AOAC SPIFAN Nutrients ERP-Final Review (Jan. 2019)

2017.03 (JAN 2019) Amino-02-(ERP Review for Pubs) Tryptophan by enz hyd_Abbott Labs_Method_10172018

FOR ERP USE ONLY DO NOT DISTRIBUTE

HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Jonathan Draher and Norman White Abbott Nutrition, Division of Abbott Laboratories (2) Note: The use of different internal standard solution preparations for the standards and samples is acceptable as long as the actual respective weights or concentrations are applied to standards and samples and the two solutions are prepared using the same lot of 5-methyl-tryptophan. c) Tryptophan Concentration in Samples

TRP g = (CTS – CTB) x Volume x 100g 100 g Sample Sample Weight (g) 1000mg/g Where:

CTS = Concentration of Tryptophan in Sample CTB = Concentration of Tryptophan in Blank Volume = Final volume in Liters i.e. 50 mL = 0.05L

2.

Sample Calculation The following example shows a typical calculation performed when only an integrator is used for sample quantitation. When a Chromatography Software is used, the software will perform the calculation shown below. Once the software processing method is properly calibrated and appropriate factors applied, the sample results will be computed in the desired units. If a data station is used to compute mg/L concentration results for the sample and blank, the equation listed in c) above will suffice for completing the g/100g calculations. With some chromatography software, such as Empower, the blank subtraction and gram/100g computation may also be executed within the software. Consult your software instruction manual. Data Obtained From Integrator Sample TRP Conc. IS Conc. Amount Ratio TRP Area IS Area Area Ratio Very Low 0.410/0.0403 10.1737 7405.8 102989 0.071909 Low 2.05/0.0403 50.8684 36760 102281 0.3594 Mid 10.3/0.0403 255.583 185145 103407 1.7904 High 20.5/0.0403 508.685 372353 103277 3.6054 Sample 105717 98633 1.0718 Blank 11323 98097 0.1154

Tryptophan Standard weight = 0.0103 grams 5-methyl-DL-tryptophan (Int Std.) = 0.0403 grams

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