50.1.25 (l) Volumetric flasks.—Low-actinic volumetric flasks, 100 mL.

AOAC Official Method 999.15

C. Reagents
Vitamin K in Milk
and Infant Formulas Use analytical grade reagents unless otherwise stated.
Liquid Chromatographic Method (a) Water.—Purified to >18 MW resistivity.
First Action 1999 (b) Nitrogen.—Cylinder N (low O content).
Final Action 2003 (c) Lipase.—From Candida rugosa, ca 1000 units/mg (Type VII,
(Ap pli ca ble to the de ter mi na tion of total vi ta min K 1 Sigma L-1754). Other sources of lipase can be used from
[phylloquinone] in infant formula and milk [fluid, ready-to-feed, Pseudomonas and Rhizopus species with the correct activity of
and powdered] containing >1 mg vitamin K1/100 g solids.) enzyme.
See Table 999.15 for the results of the interlaboratory study (d) Monobasic potassium phosphate (KH2PO4).
supporting acceptance of the method. (e) Potassium hydroxide solution.—40% (w/v). Dissolve 40 g
KOH, (j), in water and dilute to 100 mL.
Caution: Avoid skin contact with zinc dust.
(f) Phosphate buffer.—0.8M. Dissolve 54.0 g KH2PO4, (d), in
A. Principle 350 mL water. Adjust pH to 7.9–8.0 with 40% KOH solution and
Following enzymatic digestion of fat and precipitation of fatty dilute to 500 mL.
acids, vitamin K is extracted with hexane. Vitamin K is separated by (g) Ethanol.
HPLC with post-column reduction and quantitated by fluorescence (h) Methanol.—LC grade.
with an external standard. (i) Reagent alcohol.—Mix ethanol, (g), with methanol, (h),
Phylloquinone geometric isomers are quantitated either as a (95 + 5, v/v).
single unresolved peak with a C18 column, or may be estimated (j) Potassium hydroxide.
se lec tively with a C30 col umn (k¢ trans < k¢ cis ), un der the (k) Zinc chloride.
reversed-phase conditions employed. (l) Sodium acetate.—Anhydrous.
(Note: Menaquinone-4 [MK-4] will also be present in milk and (m) Acetic acid.—Glacial.
infant formulas incorporating milk fat, while (n) Potassium carbonate.—Anhydrous.
2¢,3¢-dihydrophylloquinone may be present at appreciable levels in (o) Zinc powder.—<60 mm, e.g., Merck 108774 and BDH
infant formulas containing hydrogenated soya or canola oils. If 102964L. Use freshly opened bottle.
required, both may be estimated against appropriate standards. The
(p) Hexane.—HPLC grade.
order of elution is MK-4 < K1 < 2¢,3¢-dihydroK1 [relative retention
(q) Dichloromethane.—HPLC grade.
times ca 0.6, 1.0, and 1.2, respectively].)
(r) Isopropanol.—HPLC grade.
B. Apparatus (s) Mobile phase.—To dichloromethane, (q),–methanol, (h),
(a) HPLC system.—Manual or automated isocratic system (100 + 900, v/v), add 5 mL methanol containing ZnCl2 (1.37 g), (k),
incorporating a fluorescence detector (lex 243 nm, lem 430 nm) anhydrous sodium acetate (0.41 g), (l), and glacial acetic acid
exhibiting a Raman spectra signal:noise specification of >200:1 (0.30 g), (m). Filter through 0.45 mm filter and degas.
(water). Since UV detection is insensitive to vitamin K at the (t) Zinc post-column reductor.—Dry-pack the 20 ´ 4 mm
work ing con cen tra tion lev els, con fig ure with sin gle-mode as sem bly, B(c), with Zn pow der, (o). Min i mize voids by
flu o res cence de tec tion only to min i mize ex tra-column sequentially adding small amounts with frequent gentle tapping.
band-broadening. For multi-detector LCs, configure with the With longer columns, use extra care. Re-prepare reductor column
fluorescence detector in the first position. Ensure the absence of before each analytical schedule. Reduction efficiency of Zn powder
leaks. should be monitored over time and replaced every 1–2 years as
(b) Column.—Any C18 column (monomeric or poly meric) required.
containing 5 mm spherical particle silica with ³10% carbon loading, (u) Vitamin K1 (phylloquinone).—USP grade. Con duct all
preceded by a guard column of similar packing. op er a tions un der low in can des cent light con di tions and in
(c) Post-column re duc tor.—20 ´ 4 mm stain less steel low-actinic volumetric flasks. (1) Stock solution.—ca 1.0 mg/mL.
post-column assembly placed between analytical column and Dissolve ca 100 mg, weighed accurately, in 100.0 mL isopropanol,
flu o res cence de tec tor (Part No. WAT084550, Wa ters, or (r), with warming at ca 30°C and standing. Store under N, (b), at
equivalent). Alternative devices, such as an empty (30–50 mm) LC –10°C for up to 6 months. (2) Intermediate solution I.—ca
column can be used. 50 mg/mL. Dilute 5.0 mL stock with methanol to 100.0 mL and store
(d) Spectrophotometer.—UV-Vis spectrophotometer, digital un der N at –10°C for up to 1 month. (3) In ter me di ate
readout to 0.001 A.
solution II.—ca 2.5 mg/mL. Dilute 5.0 mL Intermediate solution I
(e) Water bath.—37° ± 1°C. with methanol to 100.0 mL. Pre pare daily. (4) Working
(f) Centrifuge.—Fixed or swing-arm rotor. standards.—ca 6.25, 12.50, 18.75, 25.00, and 31.25 ng/mL. Dilute
(g) Mechanical shaker.—Orbital or wrist-action. 0.25, 0.50, 0.75, 1.00, and 1.25 mL, respectively, Intermediate
(h) Vortex mixer. solution II with methanol, (h), to 100.0 mL. Prepare daily and
(i) pH meter.—Accurate to 0.01 pH, with calibration buffers. calculate accurate concentrations as follows:
(j) Test tubes.—200 ´ 24 mm with ground-glass stoppers. Calculatephylloquinone purity from absorbance (248 nm) of a
(k) Vials.—Amber-tinted glass vials (1.8 and 8.0 mL), with solution prepared by evaporating 5.00 mL Intermediate solution I
Teflon-sealed caps. under N flow and redissolving in 25.0 mL hexane (a1% = 419):

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Table 999.15. Results of the interlaboratory study for determination of vitamin K1 in milk and infant formula
a b
Product Mean mg/100 g No. labs sr r RSDr sR R RSDR HorRat
1 0.49 31(1) 0.04 0.12 9.03 0.05 0.15 10.94 0.31
2 6.63 28(5) 0.21 0.60 3.23 0.39 1.08 5.81 0.24
3 118.07 33(0) 5.00 14.01 4.24 6.50 18.19 5.50 0.35
4 32.24 33(0) 1.54 4.31 4.77 2.14 5.98 6.63 0.35
5 78.69 33(0) 2.04 5.71 2.59 3.40 9.53 4.33 0.26
6 49.64 33(0) 2.54 7.11 5.11 3.80 10.65 7.66 0.43
7 90.94 32(1) 4.04 11.32 4.44 4.14 11.60 4.56 0.28
8 94.62 33(0) 5.38 15.05 5.68 6.41 17.95 6.78 0.42
a
Product 1 is unfortified whole liquid UHT milk; product 2 is goat milk powder; product 3 is milk-based infant formula, oil-filled; products 4 and 7 are whey-based
infant formula, partially oil-filled; product 5 is soy-based infant formula, oil-filled; product 6 is whey-based infant formula, oil-filled; product 8 is NIST SRM 1846.
b
Number of laboratories with outliers in parentheses excluded.

W 5 5 using autosampler, use a low volume vial (or insert) to minimize

Theoretical A248 = ´ ´ ´ 419 ´ 100
100 100 25 evaporation into the headspace.
(b) HPLC determination.—(1) Set-up.—Prior to connection of
where W = weight of phylloquinone in stock standard (g). post-column Zn reductor assembly, establish stable operating LC
Purity factor (PF) = measured A248/theoretical A248. conditions over ca 30 min through equilibration of column with
mobile phase (ca 1 mL/min) and set fluorescence detector to lex =
W 5 106 243 nm and lem = 430 nm (gain and sensitivity will be detector
K1 in Intermediate solution II (mg/mL) = PF ´ ´ ´5 ´ dependent). Install Zn reductor assembly between analytical
100 100 100
column and detector and equilibrate system for a further 30–60 min,
ensuring the absence of leaks (anhydrous LC eluent conditions are
Accurate vitamin K1 concentrations in the 5 working standards
required to minimize loss of reducing potential of Zn). Do not
(ng/mL) are then calculated by multiplying this value by 2.5, 5.0,
7.5, 10.0, and 12.5, respectively. recycle mobile phase. Inject a standard (20–50 mL) and set flow rate
in order to elute phylloquinone between 8–15 min, at typically
D. Procedure 1.0–1.5 mL/min (re ten tion may also be ma nip u lated by
Perform all steps of assay under incandescent lighting in the modification of dichloromethane content in eluent between
absence of direct sunlight. 8–12%). Allow total run time of ca 5 min beyond elution of K1.
(a) Preparation of test solution.—(1) Digestion.—Weigh 1.0 g (2) Calibration and analyses.—Inject methanol (standard blank)
powder or 10.0 g liquid into test tube. Include both a reagent blank and confirm absence of chromatographic activity at retention time
and a quality control material in each analytical schedule. Dissolve for phylloquinone. Inject lowest level working standard and ensure
powder in ca 15 mL warm water (<40°C) with Vortex mixer (for repeatable response, with signal:noise ³10. Inject each working
liquids, add 5 mL warm water). Add 5.0 mL phosphate buffer to each standard and ensure linear response. Sequentially inject reagent
solution and mix. Add 1.0 g lipase powder and mix on a Vortex blank and test extracts and include a working standard after every
mixer. Stopper securely and shake until well dispersed (30–60 s). 4–6 test solutions to monitor system stability. When analytical
Incubate at 37° ± 2°C for 2 h. Shake each tube for 15 s at 20 min schedule is complete, flush system with methanol. Store analytical
intervals. Cool to ambient temperature by standing in water. Add column in methanol when not in use. Dismantle and empty
10 mL reagent alcohol and mix. Add 1.0 g K2CO3 and mix. post-column assembly, soak in 5M HCl to clean frits, and rinse
(2) Ex trac tion.—(Note 1: Take pre cau tions to pre vent thoroughly with water and methanol. Dry thoroughly before
concentration of extracts through uncontrolled solvent evaporation. repacking with Zn.
Note 2: If a second 30 mL extraction shows significant residual (c) Calculations.—Construct a 5-level calibration using either
vitamin K1 or recovery is <95%, then such products need routine peak area or height and calculate slope (S) by linear regression with
multiple extractions.) Add 30.0 mL hexane, stopper, and shake forced zero. Calculate vitamin K1 (cis- and trans-) content in test
vigorously for ³10 min using mechanical shaker. Let stand in the samples as follows:
dark, preferably refrigerated, until layers separate. If this is slow
A¢ 30 100 1
(>10 min) or incomplete, centrifuge at ca 1000 rpm (ca 200 ´ g) for Vitamin K1 (mg/100 g) = ´ ´ ´
S V Wt 1000
10 min (decant a portion of supernatant into smaller centrifuge tube if
necessary). Transfer 1.00 mL (fortified products) or 5.00 mL
(nonfortified products) of upper hexane phase into vial. Evaporate where A¢ = peak area (or height) of phylloquinone in test extract;
under N flow to dryness. (Note: Larger extract volumes may be Wt = weight of test portion (g); V = volume, 1.0 mL (fortified) or
required, with the option of rotary evaporation, dependent on the 5.0 mL (nonfortified); S = slope of calibration graph.
sensitivity of the fluorescence detector.) These dried extracts may be Calibration and calculations may be achieved through data
held for 3–5 days in the dark at <0°C under N prior to LC analysis. processing within the instrument or off-line.
Re-dissolve residue in 1.00 mL methanol. Seal vial and Vortex mix. If Reference: J. AOAC Int. 83, 121(2000).

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