuge (1,000x g (2,000 rpm), 5 minutes)
MultiSep 229 Ochra
Load the supernatant
Discard the first 1 mL of eluate
2 mL eluate
Evaporate under vacuum, dry up
1 mL Acetonitrile/ water/ acetic acid (300:700:1)
Centrifuge (5,000x g (10,000 rpm), 5 minutes)
LC-postcolumn alkalization-FL (Ex. 385 nm; Em. 444 nm)
Figure 5.1.18-2 Flow sheet of the analysis method for ochratoxin A by liquid
chromatography

References: Kouji Aoyama, Eiichi Ishiguro, Naoki Kanemaru, and Masatoshi

Minamisawa: Research Report of Animal Feed, 31, 109 (2006)
History in the Feed Analysis Standards [28] New

<<Analysis method validation>>

• Spike recovery and repeatability

Spike
Spike recovery Repeatability
Sample type concentration Repeat
RSD (% or
(µg/kg) (%)
less)
Corn 1~20 3 95.8~121.3 0.4
Barley 1~20 3 96.4~118.0 2.5
Rye 1~20 3 95.9~106.2 1.6

• Collaborative study
Spike Spike recovery Intra-laboratory Inter-laboratory
Number of
Sample concentration (%) repeatability reproducibility
laboratorie HorRat
type (measured
s (µg/kg) RSDr (%) RSDR (%)
value (µg/kg))
Flour 7 5 98.2 3.7 8.8 0.40
Natural
Wheat 7 (3.83) 7.1 11.0 0.50
contamination

• Lower limit of quantification: 1 µg/kg in samples (Spike recovery 106-121 %,

repeatability 2.5 % or less)
<<Notes and precautions>>
[1] Examples of chromatograms are shown in Figure 5.1.18-3.
(Reference) Ochratoxin B Ochratoxin A
Retention time/ min

(A)
（参考）オクラトキシンB
(Reference) Ochratoxin B オクラトキシンA
Ochratoxin A
Fluorescence

intensity

0 2 4 6 8 10 12 14

Retention Time/min

(B)
Fluorescence

intensity

0 2 4 6 8 10 12 14

Retention Time/min

(C)
Fluorescence

intensity

0 2 4 6 8 10 12 14

Retention Time/min

Figure 5.1.18-3 Chromatograms of ochratoxin A

Measurement conditions were according to Example of measurement condition.
Column used was GL Sciences Inertsil ODS-2.
(A) Standard solution (0.5 ng/mL)
(B) Corn (spiked with 1 µg/kg equivalent)
(C) Barley (spiked with 1 µg/kg equivalent)
2 Simultaneous analysis of ochratoxin A and citrinin by liquid
chromatography [Feed Analysis Standards, Chapter 5, Section 1 18.2]
Analyte compounds: Ochratoxin A and citrinin (2 components)
Scope of application: Grains and formula feeds (except formula feeds for citrinin)
 A. Reagent preparation
1) Ochratoxin A standard solution. Weigh accurately 5 mg of ochratoxin A
[C20H18NO6Cl], put in a 25- mL amber volumetric flask, dissolve by the addition
of methanol, and further add the same solvent up to the graduation line (1 mL of
this solution contains 0.2 mg as ochratoxin A.). Moreover, dilute accurately a
certain amount of this solution with methanol, to prepare the ochratoxin A
standard stock solution that contains 1 µg as ochratoxin A in 1 mL.
2) Citrinin standard solution. Weigh accurately 5 mg of citrinin [C13H14O5], put in a
25- mL amber volumetric flask, dissolve by the addition of methanol, and further
add the same solvent up to the graduation line (1 mL of this solution contains 0.2
mg as citrinin.). Moreover, dilute accurately a certain amount of this solution
with methanol, to prepare the citrinin standard stock solution that contains 1 µg
as citrinin in 1 mL.
3) Mixture standard solution. Before use, mix a certain amount of ochratoxin A and
citrinin standard stock solutions, dilute accurately with acetonitrile- water (1:1),
to prepare several mixture standard solutions that contain 1-50 ng as ochratoxin
A and citrinin, respectively, in 1 mL.

B. Quantification
Extraction. Weigh 25.0 g of an analysis sample, transfer it to a 200- mL stoppered
Erlenmeyer flask, moisten by the addition of 100 mL of acetonitrile - hydrochloric
acid - water (8:1:1), then leave at rest for 5 minutes, Note 1 and further extract by
shaking for 30 minutes. Filter the extract with filter paper (No. 5A), transfer
accurately 5 mL of the filtrate to a 50- mL recovery flask, and concentrate under
vacuum to be 1 mL or less at 40°C or less. Further, remove acetonitrile by a mild
flow of nitrogen gas, [1] to be a sample solution to be subjected to purification.
Purification. Add about 0.5 g of sodium chloride to the sample solution, then add
accurately 10 mL of ethyl acetate, mix well, transfer to a 10- mL test tube, and
centrifuge at 1,500×g for 5 minutes. Transfer accurately 5 mL of the ethyl acetate
layer (upper layer) to a 50- mL recovery flask, concentrate under vacuum at 40°C
or less to be almost dried up, and then dry up by nitrogen gas flow.
Dissolve the residue by the addition of accurately 2 mL of acetonitrile- water (1:1).
Transfer this solution to a filter cup with ultrafiltration membrane (molecular
weight cutoff: 30,000) Note 2 that is attached to a plastic centrifuge tube (capacity:
1.5 mL) in advance, centrifuge at 5,000×g for 15 minutes, to obtain filtrate to be a
 sample solution to be subjected to column treatment.
Liquid chromatography. [2] Inject 20 µL each of the sample solution and respective
mixture standard solutions to a liquid chromatograph to obtain chromatograms.
Example of measurement conditions
Detector: Fluorescence detector (excitation wavelength, 335 nm; emission
wavelength, 480 nm)
Column: Octadecylsilyl silica gel column (4.6 mm in inner diameter, 250
mm in length, particle size 5 µm) Note 3[3][4]
Eluent: Acetonitrile- water/ 1 v/v% phosphoric acid (230:230:1)
Flow rate: 1.0 mL/min
Column oven temperature: 40°C
Calculation. Obtain peak heights or areas from the resulting chromatograms [5][6]to
prepare a calibration curve, and calculate the amounts of ochratoxin A and citrinin
in the sample.
Note 1 Leave at rest until it stops foaming.
2 Use Microcon YM-30 (Millipore) or equivalents.
3 Inertsil ODS-2 (GL Sciences) or equivalents.

<<Summary of analysis method>>

This is an analysis for ochratoxin A and citrinin in the case of grains, and for
ochratoxin A in the case of formula feeds.

Mycotoxins in a sample are extracted with acetonitrile acidified with hydrochloric

acid. After the solvent is replaced, the solution is passed through ultrafiltration filter
(molecular weight cutoff: 30,000), and quantitated by a liquid chromatograph with a
fluorescence detector.

The flow sheet of the analysis method is shown in Figure 5.3.9-1.

 25.0 g sample
100 mL Acetonitrile - hydrochloric acid - water (8:1:1)
Leave at rest for 5 minutes.
Shake for 30 minutes.
Filter with filter paper (5A)
5 mL Filtrate
Concentrate to be 1 mL or less
Mild nitrogen gas flow
Add 0.5 g sodium chloride and 10 mL ethyl acetate, and mix
Centrifuge (1,500x g (3,000 rpm), 5 minutes)
5 mL Upper layer
Concentrate under vacuum, and dry up
2 mL Acetonitrile- water (1:1)
Ultrafiltration membrane (molecular weight cutoff: 30,000),
Centrifuge (5,000x g (10,000 rpm), 15 minutes)
LC-FL (Ex, 335 nm; Em, 480 nm)

Figure 5.3.9-1 Flow sheet of the simultaneous analysis method for ochratoxin
A and citrinin

References: Koji Aoyama, Kaori Morifuji, Eiichi Ishiguro: Research Report of

Animal Feed, 29, 11 (2004)
History in the Feed Analysis Standards [27] New

<<Analysis method validation>>

• Spike recovery and repeatability
Spike
Name of spiked Repeatability
Sample type concentration Repeat Spike recovery (%)
component RSD (% or less)
(µg/kg)
Ochratoxin A Formula feed for adult chickens 10~100 3 96.3~97.1 12.6
Formula feed for pork pig fattening 10~100 3 98.6~103.4 13.6
Corn 10~100 3 105.6~106.5 8.9
Barley 10~100 3 98.2~103.8 10.2
Citrinin Formula feed for adult chickens 100~400 3 85.0~86.2 3.0
Formula feed for pork pig fattening 100~400 3 87.5~88.7 3.0
Corn 100~400 3 77.2~90.8 12.5
Barley 100~400 3 83.0~84.0 7.4

• Collaborative study
 Spike Spike recovery (%) Intra-laboratory Inter-laboratory
Name of analyzed Number of
Sample type concentration repeatability RSDr reproducibility HorRat
component laboratories (measured value
(µg/kg) (%) RSDR (%)
(µg/kg))

Ochratoxin A Pig formula feed 11 20 100.4 3.3 7.9 0.36

Natural
Milo 11 (18.6) 20.6 23.5 1.07
contamination
Citrinin Pig formula feed 11 200 83.6 6.1 11.8 0.58

• Lower limit of quantification: 5 µg/kg for ochratoxin A (Spike recovery

93.9~100.7 %, relative standard deviation 23.3 % or less, SN ratio 10), citrinin 20
µg/kg (Spike recovery 90.0~94.9 %, relative standard deviation 19.2 % or less, SN
ratio 10)

<<Notes and precautions>>

[1] Recovery of citrinin becomes poorer if it is dried up in this step. When it
happens to be dried up, add about 1 mL of 6 mol/L hydrochloric acid before
analysis.
[2] Conduct measurement after removing methanol by sufficiently flowing the
eluent, etc., when methanol remains in the column. The citrinin peak may
become broad if methanol remains.
[3] In addition to Inertsil ODS-2, Shodex C18M 4E (Showa Denko) can be used.
[4] The order of elution of ochratoxin A and citrinin may vary even if the same
column is used.
[5] Examples of chromatograms are shown in Figure 5.3.9-2.
 OTA
(A)
CIT

Fluorescence intensity

OTA
(B) CIT
Fluorescence intensity

0 5 10 15 20
Retention time/min

Figure 5.3.9-2 Chromatograms of ochratoxin A and citrinin

(OTA, peak of ochratoxin A; CIT, peak of citrinin.)
LC conditions are according to the Example of measurement conditions. The
column is Inertsil ODS-2 (GL Sciences).
(A) Barley (spiked with amounts equivalent to 400 µg/kg as citrinin, and 100
µg/kg as ochratoxin A)
(B) Formula feeds for pork pig fattening (spiked with amounts equivalent to 400
µg/kg as citrinin, and 100 µg/kg as ochratoxin A)

[6] Peaks can be identified by changing the acid concentration in the eluent,
emission wavelength, etc.
3 Liquid chromatography (2) [Feed Analysis Standards, Chapter 5, Section 1
18.3]
Scope of application: Grains and formula feeds

A. Reagent preparation
Ochratoxin A standard solution.[1] Weigh accurately 5 mg of ochratoxin A
[C20H10ClNO6], [2] put in a 250-mL amber volumetric flask, dissolve by the
addition of acetonitrile, and add the same solvent up to the graduation line to
prepare the ochratoxin A standard stock solution (1 mL of this solution contains 20
µg as ochratoxin A).
Before use, dilute accurately a certain amount of the standard stock solution with
acetonitrile/ dilute phosphoric acid Note 1 (11:9) to prepare several ochratoxin A
standard solutions that contain 0.01-0.05 µg of ochratoxin A in 1 mL.

B. Quantification
Extraction. Weigh 50 g of an analysis sample, transfer it to a 500-mL separatory
funnel, moisten the sample by the addition of 25 mL of acetic acid (1:19), add 250
mL of chloroform 250 mL, and extract by shaking for 30 minutes.
Transfer the extract to a stoppered centrifuge tube, and centrifuge at 1,500×g for 5
minutes. Transfer the chloroform layer (lower layer) to an Erlenmeyer flask,
dehydrate with a suitable amount of sodium sulfate (anhydrous), and filter with
filter paper (2 types).
Transfer 50 mL of the filtrate[3] to a 100-mL recovery flask, evaporate under
vacuum in a water bath at 40°C or less to be almost dried up, and then dry up by
nitrogen gas flow. [4] Add 2 mL of toluene to dissolve the residue, to be a sample
solution to be subjected to column treatment.
Column treatment.[5] Wash a silica gel mini column (690 mg) with 10 mL of toluene.
Load the sample solution to the mini column, and wash the recovery flask that
contained the sample solution twice with 2 mL each of toluene, and add the
washing to the mini column, and elute by pressurized flow. Note 2 Moreover, add 10
mL of toluene and 6 mL of chloroform/ methanol (97:3) sequentially to the mini
column, and elute by pressurized flow. Note 2
Place a 50-mL recovery flask under the mini column. Add 10 mL of toluene/ acetic
acid (9:1) to the mini column, and pressurize Note 2 to elute ochratoxin A. Evaporate
the eluate under vacuum in the water bath at 40°C or less to be almost dried up,
 and further dry up by nitrogen gas flow. Add accurately 2 mL of acetonitrile/ dilute
phosphoric acid Note 1 (11:9) to dissolve the residue, filter with membrane filter
(pore size 0.5 µm or less), to obtain a sample solution to be subjected to liquid
chromatography.
Liquid chromatography. Inject 20 µL each of the sample solution and respective
ochratoxin A standard solutions to a liquid chromatograph to obtain
chromatograms.
Example of measurement conditions
Detector: Fluorescence detector (excitation wavelength 337 nm, fluorescence
wavelength 467 nm)
Column Octadecylsilyl silica gel column (4.6 mm in inner diameter, 250 mm
in length, particle size 5 µm) Note 3 [6]
Eluent: Acetonitrile/ dilute phosphoric acid Note 1 (11:9)
Flow rate: 1.0 mL/min
Calculation. Obtain peak heights or areas from the resulting chromatograms[7] to
prepare a calibration curve, and calculate the amount of ochratoxin A in a sample.
Note 1 Phosphoric acid (1:1,000)
2 Adjust the flow rate to be 2-3 mL/min.
3 UNISIL PACK 5C18 (GL Sciences) or equivalents

<<Summary of analysis method>>

In this method, ochratoxin A in a sample is extracted with chloroform, purified with a
silica gel mini column, and quantitated by a liquid chromatograph with a
fluorescence detector.

References: Masayuki Shimomura and Eiichi Ishiguro: Research Report of Animal

Feed, 14, 1(1989)
History in the Feed Analysis Standards [11] New

<<Notes and precautions>>

[1] Accurate concentration testing for the ochratoxin A standard stock solution is
stipulated in Section 4 in this chapter.
[2] 10 µg/mL Ochratoxin A standard solution is commercially available from Kanto
Chemical, etc.
[3] Pressurizing the sample solution to flow in column treatment may be difficult in
 analysis of formula feeds etc. In that case, adjust the solution volume to about 25
mL.
[4] Eliminate the odor of acetic acid completely.
[5] The flow rate and elution rate is sufficient at around 5 mL per minute. In addition,
make sure that there is no bubble getting inside during column treatment.
[6] An example of chromatograms for ochratoxin A is shown in Figure 5.1.18-4.
[7] The column to be used only needs to be one that uses packing treated by
corresponding endcapping. The column used in the development of this analysis
method was UNISIL PACK 5C18.

Measurement conditions
Column: UNISIL PACK 5C18
Fluorescence intensity

Column temperature: 40°C

Eluent: Acetonitrile -phosphoric
acid solution (0.1 v/v%)
(11:9)
Flow: 1.0 mL/min
Detector: Hitachi
Retention Time/min
spectrofluorometer
650-10LC Ex: 337 nm,
Em: 467 nm
Figure 5.1.18-4 Chromatogram of ochratoxin A
[Other analysis methods]
4 ELISA
Products manufactured by Neogen (available from AR Brown, Kikkoman, etc.),
R-Biopharm Rhône (available from AZmax, etc.), etc. are available in Japan from
several distributors .
Table 5.1.18-1 shows major kits commercially available now in Japan, and their
summaries.
Table 5.1.18-1 ELISA kits for ochratoxin A analysis commercially available in Japan
Lower
Analysis Shelf life of
Item limit of Applicable samples Notes
time the kit
detection
®
Veratox for 1 ppb 20 6 months Corn, coffee beans, Neogen
Ochratoxin minutes bran
®
RIDASCREEN 5 ppb 25 6-9 months Grains, feeds, foods R-Biopharm Rhône
FAST minutes
Quantitation kit

Ochratoxin A
AgraQuant® 2 ppb 25 6-9 months Grains Romer Lab
Och minutes
Charm ROSA® 2 ppb 12- 25 6-9 months Grains, wine, grape Charm Science
Ochratoxin minutes juice
™
Max Signal 0.15 ppb 25 6-9 months Grains, feeds, nuts BIOO Scientific
minutes and seeds, milk