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