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Jing et al.:
J
ournal of
AOAC I
nternational
V
ol.
98, N
o
. 5, 2015
1395
Determination of Free and Total Carnitine and Choline
in Infant Formulas and Adult Nutritional Products by
UPLC/MS/MS: Single-Laboratory Validation, First Action
2014.04
Wei Jing, Joseph J. Thompson
1
, Wesley A. Jacobs,
and
Louis M. Salvati
Abbott Nutrition, 3300 Stelzer Rd, Columbus, OH 43219
Received April 26, 2015. Accepted by SG April 24, 2015.
The method was approved by the AOAC Expert Review Panel for
Infant Formula and Adult Nutritionals as First Action.
The AOAC Expert Review Panel for SPIFAN Nutrient Methods
invites method users to provide feedback on the First Action methods.
Feedback from method users will help verify that the methods are
fit for purpose and are critical to gaining global recognition and
acceptance of the methods. Comments can be sent directly to the
corresponding author or
methodfeedback@aoac.org.1
Corresponding author’s e-mail:
joseph.thompson@abbott.comDOI: 10.5740/jaoacint.15102
INFANT FORMULA AND ADULT NUTRITIONALS
A single-laboratory validation (SLV) has been
performed for a method that simultaneously
determines choline and carnitine in nutritional
products by ultra performance LC (UPLC)/MS/MS.
All 11 matrixes from the AOAC Stakeholder Panel on
Infant Formula and Adult Nutritionals (SPIFAN) were
tested. Depending on the sample preparation, either
the added (free, with a water dilution and filtering)
or total (after microwave digestion at 120
°
C in nitric
acid and subsequent neutralization with ammonia)
species can be detected. For nonmilk containing
products, the total carnitine is almost always equal
to the free carnitine. A substantial difference was
noted between free and total choline in all products.
All
Standard Method Performance Requirements
for carnitine and choline have been met. This report
summarizes the material sent to the AOAC Expert
Review Panel for SPIFAN nutrient methods for the
review of this method, as well as some additional
data from an internal validation. The method was
granted AOAC First Action status for carnitine in
2014 (2014.04), but the choline data are also being
presented. A comparison of choline results to those
from other AOAC methods is given.
O
ver the last 3 years, as part of the AOAC Stakeholder
Panel on Infant Formula and Adult Nutritionals
(SPIFAN) project, AOAC has issued regular Calls
for Methods to find suitable test methods for global dispute
resolution purposes for nutritional products. Choline was
considered early in the process.
Standard Method Performance
Requirement
(SMPR
®
)
2012.013
was developed and approved
in 2012, describing the performance requirements needed for a
choline dispute-resolution method. Three methods were chosen
for consideration from those submitted. Abbott Nutrition
submitted AOAC
2012.19
, a UPLC-MS/MS method similar to
the present submission. In September 2012, SMPR
2012.010
was developed and approved for carnitine. Abbott Nutrition
submitted AOAC
2012.17
, a carnitine-only LC/MS-based
method (1) in response to the Call for Methods, and this was the
only carnitine entry for some time.
In 2013, the Expert Review Panel (ERP) clarified that, for a
total choline method, it was not necessary to prove recovery of
the individual inherent choline species such as phosphocholine
and sphingomyelin; therefore, the choline method could be
made much simpler and combined with carnitine. Carnitine
was added as an analyte, similar to Andrieux et al. (2), but
now using MS/MS for added specificity. Fu et al. (3) reported
a choline single-laboratory validation (SLV) in infant formula
using MS/MS but did not look at simultaneous carnitine
measurement. Microwave digestion was added to speed up the
typical 3 h digestion, similar to Phillips and Sander (4), but again
the present work utilizes MS/MS instead of single quadrupole
technology. The combined method was substantially different
than either AOAC
2012.19
or
2012.17
, and so both methods
were withdrawn from the SPIFAN process. The present
submission of AOAC
2014.04
has a full set of SLV data with
the method in its final form. It is submitted in response to a
second Call for Methods for carnitine, but full SLV data are also
presented for choline in case the ERP also wants to advance it
for this use.
AOAC Official Method 2014.04
Simultaneous Determination of Choline/Carnitine
in Infant Formulas and Adult Nutritional Products
HILIC LC/MS/MS
First Action 2014
A. Principle
Reconstituted test sample is weighed into a microwave
reaction vessel. Microwave heating accelerates an acidic
hydrolysis process to release bound choline. A subsequent
alkaline degradation is performed to release L-carnitine
from inherent acylcarnitines. Choline and L-carnitine can
be determined quantitatively in nutritional products and raw
materials by hydrophilic interaction ultra-performance LC with
tandem mass spectrometry (HILIC-UPLC/MS/MS).
B. Apparatus and Materials
(
a
)
Column.
—Acquity UPLC ethylene bridged hybrid
12