115 / 670
LOD/LOQ
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.
LOD = Blank Mean + 3 Standard Deviations
LOQ = Blank Mean + 10 Standard Deviations
Ruggedness
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.
Specificity
Placebos were tested to confirm the specificity by spiking the biotin standard. This method also uses a
very specific detection system. Biotin was conjugated with streptavidin fluorescein and then detected
by fluorescence.
Acceptance Criteria
The standard method performance requirements (SMPR 2014.005) are summarized below.
Table 1. Method performance requirements: biotin
a
1
Analytical range
0.1
–
150
b
Limit of quantitation (LOQ)
≤
0.1
b
Repeatability (RSD )
r
0.1
–
1
b
≤8%
>1
b
≤5%
Recovery
0.1
–
1
b
80 to 120% of mean
spiked recovery over
the range of the assay
>1
b
90 to 110% of mean
spiked recovery over
the range of the assay
Reproducibility (RSD )
R
0.1
–
1
b
≤1
6%
>1
b
≤1
2%
a
Concentrations apply to
(a) “ready
-to-
feed” liquids “as is”;
(b) reconstituted powders (25 g into 200 g of water); and (c) liquid
concentrates diluted 1:1 by weight.
b
mcg/100 g reconstituted final product.
Bio-03 (February 2016)
FOR ERP USE ONLY
DO NOT DISTRIBUTE