AOAC Official Method 2012.20 (l) Pipet.
—Thermo Scientific Nunc Serological Pipet, 10 mL,
Choline in Infant Formula and Adult Nutritionals
Ion Chromatography and Suppressed Conductivity
First Action 2012
{Applicable to the determination of total choline (free and
bound) in all forms of infant formula/adult nutritionals/pediatric
formulas [powder, ready-to-feed (RTF), and liquid concentrates]
reported as the choline ion in mg/100 g reconstituted powder or
as-is in RTF.}
Caution: Refer to the Material Safety Data Sheets prior to working
with any chemicals. Make sure to use proper personal
protective equipment as required.
A. Principle
Microwave-assisted acid hydrolysis is used to digest and release
bound choline from infant formula and adult nutritional samples.
Following hydrolysis, separation of choline from common cations
is achieved on a Dionex IonPac CS19 column, and choline is
detected by suppressed conductivity. Total choline is measured.
B. Reagents and Standards
(a) Deionized (DI) water.—Type I reagent grade, 18 MΩ-cm
resistance or better.
(b) Hydrochloric acid (HCl).—Ultrapure reagent, Optima
[Fisher Scientific, Pittsburgh, PA; Part No. (P/N) A466250, or
equivalent].
(c) Choline bitartrate.—99% Reagent grade or better (Sigma-
Aldrich, St. Louis, MO; P/N C1629-500G, or equivalent).
C. Apparatus
(a) Chromatographic system.—Thermo Scientific (Sunnyvale,
CA) Dionex ICS-5000 system, which includes an SP Single Pump
or DP Dual Pump, DC Detector/Chromatography Compartment,
and EG Eluent Generator module, or equivalent.
(b) Autosampler.—Thermo Scientific Dionex AS-AP, or
equivalent.
(c) Software.—Thermo Scientific Dionex Chromeleon
Chromatography Data System software version 7.1, or equivalent.
(d) Eluent cartridge.—Thermo Scientific Dionex EGC III MSA
(P/N 074535).
(e) Trap column.—Thermo Scientific Dionex CR-CTC II
Continuously Regenerated Cation Trap Column (P/N 066262), or
equivalent.
(f) Guard column.—Thermo Scientific Dionex IonPac CG19, 2
× 50 mm (P/N 076029), or equivalent.
(g) Analytical column.—Thermo Scientific Dionex IonPac CS19
Analytical, 2 × 250 mm (P/N 076028), or equivalent.
(h) Autosampler vials.—Thermo Scientific Dionex Vial Kit,
10 mL polystyrene with caps and Blue Septa (package of 100; P/N
074228), or equivalent.
(i) Storage bottles.—Thermo Scientific Nalgene Narrow-
Mouth Packaging Bottles; natural high-density polyethylene with
polypropylene closures, 250 mL, 24-415 closure, bulk pack (P/N
312089-0008, or equivalent).
(j) Conical tubes.—BD Falcon (Becton Dickinson, Franklin
Lakes, NJ) 50 mL conical centrifuge tubes, polypropylene; 30 (od)
× 115 (length) mm; 25/bag (Fisher Scientific; P/N 14-432-22).
(k) Filters.—Thermo Scientific Nalgene syringe filters, 25 mm
diameter acrylic housing; 25 mm; 0.2 µm pore size; sterile (P/N
194-2520, or equivalent.)
individually wrapped, paper peel packaging, plugged, 50/bag,
200/case (P/N 170356, or equivalent).
(m) Sample prep station.—Thermo Scientific Dionex OnGuard
Sample Prep Workstation (P/N 039599), or equivalent.
(n) Needle.—Thermo Scientific Dionex OnGuard Needle,
18-gauge, 1.25 Luer (P/N 039996), or equivalent.
(o) Sample reservoir.—Thermo Scientific Dionex OnGuard
Sample Reservoir, 5 cc, package of 250 (P/N 041233), or equivalent.
(p) Valve.—Thermo Scientific Dionex OnGuard Valve, Stopcock
Luer (P/N 040896), or equivalent.
(q) Cartridge.—Thermo Scientific Dionex OnGuard II A
Cartridge, 2.5 cc, package of 48 (P/N 057092), or equivalent.
(r) Microwave system.—CEM Corp. (Matthews, NC) MARS
6 Microwave Reaction System, 230 V/60 Hz (P/N 927500), or
equivalent.
(s) Microwave temperature control.—CEM Corp. DuoTemp
Control with fiber-optic temperature option (P/N AVTC), or
equivalent.
(t) Microwave vessel.—CEM Corp. Easy Prep Vessel Full
Starter Set, pressure and temperature, 100 mL (P/N 910930), or
equivalent.
(u) Analytical balance.—Mettler Toledo (Columbus, OH)
Model XPZ05DR, or equivalent.
D. Solutions and Reagents Preparation
(a) 20 000 mg/L Choline solution.—Dry 7–10 g choline
bitartrate at 100°C for 2–3 h. Weigh 4.86 g dry choline bitartrate in
a 125 mL polypropylene bottle and tare the balance. Add 100 g DI
water to make a 20 000 mg/L choline stock solution. Cap the bottle
and shake to completely dissolve the solid material. The standard is
stable for 1 week stored at 4°C.
(b) 10 000 mg/L Choline solution.—Weigh 2.43 g dry choline
bitartrate in a 125 mL polypropylene bottle and tare the balance.
Add 100 g DI water to make a 10 000 mg/L choline stock solution.
Cap the bottle and shake to completely dissolve the solid material.
The standard is stable for 1 week stored at 4°C.
(c) 1000 mg/L Choline solution.—Weigh 0.243 g dry choline
bitartrate in a 125 mL polypropylene bottle and tare the balance.
Add 100 g DI water to make a 1000 mg/L choline stock solution.
Cap the bottle and shake to completely dissolve the solid material.
The standard is stable for 1 week stored at 4°C.
(d) Choline working standard solutions.—Deliver the
appropriate volume of the 1000 mg/L stock solution into a 125 mL
polypropylene bottle and bring to the appropriate volume (by
weight) with DI water. For this application, calibration standards
were prepared at 0.06, 5, 15, 30, 45, 60, and 75 mg/L. Aliquots
were stored at –80°C and thawed prior to use.
(e) 1.5 M HCl.—In a well-ventilated hood, weigh 435.6 g DI
water into a glass bottle and add 73.5 mL ultrapure reagent-grade
HCl (32%). Cap and swirl to mix.
(f) 3.0 M HCl.—In a well-ventilated hood, weigh 371.4 g DI
water into a glass bottle and add 147.1 mL ultrapure reagent-grade
HCl (32%). Cap and swirl to mix.
E. Chromatographic Conditions
(a) Columns.—Dionex IonPac CS19 guard and analytical
columns, 2 mm.
(b) Eluent.—Methanesulfonic acid (MSA).
(c) Gradient.— –5 to 13 min, 6.4 mM MSA; 13 min step to
25 mM MSA; 13–17 min, 25 mM MSA.
© 2013 AOAC INTERNATIONAL
Table 2012.20A. SPIFAN Single-Laboratory Validation (SLV) Test Materials Kit
Category No. Product description Container size, g Lot No. identification
Fortified 1 NIST SRM 1849a, powder 10 CLC10-b
2 Adult nutritional, milk-protein-based, powder 100 11750017V3
3 Infant formula, milk-based partially hydrolyzed, powder 360 1172572116
4 Infant formula, soy-based partially hydrolyzed, powder 360 117257651Z
5 Adult nutritional, low-fat, powder 250 00394RF00
6 Child formula, powder 250 00412RF00
7 Infant elemental, powder 250 00403RF00
8 Infant formula, milk-based, powder 360 D04HTCVV
9 Infant formula, soy-based, powder 730 E29JVLV
10 Infant formula, milk-based, RTF 60 SPIFAN control milk form
11 Adult nutritional, high-protein, RTF 250 00414RF00
12 Adult nutritional, high-fat, RTF 250 00406RF00
Placebo 13 Child formula, powder 250 00411RF00
14 Infant elemental, powder 250 00402RF00
15 Infant formula, milk-based, RTF 60 SPIFAN blank milk form
16 Adult nutritional, high-protein, RTF 250 00415RF00
17 Adult nutritional, high-fat, RTF 250 00407RF00

(d) Eluent source.—Dionex EGC III MSA with Dionex CR- (3) For reconstituted powders and RTF liquids.—Shake well
CTC II. before use. Immediately prior to use, invert the bottle at least
(e) Flow rate.—0.25 mL/min. three times to ensure the sample is mixed before taking an aliquot;
(f) Injection volume.—5 µL. however, do not shake as this will cause bubbles in the sample. Add
(g) Temperature.—30°C. 10 g of the liquid (reconstituted powder or RTF) to the microwave
(h) Autosampler temperature.—10°C. vessel and record the weight. Add 10 g of 3 M HCl and record the
(i) Detection.—Suppressed conductivity, Thermo Scientific total weight.
Dionex CSRS 300 cation self-regenerating suppressor (2 mm), (c) Run the microwave program.—
recycle mode, –5–13 min, 5 mA; 13–17 min, 19 mA. (1) Assemble the microwave vessels and place them in the rotor.
(j) System backpressure.—About 2200 psi. (2) Run the microwave program to completion (see
(k) Background conductance.—<0.2 µS. Table 2012.20B).
(l) Typical noise.—<0.2 nS. (3) Allow the vessels to cool to at least 50°C and open in a well-
(m) Run time.—17 min. ventilated hood.
F. Samples (d) Filter and dilute.—
(1) Transfer the sample to a 50 mL conical tube and cap.
Determination of total choline was conducted for the matrixes
(2) Shake the contents just before filtering a portion (about
provided in the AOAC Stakeholder Panel on Infant Formula and
3 mL) of the hydrolysate through a 0.2 µm filter disk into a clean
Adult Nutritionals (SPIFAN) Single-Laboratory Validation (SLV)
50 mL conical tube with cap.
Test Materials Kit (Table 2012.20A). All powder samples, except
(3) Transfer 0.75 g of the filtrate into a clean 50 mL conical tube
National Institute of Standards and Technolgy (NIST) Standard
and record the weight. Bring to a total weight of 12.5 g with DI
Reference Material (SRM) 1849a, were reconstituted in DI water
water and record the total weight.
(25 g powder in 200 g DI water).
(e) Dionex OnGuard II A treatment.—
G. Sample Preparation (1) Neutralize the sample using a Dionex OnGuard II A
(a) Reconstitute powders.—Reconstitute all powders Cartridge. Prepare the 2.5 cc cartridge for use by rinsing it with
except SRM 1849a in water prior to sampling. Place a 250 mL 15 mL DI water. Connect the cartridge to a 25 mL syringe and
polypropylene bottle on the balance and tare it. Add 25 g powder load the syringe with the sample prepared in G(d)(3). Push the first
and record the weight. Add 200 g DI water and record the total
weight. Cap the bottle and shake until well dissolved. Reconstituted
Table 2012.20B. Microwave program
powder samples must be used on the date of preparation.
(b) Prepare the microwave vessel with sample.— Step Time, min Temperature, °C Power, W
(1) Place a 100 mL microwave vessel on the balance and tare it. 1 Ramp 14 100 900–1050
(2) For SRM 1849a powder.—Weigh 2.5 g powder in the vessel 2 Hold 10 100 900–1050
and record the weight. Add 15 g 1.5 M HCl and record the total 3 Vent 15 — —
weight.

© 2013 AOAC INTERNATIONAL

6 mL of neutralized sample into a waste container at a flow rate of
2 mL/min. Collect the next 3 mL for injection in a 10 mL sample
vial. Up to 12 samples can be prepared in parallel using the Dionex
OnGuard Sample Prep Workstation.
(2) Samples are stable in a capped polypropylene vial at 4°C
for 3 days.
H. Quantification
(a) NIST SRM 1849a.—Choline concentration per 100 g powde:
where C = choline concentration mg/L, M1 = mass of the powder
sampled (2.5 g), M2 = total mass of powder plus acid (17.5 g),
M3 = mass portion of the filtrate used (0.75 g), and M4 = mass total
dilution (12.5 g).
Divide by a reconstitution factor of 10 to convert mg/100 g
powder to mg/100 g reconstituted powder.
(b) AOAC SLV test materials.—Choline concentration per 100 g
reconstituted powder:

where C = choline concentration mg/L, M2 = total mass of powder
plus acid (20 g), M3 = mass portion of the filtrate used (0.75 g), and
M4 = mass total dilution (12.5 g).
(c) AOAC SLV test materials.—Concentration per 100 g RTF:
where C = choline concentration mg/L, M1 = mass of the liquid
sampled (10 g), M2 = total mass of powder plus acid (20 g),
M3 = mass portion of the filtrate used (0.75 g), and M4 = mass total
dilution (12.5 g).
I. Validation Protocol
(a) SLV guidelines.—SPIFAN SLV recommended guidelines
were followed (final version 4; 1).
(b) System suitability.—Check standards at the lowest point and
midpoint of the analytical range were verified daily throughout the
batch run. The system was recalibrated when the error of the check
standards was >5%.
(c) Linearity/calibration fit.—Three separate experiments
were performed using independently prepared standards at seven
levels that span the desired working range. Relative error of back-
calculated concentrations within the desired working range were
<5%.
(d) LOD/LOQ.—LOD was calculated as blank mean plus three
times the standard deviation (SD); LOQ was calculated as blank
mean plus 10 times the SD.
(e) Precision studies.—Samples selected for precision studies
were analyzed in duplicate on each of 6 days using multiple
instruments. Fresh reagents were prepared each day.
(f) Accuracy.—
(1) Analysis of SRM 1849a.
(2) Spike recovery was determined from unfortified products.
Each selected matrix was spiked at two levels (50 and 100%) of the
amounts found in the fortified products and analyzed in duplicate
on each of 3 days. The overall mean of the unspiked samples was
used for calculating recoveries.
References: (1) Official Methods of Analysis of AOAC
INTERNATIONAL (2012) 19th Ed., Appendix L,
AOAC INTERNATIONAL Recommended Guidelines
for SPIFAN Single-Laboratory Validation (SLV),
Gaithersburg, MD, www.eoma.aoac.org
J. AOAC Int. 96, 1400(2013)
DOI: 10.5740/jaoacint.13-177
AOAC SMPR 2012.013
J. AOAC Int. 96, 492(2013)
DOI: 10.5740/jaoac.int.SMPR2012.013
Posted: December 2013
© 2013 AOAC INTERNATIONAL