130 / 258
1404
Jing
et al.
: J
ournal of
AOAC I
nternational
Vol. 98, No. 5, 2015
Precision
.—Within-day precision across the SPIFAN matrix
set generally fell in the 0–2% RSD range with the intermediate
precision across 6 days being about 1% higher. These data
are summarized in Table 9 (for total choline/carnitine) and
10 (for free choline/carnitine) and easily meet the SMPRs
for repeatability (
see
Table
1).
Reproducibility was not tested
with this SLV, but the excellent intermediate precision across
two analysts and two instruments in this study indicate that
the requirement of <15% RSD
R
would likely be met in a
multilaboratory study.
Free versus total.—
Table
11
summarizes the results from the
SPIFAN matrixes in terms of the free versus total amounts. In
all cases, the result for total choline was substantially greater
than that for free choline. Discounting the results for carnitine in
the Adult Powders, which had very low levels, it could be said
that for most products, the total carnitine level equals the free
carnitine level. The exception is the SRM 1849a material and
some of the products containing milk protein. The significance
of this is that, in most cases, manufacturing laboratories are
interested in free carnitine (howmuch is added) and total choline
results for their products. A single analysis of the final digested
solution will suffice to measure both of these parameters.
Specificity
.—The choline-d
9
and carnitine-d
3
labeled isotopes
were checked for the presence of any response at the primary
transition of the native compound, and only a negligible signal
was observed (data not shown). Conversely, during an internal
validation with 12 Abbott products (not the SPIFAN set), the
internal standards were not added to the samples on 1 day and
negligible response was observed at the primary transitions of
the internal standards, indicating these transitions are specific
(data not shown).
Throughout the course of an internal validation of this method
in the authors’ laboratory, data were collected at confirmatory
transitions for both choline and carnitine. These transitions are
included in Figure 1 along with primary transitions used for
quantitation of SRM 1849a. For carnitine, the confirmatory
transition used was 162→85 amu for the native compound and
165→85 amu for the IS. For choline, it was 104.2→45 amu
for the native compound and 113.4 → 45 amu for the IS. The
data were examined after the validation to see how the ratios
of response at the confirmatory transition relative to response
Table 9. Repeatability and intermediate precision for total
carnitine and total choline (6 days in duplicate)
Total choline
Product
Concn level
mg/100 g RTF
RSD
r
,
% RSD
iR
, %
Infant powder milk
16.9
1.2
1.7
Infant powder soy
19.2
0.9
1.7
Infant powder milk part hydrolyzed
17.9
1.0
1.8
Infant powder soy part hydrolyzed
17.0
0.9
1.8
Adult powder low fat
17.4
1.4
1.9
Adult powder milk
4.03
2.3
3.0
Child formula powder
5.45
1.3
2.5
Infant elemental powder
8.16
1.9
2.0
Infant RTF milk
20.9
0.8
2.1
Adult RTF high protein
47.2
1.4
1.4
Adult RTF high fat
51.5
1.0
1.4
Overall mean, %
1.3
1.9
Total carnitine
Infant powder milk
1.71
1.5
1.5
Infant powder soy
0.899
0.8
2.2
Infant powder milk part hydrolyzed
1.14
0.9
1.8
Infant powder soy part hydrolyzed
1.01
1.3
2.1
Adult powder low fat
0.039
<LOQ < LOQ
Adult powder milk
0.0076 < LOQ < LOQ
Child formula powder
5.44
0.7
2.0
Infant elemental powder
1.54
1.5
1.5
Infant RTF milk
2.69
1.6
1.6
Adult RTF high protein
15.6
1.0
1.4
Adult RTF high fat
21.5
1.7
1.7
Overall mean, %
1.2
1.8
Table 10. Repeatability and intermediate precision for free
carnitine and free choline (6 days in duplicate)
Product
Concn level
mg/100g RTF RSD
r
, % RSD
iR
, %
Free choline
Infant powder milk
12.8
0.7
2.1
Infant powder soy
13.9
1.3
2.7
Infant powder milk part
hydrolyzed
13.4
2.0
2.3
Infant powder soy part
hydrolyzed
14.2
1.1
2.1
Adult powder low fat
15.1
1.5
2.4
Adult powder milk
0.36
1.7
19
Child formula powder
4.39
1.4
5.4
Infant elemental powder
7.59
0.9
1.4
Infant RTF milk
12.1
0.7
0.9
Adult RTF high protein
42.3
1.3
1.3
Adult RTF high fat
46.8
1.2
1.4
Overall mean, %
1.3
2.2
Free carnitine
Infant powder milk
1.50
1.4
3.4
Infant powder soy
0.890
1.5
3.8
Infant powder milk part
hydrolyzed
0.882
1.3
1.7
Infant powder soy part
hydrolyzed
1.00
1.3
2.7
Adult powder low fat
0.0200
<LOQ <LOQ
Adult powder milk
0.0100
<LOQ <LOQ
Child formula powder
5.48
1.8
2.7
Infant elemental powder
1.54
0.9
1.4
Infant RTF milk
2.63
2.0% 2.0%
Adult RTF high protein
15.7
1.5
1.5
Adult RTF high fat
21.9
1.6
1.6
Overalll mean, %
1.5
2.3
130