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. 5, 2015
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actual LOQ might be lower due to the sensitivity of the mass
spectrometer.
LOQ
.—Tables 4 and 5 show the results of spiking a
placebo product with amounts of choline and carnitine at the
SMPR required LOQ. For choline, a recovery of 96.9% was
achieved with excellent precision (0.9% RSD). For carnitine,
recovery was 105.9% with a 1.4% RSD. The good recoveries
and precision indicate that the method is performing at a level
well above the LOQ and the SMPRs are thereby met. Indeed,
the inherent choline in this product is below the required
2 mg/100 g (6 mg/100 g powder = 0.66 mg/100 g RTF) and is
measured with a 1.1% RSD.
Accuracy (trueness)
.—SRM 1849a was analyzed by
the method on 10 days. The result for free carnitine was
13.1 mg/100 g with an RSD of 2.2%, compared to the certified
value of free carnitine of 13.6 mg/100 g, a difference of
–3.7%. The method result was within the certified range of
12.2–15.0 mg/100 g. A result of 14.9 mg/100 g (1.6% RSD) was
also obtained for total carnitine, but the SRM is not certified for
total carnitine.
The result for total choline in SRM 1849a was
102.7 mg/100 g as choline ion, compared to the certified value
of 109.0, a difference of –5.8% but within the certified range
of 98–120 mg/100 g. NIST obtained 103.2 ± 0.7 mg/100 g for
total choline and 14.9 ± 0.1 mg/100 g for free carnitine using its
LC/MS method (4). These latter values are closer to the results
of the present method, although it is unclear exactly how the
microwave digestion step included in the NIST method affects
free carnitine results (our free carnitine result was determined
after simple water dilution, and our total carnitine result agrees
exactly with the individual NIST result in reference 4).
Table 6 shows that there is generally good agreement across
the various methods for determination of choline with the
exception of the widely used AOAC
999.14
. All four methods
compared are quite independent in terms of methodology:
microwave-digestion/LC/MS/MS,
ion
chromatography,
enzyme assisted breakdown with colorimetric detection, and
HPLC-electrochemical detection. Although this is a limited
data set, the obvious recommendation is to avoid using AOAC
999.14
for the analysis of choline in infant formula or adult
nutritional products. The other AOAC First Action methods
in the table are more accurate over the full breadth of product
matrixes presently on the market.
Another LC/MS method (AOAC
2012.17
; 1) was used to
confirm the accuracy of AOAC
2014.04
total carnitine results.
Twelve internal products (not the SPIFAN set) were analyzed
on 8 days by AOAC
2014.04
and on 3–6 days by the reference
method. The differences in the mean results for each product
ranged from –3.7% to +4.6% with an average of –0.3% (data
not shown), indicating the accuracy of this present method for
total carnitine.
Tables 7 and 8 show spike recovery data from each of the
SPIFAN product matrixes at two different levels for choline
and carnitine, including a separate set of experiments to prove
the recovery of a primary inherent species, acetylcarnitine. No
systematic recovery issues were seen, with overall recoveries
close to 100% across all matrixes for choline and carnitine.
Acetylcarnitine recoveries appeared to be consistently lower,
but still within the SMPR-recommended 90–110%.
Table 4. LOQ verification in SPIFAN sample 11750017V3 for choline at the SMPR limit of 2 mg/100 g RTF
a
Sample name
Concn (OH-), mg/kg Measured spike
b
, mg/kg
Theoretical spike,
mg/kg
Recovery, %
Spike level, mg/100g
RTF
Adult powder-1
60.14
Adult powder-2
59.76
Adult powder-3
61.49
Adult powder-4
60.60
Adult powder-5
60.87
Adult powder-6
59.53
Adult powder-7
59.99
Unspiked
60.34
RSD, %
1.1
Adult powder-LOQ-1
233.5
173.1
178.9
96.8
1.92
Adult powder-LOQ-2
234.9
174.6
178.6
97.8
1.94
Adult powder-LOQ-3
233.3
172.9
176.5
97.9
1.92
Adult powder-LOQ-4
232.5
172.2
179.4
96.0
1.91
Adult powder-LOQ-5
234.1
173.7
180.7
96.1
1.93
Adult powder-LOQ-6
235.5
175.2
179.0
97.9
1.95
Adult powder-LOQ-7
232.2
171.8
179.0
96.0
1.91
Spiked
233.7
96.9
RSD, %
0.5
0.9
a
For this table, the concentrations measured are in terms of choline hydroxide.
b
The mean unspiked value is subtracted from the individual spiked result.
127