This document provides details on an AOAC official method for determining vitamin A content in foods using liquid chromatography. It describes:
1) The principle of saponifying samples to convert fats to fatty acids and retinol esters to retinol before quantifying retinol using HPLC.
2) Required apparatus including an HPLC system with UV detector, glassware, reagents, and standards for establishing calibration curves.
3) Procedures for sample extraction and saponification, as well as preparation of vitamin A working standards.
This document provides details on an AOAC official method for determining vitamin A content in foods using liquid chromatography. It describes:
1) The principle of saponifying samples to convert fats to fatty acids and retinol esters to retinol before quantifying retinol using HPLC.
2) Required apparatus including an HPLC system with UV detector, glassware, reagents, and standards for establishing calibration curves.
3) Procedures for sample extraction and saponification, as well as preparation of vitamin A working standards.
This document provides details on an AOAC official method for determining vitamin A content in foods using liquid chromatography. It describes:
1) The principle of saponifying samples to convert fats to fatty acids and retinol esters to retinol before quantifying retinol using HPLC.
2) Required apparatus including an HPLC system with UV detector, glassware, reagents, and standards for establishing calibration curves.
3) Procedures for sample extraction and saponification, as well as preparation of vitamin A working standards.
This document provides details on an AOAC official method for determining vitamin A content in foods using liquid chromatography. It describes:
1) The principle of saponifying samples to convert fats to fatty acids and retinol esters to retinol before quantifying retinol using HPLC.
2) Required apparatus including an HPLC system with UV detector, glassware, reagents, and standards for establishing calibration curves.
3) Procedures for sample extraction and saponification, as well as preparation of vitamin A working standards.
45.1.34 equivalent). Request Certificate of Lot Analysis when ordering.
If AOAC Official Method 2001.13 manufacturer’s certification is unavailable, or purity of standards Vitamin A (Retinol) in Foods needs to be verified, test vitamin A palmitate purity as follows: Liquid Chromatography Dissolve 50 mg (record to the nearest 0.1 mg) of retinol palmitate First Action 2001 standard in 2-propanol (UV-spectroscopy grade) in a 500 mL flask (Applicable to the determination of retinol from 0.15 mg/g to and dilute to volume. Dilute 10 mL of this solution to 100 mL with 1 g/g.) 2-propanol (final concentration is ca 10 mg/L). Measure maximum absorbance obtained at 325–328 nm using 1 cm pathlength cell and Caution: Potassium hydroxide is extremely caustic. This chemical 2-propanol as a blank. Calculate the purity of the retinol palmitate as can cause severe burns. Protect skin and eyes while follows: performing this method. This method involves the use of flammable liquids. Perform behind a barrier when using Percent purity = (ABS ´ 5 ´ 106) / (960 ´ W) hot water, steam, or an electric heating mantle. Use an effective fume removal device to remove flammable where ABS = absorbance maximum; 960 = absorbance of pure vapors as produced. Leave ample headroom in flask and retinol palmitate (1% solution in 1 cm cell); W = weight of test add boiling chips before heating is begun. Place all portion in mg; and 5 ´ 106 = combined dilution factors, conversion controls, unless vapor sealed, outside of vapor area. This to 1% equivalent solution, and conversion to %. method utilizes toxic chemicals. Use an effective fume Store retinol palmitate standard at 0°–4°C to allow for easier removal device to remove vapors as produced. handling while weighing. (b) Acetic acid.—Glacial. See Tables 2001.13A and B for the results of the interlaboratory (c) Methanol.—LC grade. study supporting acceptance of the method. (d) Ethanol.—95%. A. Principle (e) Tetrahydrofuran. Standards and products are saponified in basic ethanol–water (f) Hexane. solution, neutralized, and diluted, converting fats to fatty acids and (g) Pyrogallic acid.—Crystals. retinol esters to retinol. Retinol is quantitated using a high pressure (h) Mobile phase.—Combine 860 mL methanol (LC grade) and liquid chromatography (HPLC) system with UV detection at 313 or 140 mL water. Mix well. Stir overnight to degas or mechanically 328 nm. Vitamin concentration is calculated by comparison of peak degas prior to use. heights or peak areas of vitamins in test samples with those of (i) THF–ethanol (50 + 50).—Combine 500 mL tetrahydrofuran standards. and 500 mL 95% ethanol. Mix well. B. Apparatus and Materials (j) Potassium hydroxide solution.—50%. Slowly add 500 g KOH pellets to 500 mL water contained in a 2 L thick wall Erlenmeyer (a) HPLC sys tem.—(1) Pump.—A high pres sure pump flask. (Caution: The solution gives off substantial heat while KOH is operating continuously at 1.0 to 2.0 mL/min with a flow precision of dissolving; add the KOH in 100 g portions while the flask is being ±1% or better. (2) Injector.—A manual injector or autosampling cooled with cold water. Swirl the flask gently to aid in dissolution of injector with a 20 mL fixed loop having a typical sampling precision the KOH. Store in glass container with cork stopper.) o f ± 0 . 2 5 % o r b e t t e r. ( 3 ) C h ro m a t o g r a p h y (k) Vitamin A working standard (ca 15 mg/mL).—(1) Using USP column.—Reversed-phase C18, 10 m (4.6 ´ 250 mm) capable of standard.—Weigh 50 mg vitamin A acetate concentrate into a separating cis and trans isomers of retinol with a resolution of 1.0 or 100 mL low actinic volumetric flask. Record weight to nearest greater. Cis retinol typically elutes prior to trans retinol on columns 0.1 mg. Record concentration in mg/g per USP certification. Add pro vid ing effec tive sep a ra tion. (4) De tec tor.—Pho to met ric small amount of acetone (<3 mL) to aid dissolution. Dilute to detector monitoring absorbance at 328 nm. (Alternatively a volume with 95% ethanol. Store at 4°C in dark. Solution is stable for wavelength of 313 nm can be used.) (5) Recorder, integrator, or 2 weeks. (2) Using retinyl palmitate.—Weigh 55 mg of retinyl data collection system.—Compatible with detector used. palmitate into a 100 mL low actinic volumetric flask. Record weight (b) Erlenmeyer flasks.—Low actinic 125 mL with neck adapted to nearest 0.1 mg. Record purity per supplier certification or purity for connecting reflux condenser. test. Add pea-sized piece of pyrogallic acid, ca 50 mg. Dissolve and (c) Hot plate.—With sufficient heating surface area to handle dilute to volume with hexane. Pipet 5 mL of solution to second multiple reflux apparatus setups preferred. 100 mL low actinic flask and dilute to volume with 95% alcohol. (d) Reflux condensers.—With adapters (if necessary) to attach Store at 4°C in dark. Solution is stable for 2 weeks. 125 mL low actinic Erlenmeyer flasks and nitrogen lines. D. Extraction and Saponification (e) Volumetric flasks.—Low actinic 100 and 10 mL. Turn on hot plate to preheat. Start and adjust cooling water flow to (f) Nitrogen blanket apparatus.—A supply of nitrogen gas with precool reflux condensers. appropriate tubing and connectors to provide a constant nitrogen atmosphere blanket in the reflux apparatus during saponification. Prepare high standard by pipeting 5 mL vitamin A working standard, C(k), into a 125 mL low actinic Erlenmeyer flask. Add C. Reagents 25 mL of 95% ethanol. Proceed to addition of pyrogallic acid. ( a ) (1 ) C e r t i f i e d v i t a m i n A a c e t a t e c o n c e n t r a t e Prepare intermediate standard by pipeting 2 mL vitamin A (USP).—Equivalent to ca 30 mg of retinol/g of oil (content certified working solution into a second 125 mL low actinic Erlenmeyer by U.S. Pharmacopeia, Rockville, MD 20852, USA; www.usp.org); flask. Add 33 mL of 95% ethanol. Proceed to addition of pyrogallic or (2) Retinyl palmitate, all-trans.—Fluka Chemical Co. (or acid. 2006 AOAC INTERNATIONAL
ã 2005 AOAC INTERNATIONAL
Table 2001.13A. Interlaboratory study results for the determination of vitamin A in foods by LC
Prepare low standard by pipeting 0.5 mL vitamin A working Add a pea-sized piece (ca 50 mg) of pyrogallic acid (antioxidant) standard into a third 125 mL low actinic Erlenmeyer flask. Add to each standard and test flask. Add a glass bead or boiling stone to 37.5 mL of 95% ethanol. Proceed to addition of pyrogallic acid. promote even boiling. Grind solids to pass a 40 mesh sieve. Blend liquid or wet materials Swirl all flasks to ensure that all materials are thoroughly to homogeneity and store £4°C in the dark. dispersed in the solution. To prepare low fat (<40% fat) test samples, weigh enough test Turn on N2 flow and ensure N2 atmosphere for all flasks before sample (£5 g) to give ca 50 mg of vitamin A into a 125 mL low actinic and while refluxing. Erlenmeyer flask. For test samples high in sugar, add 3 mL water and Pipet 10 mL of 50% KOH solu tion into each flask and disperse the test portion as a slurry. Add 40 mL of 95% ethanol. immediately place flask on hot plate under reflux condenser. Swirl. To prepare high fat test samples, weigh test sample (£2 g) to give Reflux 45 min. Swirl flasks every 10 min. ca 50 mg of vitamin A into a 125 mL low actinic Erlenmeyer flask. Remove reflux flasks from hotplate, stopper with corks, and Add 40 mL of 95% ethanol. quickly cool flasks to room temperature using cold water or ice water.
Table 2001.13B. Interlaboratory study results for the determination of vitamin A in foods by LC (Youden pair statistical treatment)
Mean, a(b) sr RSDr, % sR RSDR, % Youden pairs Labs mg/100 g r R HorRat Dried nonfat milk (spiked) and 10(2) 1595.1 107.29 6.73 112.22 7.04 300.41 314.22 0.67 margarine/butter (50/50 mix; spiked) Corn cereal and corn cereal (spiked) 10(2) 1173.3 48.33 4.12 85.49 7.29 135.32 239.37 0.66 Margarine/butter (50/50 mix) and dried 12(0) 837.18 66.23 7.91 87.38 10.44 185.44 244.66 0.90 nonfat milk Multigrain cereal and infant formula 12(0) 609.27 87.42 14.35 105.83 17.37 244.78 296.32 1.42 (powdered) Cottage cheese (spiked) and NIST 10(2) 436.18 60.4 13.85 61.23 14.04 169.12 171.44 1.10 SRM 1846 Canned tuna in oil (spiked) and 11(1) 269.25 39.14 14.54 45.07 16.74 109.59 126.20 1.21 cheese sauce (spiked) Chicken gravy (canned; spiked) and 8(1) 137.94 22.19 16.09 36.58 26.52 62.13 102.42 1.74 whole egg powder a(b) Number of laboratories where a = number of laboratories retained after outliers removed and b = number of outlier laboratories.
2006 AOAC INTERNATIONAL
ã 2005 AOAC INTERNATIONAL
Pipet 10 mL of glacial acetic acid into each flask to neutralize the reagents section; 10 000 = combined dilution factors for vitamin A KOH. Mix well and let flasks cool again to room temperature. standard. Quantitatively transfer the solution in each flask to a 100 mL low (2) Using retinyl palmitate.—Determine the response factor for actinic volumetric flask using THF–95% ethanol (50 + 50). Dilute to vitamin A (RFA) using the following calculation: volume with the same solvent mixture. Stopper and invert volumetric flask 10 times. mgstd ´ mL std ´ purity std ´ 0.5458 RFA = Allow flasks to set for at least 1 h at room temperature and PkHTstd ´ 200 preferably overnight in refrigerator to precipitate fatty acid salts formed during saponification. In some cases, centrifugation may where puritystd = percent purity certified by supplier or determined, reduce settling time. divided by 100; mgstd = mg of retinyl palmitate weighed; PkHTstd = E. Determination peak height or area of standard from chromatogram; mLstd = mL of Start HPLC system(s) and allow to warm up and equilibrate for a working standard used in procedure; 0.5458 = ratio of retinol to minimum of 30 min with mobile phase flowing at flow rate of retinyl palmitate molecular weights; and 200 = combined dilution 1.0 mL/min. factors/ conversion from mg to mg. Inject vi ta min A stan dards that have been taken through The RFA values of the low, medium, and high standards should saponification onto HPLC system. Adjust mobile phase to achieve a agree with each other within 3% relative since the detector response resolution of 1.5 or better for cis and trans forms. All trans retinol should be linear across this concentration range. Use an average of should elute in ca 9 min. Cis retinol will elute as a small peak just RFA values calculated from high, medium, and low standards for test prior to the all trans form. sample quantitation. In ject high, me dium, and low stan dards. Ad just de tec tor Measure the peak heights or areas corresponding to retinol sensitivity to give peak heights of 50–90% of full scale for the (vitamin A) in the test sample extracts. The 13-cis isomer of retinol vitamin of interest at the high standard. Repeat injection of standard (eluting immediately proceeding the all trans isomer) might be until peak height(s) are reproducible. present in some test samples. Measure the 13-cis peak also. Inject test solutions. Intersperse with standard solution injections Multiply the height or area of the 13-cis retinol peak by 1.08 (to after every 9 tests. [If retinol in test exceeds the peak height of the compensate for difference in absorbance compared to the trans respective high standard by more than 25%, dilute test solutions isomer). using a solution of 10 mL 50% KOH, 40 mL of 95% ethanol, 10 mL Add the corrected peak height or area for the 13-cis isomer to that glacial acetic acid, and 40 mL THF–95% ethanol (50 + 50).] of the all-trans isomer to give total test sample peak height or area. F. Calculations Calculate the concentration of vitamin A (in mg/g as retinol) using (a) Vitamin A.—Calculate mg/g of vitamin A (as retinol) as the following equation: follows: Measure the peak heights or areas of the standards. RFA ´ PkHTSPLE ´ 100 (1) Using USP standard.—Determine the response factor for Vitamin A, mg/g (as retinol) = vitamin A (RFA) using the following calculation: W
mgstd ´ mL std ´ concstd where RFA = response factor for vitamin A; PkHTSPLE = total test RFA = PkHTstd ´10000 sample peak height or area of all trans and 13-cis retinol; 100 = dilution volume of test portion, mL; and W = weight of test portion, g. where PkHT std = peak height or area of stan dard from chromatogram; mLstd = mL of working standard used in procedure; Alternatively, a 3 level calibration using a zero order polynomial concstd = concentration of USP vitamin A (as retinol) per USP fit (linear) can be used to calculate vitamins A (and E). certification (mg/g); mgstd = mg of USP standard weighed in Reference: J. AOAC Int. 85, 424(2002).
