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© 2015 AOAC INTERNATIONAL
AOAC Official Method 2015.04
Monofluoroacetate
in Powdered Nutritional Products
Derivatization with 2-Nitrophenylhydrazine
and LC-MS/MS
First Action 2015
[Applicable for quantitative determination of monofluoroacetate
(MFA) in powdered nutritional product.]
Caution
: Monofluoroacetate is highly toxic and volatile below
pH 4. Acetonitrile, ethyl acetate, and pyridine are toxic
and flammable. Hydrochloric acid is toxic and corrosive.
A. Principle
The method incorporates certain elements from refs (1) and
(2). Samples are prepared by dilution in water followed by
protein precipitation with acetonitrile. An aliquot of the sample
extract is derivatized with 2-nitrophenylhydrazine (2-NPH) in
the presence of 1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide
hydrochloride (EDC). This reaction achieves the coupling of the
carboxyl group of monofluoroacetate to the amino group of 2-NPH
with the formation of an amide bond. Samples are processed
through a solid-phase extraction (SPE) step in order to exchange
sample solvent and achieve a 5-fold concentration of the extract.
A stable-isotope labeled internal standard is incorporated into the
sample preparation to correct for instrument response and losses in
sample preparation. Analysis is performed by ultra-high pressure
liquid chromatography (UPLC) coupled to tandem quadrupole
mass spectrometry (MS/MS). The MS/MS system is configured to
monitor one parent-daughter (precursor-fragment) ion pair for the
analyte and internal standard, respectively (
see
Figure
2015.04A
).
B. Apparatus
(
a
)
Balance
.—Analytical.
(
b
)
Balance.—
Micro-analytical.
(
c
)
Beakers
.—Various sizes.
(
d
)
Glass bottles
.—100 and 500 mL.
(
e
)
Graduated cylinders
.—10 and 100 mL.
(
f
)
Flasks, volumetric, glass
.—Class A; 10, 25, and 100 mL.
(
g
)
Vortex mixer
.—Vortex-Genie (Cole-Parmer, http://www.
coleparmer.ca), or equivalent.
(
h
)
Centrifuge tubes
.—2, 15, and 50 mL.
(
i
)
Spatulas and scoops
.
(
j
)
Centrifuge
.—Thermo Fisher Scientific (http://www.
thermofisher.com)Sorvall Legend XTR, or equivalent.
(
k
)
Glass centrifuge tubes with screw cap
.—VWR (Cat.
Nos. 99502-15 and 89001-048, respectively;
https://www.vwr.com).
(
l
)
Water bath
.—Thermo Fisher Scientific Precision model
2872, or equivalent.
(
m
)
Glass Pasteur pipets
.
(
n
)
Vacuum manifold for SPE
.
(
o
)
SPE cartridges
.—Supelclean Envi-Chrom P, 500 mg, 6 mL
(Cat. No. 57226; Sigma-Aldrich,
https://www.sigmaaldrich.com).
(
p
)
SPE cartridge reservoirs
.—25 mL (Supelco; Cat.
No. 54258-U).
(
q
)
Positive displacement pipets
.—Gilson
(http://www.gilson.
com) Microman (M); Model Nos. M10, M100, M250, and M1000.
Part Nos. F148501, F148504, F148505, and F148506.
(
r
)
Positive displacement pipet tips
.—Gilson Capillary Piston
(CP); Reference Nos. CP10, CP25, CP100, CP250, and CP1000.
Part Nos. F148412, F148414, F148014, and F148560.
(
s
)
Plastic syringes
.—B&D 3 mL, or equivalent.
(
t
)
Syringe filters
.—PTFE 0.2 µm, Acrodisc 13 mm, Part
No. 28143-930, or equivalent.
(
u
)
Autosampler vials
.—Glass, 2 mL, 12 × 32 mm with screw
neck (PTFE pre-split septa caps). Waters (Cat. No. 186000847C;
http://www.waters.com), or equivalent.
(
v
)
Vial inserts
.—300 µL (Waters; Cat. No. WAT094170).
(
w
)
Chromatography equipment and supplies.—
(
1
)
Mass
spectrometer
.—Waters Xevo TQ-S tandem quadrupole, or
equivalent.
(
2
)
Liquid chromatograph
.—Waters Acquity UPLC, or
equivalent.
(
3
)
LC column
.—Waters Acquity UPLC
®
BEH C
18
, 1.8 µm, 2.1
× 100 mm (Part No. 186002352).
C. Reagents
(
a
)
Sodium fluoroacetate 99.3%
.—Fluka PESTANAL (Cat.
No. 31220-100MG; Sigma-Aldrich).
(
b
)
13
C
2
,
2
H
2
-fluoroacetate
.—Cambridge Isotope Laboratories
(Cat. No. CDLM-7943-0;
http://www.isotope.com).
(
c
)
Acetonitrile
.—Fisher Scientific Optima LC/MS grade (Cat.
No. L-1693).
(
d
)
Ethyl acetate
.—Fluka LC/MS Chromasolv (Cat.
No. 34972-R).
(
e
)
Ethanol
.—ACS reagent grade (≥99.5%; Sigma-Aldrich, Cat.
No. 459844).
(
f
)
Hydrochloric acid
.—Trace metal grade (Fisher Scientific,
Cat. No. A508-P500).
(
g
)
Pyridine
.—Sigma-Aldrich (Cat. No. 270970).
(
h
)
Ammonium acetate.—
Fluka (Cat. No. 73594).
(
i
)
2
-
NPH 97%
.—Sigma-Aldrich (Cat. No. N21588).
(
j
)
EDC
.—Sigma-Aldrich (Cat. No. 03449).
(
k
)
Laboratory water
.—18 MΩ-cm.
D. Solutions
(
a
)
Monofluoroacetate
stock
standard
.—Approximately
1000 µg/mL in water.
Figure 2015.04A. Monofluoroacetate C
2
H
2
FO
2
, MW = 77.03,
CAS No. 62-74-8.
Table 2015.04A. Preparation of calibration standards
Standard
Working
standard
(1 + 10), μL
Working
standard, μL
Working
internal
standard, μL
Acetonitrile–water
(60 + 40, v/v), μL
1
10
40
950
2
20
40
940
3
50
40
910
4
10
40
950
5
50
40
910
6
100
40
860
Candidates for 2016 Method of the Year
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