AOAC SPIFAN ERP & Working Group Method Reviews (July 10, 2019)

G. HPLC Conditions ( a )  System configuration 1 .— See Figure 2012.09A . ( b )  System configuration 2 .— See Figure 2012.09B . ( c )  System settings .— ( 1 )  Run time .—25–30 min, depending on columns used. ( 2 )  Pump 1 flow rate .—ES Industries columns: 0.4 mL/min 0–11 min, 0.8 mL/min 11.1–29 min, 0.4 mL/min 29–30.0 min. Thermo Fisher Scientific columns: 0.3 mL/min 0–10 min, 0.6 mL/min 10.1–25 min, 0.3 mL/min 25–26 min. ( 3 )  Pump 2 flow rate .—0.6 mL/min. ( 4 )  Injection volume .—20 µ L. ( 5 )  Multichannel UV detector settings .—Channel 1, 325 nm; Channel 2, 285 nm. ( 6 )  Fluorescence detector settings .—Excitation, 295 nm; emission, 330 nm. ( d )  System startup .—At system startup, turn on the pumps and detector. Allow mobile phase to pass through each column for at least 30 min. After the system has equilibrated, auto-zero all detectors before beginning an analysis. ( e )  Column switch time determination, test injection .— ( 1 ) After the system has equilibrated, the column switch time is determined by injecting the high working standard solution (test injection) onto the 3 × 30 mm column with the system in configuration 2. In this configuration, the standard is only going through the 3 × 30 mm column. See Figure 2012.09B . ( 2 ) After determining the retention time of vitamin E acetate and α -tocopherol peaks from the 3 × 30 mm column, set the column switch time to approximately halfway between when the vitamin E peak returns to baseline and the beginning of the α -tocopherol peak ( see Figure 2012.09C ). The resolution (R) between α -tocopherol and vitamin E acetate should be ≥ 2.5 ( see Appendix II for an example calculation on the J. AOAC Int. website, http://aoac. ( 1 ) Standard and sample run times are approximately 30 min. Typical retention times are 6–8 and 7–9 min for 13- cis and all- trans vitamin A palmitate, respectively; 12–17 and 14–21 min for 13- cis and all- trans vitamin A acetate, respectively; 16–25 min for vitamin E acetate; and 6–8 min for α -tocopherol ( see Appendix III for example chromatograms on the J. AOAC Int. website, http:// aoac.publisher.ingentaconnect.com/content/aoac/jaoac). These times may vary somewhat from column to column or system to system, but remain fairly constant for a given column, providing care is taken in the preparation of the mobile phase and in treatment of the column. ( 2 ) Confirm that the column switch time is set correctly according to the test injection. Perform three consecutive injections of the high working standard, and confirm that peak responses publisher.ingentaconnect.com/content/aoac/jaoac). ( f )  Standard, control, and sample injections .—

Figure 2012.09C. Example test injection.

homogeneous at the subgram level should be reconstituted first and sampled immediately after the powder is completely dissolved. Sample sizes are chosen so that after dilution with laboratory water, if any, a total sample weight of about 4 g at a total solids value of <20% and a maximum oil content of about 125 mg is achieved while maintaining sample preparation concentrations of vitamin A and vitamin E within the standard concentrations. Typically sample sizes are 3.5–4.0 g ready-to-feed infant formulas and 1.75–2.0 g (plus 2.0 mL water) concentrated infant formulas. Powdered infant formulas may first be reconstituted and treated as a ready-to-feed infant formula, or approximately 0.45–0.5 g powdered formula may be weighed directly into a 50 mL centrifuge tube and diluted with 3.5 mL laboratory water. F. Procedure Test portion .—Weigh an appropriate amount of sample directly into a 50 mL centrifuge tube, and, if necessary, add enough laboratory water so that the combined weight of the sample and any water is approximately 4 g. Water should be added rapidly to powder samples, and the sample should be immediately mixed until all of the powder is dissolved. To each sample, add 25 mL (±10%) methanol using a repipet head or equivalent glassware, cap the centrifuge tube, and vortex immediately and rapidly for at least 30 s, or add a stir bar and 25 mL (±10%) methanol, and begin stirring at a rate that is fast enough to form a vortex, but does not cause sample to splash out of the centrifuge tube. Methanol should not be added to more than two samples, consecutively, without vortexing or stirring. After the addition of methanol, let samples stand for at least 10 min but not more than 40 min if vortexing, or let samples stir for at least 10 min if using a multiposition stir plate. Using a volumetric pipet, add 10.0 mL isooctane to each centrifuge tube. Cap the centrifuge tubes and vortex rapidly for at least 45 s, or cap the centrifuge tubes and stir at a rate that is fast enough to form a vortex for at least 2 min. Isooctane can be added to any number of samples, consecutively, prior to vortexing or stirring. Add 5 mL (±10%) laboratory water to each tube. Cap the centrifuge tubes and vortex rapidly for at least 20 s or stir at a rate that is fast enough to form a vortex for at least 1 min. Water can be added to any number of samples, consecutively, prior to vortexing. If samples cannot be effectively vortexed or stirred, they can be shaken by hand for at least 20 s. A sample must be repeated from the beginning of the procedure if spillage or leakage occurs anytime during these steps. Centrifuge samples until there is a clean phase separation between the isooctane and the water–methanol phase. Using a glass transfer pipet, transfer about 1 mL of the clear upper layer into appropriate autosampler vials.

Table 2012.09. Standard dilution factors

Working standard dilution factors

Vitamin Low Very low Vitamin A palmitate 0.125 0.312500 0.625000 2.500000 Vitamin A acetate 0.0125 0.0312500 0.0625000 0.2500000 Vitamin E acetate 0.005000 0.012500 0.025000 0.100000 α -Tocopherol 0.005000 0.008333 0.025000 0.100000 High Middle

© 2012 AOAC INTERNATIONAL