OMA Consulting Protocol Review: OMACON-2019-2

2.5.1 Prepare and homogenize each matrix such that there are a minimum 6 known concentration levels of lactose, including zero (if possible), to cover the analytical range of the method. 2.5.2 Aliquots of 50 mL of each matrix will be spiked with the appropriate volume of a stock solution of D-lactose of 4 g/L or 60 g/L in LAC Measurement Solution, in order to achieve the desired concentration. 2.5.3 For liquid samples analyzed at the 0-200 mg/L range, the pretreatment with activated charcoal will be performed before the addition of lactose. 2.5.4 For semi solid samples analyzed at 0-2 g/L or 0-6 g/L range, the addition will be performed before the 1/10 dilution required for analysis. 2.5.5 Each of the three analytical ranges will be evaluated for each matrix. 2.5.5.1 For the 0-200 mg/L calibration range a minimum of 6 contamination levels between 0 and 200 mg/L should be used. 2.5.5.2 For the 0-2 g/L calibration range a minimum of 6 contamination levels between 0 and 2 g/L should be used. 2.5.5.3 For the 0-6 g/L calibration range a minimum of 6 contamination levels between 0 and 6 g/L should be used. 2.5.6 Randomize and blind code 2 sample replicates per concentration per matrix. 2.5.7 From each blinded sample, analyze according to the candidate method (2.4 and Appendix 1). 2.5.8 Decode and tabulate the replicate data and calculate the mean at each concentration. 2.5.9.1 Graph replicate candidate method results vs. known concentration and perform regression analysis. 2.5.9.2 Determine recovery and bias at each concentration from the comparison of the candidate method means to the known histamine concentration as follows: 2.5.9.2.1 Recovery = [mean cand /known spike] x 100 2.5.9.2.2 Bias = mean cand – known spike 2.5.9.3 Determine accuracy and precision for the candidate method at each concentration for each matrix as follows: 2.5.9 Bias, Recovery and Repeatability Precision Determination

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2.5.9.3.1 s r =

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