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Infant Formula and Adult Nutritionals
204
E
llingson
et al
.:
J
ournal of
AOAC I
nternational
V
ol
.
99, N
o
.
1, 2016
Analytical methods for the analysis of both L-carnitine and
choline are needed for reliable and accurate determination in
infant formula and adult/pediatric nutritional formula. These
compounds are different in how they are utilized by the human
body, but are structurally similar. L-carnitine and choline
are quaternary ammonium compounds, enabling both to be
retained under acidic conditions with strong cation exchange
(SCX)chromatography.Thismethodanalyzesbothcompounds
simultaneously as either the free forms or as a total amount
that includes bound sources such as phosphatidylcholine or
acetylcarnitine. The free analysis consists of water extraction
and analysis by LC/MS/MS, while the total analysis consists of
extraction by acid assisted microwave hydrolysis and analysis
by LC/MS/MS. Calibration standards used for calculations
are extracted with all samples in the batch. Asingle laboratory
validation (SLV) was performed following the guidelines of
the AOAC Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN) utilizing the kit of materials provided.
The results achievedmeet the requirements of SMPR 2012.010
and 2012.013 for L-carnitine and total choline, respectively.
A
n analytical method for the analysis of
l
-carnitine and
total choline in infant formula (IF) and adult/pediatric
nutritional formula is needed tomeet the
StandardMethod
Performance Requirements
(SMPRs
®
) of the AOAC Stakeholder
Panel on Infant Formula and Adult Nutritionals (SPIFAN; 1, 2).
There are currently other methods that can either quantify
carnitine or total choline in IFs (3–5). We developed a method
that can analyze these compounds simultaneously using strong
cation-exchange (SCX) chromatography because both are
quaternary ammonium compounds and can be retained under
acidic conditions. This method can be used to quantify the free or
total choline and carnitine content of a sample. The free portion is
analyzed using water extraction, whereas the total extraction uses
acid-assisted microwave hydrolysis. The total amount can include
contribution from bound sources such as phosphatidylcholine or
acetylcarnitine. Both extraction methods use LC tandem mass
spectrometry (MS/MS) analysis with electrospray ionization
(ESI). Calibration standards are included through each extraction
procedure for greater assurance with quantification, and the acidic
conditions of the total extraction allowdirect injection after dilution
with acetonitrile for fast analysis. A single-laboratory validation
(SLV) was performed with this method using the SPIFAN SLV
kit of materials. All requirements of SMPRs were met, except the
ability to differentiate
l
- and
d
-carnitine. Verification for recovery
of the bound forms of choline and carnitine commonly found in
these matrixes was also completed.
AOAC Official Method 2015.10
Determination of Free and Total Choline and Carnitine
in Infant Formula and Adult/Pediatric
Nutritional Formula
Liquid Chromatography/Tandem Mass Spectrometry
(LC/MS/MS)
First Action 2015
[Applicable to the determination of free and total choline and
carnitine in infant formula and adult/pediatric nutritional formula.]
Caution
: It is recommended that all preparation steps with
nitric acid be performed within a fume hood, and
the necessary personal protective equipment used
when handling.
A. Principle
The method uses a water extraction for free analysis and
acid-assisted microwave hydrolysis for total analysis. Both
compounds are simultaneously analyzed and quantified by
LC/MS/MS with ESI.
B. Apparatus
(a)
LC system
.—Prominence, Shimadzu (Kyoto, Japan) or
equivalent.
(b)
MS/MS system
.—API 4000 with ESI, ABSciex
(Framingham, MA) or equivalent.
(c)
Mass spectrometry software
.—Analyst (ABSciex) or
equivalent.
(d)
Analytical column
.—Zorbax 300-SCX, 3.0 × 50 mm,
5 μm, Agilent (Santa Clara, CA) or equivalent.
Determination of Free and Total Choline and Carnitine
in Infant Formula and Adult/Pediatric Nutritional
Formula by Liquid Chromatography/Tandem Mass
Spectrometry (LC/MS/MS): Single-Laboratory Validation,
First Action 2015.10
D
avid
J. E
llingson
, J
effrey
J. S
hippar
,
and
J
ustin
M. G
ilmore
Covance Laboratories, Nutritional Chemistry and Food Safety, 3301 Kinsman Blvd, Madison, WI 53704-2523
Received June 1, 2015. Accepted by AK July 31, 2015.
This method was approved by the Expert Review Panel for Infant
Formula and Adult Nutritionals as First Action.
The Expert Review Panel for Infant Formula and Adult Nutritionals
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 for gaining global recognition and
acceptance of the methods. Comments can be sent directly to the
corresponding author or
methodfeedback@aoac.org.Corresponding author’s e-mail:
david.ellingson@covance.comDOI: 10.5740/jaoacint.15-0144
99