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Abbott Nutrition Division
Simultaneous Determination of Total Vitamin B
6,
B
2,
B
3
and B
1
in Infant
Formula Products by LC-MS/MS Using Enzymatic Digestion
B.
THEORY
1.
Method
a)
This method via an enzymatic digestion facilitates the simultaneous quantitation of the total
content of three vitamins, B6, B2, and B1 from Abbott Nutrition Milk Based Pediatric products.
b)
The B6 is quantitated as a sum of three vitamers Pyridoxine, Pyridoxal, and Pyridoxamine.
c)
Samples are prepared by the addition of an enzyme mixture consisting of Papain, Alpha-Amylase,
and Acid Phosphotase. The mixture breaks down the matrix freeing any bound vitamins, and
dephosphorylates the vitamin phosphate forms to the free forms
d)
Stable-isotope labeled internal standards are incorporated into the sample preparation and are used
to correct for variability in both the sample preparation and instrument operation.
e)
A series of 6 mixed working standard solutions spanning two orders of magnitude in concentration
are used to generate calibration curves. A response ratio (ratio of analyte to stable-isotope internal
standard peak responses) is calculated for all analytes and used together with known analyte
concentration to generate calibration curves used for quantitation.
f)
Prepared samples and working standard solutions are injected onto an Acquity UPLC (Waters,
Corp) interfaced to a Triple-Quadrupole Mass Spectrometer (MS/MS) for analysis using a 2.1mm x
50mm x 1.7
m
m BEH C-18 (Waters, Corp) and the analytes of interest are ionized via Electrospray
Ionization (ESI). Analytes elute in less than 5 minutes with an additional 2 minutes required for
column re-equilibration.
g)
The analytes are detected by tandem mass spectrometry. The MS/MS is configured to monitor
parent-daughter (precursor-fragment) ion pairs for each analyte and internal standard and form the
basis for analyte detection and quantitation and form the basis for method selectivity.
h)
Analytes are quantified by interpolation of the analyte-stable isotope internal standard response
ratio for the sample against a calibration curve.
i)
Analysis of the seven analytes is accomplished by a single injection of the prepared sample on the
Quattro Premier XE instrument platforms in most matrices.
2.
Stable Isotope Internal Standards in Mass Spectrometry
The addition of an appropriate internal standard to a quantitative assay has several significant
advantages. An internal standard can correct for variations in the extraction efficiency during sample
preparation, correct for errors or losses in volumes during sample handling and in the case of mass
spectrometry compensate for competitive ionization in the source caused by co-eluting matrix
components. An appropriate internal standard must be a compound that has similar chemical and
physical properties to the analyte of interest. The closer the match between the internal standard and
the compound of interest the better the performance of the internal standard. In the case of mass
spectrometry the ideal internal standard can be utilized, the compound itself. This can be
accomplished by synthesizing the compound with a stable isotope, most commonly of hydrogen (
2
H),
carbon (
13
C) or nitrogen (
15
N). The resulting compound will behave almost identically to the native
20
BVIT-02
FOR ERP/WORKING GROUP USE ONLY
DO NOT DISTRIBUTE