119 / 129
M
eisser
-R
edeuil
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
97, N
o
. 4, 2014
1125
(= 200 g); m
3
=mass of the test portion, in g (= 15 g); V
1
=volume
of the of sample extract, in mL (= 100 mL); V
2
=volume of
sample loaded on SPE, in mL (= 3.0 mL); V
3
=volume of the
reconstituted sample, in mL (= 1.5 mL); 100 = conversion to
100 g basis; 1000 = conversion from ng to µg.
For liquid samples:
1000 x 2 x V3m
100 x3x V1V
C w1
×=
where C = concentration in the test solution (ng/mL) of FA or
5-Me THF, calculated using the dedicated calibration curve;
m
3
=mass of the test portion, in g (= 15 g); V
1
=volume of
the of sample extract, in mL (= 100 mL); V
2
=volume of
sample loaded on SPE, in mL (= 3.0 mL); V
3
=volume of the
reconstituted sample, in mL (= 1.5 mL); 100 = conversion to
100 g basis; 1000 = conversion from ng to µg.
Verify that FA and 5-Me THF software calculated
concentrations are >lower LOQ (LLOQ) and <upper LOQ
(ULOQ).
If calculated concentration is <LLOQ, then this concentration
cannot be taken into account for vitamin B
9
concentration.
Folate (vitamin B
9
) concentration is the sum of folic acid plus
5-Me THF. Results in µg/100 g are expressed as folic acid in
reconstituted product.
M. System Suitability
RSD of retention time and peak area should not be higher
than 5% for FA and 5-Me THF.
N. Specificity
A tandem mass spectrometer was chosen as detection mode.
Optimization consisted of selecting the precursor ion as well
as the two main product ions for each analyte. The transition
precursor ion/main product was defined as quantifier. The
transition precursor ion/second main product ion was defined as
qualifier. The ratio quantifier/qualifier was followed in all series
with a defined limit to confirm peak identification.
Results
The validation study was conducted in accordance with the
SPIFAN SLV Guidelines (3). System suitability was determined
by checking retention times and peak shape for each analyte. RSD
of retention time was below 0.5% for both folic acid and 5-Me
THF in all analytical series. For specificity, native folate content
was analyzed and calculated in three placebo samples supplied
with the SPIFAN test material kit. A tandem mass spectrometer
was chosen as detection mode. To confirm peak identification,
the quantifier/qualifier ratio was followed in all series with a
defined limit. Linearity was demonstrated by testing working
solutions at 10 levels ranging from 0.3 to 9.0 ng/mL, each
injected once, and resulting in
R
2
> 0.99.Acalibration range from
1 to 400 ng/mL was injected at the beginning of each analytical
series. The SD of calibration point “STD 1” was <20%, while
points “STD 2 to 8” were <15%. LOD and LOQ were estimated
by performing 10 independent analysis of nonfortified sample
(infant formula RTF, milk-based), spiked at low levels. LODs
Table 1. Precision results expressed in reconstituted
product
Matrix
a
Folate mean,
µg/100 g
RSD
r
,
%
b
RSD
iR
,
%
c
SRM 1849
22.4
1.3 4.2
Adult nutritional powder milk protein-based 27.9
1.9 4.6
Infant formula powder part hyd milk-based 15.1
1.4 2.8
Infant formula powder part hyd soy-based 16.8
1.5 3.6
Adult nutritional powder low-fat
33.5
0.84 4.4
Child formula powder
18.6
4.0 5.1
Infant elemental powder
28.2
0.93 4.4
Infant formula powder milk-based
17.0
2.1 3.7
Infant formula powder soy-based
16.0
2.2 3.8
Infant formula RTF milk-based
16.1
2.0 5.3
Adult nutritional RTF high-protein
51.4
5.2 5.4
Adult nutritional RTF high-fat
57.9
2.4 5.1
a
Fortified samples provided in the SPIFAN test material kit.
b
RSD
r
is the RSD of repeatability.
c
RSD
iR
is the RSD of intermediate reproducibility.
Table 2. Recovery results of spiking experiments for folic
acid
+50% of
target
+100% of
target
Target,
µg/100 g
Avg.,
%
RSD,
%
Avg.,
%
RSD,
%
Nonfortified products
Child formula powder
165
91 4.2
90 2.7
Infant elemental powder
240
94 0.36 94 3.1
Infant formula RTF, milk
20
90 10.4 95 3.8
Fortified products
AN RTF high-fat
60
90 8.0
89 6.2
AN RTF high-protein
16.7
111 28 102 6.8
Table 3. Recovery results of spiking experiments for 5-Me
THF
+50% of
target
+100% of
target
Target
(µg/100 g)
Avg.,
%
RSD,
%
Avg.,
%
RSD,
%
Nonfortified products
Child formula powder
165
103 14.3 95 1.2
Infant elemental powder
240
98 1.5
99 1.7
Infant formula RTF, milk
20
96 7.4 101 2.1
Fortified products
AN RTF high-fat
60
93 2.2
92 2.8
AN RTF high-protein
16.7
110 14 114 18
110