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average of 3 independent testing of the sample, and the 50% and 100% of the background

values of samples were used as the two spike levels in this study. As no VD2 was detected in

the selected samples, lower spike levels were used. Three replicates were done for all the

samples, and the recoveries of VD were calculated. The results in Table 7 showed that the

recoveries of VD2 and VD3 were all within 89-108% at the two spike levels.

Table 7. Contents and recoveries of VD in samples with different matrices

Sample

number

Content

(n=3),

ng/g

VD3

Content

(n=3),

ng/g

VD2

Spike level,

ng

Recovery

RSD

(n=3)

Spike level,

ng

Recovery

RSD

(n=3)

1

˄

powder

˅

82±1

50

101% 7.48%

ND

5.0

90% 2.93%

100

97% 8.18%

10

95% 7.82%

2

˄

powder

˅

93±2

50

99% 4.79%

ND

5.0

89% 2.96%

100

98% 7.36%

10

94% 3.42%

3

˄

powder

˅

91±1

50

108% 2.68%

ND

5.0

93% 3.02%

100

105% 0.95%

10

93% 0.91%

4

˄

liquid

˅

12.3±0.3

70

102% 4.82%

ND

3.5

97% 3.88%

140

102% 2.48%

7.0

91% 2.03%

Note

˖

Samples 1, 2, 3, and 4 are amino acid based, milk protein based, soy protein based, and adult nutritional

liquid (high fat) products, respectively; ND: no detection.

3.3 Limit of Detection (LOD) and Limit of Quantitation (LOQ)

3.3.1 Calculation method

A blank sample was prepared by the same procedure described in 2.2, and analyzed 10 times

by UPLC-MS-MS using the same parameters as samples. The mean value (x) and standard

deviation (SD) of the 10 testing results were calculated, and

the values of “x + 3SD” and “x +

10SD” were determined as the LOD and LOQ of the method, respectivel

y. In this validation

study, the “x” and “SD” values for VD3 determination were 1.34 and 0.83 pg/mL, respectively,

and the LOD and LOQ of this method for VD3 determination were calculated as 0.382 and 0.961

ng/100 g reconstituted/RTF samples, respectively.

For the VD2 determination, the “x” and “SD”

values were 0.782 and 0.734 pg/mL, respectively, and the LOD and LOQ of this method were

calculated as 0.299 and 0.813 ng/100 g reconstituted/RTF samples, respectively.

3.3.2 Dilution method

A blank sample was prepared by the same procedure described in 2.2, and analyzed by

UPLC-MS-MS using the same parameters as samples. The signal/noise ratio (S/N) was then

calculated, and the 3 and 10 times of the S/N were defined as the LOD and LOQ of the method,

respectively. By the dilution method, the 3 and 10 times of S/N for the VD3 determination were

0.0105 and 0.0343 ng/mL, respectively, corresponding to 1.05 (LOD) and 3.43 (LOQ) ng/100 g

reconstituted/RTF samples, respectively. The 3 and 10 times of S/N for the VD2 determination

were 0.0137 and 0.0458 ng/mL, respectively, corresponding to 1.37 (LOD) and 4.58 (LOQ)

ng/100 g reconstituted/RTF samples, respectively.

VitD-17 (February 2016)

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