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206
E
llingson
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
1, 2016
Viscous ready-to-feed (RTF) products that were being analyzed
for total choline and carnitine were prediluted by weighing
1.0 g and diluting with water to a final weight of 5.0 g.
(a)
Free choline and carnitine
.
—
Samples were prepared
by weighing 1.0 g of reconstituted product into a 50 mL
polypropylene tube. Six additional tubes were designated for
the working standards along with two tubes for the reagent
blank and reagent blank + internal standard to monitor any
interference or carryover. The working standards, reagent
blank, and reagent blank + internal standard were included with
each free analysis and treated the same as samples through the
sample preparation. The working standard tubes received 50 μL
of the appropriate intermediate working standard level. All tubes
except the reagent blank received 50 μL of the intermediate
internal standard solution. The tubes were diluted to 25 mL with
water and thoroughly mixed on a horizontal shaker. The reagent
blank + internal standard solution was used as the diluent if
dilutions were needed. A 0.5 mL aliquot the sample solution
was mixed with 0.5 mL of acetonitrile in a microcentrifuge
tube, and then filtered through a 0.45 μm GHP syringe filter into
a silanized injection vial. Aliquots of 0.5 mL of the working
standard and reagent blank solutions were mixed with 0.5 mL
acetonitrile directly in the silanized injection vials.
(b)
Total choline and carnitine
.
—
Samples were prepared by
weighing 1.0 g of reconstituted or diluted product into a 55 mL
MARSXpress liner. Six additional liners were designated for the
working standards along with two liners for the reagent blank
and reagent blank + internal standard to monitor any interference
or carryover. The working standards, reagent blank, and reagent
blank + internal standard were included with each total analysis
and treated the same as samples through the sample preparation.
Liners designated for the working standards received 50 μL of the
appropriate intermediate working standard level. All liners except
the reagent blank received 50 μL of the intermediate internal
standard solution. A 5 mL volume of water followed by 2.5 mL
of 70% (w/w) nitric acid delivered with a bottle top dispenser
were then added to each liner, capped, and vortexed to mix. The
microwave program used was a ramp to temperature of 120°C
over 10 min, followed by a 40 min hold at a power of 1000 W,
ending in a cool down (6). The contents of the vessels were
transferred into 50 mL polypropylene tubes with water and diluted
to a volume of 25 mL with water. A 0.5 mL aliquot of the sample
solution was mixed with 0.5 mL acetonitrile in a microcentrifuge
tube, and then filtered through a 0.45 μm PTFE syringe filter
into a silanized injection vial. Aliquots of 0.5 mL of the working
standard and reagent blank solutions were mixed with 0.5 mL of
acetonitrile directly in the silanized injection vials.
G. LC/MS/MS Parameters
A Shimadzu Prominence LC system equipped with an Agilent
Zorbax 300-SCX column (3.0 × 50 mm, 5 μm) was used. A flow
rate of 1.0 mL/min was maintained over the 4.2 min total run time.
The mobile phase conditions were 100% mobile phase A until
1.0 min, ramped to 100% mobile phase B by 1.5 min, and ramped
back to 100% phaseAby 3.0 min. A column temperature of 40°C,
and an autosampler temperature of 5°C was maintained. A 1 μL
injection was used. Autosampler rinse settings were adjusted to
eliminate carryover as much as possible. An ABSciex API 4000
mass spectrometer with positive ESI was used in multiple reaction
monitoring (MRM) mode. The MS/MS overall settings used are
described in Table
2015.10A
. The MS/MS settings may need to
be modified except for ionization, mode, and gas types to obtain
optimum chromatography and sensitivity. Figures
2015.10A
and
2015.10B
show typical extracted ion chromatograms (XICs) from
National Institute of Standards and Technology (NIST) Standard
Reference Material (SRM) 1849a for choline and carnitine.
H. Quantification and Confirmation
The quantification of choline and carnitine was accomplished
by the generation of calibration curves using the peak area ratio of
the chosen transition (Table
2015.10B
) versus the corresponding
deuterated internal standards. Least-squares regression analysis
using a linear model with 1/
x
2
weighting was used for both
analytes. Confirmation was achieved through the analysis of ion
ratios between samples and reference standards for at least one
additional transition listed in the table. The concentration of each
analyte in a sample was calculated by the following equation:
C V D
100
10 ng mg
6
R
S
= × × ×
Figure 2015.10A Extracted ion chromatogram (XIC) of choline.
Table 2015.10A. MS/MS settings
Ionization
Positive ion electrospray (ESI+)
Mode
MRM
Ion spray voltage
1000 V
Turbo ion spray temp.
550°C
Declustering potential
120 V
Dwell time
100 msec
Entrance potential
10 V
Collision cell exit potential
25 V
Collision gas
Nitrogen, 5 psig
Curtain gas
Nitrogen, 20 psig
Nebulizing gas (gas 1)
Nitrogen, 60 psig
Auxiliary gas (gas 2)
Nitrogen, 60 psig
Needle position
Y
= 5 mm,
X
= 5 mm
101