Silliker Laboratories of IL, Crete, IL60417
a.) PhosphateBuffer: 54gPotassiumphosphatemonobasic in 350mLH2O. Adjust
pH to8.0 (+/-0.1) by slowly adding40%KOH.Make up to volume to500mLwith
H2O.
b.) 6NNaOH: 10NNaOHdiluted to6Nwithwater.
c.) 75:25MeOH:IPA solution: Mixmethanol 75ml with 25mLof iso-propanol and
mix.
d.) VitaminK1StockStandardSolution: Prepare vitaminK1 standard stock solution
in 75:25MeOH:IPA solution. 10mgof vitaminK1cis andall-trans is dissolved in
75:25MeOH:IPAandmakeup volume to10mL.
e.) VitaminK2-4StockStandardSolution: Prepare vitaminK2-4 standard stock
solution in 75:25MeOH:IPA solution. 10mgof vitaminK1 is dissolved in75:25
MeOH:IPAandmakeup volume to10mL.
f.) VitaminK1 Internal standardSolution: DissolveVitaminK1-d7 (5,6,7,8-d4, 2-
methyl-d4) in 75:25MeOH:IPA tomake 20ppm solution.
g.) Calibration standard solutions: MakeVitaminK1 andK2-4 calibration solutions at
5-250ng/mL.
Procedure:
a.) weight sample in50mL centrifuge tube (0.25gpowder / 2.0 g liquid)
b.) Std. solution aliquot containing250ng of VitaminK1 and25ngof vitaminK2-4 is
also processed (extracted) like of sample.
c.) add20uL IS (10ppm) (200ng)
d.) Add2.5mLPhosphateBuffer
e.) Addapprox 0.25gLipase
f.) Add1.5mLH2O
g.) FlushwithN2 and cap
h.) Incubate in shakingwater bath for 2hr at 37
o
C
i.) Add1.67mL 6NNaOH
j.) Add10mL Iso-Octane
k.) Shake30minon low speed
l.) Add5mLEthanol
m.)Shake30minon low speed
n.) Centrifuge 5minat 5000rpm
o.) Pipet off organic layer into culture tube
p.) Evaporate todryness
q.) Reconstitute in2mL75:25MeOH:IPA
r.) Filter and inject 10uL
LCMS/MSConditions:
ShimadzuUFLC system coupledwithAPI 3200TripleQuadMass Spectrometer
VitK-01
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
