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Silliker
Chem, Res. Center Crete, IL – Validation of a LC-MS/MS Method for Vitamin K Analysis
Silliker Laboratories, Chemistry Research Center, Date: 1/30/15
6
n.)
Calibration standard solutions: Make Vitamin K1, K2-7 and K2-4 calibration solutions at 5-250 ng/mL.
Preparation of calibration standards is summarized in Table 1.VitaminK1 Internal standard (10 ppm) is
added in volume of 10 µL / mL of every calibrant standard solution (conc. = 100 ng/mL).
Table 1: Preparations of the standard solutions
Calibration
Std.
Targeted conc.
Of Vit K1, K2-4
and K2-7
(ng/mL)
Working
Std.Sol
(µg/mL)
Working
Std.Sol
(mL)
Final Volume
to (mL) with
IPA: MeOH
Final
Conc.
(ng/mL)
Internal
std.
soln*
(µL)/mL
Internal
Std
(ng/mL)
1
250
1
2.5
10
250
10
100
2
100
1
1
10
100
10
100
3
50
1
0.5
10
50
10
100
4
25
1
0.25
10
25
10
100
5
10
1
0.1
10
10
10
100
6
5
1
0.05
10
5
10
100
*Internal std. solution conc. =
10000
ng/mL
All Calibrant standard solution were spiked with vitamin K1 internal standard solution in volume of
10 µL / mL (conc. = 100 ppb).
Procedure:
a.)
weight sample in a 50mL centrifuge tube (0.25-2.0 g powder / 0.25-2.0 g liquid)
b.)
Std. solution aliquot containing an aliquot of a mixture of 50-250 ng each of Vitamin K1, vitamin K2-4
and vitamin K2-7 is also processed (extracted) and analyzed like a sample. A method blank with no
sample and standard solution is also processed (extracted) like a sample.
c.)
add 20uL IS (10ppm) (200 ng)
d.)
Add 2.5mL Phosphate Buffer
e.)
Add approx 0.25g Lipase
f.)
Add 1.5mL H2O
g.)
Flush with N2 and cap
h.)
Incubate in shaking water bath for 2hr at 37
o
C
i.)
Add 1.67mL 6N NaOH
j.)
Add 10mL Iso-Octane
k.)
Shake 30min at low speed
l.)
Add 5mL Ethanol
m.)
Shake 30min on low speed
n.)
Centrifuge 5min at 5000rpm
o.)
Pipette off organic layer into culture tube
p.)
Evaporate to dryness
q.)
Reconstitute in 1mL 75:25 MeOH:IPA (Internal std. conc. = 25 ng/mL).
r.)
Filter and inject 10uL
LCMS/MS Conditions:
Shimadzu UFLC system coupled with API 3200 Triple Quad Mass Spectrometer
LC Conditions
Column: YMC CAROTENOID S-3 2.0X150mm Part# CT99S03-1502WT
Mobile Phase: A: 5mMAmmoniumAcetate in Water
B: 5mMAmmoniumAcetate in 75:25 MeOH:IPA
VitK-01 w/SLV
FOR ERP USE ONLY
DO NOT DISTRIBUTE