Review Team (February 2016)

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

Results

Precision

Repeatability requirements were met for all SPIFAN matrices. Repeatability ranged from 0.5-3.0% RSD. See attached Table 1.

Accuracy

Spike recovery requirements were met for 10 out of 11 tested SPIFAN matrices. Mean spike recovery data ranged from 95.1-111%. See attached Table 2. Recovery of SRM 1849a met requirement. See attached Table 3.

Calibration Fit

The method demonstrated good polynomial regression (cubic) fit, over a standard range of ~5-100 ng/mL biotin with r 2 greater than 0.9990. The calibration error for the lowest 2 levels (near LOQ level) is around 25% and 10% respectively. The calibration error for the rest levels is less than 8%. See the attached Table 4 and Figure 1.

Suitable calibration curve range

Due to the characteristics of the post column protein binding reaction, the calibration is not a linear regression and the protein binding reaches saturation at high concentration. The method requests appropriate sample size for analysis. Based on the recovery data of SRM 1849a with different sample sizes, the suitable range of the calibration curve for use is 8.5 – 60 ng/mL. See attached Table 5. If the concentration of the prepared sample is higher than 60 ng/mL, a further dilution with mobile phase is required.

LOQ/LOD

The limit of quantitation requirement (0.1 mcg/100 reconstituted final product) was not met. The quantitation limits 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. However, the biotin levels in all the SPIFAN matrices are above the LOQ of this method.

Specificity

Chromatograms from all of the SPIFAN matrices help to establish the specificity of this method for biotin. See attached chromatograms of all of the SPIFAN matrices.