49.5.

01
AOAC Official Method 973.37
Ochratoxins in Barley
Thin Layer Chromatographic Method
First Action 1973
Final Action 1988

IUPAC-AOAC Method

(Ochratoxin A causes kidney and liver damage and is carcinogenic

in some animals. Observe precautions given in introductory statement of this chapter. See also Appendix B, safety notes on distillation, hazardous radiations, benzene, chloroform, and hexane.)
(Quantitative for ochratoxin A, qualitative for B and for esters of A
and B.)
A. Principle

Ochratoxin acids and esters are extracted from barley by CHCl3aqueous H3PO4. Acids are entrapped on aqueous NaHCO3-diatomaceous earth column, esters and fat are removed with hexane and
CHCl3, and acids are eluted with HCOOH-CHCl3. Esters are isolated
by entrapment on methanol-aqueous NaHCO3-diatomaceous earth
column, fats are removed with hexane-benzene, and esters are eluted
with HCOOH-hexane-benzene. Compounds are determined from
fluorescence intensity on thin layer chromatograms.
All glassware must be free of alkaline soap or detergent residues
to avoid loss of the toxins from neutral solvent by salt formation,
precipitation, and/or adsorption onto glassware.
B. Apparatus

(a) Chromatographic tubes.700 17 (od) mm and 350 25

mm, with stopcocks.
(b) Bchner funnels.Glass, 9 cm diameter fitted with Whatman
GF/B glass fiber paper, or equivalent; 24 cm diameter fitted with
Whatman No. 1 paper, or equivalent.
(c) Thin layer chromatographic apparatus.See 970.43A(k)
(see 49.1.01).
(d) Densitometer.See 970.43A(e) (see 49.1.01).
C. Reagents

See 970.43B(b) (see 49.1.01), plus the following:

(a) Diatomaceous earth.Soak ca 900 g acid-washed Celite 545
overnight in methanol. Filter through double thickness Whatman
No. 1 paper in 24 cm Bchner, wash with 8 L H2O, and dry 12 h at
150.
(b) Silica gel for thin layer chromatography.See 970.43B(d)
(see 49.1.01). Test adsorbent for resolution and fading of ochratoxins
as in 970.43B(d) (see 49.1.01), using ochratoxins A and B and their
ethyl esters as in 973.37G. Develop with benzene-methanolCH3COOH, (h)(1). TLC spots should be round or slightly elliptical,
well separated, in Rf range 0.40.9, and located away from primary
and secondary solvent fronts. Ochratoxins on occasion fade rapidly
on some silica gel plates, especially when exposed to 60% humidity. Protect plate from humidity during spotting by placing in chamber under N2 or under stream of warm air from hair dryer, or by
covering with clean glass plate, using tape on sides as spacers. After
development, dry plate 15 min at 50 and immediately cover with
clean glass plate, using tape on sides as spacers, for protection
during scanning densitometry.
(c) Solvents.ACS, in glass: CHCl3, hexane, CH3COOH,
methanol, HCOOH (90%), and H3PO4 (85%).

(d) Methanolic sodium bicarbonate solution.Dissolve 0.3 g

NaHCO3 in 30 mL H2O and add 70 mL methanol.
(e) Alcoholic sodium bicarbonate solution.Dissolve 6.0 g NaHCO3 in 100 mL H2O and add 20 mL alcohol.
(f) Formic acid-benzene-hexane solution.Shake 100 mL benzene-hexane (20 + 80) with 10 mL H2O-methanol (30 + 70), let
layers separate, and discard lower layer. Shake upper layer with 5
mL HCOOH, let separate, and discard lower layer.
(g) Boron trifluoride.14% (weight/volume). Bubble gaseous
BF3 into chilled alcohol. (Caution: Perform in hood. Avoid contact
with skin, eyes, and respiratory tract.)
(h) Developing solvents.(1) Benzene-methanol-acetic acid
(18 + 1 + 1).Combine 2 volumes methanol-CH3COOH (1 + 1)
with 18 volumes benzene. Adjust benzene:(methanol-CH3COOH)
ratio, if necessary, to produce development described in 973.37G.
Decrease benzene to increase Rf. (2) Hexane-acetone-acetic acid (18
+ 2 + 1).Combine 3 volumes acetone-CH3COOH (2 + 1) with 18
volumes hexane. Adjust hexane:(acetone-CH3COOH) ratio, if necessary, to produce development described in 973.37G. Decrease
hexane to increase Rf.
(i) Ochratoxin standard solutions.See 970.44 (see 49.2.02) and
971.22 (see 49.2.03). Prepare original solutions, each ca 40 g/mL,
in CH3COOH-benzene (1 + 99). Determine concentration as in
970.44 (see 49.2.02) and 971.22 (see 49.2.03), using information
found in Table 973.33.

Table 973.33

Molecular Weights and Absorptivities of

Ochratoxins.
maximum, nm
Ochratoxin
MW

A
B
A Ethyl Ester
B Ethyl Ester

333
320
333
320

403
369
431
397

5550
6000
6200
6500

Combine portions of ochratoxin solutions and dilute with benzene

to give concentration in range 15 g/mL for each ochratoxin.
(j) Purified cotton.Wash 50 g absorbent cotton in beaker with
1 L CHCl3. Decant solution., evaporate residual CHCl3, and store
cotton in closed container.
D. Preparation and Extraction of Sample

Prepare entire sample as in 977.16 (see 49.2.01).

Weigh 50 g into 500 mL glass-stoppered Erlenmeyer. Add 25 mL
0.1M H3PO4 and 250 mL CHCl3, and secure stopper with masking
tape. Shake 30 min on wrist-action shaker, 970.43A(o) (see 49.1.01),
and filter through glass fiber paper, covered with ca 10 g diatomaceous earth, on 9 cm Bchner.
E. Separation of Ochratoxin Acids

(a) Removal of esters.Place plug of purified cotton, (j), in

bottom of 700 17 mm chromatographic tube. Mix 2.0 g diatomaceous earth with 1 mL 1.25% NaHCO3 solution in 50 mL beaker.
Add to chromatographic tube and tamp firmly. Mix 50 mL sample
extract with 40 mL hexane, and add to column. Reserve remainder
of CHCl3 sample extract for confirmation of identity, 973.37H.
Elute at maximum flow rate; then elute with 75 mL CHCl3. Combine
eluates, evaporate to dryness on steam bath, and reserve for ochratoxin ester separation, 973.37F.

Copyright 1998 AOAC INTERNATIONAL

(b) Removal of acids.Elute ochratoxins A and B with 75 mL

freshly prepared HCOOH-CHCl3 (1 + 99), and collect in 250 mL
Erlenmeyer. Immediately add 2 boiling chips, 970.43B(b) (see
49.1.01), evaporate nearly to dryness on steam bath, and quantitatively transfer residue to 15 mL conical centrifuge tube with CHCl3.
Evaporate to dryness under gentle stream of N2 on steam bath.
(Note: Delay in evaporation of HCOOH-CHCl3 may result in loss
of ochratoxins.) Reserve extract for TLC, 973.37G.

Plot standard curve from instrument response, drawing line

through origin to check for linearity and system performance. Dissolve sample extract in 0.5 mL CH3COOH-benzene (1 + 99) and
spot 2 replicates of 3 L sample extract and standard solutions.
Amount sample spotted must contain amount ochratoxin close to or
within range of standard curve.

F. Separation of Ochratoxin Esters

Purify reserved CHCl3 extract as in 973.37E(a) and (b). Dissolve

portion of extract, 973.37E(b), containing equivalent of 10 g
sample, in 5 mL CHCl3 in 25 mL Erlenmeyer. Into separate 25 mL
Erlenmeyer, add 250 ng ochratoxin A and B standard solution. (This
step may be omitted when ester standards are available.) Add 10 mL
14% BF3 in alcohol. Heat to bp and hold on steam bath 5 min.
Transfer to 125 mL separator containing 30 mL H2O. Extract with
three 10 mL portions CHCl3. Combine CHCl3 extracts, wash with
three 10 mL portions H2O, evaporate to dryness, quantitatively
transfer to 15 mL centrifuge tube with CHCl3, and evaporate to
dryness under gentle stream of N2. Dissolve residue in 250 L
CH3COOH-benzene (1 + 99) and proceed as in 973.37G(a), modified as follows:
Spot 10 L unmodified extract, 10 L esterified sample extract,
10 L standard ochratoxin esters, and 10 L esterified sample
extract plus 10 L standard ochratoxin esters. Develop plate with
benzene-methanol-CH3COOH, 973.37C(h)(1). Examine plate
under long- and shortwave UV light. Rf of ochratoxin A ester is
greater than that of ochratoxin B ester, typically 0.8 and 0.7,
respectively. Ethyl esters have much higher Rf values than ochratoxins A and B, but approximately same fluorescence. For positive confirmation, presumptive ochratoxin A or B spots should be
absent after esterfication and spots at Rf values of esters should
be present.
Illuminate developed plate with long- and shortwave UV lamps
used for viewing. Photograph plate, using 200 (ASA) speed Polaroid
black and white film. Operate camera with Kodak 3A lens filter, or
equivalent, cutting off all light below 380 nm, and lens opening f /8
and shutter speed of 5 s, or other combination of lens opening and
shutter speed which produces proper exposure. Cover developed
area of plate with black mask, add legend strip to identify each spot,
and superimpose legend on original picture by double exposure,
using white light for second exposure.

Prepare column as in 973.37E(a), using 350 25 mm chromatographic tube, 2.5 mL methanolic NaHCO3 solution, (d), and 4 g
diatomaceous earth. Dissolve residue, 973.37E(a), in 50 mL hexane
and add to column. Rinse extraction vessel with each subsequent
solvent in turn; add rinses to column. Force eluting solvents through
column at convenient rate with compressed gas at 12 psi (6.913.8
kPa). Do not let liquid fall below top of column. Elute with 50 mL
benzene-hexane (1 + 9) previously equilibrated with 2.5 mL
methanolic NaHCO3 solution (discard). Then elute with 100 mL
HCOOH-benzene-hexane mixture. Immediately evaporate eluate to
dryness, quantitatively transfer to conical centrifuge tube with
CHCl3, evaporate to dryness under gentle stream of N2 on steam bath,
and reserve for TLC, 973.37G.
G. Thin Layer Chromatography

(a) Visual analysis.Proceed as in 968.22F (see 49.2.08), with

following modifications: Use silica gel, 973.37C(b), and dissolve
extract from 973.37E(b) in 750 L CH3COOH-benzene (1 + 99).
Spot 3, 5, 7.5, and 10 L. On same plate, spot 10 L extract
superimposed with 10 ng each ochratoxin A and B standard solutions
as internal standard. Also spot 5, 7.5, and 10 L ochratoxin A and B
standard solutions. Develop plate to solvent stop line, but <90 min,
with benzene-methanol-CH3COOH (18 + 1 + 1) in unlined, unequilibrated tank. Remove plate, let solvent evaporate at room temperature, and view in dark under long- and shortwave UV lamps.
Ochratoxins A and B should be found in Rf range 0.40.8 with
ochratoxin A above B, typically at 0.65 and 0.5, respectively. Ochratoxin A fluoresces most intensely under longwave UV light, while
ochratoxin B is brightest under shortwave light. Examine pattern
from sample for fluorescent spots having Rf values close to those of
standards and with similar appearance. Compare fluorescence intensities of ochratoxin A spots in sample with those of standard spots,
and determine standard and sample which match most closely,
interpolating, if necessary. If sample concentration is outside range
of standards, concentrate or dilute sample extract and rechromatograph. Calculate concentration of ochratoxin A in g/kg. Spray
plate with alcoholic NaHCO3 solution, (e), dry at room temperature,
and view in dark under longwave UV light. Fluorescence should
have changed from greenish blue to blue and increased in intensity.
Again estimate ochratoxin A in sample. In case of disagreement, use
estimate obtained before spraying.
Proceed with TLC of ochratoxin A and B esters from 973.37F on
separate plate in same manner as above for acids, and develop plate
with hexane-acetone-CH3COOH (18 + 2 + 1). Rf value of ochratoxin
A ester is ca 0.5, above ochratoxin B ester.
(b) Densitometric analysis.Prepare and develop TLC plate as
in 973.37C(b) and 973.37G(a). In separate channels, spot 4 spots
with increasing amounts standard ochratoxin A in range 310
ng/spot. Scan TLC plate with densitometer according to instructions of manufacturer. Optimum spectral settings for ochratoxin A
are excitation 310340 nm; emission, 440475 nm.

H. Confirmation of Identity of Ochratoxins A and B by Formation

of Ethyl Esters

Reference: JAOAC 56, 817, 822(1973).

CAS-303-47-9 (ochratoxin A)
CAS-4825-86-9 (ochratoxin B)
CAS-4865-85-4 (ochratoxin A ethyl ester)
CAS-18420-71-8 (ochratoxin B ethyl ester)

Copyright 1998 AOAC INTERNATIONAL