45.1.31
For premixes with
AOAC Official Method 974.30
Menadione Sodium Bisulfite
(Water-Soluble Vitamin K3) in Feed Premixes
Gas Chromatographic Method
First Action 1974
Final Action 1975
A. Principle
Menadione is extracted as menadione×NaHSO3×3H2O with
methanol and converted to menadione by on-column pyrolytic GC.
B. Apparatus
(a) Gas chromatograph.—With flame ionization detector.
Operating conditions: temperatures—column 135°C, injection port
250°C, detector block 235°C; flow rates (mL/min)–N2 25, H2 25, air
300. Adjust conditions to give ³0.5 full scale deflection for 0.5 mg
menadione/mL.
(b) Column.—Pyrex or stainless steel 120 ´ 0.64 (id) cm
(4 ft ´ 14 in. id) or 180 ´ 0.32 (id) cm (6 ft ´ 18 in. id), with glass-lined
or on-column injection, packed with (1) 2% OV-17 on 80–100 mesh
acid-washed Chromosorb W (Supelco, Inc.) silylated with BSA
[N,O-bis-(trimethylsily l) acetamide]-TMCS (trimethy l
chlorosilane), 5 + 1; or (2) 3% Dexsil 300 on 100–120 mesh Gas
Chrom Q. Condition 2% OV-17 column (1) 12 h at 250°C with N2.
(Caution: Silanes are toxic. Wear disposable gloves and use
effective fume removal device when handling.)
C. Standard Solutions
( a ) M e n a d i o n e s t a n d a rd s o l u t i o n . — ( 1 ) St o c k
solution.—4 mg/mL. Dissolve 200 mg USP Menadione Reference
Standard (www.usp.org) in methanol and dilute to 50 mL with
meth a nol. Store in dark; pre pare fresh daily. (2) Working
solutions.—Prepare standard solutions, each containing 1.0 mg
diethyl phthalate (DEP)/mL, as shown in Table 974.30.
(b) Menadione sodium bisulfite standard solution.—(1) Stock
solution.—2 mg/mL. Dissolve 200 mg menadione×NaHSO3×3H2O
(94+%, Sigma No. M5750) in methanol and dilute to 100 mL with
methanol. Prepare weekly. (2) Check solution.—Mix equal parts
so lu tion (b)(1) and (c); fi nal con cen tra tion is 1.0 mg
menadione×NaHSO3×3H2O and 1.0 mg DEP/mL.
( c ) D i e t h y l p h t h a l a t e ( D E P ) i n t e r n a l s t a n d a rd
solution.—2 mg/mL. Dissolve 1.0 g DEP (Aldrich Chemical Co.,
Inc., No. 49,222-1) in methanol and dilute to 500 mL with methanol.
D. Preparation of Test Solution
Accurately weigh 5.0 g feed premix contain ing ca 5.0 g
menadione/lb (16 g menadione×NaHSO3×3H2O/lb) and transfer to
200 mL glass-stoppered Erlenmeyer. Add 50 mL internal standard
solution, C(c), mix gently 10 min, and let stand 5 min. Dilute 5 mL
aliquot with 5 mL methanol, mix, and centrifuge.
different menadione levels, adjust extraction technique to obtain
final concentration so that equivalent menadione content is ca
0.5 mg/mL and test solution contains 1.0 mg DEP/mL.
E. Calibration
Inject 2 mL 0.5 mg menadione–DEP working standard solution
into gas chromatograph. Adjust to give ³0.5 full scale deflection.
De ter mine menadione/DEP peak response ratio (R 1 ), us ing
integrator or peak areas as follows:
PA1
R1 =
PA2
where PA1 and PA2 = peak areas of menadione (0.5 mg/mL) and
DEP (1.0 mg/mL), respectively.
Inject 2 mL aliquot of each menadione standard solution and
determine menadione/DEP peak response ratios. Prepare standard
curve by plotting mg menadione/mL against menadione/DEP peak
response ratio for each standard solution. Adjust instrument to
obtain linear standard curve.
Prior to test solution analysis, check injection port temperature for
on-column pyrolysis of menadione×NaHSO3×3H2O compound to
menadione by injecting 2 mL aliquot menadione×NaHSO3×3H2O.
Check stan dard so lu tion. De ter mine menadione (pyrolytic
product)/DEP peak response ratio (R2) and calculate % menadione
in menadione×NaHSO3×3H2O check standard solution.
= (MD1 /MSB2) ´ (R2/R1) ´ 100
where MD1 = mg menadione/mL in standard solution, and MSB2 =
mg menadione×NaHSO3×3H2O/mL in check standard solution.
F. Determination
Check chromatographic conditions daily by injecting 2 mL
0.5 mg/mL standard solution ³3 times and determine average peak
response ratio, R1, which should be within ±10% of standard curve.
If not, readjust temperatures and flow rates.
Inject 2 mL test solution ³2 times and determine average
menadione/DEP peak response ratio, Rx.
g Menadione/g test portion = (MD1/PMx) ´ (Rx/R1)
where PMx = mg product/mL test solution.
g Menadione/lb product =
(g menadione/g premix) ´ (453.7 g/lb)
Reference: JAOAC 56, 1277(1973).
CAS-130-37-0 (menadione sodium bisulfite, anhydrous)
CAS-58-27-5 (menadione)
2006 AOAC INTERNATIONAL
ã 2005 AOAC INTERNATIONAL