Review Team (February 2016)

Bio-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

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 90 to 110% of mean spiked recovery over the range of the assay

>1 b

0.1 – 1 b

≤1 6% ≤1 2%

Reproducibility (RSD ) R

>1 b

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.