AOAC SPIFAN Nutrients ERP-Final Review (Jan. 2019)

Chol-08/Carn-07 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Validation Results Limit of Detection (LOD)

The Limit of Detection for the analysis was determined by LOD = ((3 x standard deviation of blank) + mean of blank). Ten blank replicates were utilized to calculate the LOD. An instrumental LOD of 680 pg for carnitine and 940 pg for choline was obtained. This equates to an LOD of 0.0034 mg/100g for carnitine and 0.0047 mg/100g for choline by this method when utilizing the default weights and dilution factors. This is well below the required LOD of both SMPR. Linearity The linearity was assessed by the percent deviation from theoretical concentration across the calibration (Table 4). The calibration range was from 10 ng/mL to 5000 ng/mL. A linear model with a weighting of 1/x 2 was used to best represent the response across this range. The correlation coefficient across these same three analyses is included in Table 5. Both tests show acceptable linearity across the calibration range. Table 4. Deviation from Theoretical, n = 3 analyses

Conc. (ng/mL)

10

20

500

2000

4000

5000

Analyte Carnitine Choline

Percent Deviation from Theoretical

Mean

2.2 1.9

2.9 2.5

3.0 4.1

2.0 1.4

2.4 1.1

1.2 1.0

2.0 1.7

Table 5. Correlation Coefficient (r), n = 3 analyses

Analyte Carnitine Choline

Mean Correlation Coefficient

0.9994 0.9993

Specificity Specificity was determined through equivalence of the ion ratios across two different transitions in both the free and total analyses for all non-placebo materials (Tables 6-9). The comparison of the ion ratio between sample and reference standards showed ≤ 7.5% across all matrices, with the majority under 5%. This method can not determine between D and L-carnitine. The majority of these products are fortified with L-carnitine, and all concentrations were below quantitation levels in the placebo products. There was really no considerable increases from free to total analysis except in the milk based products, which supports the presence of bound forms of carnitine.