AOAC2001.13DeterminationofVitaminA(Retinol)inFoods(食品中维生素A的测定).pdf

AOAC Official Method 2001.13 Determination of Vitamin A (Retinol) in Foods Liquid Chromatography First Action 2001

(Applicable for the determination of retinol from 0.15 g/g to 1 g/g.)

Caution: Potassium hydroxide is extremely caustic. This chemical can cause severe burns. Protect skin and eyes while performing this method. This method involves the use of flammable liquids. Perform behind a barrier when using hot water, steam, or an electric heating mantle. Use an effective fume removal device to remove flammable vapors as produced. Leave ample headroom in flask and add boiling chips before heating is begun. Place all controls, unless vapor sealed, outside of vapor area. This method utilizes toxic chemicals. Use an effective fume removal device to remove vapors as produced.

See Tables 2001.13A and 2001.13B for the results of the interlaboratory studies supporting acceptance of the method.

A. Principle

Standards and test samples are saponified in basic ethanol–water solution, neutralized, and diluted, converting fats to fatty acids and retinol esters to retinol. Retinol is quantitated using a high pressure liquid chromatography (HPLC) system with UV detection at 313 or 328 nm. Vitamin concentration is calculated by comparison of peak heights or peak areas of vitamins in test samples with those of standards.

B. Apparatus and Materials

(a) HPLC system.—(1) Pump.—A high pressure pump operating continuously at 1.0 to 2.0 mL/min with a flow precision of 1% or better. (2) Injector.—A manual injector or autosampling injector with a 20 L fixed loop having a typical sampling precision of 0.25% or better. (3) Chromatography column.—Reverse phase C18, 10 (4.6 250 mm) capable of separating cis and trans isomers of retinol with a resolution of 1.0 or greater. Cis retinol typically elutes prior to trans retinol on columns providing effective separation. (4) Detector.—Photometric detector monitoring absorbance at 328 nm. (Alternatively a wavelength of 313 nm can be used.) (5) Recorder, intergrator, or data collection system.—Compatible with detector used.

(b) Erlenmeyer flasks.—Low actinic 125 mL with neck adapted for connecting reflux condenser.

(d) Reflux condensers.—With adapters (if necessary) to attach 125 mL low actinic Erlenmeyer flasks and nitrogen lines.

(e) Volumetricflasks.—Low actinic 100 and 10 mL.

(f) Nitrogen blanket apparatus.—A supply of nitrogen gas with appropriate tubing and connectors to provide a constant nitrogen atmosphere blanket in the reflux apparatus during saponification.

C. Reagents

(a) (1) Certified vitamin A acetate concentrate (USP).—Equivalent to ca 30 mg of retinol/g of oil (content certified by United States Pharmacopeia, Rockville, MD 20852 USA; www.usp.org); or (2) Retinyl palmitate, all-trans.—Fluka Chemical Co., Ronkonkoma, NY, +1-800-358-5287. Request Certificate of Lot Analysis when ordering. If manufacturer’s certification is unavailable, or purity of standards needs to be verified, test vitamin A palmitate purity as follows: Dissolve 50 mg (record to the nearest 0.1 mg) of retinol palmitate standard in 2-propanol (UV-spectroscopy grade) in a 500 mL flask and dilute to volume. Dilute 10 mL of this solution to 100 mL with 2-propanol (final concentration is ca 10 mg/L). Measure maximum absorbance obtained at 325–328 nm using 1 cm pathlength cell and 2-propanol as a blank. Calculate the purity of the retinol palmitate as follows:

(Table 2001.13A Interlaboratory study results for the determination of vitamin A in foods by LC)

Food matrix sector No.		${ \mathsf { L a b s } } ^ { a ( b ) }$	Mean, g/100 g	$\mathsf { R e c . , \% }$	$\mathsf { s } _ { \mathsf { R } }$	${ \sf R S D } _ { \sf R } ,$ %	R	HORRAT
1	Matrix Margarine/butter (50/50 mix)	${ \mathsf { L a b s } } ^ { a ( b ) }$	Mean, g/100 g	$\mathsf { R e c . , \% }$	$\mathsf { s } _ { \mathsf { R } }$	${ \sf R S D } _ { \sf R } ,$ %	R	HORRAT
		12(0) 10(2)	857.08 1457.20	106.6	68.36 89.90	7.98 6.17	191.41 251.72	0.69 0.58
1	Margarine/butter (50/50 mix; spiked)					14.62
2	Chicken gravy (canned; spiked)	9(1)	131.13	97.1	19.17		53.68	0.95
3	Cheese sauce (spiked)	11(0)	271.45	103.7	34.96	12.88	97.89	0.94
4	Whole egg powder	10(0)	161.98		62.48	38.57	174.94	2.59
4	Whole egg powder (spiked)	12(0)	426.12	91.7	205.71	48.28	575.99	3.75
5	Multigrain cereal	12(0)	634.38		114.85	18.10	321.58	1.49
5 5	Corn cereal	12(1)	945.52		86.78	9.18	242.98	0.80
	Corn cereal (spiked)	12(1)	1395.91	108.5	76.11	5.45	213.11	0.51
6	Infant formula (powdered)	12(0)	584.15		95.98	16.43	268.74	1.34
6	NIST SRM 1846	12(0)	464.28	79.5	49.06	10.57	137.37	0.83
7	Dried nonfat milk	12(0)	817.27		102.94	12.60	288.23	1.08
7	Dried nonfat milk (spiked)	12(0)	1708.25	125.5	195.68	11.46	547.90	1.10
8	Cottage cheese	8(1)	46.02		8.19	17.80	22.93	0.99
8	Cottage cheese (spiked)	10(2)	411.03	98.9	69.29	16.86	194.01	1.30
9	Canned tuna in oil (spiked)	11(1)	262.36	89.5 100.1 13.2	56.11	21.39	157.11	1.55
Avg.

45.1.34

(Table 2001.13B Interlaboratory study results for the determination of vitamin A in foods by LC (Youden pair statistical treatment))

Youden pairs	${ \mathsf { L a b s } } ^ { a ( b ) }$	Mean, g/100 g	$\mathsf { s } _ { \mathsf { r } }$	${ \sf R S D } _ { \Gamma } , \ \%$	$\mathsf { s } _ { \mathsf { R } }$	${ \sf R S D _ { R } } , \ \%$	r	R	HORRAT
Dried nonfat milk (spiked) and margarine/butter (50/50 mix; spiked)	10(2)	1595.1	107.29	6.73	112.22	7.04	300.41	314.22	0.67
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 nonfat milk	12(0)	837.18	66.23	7.91	87.38	10.44	185.44	244.66	0.90
Multigrain cereal and infant formula (powdered)	12(0)	609.27	87.42	14.35	105.83	17.37	244.78	296.32	1.42
Cottage cheese (spiked) and NIST SRM 1846	10(2)	436.18	60.4	13.85	61.23	14.04	169.12	171.44	1.10
Canned tuna in oil (spiked) and cheese sauce (spiked)	11(1)	269.25	39.14	14.54	45.07	16.74	109.59	126.20	1.21
Chicken gravy (canned; spiked) and whole egg powder	8(1)	137.94	22.19	16.09	36.58	26.52	62.13	102.42	1.74

$$ \mathrm { P e r c e n t p u r i t y } = ( \mathrm { A B S } \quad 5 \quad 1 0 ^ { 6 } ) / ( 9 6 0 \quad \mathrm { W } ) $$

where ABS = absorbance maximum; 960 = absorbance of pure retinol palmitate (1% solution in 1 cm cell); W = weight of test portion in mg; and 5 10⁶ = combined dilution factors, conversion to 1% equivalent solution, and conversion to %.

Store retinol palmitate standard at 0–4 C to allow for easier handling while weighing.

(b) Acetic acid.—Glacial.

(d) Ethanol.—95%.

(e) Tetrahydrofuran.

(f) Hexane.

(g) Pyrogallic acid.—Crystals.

(h) Mobile phase.—Combine 860 mL methanol (HPLC grade) and 140 mL water. Mix well. Stir overnight to degas or mechanically degas prior to use.

(i) THF–ethanol (50 + 50).—Combine 500 mL tetrahydrofuran and 500 mL 95% ethanol. Mix well.

(j) Potassium hydroxide solution, 50%.—Slowly add 500 g of KOH pellets to 500 mL water contained in a 2 L thick wall Erlenmeyer flask. (Caution: The solution gives off substantial heat while KOH is dissolving; add the KOH in 100 g portions while the flask is being cooled with cold water. Swirl the flask gently to aid in dissolution ofthe KOH. Store in glass container with cork stopper.)

(k) Vitamin A working standard (ca 15 g/mL).—(1) Using USP standard.—Weigh 50 mg vitamin A acetate concentrate into a 100 mL low actinic volumetric flask. Record weight to nearest 0.1 mg. Record concentration in mg/g per USP certification. Add small amount of acetone (<3 mL) to aid dissolution. Dilute to volume with 95% ethanol. Store at 4 C in dark. Solution is stable for 2 weeks. (2) Using retinyl palmitate.—Weigh 55 mg of retinyl palmitate into a 100 mL low actinic volumetric flask. Record weight to nearest 0.1 mg. Record purity per supplier certification or purity test. Add pea-sized piece of pyrogallic acid, ca 50 mg. Dissolve and dilute to volume with hexane. Pipet 5 mL of solution to second 100 mL low actinic flask and dilute to volume with 95% alcohol. Store at 4 C in dark. Solution is stable for 2 weeks.

D. Extraction and Saponification

Turn on hot plate to preheat. Start and adjust cooling water flow to precool reflux condensers.

Prepare high standard by pipeting 5 mL vitamin A working standard, C(k), into a 125 mL low actinic Erlenmeyer flask. Add 25 mL of 95% ethanol. Proceed to addition of pyrogallic acid.

Prepare intermediate standard by pipeting 2 mL vitamin A working solution into a second 125 mL low actinic Erlenmeyer flask. Add 33 mL of 95% ethanol. Proceed to addition of pyrogallic acid.

Prepare low standard by pipeting 0.5 mL vitamin A working standard into a third 125 mL low actinic Erlenmeyer flask. Add 37.5 mL of 95% ethanol. Proceed to addition of pyrogallic acid.

Grind solids to pass a 40 mesh sieve. Blend liquid or wet materials to homogeneity and store 4 C in the dark.

To prepare low fat (<40% fat) test samples, weigh enough test sample ( 5 g) to give ca 50 g of vitamin A into a 125 mL low actinic Erlenmeyer flask. For test samples high in sugar, add 3 mL water and disperse the test portion as a slurry. Add 40 mL of 95% ethanol.

To prepare high fat test samples, weigh test sample ( 2 g) to give ca 50 g of vitamin A into a 125 mL low actinic Erlenmeyer flask. Add 40 mL of 95% ethanol.

Add a pea-sized piece (ca 50 mg) of pyrogallic acid (antioxidant) to each standard and test flask. Add a glass bead or boiling stone to promote even boiling.

Swirl all flasks to ensure that all materials are thoroughly dispersed in the solution.

Turn on N flow and ensure N atmosphere for all flasks before and while refluxing.

Pipet 10 mL of 50% KOH solution into each flask and immediately place flask on hot plate under reflux condenser. Swirl.

Reflux 45 min. Swirl flasks every 10 min.

Remove reflux flasks from hotplate, stopper with corks, and quickly cool flasks to room temperature using cold water or ice water.

Pipet 10 mL of glacial acetic acid into each flask to neutralize the KOH. Mix well and let flasks cool again to room temperature.

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