50.1.05
00 mL each stock
AOAC Official Method 992.26
Vitamin D3 (Cholecalciferol)
in Ready-To-Feed Milk-Based Infant Formula
Liquid Chromatographic Method
First Action 1992
Final Action 1995
Codex–AOAC Method*
(Ap pli ca ble to ready-to-feed milk-based in fant for mu las
containing 488–533 IU/L vitamin D3.)
A. Principle
Test portion is saponified and ether extracted. Extract is injected
for cleanup on silica liquid chromatographic (LC) column. Eluent
fraction containing vitamin D3 is collected and quantified by LC on
silica column with ultraviolet (UV) detection at 254 nm.
B. Apparatus
(a) LC system for cleanup.—With constant-flow pump capable
of 3000 psi and maintaining 2 mL/min eluant flow rate; 30 ´ 4.6 mm
id guard column with amino packing (Brownlee Spheri-5 Amino,
Rainin, Woburn, MA, USA, is suitable); 250 ´ 4.6 mm id
normal-phase cleanup column packed with silica (Whatman
Partisil-5 PAC is suitable); UV detector, sensitivity 0.1 AUFS,
254 nm filter; injector with 320–370 mL loop volume; chart recorder,
speed 0.5 cm/min; mobile phase, hexane–amyl alcohol (99.2 + 0.8).
(b) LC system for quantitation.—Dual-piston pump with
3000 psi and maintaining 3 mL/min eluant flow rate; 150 ´ 4.6 mm
id normal-phase analytical column packed with 3 mm silica (Apex I,
Jones Chromatography, Lakewood, CO, USA, is suitable); UV
detector, sensitivity ca 0.003 AUFS, 254 nm filter; valve injector
with 250 mL loop volume; chart recorder, speed 0.5 cm/min; mobile
phase, hexane–amyl alcohol (99.85 + 0.15).
(c) Timing device.—For collecting eluent fractions from LC
cleanup system.
(d) Water bath.—Maintaining 75° ± 2.0°C.
(e) Boiling flask.—Glass, 500 mL, flat bottomed, with 20/40
ground glass joint.
(f) Separatory funnels.—500 mL, with Teflon stopcocks.
(g) Rotary evaporator.—500 mL capacity, minimum.
(h) Filters.—Disposable cellulose filter, 0.45 mm.
C. Reagents
(a) Solvents (v/v).—(1) Reagent alcohol.—5% 2-propanol,
95% spe cially de na tured al co hol (for mula 3A). (2) Ethyl
ether.—98% minimum purity, anhydrous. (3) Petroleum ether. (4)
Amyl al co hol.—n-Pen ta nol, 99% min i mum pu rity. (5)
Hexane.—n-Hexane, 85–100% purity, LC grade. (6) Acetone.—LC
grade.
(b) Potassium hydroxide.
(c) Ascorbic acid.
(d) Vitamin D3 standard solutions.—(Note: Protect vitamin D
solutions from exposure to white light; wrap flasks with aluminum
foil and store at 4°C. Prepare complete fresh set of standards every
7 days.)
(1) Stock standard solutions.—Accurately weigh ca 10 mg
vitamin D3, USP reference standard, to nearest 0.1 mg, into each of 3
labeled 50 mL volumetric flasks. Dissolve and dilute to volume with
hexane. Mix well. Label as stock standards 1, 2, and 3.
(2) Intermediate standard solutions.—Pipet 1.
standard solution into separate 200 mL volumetric flasks. Dilute to
volume with hexane. Mix well. Label as intermediate standards 1, 2,
and 3.
(3) Working standard solutions.—Pipet 25.0, 15.0, and 7.0 mL
intermediate standard solutions 1, 2, and 3, respectively, into
separate 100 mL volumetric flasks, labeled I, II, and III, respectively
(ca 10, 6, and 3 IU/mL). Dilute to volume with hexane and mix well.
(e) Compressed nitrogen.
D. Saponification and Extraction of Test Portion
Preheat water bath to 75°C. Accurately weigh amount of infant
formula equivalent to ca 12 IU vitamin D3 to nearest mg and
transfer test portion to centrifuge tube. Add 15 mL reagent
alcohol and ca 400 mg ascorbic acid to each tube. Cap tubes and
shake vigorously 5 s. Add 7.5 g KOH pellets to each tube, cap
tightly, and shake vig orously ca 20 s to dissolve pellets. Loosen
caps to vent pressure and place tubes in bath. After 30 min,
remove tubes from bath and bring rapidly to room temperature in
cold tap water bath.
Transfer tube con tents to 500 mL separatory funnel, rinsing
tube once with 5 mL reagent al cohol and adding rinse to funnel.
Add 130 mL ethyl ether to funnel, stopper tightly, and shake
vigorously 30 s. Add 130 mL petroleum ether to funnel, stopper
tightly, and shake vig orously 30 s. Let lay ers separate ³2 min.
Drain and discard all but ca 1 mL brown aqueous layer. Leave any
emulsion layer in funnel. Add 50 mL H2O to funnel, stopper
tightly, and shake vig orously 15 s. Vent, let lay ers separate, and
drain off all but ca 1 mL aqueous layer, leav ing any emulsion
layer in funnel. Wash ether ex tract with another 50 mL H2O and
drain as before. Add 15 mL reagent al cohol and 50 mL H2O and
shake vigorously 15 s. Drain all of aqueous layer plus ca 1 mL
ether layer. Discard all aqueous layers. Transfer ether layer into
labeled 500 mL boiling flask. Close stopcock immediately after
all ether layer has been drained to prevent any water on funnel
walls from transferring to boiling flask.
Connect boiling flask to rotary evaporator and evaporate
contents (50°C maximum) to dryness. Remove flask
immediately, add 50 mL acetone, and evaporate contents to
dry ness again. Immediately remove flask, dissolve dried extract
by swirling with 10 mL ethyl ether, and care fully trans fer
reconstituted extract to clean centrifuge tube. Rinse boiling flask
with two 10 mL portions ether and add rinses to tube. Immerse
end of tube in warm (50°C max i mum) wa ter bath and evap o rate
to dry ness un der nitrogen. If water droplets remain in tube, add
few mL acetone and evaporate to dry ness under nitrogen again.
Tightly cap tube containing dried ex tract.
E. System Suitability Test for Cleanup Chromatographic
System
Equilibrate LC cleanup system with mobile phase, B(a), at
2 mL/min flow rate until baseline stabilizes. Inject intermediate
standard solution into LC system to obtain reproducible retention
time (±15 s) for vitamin D3 peak, eluting within 14–18 min. Based
on vitamin D peak, efficiency, N, of guard and cleanup col umns
together is typically 5000–7000 theoretical plates, 4000 minimum.
Reinject intermediate standard solution after every 4 test solution
injections to update vitamin D3 retention time.
ã 2005 AOAC INTERNATIONAL
F. LC Cleanup of Test Solution
Referring to chromatogram of intermediate standard solution,
determine time vitamin D3 peak reaches UV detector and set timing
device to collect fractions from 30 s before to 30 s after peak
reaches detector.
Pipet 1.00 mL hexane into tube containing dried extract, D, cap
tube, and mix 5 s on Vortex mixer. Use clean, dry 1 mL glass syringe
to withdraw hexane solution from tube, invert syringe, and expel air.
If solution appears cloudy or contains precipitate, clarify solution
using disposable filter, B(h).
Fill LC injection loop, inject solution, and start timing device,
col lect ing vi ta min D 3 -con tain ing frac tion in vial. Cap vial
immediately.
Rinse syringe with acetone and dry thoroughly before next test
solution injection. After collecting fraction and before next
injection, elute remaining peaks at 4 mL/min flow rate. When
baseline stabilizes, adjust flow rate to 2 mL/min, and wait until
baseline again stabilizes before next injection.
Evaporate each collected fraction to dryness under nitrogen, with
warm water bath (50°C maximum) as before. Unevaporated amyl
alcohol remaining in fraction may result in excessively broad
vitamin D3 peak during quantitative LC. Cap vial immediately and
perform quantitative analysis as soon as possible. Vials may be
stored 24 h, at 4°C, protected from white light. Bring vials to room
temperature before opening.
G. System Suitability for Quantitative LC
Equilibrate quantitative LC system with mobile phase, B(b), at
3 mL/min flow rate until baseline stabilizes. Short-term noise
should be less than 1% full scale. Inject work ing stan dard
solution I until replicate peak heights agree ±2%. Adjust injection
volume and/or detector at ten u a tion un til stan dard so lu tion I
peak height is 50–80% full scale. Vitamin D3 retention time
should be within 14–18 min.
H. Quantitative LC Determination
Reconstitute dried test extract in 1.00 mL hexane as described in
F. Inject working standard solutions I, II, and III prior to injection
of every 8 test solutions.
I. Calculations
Calculate vitamin D3 concentration, Cstd (IU/mL) for working
standard solutions I, II, and III as follows:
ã 2005 AOAC INTERNATIONAL
W std ´ K ´ D
Cstd =
10000
where Wstd = mg vitamin D3 used for stock standard solution; K =
40 000 IU/mg (weight conversion factor for vitamin D3); D =
di lu tion fac tor for work ing stan dard solu tions (25/100 for
standard I, 15/100 for standard II, and 7/100 for standard III); and
10 000 = combined dilution factor for dry vitamin D stock (Wstd to
50 mL) and intermediate (1 mL stock to 200 mL) standard
solutions.
Ob tain le ast-squares re gres sion of peak height s vs
con cen tra tion for work ing stan dard so lu tion in jec tions
brack et ing each test so lu tion group (8 max i mum) from
quan ti ta tive LC anal y ses. Determination co effi cient, r, for
re gres sion anal y sis is typ i cally ³0.995.
Calculate vitamin D3 concentration, C, (IU/L) for test solution
from peak heights and regression line as follows:
C s ln ´ 100
. ´ 0.97 ´ 1000
C=
VL ´ 0.86 ´ VP
C s ln ´ 1130
C=
VL ´ VP
where Csln = concentration (IU/mL) vitamin D3 in injected test
so lu tion; 1.00 = mL re con sti tuted test so lu tion be fore
quantitative LC; 0.97 = mL of reconstituted extract, corrected for
evaporation in 50 mL tube; 1000 = mL to L conversion factor;
V L = mL con tained in cleanup in jec tion loop, mea sured by
weighing loop filled with H2O of known density; 0.86 = fraction
of vi ta min D 3 not con verted to previtamin D dur ing
saponification; VP = mL product saponified.
References: JAOAC 68, 177(1985).
J. AOAC Int. 76, 1042(1993).
CAS-67-97-0 (cholecalciferol, vitamin D3)
Revised: March 1996
* Adopted as a Codex Reference Method (Type II) for liquid
chromatography of vitamin D (D3, milk-based infant formula) in special
foods.