SimultaneousDetermination of SevenWater SolubleVitamins inProducts byLC-MS/MS
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APPENDIX–DETAILEDMETHOD (WRITTENFORWATERSXEVOTQ-S)
1)
APPROXIMATESAMPLESIZE
a)
Nominally, use 1.0 g for a ready to feed infant formula and 0.25 gof a ready to feed adult nutritional
product. Powder products and concentrated liquids shouldbe reconstituted to a 10%w/w level prior to
analysis. Due tomatrix suppression samples sizes above 1 gram are not recommended.
2)
THEORY
a)
Thismethod facilitates the simultaneous quantitationof sevenwater-soluble vitamins (WSV) inAbbott
Nutrition products, includingbiotin, pantothenic acid, folic acid, niacinamide (includingnicotinic acid in
infant formula powders), riboflavin, pyridoxine and thiamine. Samples are prepared by the addition of
1% ascorbic acid topartiallyprecipitate proteins aswell as protect folic acid fromdegradation. Stable-
isotope labeled internal standards are incorporated into the sample preparation and are used to correct for
variability inboth the sample preparation and instrument operation. A series of fivemixedworking
standard solutions spanning twoorders ofmagnitude invitamin concentration are used togenerate
calibration curves basedon the peak response ratioof the analyte to its stable-isotope labeled internal
standard
Prepared samples andworking standard solutions are injectedontoultra-high pressure liquid
chromatograph (UPLC) interfaced to a triple-quadrupolemass spectrometer (MS/MS) for analysis. A
WatersAcquityUPLCwith aXevoTQ-Shas been demonstrated as suitable for thismethod.
Chromatographic separation is executedusing a 1.0mmx 100mmx 1.8µmHSS-T3 column (Waters,
Corp), and the analytes of interest are ionizedvia electrospray ionization (ESI). Analytes elute in less
than6minuteswith an additional 4minutes required for column cleaning and re-equilibration. The
analytes are detected by tandemmass spectrometry. TheMS/MS is configured tomonitor parent-daughter
(precursor-fragment) ionpairs for each analyte and internal standard. This reaction forms the basis for
method selectivity. Analytes are quantifiedby interpolation of the response ratiowith the calibration
curve.
3)
APPARATUS
a)
Column, -AcquityUPLCHSS-T31.8µm1.0 x 100mm (WatersCorp.,Milford,MA) or equivalent
b)
Liquid chromatograph.—WatersAcquityUPLCBinary
c)
SolventManager - capable of 15 000psi or equivalent.
d)
Detector.—Waters Quattro Premier XE, Xevo triple quadrupolemass spectrometer, or equivalent.
e)
Injector.—Waters Acquity sample manager with integrated columnoven or equivalent.
f)
Nitrogen generator.—Peak Scientific (Billerica, MA)Model NM30LAor equivalent.
g)
Data system.—Waters MassLynx, latest revision or equivalent.
h)
Centrifuge tubes.—PP (Polypropylene), 50mL capacitydisposable.
i)
Vortexmixer.
j)
Balance.—Readable to at least 0.001 g. (Mettler-ToledoXS-204, Columbus, OH, or equivalent).
k)
Balance.—Readable to at least 0.001mg. (Mettler-ToledoXS-204, Columbus, OH, or equivalent).
l)
Stir Plate
m)
Syringe –1mL luer tip.
n)
Syringe Filters PTFE0.45µm syringe filters, Acrodisc 25-mm, or equivalent.
o)
Filters –0.45µmPTFE syringe tip
BIO-03
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