AOAC Official Method
Aflatoxins M1 and M2 in Fluid Milk
Liquid Chromatographic Method
First Action 1986
Final Action 1990
A. Principle
Aflatoxins M1 and M2 are extracted from milk on C18 cartridge,
eluted with ether onto silica column, eluted with CH2Cl2–alcohol,
and M1 is derivatized with trifluoroacetic acid. Liquid chromato-
graphic peaks are detected fluorometrically and compared with stan-
dard–TFA derivatives.
B. Reagents
(a) Solvents.—Distilled in glass CH3CN, CH2Cl2, isopropyl al-
cohol; reagent grade alcohol, ether (% ethyl alcohol preserva-
tive), hexane, methanol, trifluoroacetic acid, and H2O (deionized,
filtered through m m filter).
(b) Water–acetonitrile wash solution.—95 + 5.
(c) Methylene chloride–alcohol elution solution.—95 + 5.
(d) Mobile phase.—Prepare H2O–isopropyl alcohol–CH3CN
(80 + 12 + 8). Degas in ultrasonic bath, or equivalent. Alternative
solvent proportions may be used to give optimum resolution (.,
84 + 11 + 5).
(e) Aflatoxin standard solutions.—Aflatoxins M1 and M2 (Sigma
Chemical Co., or other suitable source). Prepare stock solutions (ca
200 m g M1/mL and 100 m g M2/mL) in CH3CN and determine con-
centrations according to (see ) and (see
), using extinction coefficients of 19 850 and 21 400 for M1
and M2, respectively, in CH3CN. Make working standard solution
containing m g M1 and m g M2/mL in CH3CN–benzene
(1 + 9) for use in preparing M1–TFA derivative.
(f) Dichlorodimethylsilane (DDS).—5% in toluene. Add 5 mL
DDS (99%) to toluene and dilute to 100 mL. Store in glass-stoppered
flask in cold. (Caution: DDS is a lachrymator and is flammable.)
C. Apparatus
(a) Silica gel cleanup columns.— · cm polypropylene
Econo-Column with Luer tip, 35 m m, porous polypropylene bed
support disk, and 10 mL reservoir (Bio-Rad Laboratories, Cat.
No. 731-1550, or equivalent).
(b) Silica gel cleanup column packing and preparation.—Dry
silica gel 60, particle size – mm (E. Merck, No. 9385) in
105°C oven for 1 h. Cool and add 1% H2O by weight. Shake in sealed
container and equilibrate overnight before use. Assemble poly-
propylene column and 25 mL vacuum flask fitted with 1-hole stop-
per as shown in Figure . Fill column to ca 2 mL mark with
silica gel (ca 1 g). Pull gentle vacuum to pack bed and add ca 1 g an-
hydrous Na2SO4 to top of silica gel bed.
(c) Extraction cartridges.—C18 Sep-Pak sample preparation car-
tridges (Waters Associates, Inc.).
(d) Disposable pipet tips.—50 and 200 m L Eppendorf or
equivalent.
(e) Liquid chromatograph.—Any pulse-free or pulse-dampened
liquid chromatographic system which includes pump(s), injector,
and compatible recorder.
(f) Fluorescence detector.—Any fluorescence detector capable
of providing 365 nm excitation and >400 nm emission wavelengths
and sensitivity of 50–100% full-scale response for 1 ng M1–TFA de-
rivative (., Spectroflow 980, Applied Biosystems, Inc., 170 Wil-
liams Dr, Ramsey, NJ 07446, USA).
(g) LC analytical column.—Any · 25 cm column containing
spherical 5 m m particle size C18 bonded silica gel (., DuPont ODS
[MAC-MOD Analytical, Inc., Chads Ford, PA 19317, USA],
Spherisorb 5 ODS 2 [Phase Separations Ltd., Deeside Industrial Es-
tate, Queensferry, Clwyd, UK]).
(h) Vacuum regulator.—Any commercial or custom device ca-
pable of creating and controlling partial and full vacuum with side
arm vacuum flask.
(i) Silylated vials for aflatoxin standard solutions.—Fill 1 or 112
dram (4 or 6 mL) glass vials nearly full with 5% DDS and heat ca
Figure —Diagram of apparatus for extraction and cleanup of milk extracts.
40 min at 45–55°C. Discard solution, and rinse vials 3 times with to-
luene and then 3 times with methanol. Heat vials in oven at 75°C for
20–30 min to evaporate methanol. Cap vials (with Teflon liners) and
store for aflatoxin standard solutions.
D. Extraction
Attach inlet (longer) stem of C18 cartridge to Luer tip of 30–50 mL
syringe. Assemble syringe, cartridge, and vacuum flask as shown in
Figure . Adjust vacuum to pull solvents through cartridge in
fast dropwise manner (ca 5 mm Hg). Prime cartridge by adding 5 mL
methanol, then 5 mL H2O (do not pull cartridge dry; leave small ex-
cess H2O in stem). Discontinue vacuum and remove cartridge–sy-
ringe assembly from stopper to prevent loss of prime.
Warm milk (test sample) to room temperature. Gently invert test
sample ‡ 10 times to evenly distribute cream. Transfer 20 mL milk to
graduate containing 20 mL hot (ca 80°C) H2O. (If necessary, more
hot H2O may be used to thin milk solution.)
Replace cartridge–syringe assembly in stopper. Pour entire 40 mL
warm, diluted milk into syringe and gently pull sample through car-
tridge at flow rate ca 30 mL/min (very fast drops). (Caution: Too fast
a flow will not allow sufficient time for aflatoxin to adsorb, resulting
in low recoveries.) Add 10 mL H2O–CH3CN wash solution to sy-
ringe and pull through. Plug syringe barrel with stopper and pull
hard vacuum on cartridge for ca 30 s to remove as much wash solu-
tion as possible from packing. Remove cartridge and dry inside of
both stems with cotton swab or tissue paper to eliminate any remain-
ing wash solution. Reprime cartridge by adding 150 m L CH3CN to
inlet bed support disk and let solvent soak into packing for 30 s. At-
tach cartridge to dry glass or plastic 10 mL Luer tip syringe, retaining
same stem as inlet.
Insert silica gel cleanup column into 250 mL vacuum flask fit-
ted with 1-hole rubber stopper (Figure ). Wash column
with 5 mL ether. Add 7 mL ether to syringe-cartridge positioned
above silica gel cleanup column. With plunger, slowly force
through cartridge (in portions), collecting eluate in column reser-
voir. Pull ether slowly through silica cleanup column, using vac-
uum to maintain flow rate ca 10 mL/min (fast drops). Rinse silica
column with 2 mL additional ether, continuing to use vacuum.
Discard ether.
Remove column and stopper from flask and place 16 · 125 mm
collection tube in flask to catch eluate from column. Add 7 mL elu-
tion solvent (CH2Cl2–alcohol) to column reservoir. Pull solvent
through column with vacuum at ca 10 mL/min flow rate, collecting
eluate in tube.
Discontinue vacuum and remove collection tube from assembly.
Evaporate eluate just to dryness under N2 stream, using heat to keep
collection tube near room temperature or under vacuum at <35°C.
Transfer residue to 1 dram vial with CH2Cl2 and evaporate to dry-
ness under N2 on steam bath or in heating block <50°C. (Do not over-
heat dry residue.) Save for derivative preparation.
E. Liquid Chromatography
Prepare derivative of residue from D by adding 200 m L hexane
and 200 m L trifluoroacetic acid to dry residue in vial. Shake on vor-
tex mixer ca 5–10 s. Let mixture sit 10 min at 40°C, in heating block
or bath; then evaporate to dryness under N2 on steam bath or heat-
ing block (<50°C). Add 2 mL H2O–CH3CN (75 + 25) to vial to dis-
solve residue and shake well in Vortex mixer for LC analysis. For
derivative of standard M1, add 200 m L hexane and 50 m L
trifluoroacetic acid to silylated vial and mix. Add 50 m L M1–M2
working standard solution directly into hexane-TFA mixture and
shake on Vortex mixer 5–10 s. Treat as described for test solution.
Stabilize instrument and detector for suitable period at flow rate of
mL/min with H2O–isopropanol–CH3CN (80 + 12 + 8). Adjust
detector attenuator so that 50–100 m L injection of standard
(– ng M1, – ng M2) gives 50–75% full-scale re-
corder pen deflection for aflatoxin M1. Inject LC standard
2–3 times until peak heights are constant. Prepare standard curve
from either peak heights or peak areas to ensure linear relationship.
Inject test solutions (typically 50–100 m L) with standard injections
interspersed to ensure accurate quantitation. Retention times of M1
(as TFA derivative) and M2 are ca 4–5 min and ca 7 min, respec-
tively. Calculate aflatoxin concentration:
ppb (M1 or M2) =
H C VI V
H VI W
·
¢
·
¢
·
¢
· ·
where H and H ¢ = peak height of injected test solution and LC stan-
dard, respectively; C ¢ = concentration of standard (ng/mL); VI ¢ and
VI = volume injected of standard and test solution, respectively;
V = final total test solution volume (m L); and W = volume of milk
represented by test solution (typically 20 mL). Separately calcu-
late concentration for M1 and M2.
Reference: JAOAC 69, 880(1986).
