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5841 v2
Page 7 of 9
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The placebos or unfortified SPIFAN matrices were spiked at 50% and 150% of the typical
target level. In absence of a placebo, fully fortified product was over-spiked at 50% and
100%.
The biotin results in both SRM 1849a and IRM were close to 100% of the respective target
values confirming the accuracy of the method (Table 2).
All the biotin spiked recoveries were within 95% to 105% of added value confirming
accuracy of the method (Table 3). The recoveries well exceeds the SMPR 2014.005
requirements of 90% to 110% in all instances.
Table 2: NIST SRM 1849a and IRM
Reference
Materials
Biotin (μg/100g)
Limits
Day 1
Day 2
Day 3
Day 4
Day 5 Day 6
NIST SRM 1849a
199 ± 13
199.97
199.65
196.50
197.87 198.37 198.42
IRM
26.55 ± 7.7
26.12
27.58
25.36
25.61
25.13
25.39
Table 3: Spiked recovery
Placebo
Spiked
Levels
Recovery (%)
Day 1
Day 2
Day 3
Average
Child Formula
Powder
50%
99.18
99.66
101.00 100.95 99.39
99.72
99.98
150%
99.40
98.89
99.28
98.05
99.97
96.65
98.71
Infant Elemental
Powder
50%
99.42
99.94
100.44
98.90
99.91
99.14
99.63
150%
94.91
94.38
95.28
95.33
94.47
94.47
94.81
Adult Nutritional
RTF, High Fat
50%
97.98
98.56
98.17
97.58
99.21
97.85
98.23
150%
95.41
95.25
95.11
94.61
97.35
94.66
95.40
Infant Formula RTF,
Milk Based
50%
97.15
97.73
96.51
97.50
97.18
97.85
97.32
150%
94.87
95.13
95.51
95.86
95.06
94.77
95.20
*Infant Formula
RTF, Milk Based
50%
97.49
98.07
93.52
94.50
97.51
98.19
96.55
Sample
Spiked
Levels
Day 1
Day 2
Day 3
Average
**Adult Nutritional
RTF High Protein
50%
103.00
103.50
103.98 105.21 103.42 103.01
103.69
100%
96.25
96.95
97.65
97.04
97.24
95.24
96.73
*
Calibration forced through origin as the 50% levels were lower than the lowest calibration.
**Placebo for Adult Nutritional RTF, High Protein Lot#/ID 00415RF00 could not be used for
spiked recovery studies due to the microbial growth observed in the milk samples. Instead
the fully fortified sample of the same matrix Lot#/ID00414RF00 was used for over spiking.
5.6 Precision (Repeatability)
Repeatability was determined by analysing the 12 different matrices in the SLV test
materials kit in duplicate on each of the 6 days by two different analysts. In addition two
different liquid chromatography equipment were utilized during the course of validation.
Precision data for all the samples can be found in Table 4. Coefficient of variation (%RSD)
was calculated on all the 12 data points on all the 12 SLV matrices. The %RSD was not
more than 3.2% and is well below the set limit of 6% as per SMPR 2014.005.
Bio-02 (February 2016)
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