AOAC Methods for Review in Codex STAN 234_11-2018

AOAC Official Methods Listed in CXS 234 for Milk and Milk Products

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D. Ashing Procedure Note : For liquid formulaes, shake container before weighing. Place composite portion in previously cleaned Vycor evapo- rating dish (which may contain 5 g filter pulp for ease of han- dling). Exact amount of composite required will depend on concentration of minerals present. (For powders, take ≥ 1.5 g.) In general, 25 mL will be adequate. If some minerals, in par- ticular Fe, Cu, or Mn, are at very low levels, a larger portion ( ≤ 50 mL) may be necessary. Dry portion in 100°C oven overnight or in microwave oven (programmed over ca 30 min). When dry, heat on hot plate until smoking ceases, and then place dish in 525°C furnace (care- fully avoiding ignition) for minimum time necessary to obtain ash that is white and free from C, normally 3–5 h, but ≤ 8 h. Remove dish from furnace and let cool. Ash should be white and free from C. If ash contains C particles (i.e., it is gray), wet with H 2 O and add 0.5–3 mLHNO 3 . Dry on hot plate or steam bath and return dish to 525°C furnace 1–2 h. Dissolve ash in 5 mL 1N HNO 3 , warming on steam bath or hot plate 2–3 min to aid solution. Add solution to 50 mL volu- metric flask and repeat with 2 additional portions of 1N HNO 3 . Dilute to 50 mL with 1N HNO 3 . ( Note : Additional dilutions may be necessary to bring concentrations within the linear range of instrument.) solution to final dilution of each standard and test solution to make 0.1 % (w/v) La for determination of Ca and Mg only. Add CsCl solution to final dilution of each standard and test solution to make 0.5 % (w/v) Cs (0.04M) for determi- nation of Na and K only. Prepare blanks representing all reagents and glassware, and carry through entire procedure. Prepare calibration curve (concentration vs absorbance) for each mineral to be determined, using wavelength and flame specified in Table 985.35B . Optimize flame parameters in ac- cordance with instrument manufacturer’s instructions. Prepare solutions for calibration of instrument to cover linear range of calibration curve. See instrument instruction manual. Assay samples in similar manner. Determine concentration of each mineral from its calibration curve, and calculate con- centration in test sample, taking into account test portion size and dilutions. Ref.: J. Assoc. Off. Anal. Chem. 68 , 514(1985); J. AOAC Int . 80 , 834–844(1997) E. Determination Add LaCl 3

Method Performance: See Table 986.24 for method performance data. A. Principle

Phosphorus is determined by spectrophotometry on ashed test portion by complexing with molybdovanadate reagent. B. Apparatus ( a ) Spectrophotometer .—Capable of operation at 400 nm. ( b ) Muffle furnace .—Equipped with pyrometer and con- troller. ( c ) Ashing dishes .—Silica or porcelain. C. Reagents ( a ) Hydrochloric acid solution. —(1 + 3, v/v). Add 250 mL HCl to 750 mL H 2 O. ( b ) Molybdovanadate reagent .—Dissolve 20 g ammo- nium molybdate in 200 mL hot H 2 O and cool. Dissolve 1.0 g ammonium metavanadate in 125 mL hot H 2 O, cool, and add 160 mL HCl. Gradually add, with stirring, molybdate solution to vanadate solution and dilute with H 2 O to 1.0 L. ( c ) Phosphorus standard solutions. —( 1 ) Stock standard solution. —2 mg P/mL. Weigh 8.7874 g KH 2 PO 4 previously dried 2 h at 105°C. Quantitatively transfer to 1 L volumetric flask and add ca 750 mL H 2 O to dissolve. Dilute to volume with H 2 O. Store in refrigerator. ( 2 ) Working standard solu- tion. —0.1 mg P/mL. Dilute 50 mL stock standard solution with H 2 O to 1 L. Store in refrigerator. Prepare fresh on day of analysis. D. Preparation of Test Solution Note : For liquid formulae, shake container before weighing. Accurately weigh amount of test portion to contain ca 4.0 mg P into ashing dish and evaporate to dryness on hot plate or steam bath. Ignite in muffle furnace at maximum tempera- ture of 600°C until free of C (3–4 h). Cool; add 40 mL HCl solution, C ( a ), and several drops of HNO 3 ; and bring to boil on hot plate. Cool, transfer quantitatively to 100 mL volumetric flask, and dilute to volume with H 2 O. E. Determination Transfer portions of 0.0, 5.0, 8.0, 10.0, and 15.0mLworking standard solution to respective 100 mL volumetric flasks. These represent 0.0, 0.5, 0.8, 1.0, and 1.5 mg P. Pipet 20.0 mL test solution into each 100 mL volumetric flask. To each stand- ard and test flask, add 20.0 mL molybdovanadate reagent, di- lute to volume with H 2 O, and mix well. Let flasks stand 10 min for complete color development. Determine absorbance of standards and sample in 1 cm cells at maximum near 400 nm. Use 0.0 mg standard to zero spec- trophotometer. Use linear regression of standard absorbance vs mg P of standards to determine mg P for each sample. F. Calculations Calculate content of phosphorus in test sample as follows: P, mg/L = mg P × 5000 × test sample density/g sample

986.24 Phosphorus in Infant Formula and Enteral Products, Spectrophotometric Method

First Action 1986 Final Action 1988 Revised First Action 1997

Caution: See Appendix B: safety notes on safe handling of acids. Dispose of waste solvents in an appropriatemanner com- patible with applicable environmental rules and regulations.

10/9/2018