AOAC Final Action Methods in 2017

(f)  Analyze a reference sample (e.g., National Institute of Standards and Technology Standard Reference Material 1849a) in duplicate. (g)  Identification of biotin peak is based on absolute retention time. Spectrum scan can be used for peak purity confirmation if required. ( h ) Perform three high-level recoveries with every new batch of IACs. I. Calculation and Reporting The chromatography software will automatically calculate the concentration of the sample (μg/100 g), provided the concentration of the standards (μg/100 mL), sample weight (g), and dilution are entered correctly. Manual calculation can also be performed by using the following equation: where dilution = 10 (i.e., 100 × 1 ÷ 10): sample is diluted with water to 100 mL, with 10 mL used for IAC cleanup, to a final volume of 1 mL for HPLC analysis (the dilution will be 20 if 5 mL is used for IAC cleanup); slope = the valid slope calculation based on concentration on the x -axis and peak area on the y -axis; and sample weight = the calculated powder equivalent (g) using the following equation for reconstituted powder samples: Sample weight (powder equivalent) in grams = ( W 1 × W 2)/ W 3 where W 1 = weight of powder sample (g); W 2 = weight of powder and water or total slurry weight (g); and W 3 = weight of slurry taken for analysis (g). For ready-to-feed liquid samples, the sample weight used for extraction is used for the calculation.

Report results to three significant figures (i.e., μg/100 g), or convert to other units as required. J. Repeatability The difference between the results of duplicate portions of the same sample tested at the same sequence should not exceed 6% of the mean result. K. Reproducibility The difference between the results of duplicate determinations tested on different days should not exceed 12% of the mean result. L. Uncertainty of Measurement Uncertainty of the method is calculated as 7%, using appropriate statistical procedure (i.e., square root of the sum of squares of the errors, expressed as a percentage). M. LOQ LOQ was calculated based on the lowest working standard and the dilution factor as follows: LOQ = (1 × 100)/(20 × 50) = 0.1 μg/100 g (1 ppb) where 1 = the lowest working standard concentration of 1 μg/100 mL; 100 = volume (mL); 20 = 20 g sample; 50 = volume (mL) loaded on the IAC; and 1 = final volume (mL). References: J. AOAC Int . 99 , 1110(2016) DOI: 10.5740/jaoacint.16-0155 (First Action) J. AOAC Int . 101 , 831(2018) DOI: 10.5740/jaoacint.17-0242 (Final Action) AOAC SMPR 2014.005 J. AOAC Int . 98 , 1044(2015) DOI: 10.5740/jaoac.int.SMPR2014.005 * Method adopted by AOAC as an Official Method of Analysis SM and by the International Organization for Standardization as ISO 23305:2018. Posted: February 2018, July 2018

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