6. AOACSPIFANMethods-2018Awards

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1404  Jing et al. : J ournal of AOAC I nternational Vol. 98, No. 5, 2015

Table 9. Repeatability and intermediate precision for total carnitine and total choline (6 days in duplicate) Total choline

Specificity .—The choline-d 9 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 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

Concn level mg/100 g RTF

RSD r

, % RSD iR

Product

, %

Infant powder milk Infant powder soy

16.9 19.2 17.9 17.0 17.4 4.03 5.45 8.16 20.9 47.2 51.5

1.2 0.9 1.0 0.9 1.4 2.3 1.3 1.9 0.8 1.4 1.0 1.3 1.5 0.8 0.9 1.3 0.7 1.5 1.6 1.0 1.7 1.2

1.7 1.7 1.8 1.8 1.9 3.0 2.5 2.0 2.1 1.4 1.4 1.9 1.5 2.2 1.8 2.1 2.0 1.5 1.6 1.4 1.7 1.8

Infant powder milk part hydrolyzed Infant powder soy part hydrolyzed

Adult powder low fat

Adult powder milk

Child formula powder

Infant elemental powder

Infant RTF milk

Adult RTF high protein

Adult RTF high fat

Overall mean, %

Total carnitine

Infant powder milk Infant powder soy

1.71

0.899

Infant powder milk part hydrolyzed Infant powder soy part hydrolyzed

1.14 1.01

Adult powder low fat

0.039

Adult powder milk

0.0076 < LOQ < LOQ

Child formula powder

5.44 1.54 2.69 15.6 21.5

Infant elemental powder

Infant RTF milk

Adult RTF high protein

Adult RTF high fat

Overall mean, %

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.