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

( f )  Light shields .—Clear UV shields with a minimum UV cutoff of 385 nm. ( g )  Centrifuge tubes .—Glass, 50 mL, fitted with PTFE-lined screw cap. ( h )  Vortexer. ( i )  Transfer pipets .—Glass; Pasteur. ( j )  Measuring pipets.— Glass, reusable, Class A, 2 mL. ( k )  Centrifuge .—Equipped with adapters for 50 mL centrifuge tubes. ( l )  HPLC system .—One isocratic pump, one programmable pump with flow rates ranging from 0.3 to 0.8 mL/min, one six-port switching valve, one autosampler capable of 20 µL injections, one multichannel UV detector (vitamin A, 325 nm; and vitamin E, 285 nm), a fluorescence detector (α-tocopherol, excitation 295 nm; emission 330 nm), and data collection system. ( m )  HPLC columns .—ES Industries (West Berlin, NJ) Chromegasphere Si-60, 3 µm, 3.0 × 30 mm, Part No. 183111 and Si-60, 3 µm, 3.0 × 200 mm, Part No. 143111 or Thermo Scientific (Bellefonte, PA) BETASIL Silica-100, 3 µm, 3.0 × 30 mm, Part No. 70003-033030 and BETASIL Silica-100, 3 µm, 3.0 × 200 mm, Part No. 70003-203030, or equivalent. C. Standards and Reagents Use only reagents of recognized analytical grade, unless otherwise specified, and distilled or demineralized water or water of equivalent purity. ( a )  Vitamin A acetate standard .—USP retinyl acetate, official lot with specified potency, or equivalent. Store per label instructions. ( b )  Vitamin A palmitate standard .—USP retinyl palmitate, official lot with specified potency, or equivalent. Store per label instructions. If potency information is not available it can be determined experimentally ( see Appendix I on the J. AOAC Int. website, http://aoac.publisher.ingentaconnect.com/content/aoac/ jaoac). ( c )  Vitamin E acetate standard .—USP all-rac-α-tocopheryl acetate, official lot with specified purity or equivalent. Store per label instructions. ( d )  α-Tocopherol standard .—USP α-tocopherol, official lot with specified purity or equivalent. Store per label instructions. ( e )  Methanol .—HPLC (distilled-in-glass) grade. ( f )  Methylene chloride (dichloromethane) .—HPLC (distilled- in-glass) grade. ( g )  Isopropanol .—HPLC (distilled-in-glass) grade. ( h )  2,2,4-Trimethylpentane (isooctane) .—HPLC (distilled-in- glass) grade. ( i )  Reagent alcohol. ( j )  Coconut oil .—Food grade. D. Preparation of Reagents and Standard Solutions All sources of contamination must be eliminated. This includes reagents, glassware, and all plastics except fluorinated polymers such as Teflon, which do not contain plasticizers. Also, traces of water, except that specifically required during the sample preparation, must be avoided. Solutions should be prepared fresh or whenever indications of contamination, chemical degradation, or changes in concentrations are evident. Expiration dates and storage conditions are stated. All solutions listed below may be scaled up or down for convenience provided good laboratory practices are observed. Preparation should be performed under shielded fluorescent lighting with a minimum UV cutoff of 385 nm. Avoid

AOAC Official Method 2012.09 Vitamins A and E in Infant Formula and Adult/Pediatric Nutritional Formula HPLC with UV and Fluorescence Detection First Action 2012

[The method involves the quantification of vitamin A palmitate, vitamin A acetate, vitamin E acetate, and α-tocopherol in infant formula and adult/pediatric nutritional formula samples. All- trans vitamin A palmitate and all- trans vitamin A acetate, like the free alcohol, are susceptible to cis-trans isomerization. The 13- cis isomer is the most common cis isomer and has 75% of the biological potency of the all- trans isomer. One 1 μg all- trans vitamin A palmitate is equivalent to 1.817 International Units (IU). One 1 μg vitamin A acetate is equivalent to 2.904 IU, and 1 μg free alcohol is equivalent to 3.33 IU. Similarly 1 mg dl-α-tocopheryl acetate is equal to 1 IU vitamin E. One 1 mg d-α-tocopheryl acetate, which is more active, is equal to 1.36 IU. One 1 mg d-α-tocopherol is equivalent to 1.49 IU.] A. Principle Samples are initially mixed with methanol, which precipitates proteins and disrupts micelles freeing lipids for extraction. Samples are then extracted with isooctane and centrifuged to separate the isooctane layer from the alcohol–water layer. A20 µL aliquot of the upper isooctane layer is injected onto the HPLC system. The HPLC system comprises two midbore (3.0 mm id) silica columns placed in series and separated by a switching valve. Following sample injection, vitamins A palmitate, vitamin A acetate, and vitamin E acetate are separated from α-tocopherol and other extraneous compounds present in sample extracts with a 3 cm × 3.0 mm id column and a 1% methylene chloride–0.06% isopropanol in isooctane mobile phase. After vitamin A palmitate, vitamin A acetate, and vitamin E acetate elute from the 3.0 cm column onto a 20 cm × 3.0 mm id column, a column switch takes place, and vitamin A palmitate, vitamin  A acetate, and vitamin E acetate are further separated on a 20 cm column and detected by UV at 325 nm (vitamin A palmitate and vitamin A acetate) and 285 nm (vitamin E acetate). α-Tocopherol is further separated from other extraneous compounds on the 3 cm column and detected by fluorescence at excitation and emission wavelengths of 295 and 330 nm, respectively. The concentration of trans vitamin A palmitate, trans vitamin A acetate, vitamin E acetate, and α-tocopherol are calculated by comparing peak areas of samples to peak areas of standards of known concentration. Quantitation of 13- cis vitamin A, the major cis isomer of vitamin A, is accomplished by using a response factor relative to that for the all- trans isomer. Results of the cis vitamin A determination are added to the all- trans vitamin A results to give total vitamin A results. B. Apparatus Common laboratory equipment and, in particular, the following: ( a )  Analytical balance .—Capable of weighing to the nearest 1 mg, with a readability of 0.1 mg. ( b )  Volumetric flasks .—Class A; various sizes. ( c )  Volumetric pipets .—Class A; various sizes. ( d )  Pipettors .—1–5 mL variable volume with tips, repipet heads, 5 and 25 mL, or equivalent. ( e )  Stir plate .—Multiposition with appropriate size test tube rack.

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