C. Alumina Column 45.1.22 AOAC Official Method 982.29 Vitamin D in Mixed Feeds, Premixes, and Pet Foods Liquid Chromatographic Method First Action 1982 Final Action 1983 [Applicable to all food products containing >2 IU and <200 IU vitamin D/g. For products containing ≥200 IU vitamin D/g, use 980.26 (see 45.1.19).] A. Principle Products are saponified and extracted, and unsaponifiable material is chromatographed successively on alumina to remove tocopherols and carotenes, if present, and on LC cleanup column to separate from interfering substances. Second LC column packed with silica separates vitamin D from impurities. Vitamin D is corrected for amount previtamin D formed during saponification. Vitamin D is sum of vitamin D and previtamin D. B. Reagents (a) Solvents.—Methanol, alcohol, CH3CN, toluene, peroxideand acid-free ether, n-hexane (spectroquality). Dry n-hexane by passing through column 60 × 8 cm diameter containing 500 g 50–250 µm silica dried 4 h at 150°C. (b) Sodium ascorbate solution.—Dissolve 3.5 g ascorbic acid in 20 mL 1M NaOH. Prepare fresh daily. (c) Antioxidant solution.—1 mg butylated hydroxytoluene (BHT)/mL hexane. (d) Petroleum ether.—Reflux over KOH pellets and collect fraction distilling between 40° and 60°C. (e) Ether–petroleum ether eluants.—8 + 92 and 40 + 60. (f) Alumina.—Neutral, type 1097 (E. Merck). Seal coarse fritted glass disk in lower end of 150 × 20 mm (id) tube and 250 mL bulb at upper end. Fit constricted portion at lower end with Teflon stopcock. Heat 250 g alumina overnight at 750°C. Cool and store in vacuum desiccator. Weigh 30 g dried alumina into 100 mL Erlenmeyer. Pipet 2.7 mL H2O into flask, and stopper. Heat 5 min on steam bath. Vigorously shake warm flask until powder is free-flowing. Cool and let stand 30 min. Add 40 mL petroleum ether to deactivated alumina, swirl, and transfer to tube, using petroleum ether. Let packing settle. Maintain head of >0.5 cm liquid on column throughout assay (alumina column can be used for only 1 assay). D. Liquid Chromatography (a) Liquid chromatograph.—Hewlett-Packard 1010A (replaced by HP 1050 or HP 1090), or equivalent, with 254 nm UV detector with 2 columns: cleanup and analytical. (b) Cleanup column.—Stainless steel, 250 × 4.6 (id) mm, packed with 10 µm particle size Li-Chrosorb RP-18, or equivalent. Typical operating conditions: chart speed, 1 cm/min; eluant flow rate, 1.4 mL/min; detector sensitivity, 0.08 AUFS; temperature, ambient; valve injection volume, 500 µL; solvent system, CH3CN–methanol–H2O (50 + 50 + 5). (c) Analytical column.—Stainless steel, 250 × 4.6 (id) mm, packed with 5 µm particle size Partisil-5,or equivalent, passing system suitability test. Typical operating conditions: chart speed, 1 cm/min; eluant flow rate, 2.6 mL/min (ca 1500 psi); detector sensitivity, 0.008 AUFS; temperature, ambient; valve injection volume, 200 µL; solvent system, n-hexane containing 0.35% (v/v) n-amyl alcohol. E. System Suitability Test for Analytical Column Dissolve 0.1 g system suitability standard solution in 100 mL toluene–mobile phase (5 + 95) and inject 200 µL. Determine peak resolution between previtamin D3 and trans-vitamin D3 as: (g) Mobile phase (for cleanup column).—CH3CN–methanol–H2O (50 + 50 + 5). (h) Mobile phase (for analytical column).—n-Hexane containing 0.35% (v/v) n-amyl alcohol. (i) Vitamin D standard solutions.—USP Reference Standard Ergocalciferol (if product labeled as containing vitamin D2) or Cholecalciferol (if la beled as con tain ing vitamin D or D3). Accurately weigh ca 12.5 mg vitamin D standard (W′) in 100 mL amber volumetric flask. Dissolve without heat in toluene and dilute to volume with toluene (125 µg/mL, solution A). Dilute 10 mL solution A to 100 mL with mobile phase (h). Dilute 10 mL of this solution to 100 mL with toluene–mobile phase (h) (5 + 95) for vitamin D standard (solution B) (1.25 µg/mL; 50 IU/mL). Also dilute 10 mL solution A to 100 mL with mobile phase (g); dilute 20 mL of this solution to 100 mL with mobile phase (g) (2.5 µg/mL, solution C). Prepare fresh daily. (j) System suitability standard solution.—Use USP Vitamin D Assay System Suitability Reference Standard, or prepare solution containing 2 mg vitamin D3 and 0.2 mg trans-vitamin D3/g in vegetable oil. Peaks of trans-vitamin D3 and previtamin D3 must have ca same peak heights. If necessary, increase previtamin D3 content by warming oil solution ca 45 min at 90°C. Store solution at 5°C. R= 2D B+C where D = distance between peak maximum of previtamin D3 and trans-vitamin D3; B = peak width of previtamin D3; and C = peak width of trans-vitamin D3. Performance is satisfactory if R is ≥1.0. F. Calibration Inject 500 µL vitamin D standard solution (solution C) onto cleanup column through sampling valve and 200 µL solution B onto analytical column, and adjust operating conditions of detector to give largest possible on-scale peaks of vitamin D. Determine retention time of vitamin D on cleanup and analytical columns and peak height of vitamin D on analytical column. Retention time of vitamin D on clean-up column should be between 15 and 25 min; adjust H2O content of mobile phase, if necessary, to achieve this situation. Retention time of vitamin D on analytical column should be between 15 and 20 min; adjust amyl alcohol content in mobile phase, if necessary, to achieve this situation. G. Preparation of Test Solution Isolation of unsaponifiable matter from powder.—Accurately weigh ca 25 g powdered test portion (preferably particle size <1 mm) into saponification flask. Add 80 mL alcohol, 2 mL Na 2005 AOAC INTERNATIONAL ascorbate solution, a pinch of Na2EDTA, and 10 mL 50% aqueous KOH solution. Reflux 30 min on steam bath under N2 with magnetic stirring. Cool and extract with five 60 mL portions of ether in saponification flask; decant each time and transfer ether layer to 1 L separator containing 100 mL H2O. Shake ether layer in separator (A), let separate, and transfer aqueous phase to 500 mL separator (B). Extract aqueous phase with 60 mL ether and transfer ether layer to separator (A). Wash combined ether extracts with 100 mL 0.5M KOH solution and then 100 mL portions of H2O until last washing is neutral to phenolphthalein. Add 150 mL petroleum ether, wait 0.5 h, separate from last drops of H2O, and add 2 sheets of 9 cm filter paper in strips to separator. Shake, add 1 mg BHT, and transfer to round-bottom flask, rinsing separator and paper with petroleum ether. Evaporate test solution by swirling (Rotavapor) under N2 stream in 40°C water bath. Dissolve residue immediately in 5 mL hexane. I. Determination (a) Cleanup.—Inject 500 µL test solution onto cleanup column through sampling valve and adjust operating conditions of detector to give largest possible on-scale peaks for vitamin D. Collect fraction between 3 min before and 3 min after vitamin D peak in 10 mL volumetric flask. Add 1 mL antioxidant solution and evaporate to dryness under N2 stream. Dissolve residue immediately in 2.0 mL toluene–mobile phase (5 + 95). Use this test solution for injection onto analytical column. (b) Assay.—Inject 200 µL cleaned up test solution (a) onto analytical column through sampling valve, and adjust operating conditions of detector to give largest possible on-scale peaks of vitamin D. Measure peak height of vitamin D. Use same operating conditions and inject standard solution B. Measure peak height of vitamin D. (c) Calculations.— H. Alumina Column Chromatography Transfer test solution to column with aid of three 10 mL portions of hexane. Discard eluate (contains carotenoids). Elute column with seven 10 mL portions of ether–hexane (8 + 92) and discard eluate (contains tocopherols and ethoxyquin). Elute column with seven 10 mL portions of ether–hexane (40 + 60), discard first 20–25 mL, collecting rest of eluate in round-bottom flask (contains vitamins A and D) when front of fluorescent vitamin A band is located 3 cm from bottom of column. Examine column <1 s under UV light (360 nm) with portable UV lamp to verify elution of vitamin A. Evaporate solution by swirling (Rotavapor) under N2 stream in 40°C water bath. Transfer to centrifuge tube, rinsing flask with 2–3 mL ether, evaporate ether, and dissolve in 1.0 mL methanol with warming. Add 1.0 mL CH3CN and cool. Centrifuge and use clear supernate for injection onto cleanup column. 2005 AOAC INTERNATIONAL Vitamin D potency in IU/g test portion = 125 . × P × W ′ × V × 40000 P′ × W × V ′ where P = peak height of cleaned up vitamin D in test solution; 1.25 = correction factor for previtamin D formed during refluxing for saponification; P′ = peak height of vitamin D in reference solution; W = g test portion weighed; W′ = mg reference standard; V = total mL test solution; V′ = total mL reference standard solution; 40 000 = IU vitamin D/mg USP Reference Standard. Reference: JAOAC 66, 751(1983). CAS-67-97-0 (cholecalciferol; vitamin D3) CAS-50-14-6 (ergocalciferol; vitamin D2)