SimultaneousDetermination of SevenWater SolubleVitamins inProducts byLC-MS/MS
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(1) Weigh14.0± 0.1mg into a tarredweighingvessel. Quantitatively transfer to a 25-mL
volumetric flaskwith laboratorywater and dilute to volumewith laboratorywater. Mixwell and
transfer to a 50-mL amber bottle andStore refrigerated (2˚-8˚C).
(2)
Expiration: Until exhausted or evidence of contamination.
v)
13
C
4
-PyridoxineStockSolution (ApproximateConcentration: 70µg/mL)
(1) Weigh7.0± 0.1mg into a tarredweighingvessel. Quantitatively transfer to a 100-mL
volumetric flaskwith laboratorywater and dilute to volumewith laboratorywater. Mixwell and
transfer to a 100-mL amber bottle andStore refrigerated (2˚-8˚C).
(2)
Expiration: Until exhausted or evidence of contamination.
vi)
13
C
4
-ThiamineChlorideStockSolution (ApproximateConcentration: 100µg/mL)
(1)
Weigh5.0± 0.1mg into a tarredweighingvessel. Quantitatively transfer to a 50-mL volumetric
flaskwith0.12NHCl anddilute tovolumewith 0.12NHCl. Mixwell and transfer to a 100-mL
amber bottle andStore refrigerated (2˚-8˚C).
(2)
Expiration: Until exhausted or evidence of contamination.
vii)
13
C
4
,
15
N
2
-RiboflavinStockSolution (ApproximateConcentration: 73µg/mL)
(1) Weigh7.3± 0.1mg into a tarredweighingvessel. Quantitatively transfer to a 100-mL
volumetric flaskwith1.0% acetic acid anddilute to volumewith 1.0% acetic acid. Mixwell and
transfer to a 100-mL amber bottle andStore refrigerated (2˚-8˚C).
(2)
Expiration: Until exhaustedor evidence of contamination.
k)
Internal StandardStockMixture (ISSM)
i)
Prepare a sufficient volume of ISSM for theworklist by combining equivalent volumes of 1%
ascorbic acid and each stable-isotope labeled internal standard stock solution. Prepare enough ISSM
for theworklist plus an additional 200-300μL. For example, aworklist containing30 samples and 5
working standards requires aminimum ISSM volume of 1750µL (50µLx (30+5)) because 50µL of
ISSM is required for eachof the 5 standards and for each sample. Round up to 2000μL to allow250
μL excess ISSM. Toprepare 2000µL ISSM,mix250µLof each solution (2000µL/8 solutions). To
maintain ISSM stability,minimize container volume and follow the order of addition given below:
(1)
1%Ascorbic acid
(2)
13
C
5
-Folic acid
(3)
13
C
5
-Biotin
(4)
13
C
3
,
15
N-Pantothenic acid
(5)
2
H
4
-Niacin
(6)
13
C
4
-Pyridoxine
(7)
13
C
4
-Thiamine chloride
(8)
13
C
4
,
15
N
2
-Riboflavin.
ii)
Mix thoroughly. Expiration: Prepare fresh daily.
l)
IntermediateWorkingStandardSolution (IWSS) –Prepare freshon the dayof analysis
i)
Combine the followingvolumes of the stock standard solutions in a 10-mL volumetric flask
containing approximately5mL1% ascorbic acid.
TableA1. StockStandard solutions volumes needed tomake IWSS.
VitaminStockStandardSolution
Volume (µL)
FolicAcid
125
VitaminStockStandardMixture (VSSM)
500
ii)
Dilute to themarkwith1% ascorbic acid solution.
m)
WorkingStandardSolution–Prepare fresh on the dayof analysis
i)
Aliquot 25± 1mLof 1% ascorbic acid in each of five disposable 50mL centrifuge tubes. Combine
the IWSS and ISSM solutionvolumes shown inTableA2 into each of the tubeswith25mL 1%
ascorbic acid. Swirl tomix.
BIO-03
FORWORKINGGROUP/ERPUSEONLY
DONOTDISTRIBUTE
