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1384 Bidlack et al.: J ournal of AOAC I nternational V ol. 98, N o. 5, 2015

Table 3. Trans vitamin K 1

SLV data–accuracy

Spike level

100%

50%

No. of replicates (duplicates on multiple days)

Native level, µg/100 g RTF Recovery, % RSD, % Recovery, % RSD, %

Sample type

Child formula powder Infant elemental powder

6 6 6 6 6 6 6 6 6 6 6 6

2.66 7.57 1.11 a 3.26 7.69 8.99 2.92 6.09 6.26 9.01 9.10 10.7

98.2 93.2 104 96.4 96.6 97.9 98.0 97.6 97.9 100 96.7 98.2

5.9 7.6 2.9 2.0 3.6 1.0 2.5 1.0 1.7 1.3 2.7 1.2

96.2 94.0 95.5 95.1 91.9 96.1 95.2 102 102 104 106 93.8

7.1 2.6 1.8 1.8 3.0 2.6 2.6 2.3 0.3 0.4 0.7 4.0

SRM 1849a

Adult nutritional powder milk protein based

Infant formula powder partially hydrolyzed milk based Infant formula powder partially hydrolyzed soy based

Adult nutritional powder low fat Infant formula powder milk based Infant formula powder soy based Infant formula RTF milk based Adult nutritional RTF high protein Adult nutritional RTF high fat a  Results reported as mg/kg powder.

extrapolated from the standard LOD and LOQ using a typical sample weight and dilution volume. Ruggedness (or robustness) was not explicitly studied; however, several parameters relevant to this were varied during the SLV in order to factor as much uncertainty as possible into the method performance metrics. Samples were prepared by two analysts and analyzed with silica columns from three different vendors. New mobile phase and postcolumn reagents were made daily, and two sets of stock, intermediate, and working standards were prepared and used during validation. All of the unfortified matrixes were expected to contain some trans vitamin K 1 and could not be used to unambiguously establish method specificity; however, this method uses a very specific detection technique. Relatively few compounds

constructed from these standards, and the regression parameters from least-squares fittings were used to back calculate the concentration of each working standard to determine calibration errors at each level. It should be noted that all commercially available vitamin K 1 standards contain a mixture of cis and trans vitamin K 1 . The percentage of trans vitamin K 1 in the standard was determined experimentally for each run using cis and trans peak areas from all working standard chromatograms. The experimentally determined ratio of trans vitamin K 1 was then used to calculate the trans vitamin K 1 standard concentrations of the working standards. Trans vitamin K 1 LOD and LOQ were determined experimentally by injecting a very low level vitamin K 1 standard of known concentration and measuring the S/N. Trans vitamin K 1 LOD and LOQ in the standard solution were calculated by multiplying the background noise by 3 (LOD) or 10 (LOQ) and dividing by the sensitivity, which was defined as the ratio of the analytical signal to the concentration of the analyte producing the signal. Product LOD and LOQ were

relative (%) calibration errors by level (30 curves) a

Table 4. Summary of trans vitamin K 1

P c

Calibration level b

Mean 0.247 0.484 0.117

Median

Minimum

Maximum

1 2 3 4 5 6

1.55

–7.94 –3.65 –1.00 –1.08 –1.70

5.04 2.93 2.21

0.698

0.879

0.0930

0.0280 –0.0710 –0.271

0.350 0.443

0.0972 –0.328 0.0561

1.9

1.47

0.0460

0.101 0.364

–0.421

0.372

0.201 0.428

Run average

0.104

–1.73

1.18

a  r 2 for the 30 curves ranged from 0.99985 to 1.00000, with an average of 0.99994. b  Levels 1–6 corresponds to trans vitamin K 1

concentrations of 2–3, 6–8, 11–13, 22–30, 37–45, and 74–88 µg/L.

c P value for one sample t -test relative to zero.