AOAC Final Action Methods in 2019

extraction are dried under nitrogen and reconstituted in 2-propanol (isopropyl alcohol; IPA). Separation is performed by reversed-phase chromatography on a C30 column. All- trans lutein, β-carotene, and lycopene are separated from their major cis isomers, as well as from zeaxanthin and α-carotene. Although the method does not involve high system backpressure normally associated with UHPLC, the low system volume is recommended for resolution with a 2.0mm internal diameter (id) column. B. Apparatus Note : Carotenoids are light sensitive, and all work should be done under protection from UV light. ( a )  UHPLC system .—Consisting of binary pump, autosampler, thermostatted column compartment, UV-Vis detector, and data acquisition software. Note : Although initial validation was done with a 60 mm flow cell, a standard 10 mm cell may be used if system suitability criteria in G ( b ) can be met. ( b )  Analytical column .—C30 carotenoid column, 3 μm, 2.0 × 250 mm (Part No. CT99S03-2502WT; YMC, Kyoto, Japan). Other columns may be used if the system suitability criteria in G ( b ) can be met. ( c )  Guard column .—C30, 3 μm, 2.1 × 10 mm (Part No. CT99S03-01Q1GC; YMC). ( d )  Guard cartridge holder .—Part No. XPGCH-Q1 (YMC). ( e )  Spectrophotometer .—Wavelength range of 200–700 nm, with 1 cm quartz cells. ( f )  Top-loading balance .—Capable of weighing to 0.1 g. ( g )  Analytical balance .—Capable of weighing to 0.01 mg. ( h )  Ultrasonic water bath .—40 kHz. ( i )  Reciprocating shaker .—Capable of 200 rpm. ( j )  Evaporator .—With pure nitrogen supply. ( k )  Laboratory centrifuge .—With adapters for 50 mL centrifuge tubes. ( l )  Centrifuge tubes .—50 mL, polypropylene. ( m )  Syringes .—1 mL, disposable. ( n )  Syringe filters .—0.2 μm, PTFE. ( o )  Class A volumetric flasks .—Various sizes; clear and amber. ( p )  Scintillation vials .—12 mL, amber.

AOAC Official Method 2016.13 Lutein, β-Carotene, and Lycopene in Infant Formula and Adult Nutritionals Reversed-Phase Ultra-High-Performance Liquid Chromatography (UHPLC) First Action 2016 Revised First Action 2017 Final Action 2019 ( β -Carotene and Lycopene Only)

[Applicable to the determination of all- trans lutein, cis isomers of lutein, all- trans β-carotene, cis isomers of β-carotene, and lycopene in infant formula and adult nutritionals. Materials tested must not contain measurable levels of β-apo-8′-carotenal. Because apocarotenal is used as an internal standard (ISTD), its presence in the test material would inflate the amount of ISTD measured in the samples, leading to artificially low results for the analytes. Unknown samples should be prepared as blanks, using 5 mL pyrogallol solution in place of ISTD solution, F ( c ), to demonstrate that they do not contain apocarotenal.] Caution : Tetrahydrofuran (THF) can form peroxides, and only THF stabilized with butylated hydroxytoluene (BHT) should be used. Refer to safety data sheets when using any reagent, and use appropriate personal protective equipment when performing analyses. Note: Throughout the method, estimated sample concentrations for standard and sample preparations are stated per 100 g on a reconstituted basis [as-is for ready-to-feed (RTF) liquids, 25 g sample plus 200 g water for powder samples, or diluted 1:1 by weight for liquid concentrates] in accordance with Standard Method Performance Requirements (SMPRs ® ) 2017.006–2017.008. Test sample concentrations calculated in H(j) – (l) are expressed per 100 g on an as-is basis for all samples, allowing each laboratory to apply appropriate reconstitution factors. See Tables 2016.13A – E for method performance information supporting acceptance of the method. A. Principle Test samples (reconstituted powders, liquid RTFs, and liquid concentrates) are spiked with an ISTD and treated briefly with potassium hydroxide. This liberates carotenoids from the sample matrix without causing isomerization of the analytes, although it may not result in the complete hydrolysis of lipids. Samples are then extracted with methyl tert -butyl ether (MTBE) and THF, followed by hexane. The supernatants from the liquid–liquid

( q )  HPLC vials .—Amber, with 300 μL inserts. ( r )  Class A volumetric pipets .—Various sizes.

Table 2016.13A. Interlaboratory study results for determination of all- trans lutein a Sample No. of labs used No. of outlier labs b Mean s r c s R d

RSD r

RSD R

HorRat

NIST SRM 1869

10 10

0 0 1 0 1 1

24.6 25.0 72.0

0.6 2.0 1.1 1.0 0.1

3.5 3.7 7.8 1.1 0.5

2.4 8.0 1.5

14.0 14.8 10.9 10.9 10.5 13.4

0.71 0.75 0.65 0.48 0.41 0.86

Milk-based toddler formula powder

Adult nutritional powder

9

Milk-based infant formula powder 1 Milk-based infant formula powder 2 Chocolate pediatric nutritional RTF

10

9.7 4.7

10.0

9 9

2.3 2.8

123.1 3.5 16.5

a  Results expressed as µg/100 g reconstituted final product. b  Values from outlier laboratories were not included in the statistical analysis. c  s r = SD for repeatability. d  s R = SD for reproducibility.

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