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FOR EXPERT REVIEW PANEL USE ONLY
NOT FOR DISTRIBUTION
2
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Method Optimization
:
Several parameters were varied during validation to establish method ruggedness.
Samples were prepared by three analysts and analyzed with C18 columns with 4 different lots.
New mobile phase, post column reagents, intermediate standards and working standards were made daily
and used during validation.
Performance Characteristics:
Analytical Range: No data
SMPR requirements : 0.1–150 mcg/100g
LOQ:
0.8 and 1.5 mcg/100g
SMPR Requirements : ≤0.1 mcg/100 g reconstituted final product
The limit of quantitation 0.1 mcg/100 g was not met.
Biotin detection and quantitation limits were determined experimentally by spiking a very low level
biotin into placebos. Blank mean and standard deviation were obtained from 8 injections.
The LOQ was estimated to be 0.8 mcg/100g
reconstituted final product for powder assuming 4 gram
sample was diluted to 50 ml and
1.5 mcg/100g for RTF
assuming 20 gram sample was diluted to 50 ml.
Accuracy/Recovery: 95 – 109 %
SMPR Requirements: 0.1 – 1 mcg/100g : 80 – 120 %
˃ 1.0 mcg/100g : 90 – 110 %
Sample
Replicates
Native level
(mcg/100g
reconstituted)
Level 1 (50%)
Avg (%)
Level 2 (150% for
placebo, 100% for
fortified)
Avg (%)
Child formula powder, placebo
6
Not detected
103
105
Adult nutritional RTF high protein,
placebo
6
Not detected
102
103
Adult nutritional RTF high fat,
placebo
6
Not detected
102
104
Infant formula powder partially
hydrolyzed soy based
6
4.43
105
101
Infant formula powder milk based
6
5.11
104
103
Adult nutritional powder low fat
6
31.9
111
102
Child formula powder
6
21.6
109
104
Infant elemental powder
6
10.7
106
95.1
Infant formula powder FOS/GOS
based
6
1.66
105
99.9
Infant formula powder milk based
6
5.11
104
103
Adult nutritional RTF high protein
6
56.8
109
102
Adult nutritional RTF high fat
6
76.2
109
101
Bio-03 Review Forms