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1130 

Mottier et al.

: J

ournal of

AOAC I

nternational

Vol. 98, No. 4, 2015

along with each series of routine samples. Water is used instead

of milk. Proceeded exactly as described in

E

(

a

) and (

b

).

F. Instrumental Conditions

(

a

) 

LC-MS/MS analysis

.—Where a specific instrument is

cited, an alternative may be used provided it has the same or

better characteristics. As well, an alternative HPLC column may

be used provided it allows a retention time of the eluting analyte

that is at least twice the retention time corresponding to the void

volume of the column.

(

1

) 

HPLC conditions.—

Using an Agilent 1200 SL HPLC

system (

see

Table

2015.03B

).

See

Table

2015.03C

for LC

gradient.

Using these conditions, the compound elutes at approximately

1.7 min (

see

Figures

2015.03B

E

).

(

2

)

 MS parameters.

—MS parameters (Tables

2015.03D

and 

E

) are obtained by separately syringe-infusing standard

solution (approximately 1 µg/mL) of each unlabeled and

labeled compounds (syringe flow rate of 10 µL/min) along with

the HPLC flow at 0.45 mL/min using a T connector. The HPLC

flow is constituted with 10%A,

C

(

i

)(

1

), and 90% B,

C

(

i

)(

2

).

(

b

)

Instrument check test

.—Before routine analysis, ensure

that the LC-MS/MS apparatus is working in conditions such as

the method remains fit for purposes. This involves to inject a

low concentration calibrant [e.g., STD 2,

C

(

h

)] to check that

sensitivity of the instrument is adequate.

G. Operating Procedure and Determination

(

a

) 

Sequence setup

.—Inject solutions in the following order:

acetonitrile (as blank solvent) at least three times, standard

solutions,

C

(

h

), acetonitrile at least three times, reagent blank,

E

(

c

), extract solutions,

E

(

b

), and standard solutions,

C

(

h

),

again. Inject acetonitrile after each three to four extract solutions

to check for any carry-over.

(

b

) 

Calibration

.—Draw a calibration curve by plotting peak

area ratio of the analyte and its IS (=

y

axis) against concentration

ratio of the analyte and its IS (=

x

axis). Calculate the slope

and intercept by linear regression. Check the linearity of the

calibration [regression coefficient R

2

should be higher than

0.98 and relative standard deviation of the average of response

factors (=

y/x

) should be <15%].

(

c

) 

Identification and confirmation

.—Sodium fluoroacetate

is identified and confirmed when the following criteria are

fulfilled (1).

(

1

) The ratio of the chromatographic retention time of

the analyte to that of its IS, i.e., the relative retention time,

corresponds to that of the averaged relative retention time of the

calibration solutions within a ±2.5% tolerance.

(

2

) The peak area ratios from the different transition reactions

recorded for the analyte and its IS are within the tolerances fixed

by the EU criteria (1) as shown in Table

2015.03E

.

(

d

) 

Time of analysis

.—Following this procedure, 20 samples

can be analyzed within 24 h.

H. Calculations and Expression of Results

(

a

) 

Calculation

.—Calculate the mass fraction,

w

, of sodium

fluoroacetate in microgram per kilogram of sample (µg/kg),

using the equation:

a

is

is

a

m

m

S

I

A

A

x

w

−



=

where A

a

= peak area of the analyte in the sample (transition

reaction used for quantification); A

is

= peak area of the IS in the

sample (transition reaction used for quantification); I = intercept

of the regression line for the transition reaction used for

quantification; S = slope of the regression line for the transition

reaction used for quantification; m

is

= mass of IS added to the

test portion, in ng (i.e., 10 ng for powdered sample and 50 ng for

liquid sample); m

a

= mass of the test portion, in g (i.e., 1 g for

powdered sample and 5 g for liquid sample).

(

b

) 

Expression of results

.—Report the result of sodium

fluoroacetate in µg/kg with one significant figure. Nondetected

amount must be expressed as <1 µg/kg.

I. Performance Characteristics

The method was validated using samples provided by

the AOAC Stakeholder Panel on Infant Formula and Adult

Nutritionals (SPIFAN). Infant formulae considered are

described in Table 

2015.03F

.

Validation was performed according to the protocol described

in SANCO/12571/2013 (2): precision data were obtained by

spiking each sample at 1 and 10 µg/kg concentration levels

(respectively corresponding to 1 × LOQ and 10 × LOQ level).

At least two operators were involved in these experiments,

each performing five replicates at the mentioned fortification

levels on two different days (leading to a total of 10 separate

experiments for each fortification level). Nonfortified samples

were analyzed as well to verify absence of the pesticide before

fortification trials.

Table 2015.03C. LC gradient used for analysis of sodium

fluoroacetate

Time, min

A, %

B, %

0

10

90

2.0

10

90

3.0

60

40

4.5

60

40

4.6

10

90

8.0

10

90

Table 2015.03B. HPLC conditions for the analysis of

sodium fluoroacetate

Mobile phase A

Water containing 5 mM ammonium

formate and 0.01% formic acid,

C

(

i

)(

1

)

Mobile phase B

Acetonitrile,

C

(

i

)(

2

)

Injection volume 

20 µL

Column 

Waters Acquity UPLC BEH Amide,

2.1

×

100 mm, 1.7 µm

Column oven temp.

45°C

Flow rate

0.45 mL/min

Needle wash

In flush port for 20 s using acetonitrile–water

(1 + 1) solution,

C

(

i

)(

3

)

Diverter valve

HPLC flow is directed into the MS detector

between 1.0 and 2.5 min

Gradient

LC gradient is described in Table

2015.03C

249