SimultaneousDetermination of SevenWater SolubleVitamins inProducts byLC-MS/MS
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4)
SolutionPreparation
a)
Mobile phase.
i)
Mobile phaseA.— 0.05%FormicAcid, 0.04%HFBA inWater
(1)
Using a graduated cylinder, transfer 1000mL laboratorywater to amobile phase reservoir. Add
500µLof concentrated formic acid and 400µL concentratedHFBAusing a 1000μL positive
displacement pipette. Mixwell. Expiration: 10days.
ii)
Mobile phaseB.— 5mMAmmoniumHydroxide inMethanol
(1)
Using a graduated cylinder, transfer 500mLLCMS-grademethanol to amobile phase reservoir.
Add 335µL concentrated ammoniumhydroxide using a 1000μLpositive displacement pipette.
Mixwell. Expiration: 10days.
b)
1%AscorbicAcidSolutionPreparation
i)
Weigh10.0± 0.1 gof ascorbic acid into a tarredweighboat; quantitatively transfer to a 1000-mL
volumetric flask andQCwith laboratorywater. The amount of prepared solution can be scaled as
necessary to suit the needs (size) of the sample queue. 260mL is required for standard preparation,
and each sample requires an additional 25mL. Expiration: Prepare freshdaily.
c)
AscorbicAcidDiluent Solution
i)
Pipette 25mL1% ascorbic acid solution into a 50mL centrifuge tube. Add 80µL ammonium
hydroxide andmixwell.Volumesmaybe adjusted basedon the number of samples and standards on
theworklist. Each samplewill require 2.25mL of diluent. Expiration: Prepare fresh daily.
d)
0.3%AmmoniumHydroxideSolution
i)
This solution is only required for preparing folic acid and
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-folic acid stock standards. It is not
preparedwith everyworklist.
ii)
Using a volumetric pipette, deliver 3mLof ammoniumhydroxide (28%-30%Solution) to a 1000-mL
graduated cylinder containing approximately250mL of laboratorywater andgently swirl tomix
well. Add laboratorywater to bring total volume to approximately300mL andgently swirl to
provide additionalmixing. Expiration: Prepare fresh daily.
e)
50%Methanol
i)
This solution is only required for preparing
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-biotin stock standard. It is not preparedwith every
worklist.
ii)
Using a graduated cylindermeasure and then transfer approx. 100± 2mLmethanol and then 100± 2
mL laboratorywater into a 250-mLbottle andmix thoroughly. Expiration: 1month.
f)
1.0%AceticAcid
i)
This solution is only required for preparing
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,
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2
-riboflavin stock standard. It is not prepared
with everyworklist.
ii)
Add approximately30mLwater to a 500-mL volumetric flask. Add 5.0± 0.1mL glacial acetic acid
and swirl tomix. Bring tovolumewith laboratorywater andmixwell. Expiration: 3months.
g)
0.12MHCl
i)
This solution is only required for preparing
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-thiamine stock standard. It is not preparedwith
everyworklist.
ii)
Add approximately300mLwater to a 500-mL volumetric flask. Add 5.0±0.1mL conc. HCl and
swirl tomix. Bring to volumewith laboratorywater andmixwell. Expiration: 3months.
h)
PhosphateBuffer Solution, pH5.0 (0.10MPotassiumPhosphateDibasic, 1%EDTA, 2%Metaphosphoric
Acid)
i)
This solution is only required for preparing the vitamin stock standardmixture (VSSM). It is not
preparedwith everyworklist.
ii)
Weigh 20.0± 0.2 gof EDTA into a tarredweighingvessel and quantitatively transfer to a 2000-mL
beaker containing approximately1800mL laboratorywater and add amagnetic stir bar.
iii) Weigh 34.8± 0.1 gof potassiumphosphate dibasic into a tarredweighingvessel andquantitatively
transfer to the 2000-mLbeaker already containing approximately1800mL laboratorywater and
BIO-03
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
