Inoue & Dowell: Journal of AOAC International Vol. 95, No.
3, 2012  1
INFANT FORMULA AND ADULT NUTRITIONALS
HILIC-MS/MS Method for the Quantitation of Nucleotides in
Infant Formula and Adult Nutritional Formula:
First Action 2011.21
Koichi Inoue1
Kinjo Gakuin University, School of Pharmacy, Department of Physical and Analytical Chemistry, Nagoya, Japan
Dawn Dowell
AOAC INTERNATIONAL, 481 N. Frederick Ave, Suite 500, Gaithersburg, MD 20877-2417
Official MethodSM 2011.21 is for the quantitation of the
following nucleotides: adenosine 5′-monophosphate
(AMP), guanosine 5′-monophosphate (GMP), uridine
5′-monophosphate (UMP), cytidine 5′-monophosphate
(CMP), and inosine 5′-monophosphate (IMP) in infant
formula and adult/pediatric nutritional formula. It
uses hydrophilic interaction liquid chromatography
with tandem mass spectrometry (HILIC-MS/MS).
Preparation of the internal standards was conducted
using centrifugal ultrafiltration and the standards are
13 15 13 15 13 15
AMP- C10, N5; GMP- C10, N5; UMP- C9, N2; and
13 15
CMP- C9, N3. Data were collected by using multiple
reaction monitoring of the product ions of protonated
molecules of the five nucleotides generated by
positive-electrospray ionization. The HILIC conditions
were conducted with ammonium formate (30 mmol/L)
in water (pH 2.5, adjusted with formic acid) and
methanol. The LOD and LOQ of the standard solution
were 0.005–0.01 and 0.01–0.03 µg/mL, respectively.
Recovery data were collected for intraday and
interday testing and ranged from 98.1 to 108.9%
with an RSD of 0.7–5.4%. The analytical range of
the method is between 0.04 to 5 μg/mL for standard
solution.
I
n August 2011, the Expert Review Panel (ERP) for Infant
Formula and Adult Nutritionals reviewed the manuscript
“Development and Application of an HILIC-MS/MS
Method for the Quantitation of Nucleotides in Infant Formula”
for AOAC Official First Action consideration (1). After
reviewing the single-laboratory validation data and evaluating
against consensus-based standard method performance
Submitted for publication February 13, 2012.
The method was approved by the Expert Review Panel on Infant
Formula and Adult Nutritionals as First Action. See “Standards News,”
(2011) Inside Laboratory Management, September/October issue.
The AOAC Stakeholder Panel on Infant Formula and Adult
Nutritionals (SPIFAN) 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.
1 (e)  Centrifuge tubes.
Current address: University of Shizuoka, School of
Pharmaceutical Science, Laboratory of Analytical and Bio-Analytical
Chemistry, Shizuoka, Japan.
Corresponding author’s e-mail: kinoue@u-shizuoka-ken.ac.jp
DOI: 10.5740/jaoacint.CS2011_21
requirements, the study was determined to be appropriate for
Official First Action status and was identified as AOAC Official
SM
Method 2011.21, applicable to infant formula and adult
nutritional formula. The method was brought forward for review
as a quantitative hydrophilic interaction liquid chromatography
with tandem mass spectrometry (HILIC-MS/MS) method that
would prove useful for regulatory analysis of AMP, GMP, UMP,
CMP, and IMP.
AOAC Official Method 2011.21
Nucleotides in Infant Formula
and Adult Nutritional Formula
HILIC-MS/MS Method
First Action 2011
[Applicable to the determination of adenosine
5′-monophosphate (AMP), guanosine 5′-monophosphate
(GMP), uridine 5′-monophosphate (UMP), cytidine
5′-monophosphate (CMP), and inosine 5′-monophosphate
(IMP) in infant formula and adult nutritional formula.]
Caution: Consult the Material Safety Data Sheet (MSDS) for
all materials prior to use. Use personal protective
equipment where required.
A.  Principle
Hydrophilic interaction chromatography with tandem mass
spectrometry (HILIC-MS/MS) uses ultrafiltration and multiple
reaction monitoring (MRM) of the product ions of protonated
molecules of the five nucleotides generated by the positive-ion
ESI.
B.  Apparatus
(a)  LC-MS/MS system.—Waters Alliance 2965/Micromass
Quattro Premier (Waters, Milford, MA) or equivalent.
(b)  TSK-gel NH2-100 column.—3 µm, 2.0 × 150 or 50 mm
(Tosoh Co., Tokyo, Japan); 40°C, injection volume = 10 µL, or
equivalent.
(c)  Amicon filter.—Ultra-4, Ultra-3k, regenerated cellulose
3000 M.W. (molar weight) for volumes <4 mL (Millipore Co.,
Ltd, Billerica, MA), or equivalent.
(d)  Centrifuge.—Kubota 5420 (Kubota Co., Tokyo, Japan)
or equivalent.
C.  Reagents
(a)  AMP.—Purity, 98.1% (Oriental Yeast Co., Tokyo, Japan)
or equivalent.
2  Inoue & Dowell: Journal of AOAC International Vol. 95, No. 3, 2012

Table  2011.21A.  MS/MS conditions of nucleotides and corresponding internal standardsa

Analyte Identity Precursor ion (m/z) Product ion (m/z) Cone voltage, V Collision energy, eV Dwell time, ms
+ b
AMP [M + H] 348 136 /97 22 20b/33 100
13 15 +
AMP- C10, N5 [M+ H] 363 146 22 20 100
GMP [M + H]+ 364 152b/135 20 20b/45 100
13 15 +
GMP- C10, N5 [M + H] 379 162 20 20 100
UMP [M+ H]+ 325 97b/213 20 18b/10 100
UMP-13C9, 15N2 [M + H]+ 336 102 20 18 100
CMP [M+ H]+ 324 112b/97 23 15b/35 100
13 15 +
CMP- C9, N3 [M + H] 336 119 23 15 100
+ b b
IMP [M + H] 349 137 /97 20 15 /30 100
a
 LC-MS/MS system: Waters Alliance 2965/Micromass Quattro Premier (Waters, Milford, MA). HILIC: TSK-gel NH2-100 column
(3 µm, 2.0 × 150 mm; Tosoh, Tokyo, Japan). Mobile phase: solvent A, 30 mmol/L ammonium formate in water (pH 2.5, adjusted by
formic acid), and solvent B, methanol. Ionization: ESI (positive ionization mode) condition.
b
  Most abundant ion (also used for analyte quantification).

(b)  GMP.—Purity, 97.0% (Wako Pure Chemical Industries (o)  Argon gas.—99.99%.
Co., Osaka, Japan) or equivalent. D.  Preparation of Standard, Internal Standard, and Mobile
(c)  UMP.—Purity, 98–100% (Sigma-Aldrich Co., St. Louis, Phase Solutions
MO) or equivalent.
(a)  Pure standard solutions.—Prepare stock solution of
(d)  CMP.—Purity, 98–100% (Sigma-Aldrich Co.) or
equivalent. 1.0 mg/mL by dissolution of 10 mg of each nucleotide in 10 mL
(e)  IMP.—Purity, 98–100% (Sigma-Aldrich Co.) or water, and store at −20°C for 6 months.
equivalent. (b)  Mixed standard.—Prepare mixed standard solution of
13 15
(f)  Adenosine- C10, N5 5′-monophosphate (AMP- C10,
13 0.1  mg/mL by dissolution of each nucleotide in 1 mL pure
15
N5).—Purity, 98% (Sigma-Aldrich Co.) or equivalent. standard solutions (1.0 mg/mL) in 10 mL water–methanol
13 15
(g)  Guanosine- C10, N5 5′-monophosphate (GMP- C10,
13 (50 + 50, v/v). Prepare dilutions of this mixed standard solution
15
N5).—Purity, 98% (Sigma-Aldrich Co.) or equivalent. (0.1  mg/mL) for each concentration level (between 0.04, 0.1,
13 15
(h)  Uridine- C9, N2 5′-monophosphate (UMP-13C9, 0.2, 0.4, 1, 2, and 5 mg/mL) for calibration curve for each run.
15
N2).—Purity, 98% (Sigma-Aldrich Co.) or equivalent. (c)  Internal standard solutions.—Prepare stock solutions
13 15
(i)  Cytidine- C9, N3 5′-monophosphate (CMP-13C9, of each nucleotide of 0.1 mg/mL by dissolution of 1 mg
15 13 15 13 15
N3).—Purity, 98% (Sigma-Aldrich Co.) or equivalent. labeled nucleotides (AMP- C10, N5, GMP- C10, N5,
13 15 13 15
(j)  Ammonium formate.—Wako Pure Chemical Industries UMP- C9, N2, and CMP- C9, N3) in 10 mL water. Prepare
Co., or equivalent. standard solution of 10 mg/mL by dissolution of each at 1 mL
(k)  Formic acid.—LC/MS grade (Wako Pure Chemical pure standard solutions in 10 mL water–methanol (50 + 50, v/v).
Industries Co.) or equivalent. Solutions are good for 3 months.
(l)  Methanol.—HPLC grade (Wako Pure Chemical Industries (d)  Mobile phase.—Combine solvent A, 30  mmol/L
Co.) or equivalent. ammonium formate in water (pH 2.5, adjusted by formic acid),
(m)  Purified water.—Milli-Q purifying system (Millipore, and solvent B, methanol (deliver at a flow rate 0.2 mL/min).
Bedford, MA) or equivalent.
E.  Sample Preparation and Determination
(n)  Nitrogen gas.—N2 Supplier 24 s (SLP-17; Anest Iwata
Co., Tokyo, Japan) or equivalent. (a)  HILIC analysis.—(1)  Elution.—0.0 min [A/B: 85/15],

Table  2011.21B.  HILIC-MS/MS analytical factors of nucleotidesa

Calibration curve
2
Analyte LOD, µg/mL LOQ, µg/mL Linearity (r ) Calibration range, µg/mL
AMP 0.005 0.010 0.999 0.04–5
GMP 0.005 0.025 0.999 0.04–5
UMP 0.010 0.020 0.999 0.04–5
CMP 0.005 0.010 0.999 0.04–5
IMP 0.005 0.030 0.999 0.04–5
a
 Calculation of LOD and LOQ: signal-to-noise ratio (S/N) = 3 and 10, respectively. Calibration curves: peak area ratios of
nucleotides to each corresponding stable isotope nucleotide.
 Inoue & Dowell: Journal of AOAC International Vol. 95, No. 3, 2012  3
10.0 min [A/B: 85/15], 10.1 min [A/B: 98/2], 30.0 min [A/B:
98/2], 30.1 min [A/B: 85/15], and 40.0 min [A/B: 85/15].
(2)  ESI (positive ionization mode) conditions.—Capillary
voltage, 2.5 kV; extractor, 4.0 V; RF lens voltage, 0.0 V;
source temperature, 110°C; desolvation temperature, 390°C;
cone and desolvation gas flows, 50 and 900 L/h, respectively
(obtained using a nitrogen source). Use argon as collision gas
and regulate at 21 mL/h, setting the multipliers to 650 V. Cone
voltages and the collision energies of analytes are summarized
in Table 2011.21A.
(b)  Sample preparation using centrifugal ultrafiltration
(CFU).—(1) Powder.—Add 1.0 g sample to 50 mL water.
Liquid.—If liquid sample is out of the range of the calibration
standards, dilute sample with water by 0–50%.
(2)  Transfer 0.5 mL sample to plastic tube.
(3)  Add 10 µg/mL, 50 µL internal standard.
(4)  Vortex-mix for 5 min.
(5)  Transfer mixed-sample solution (0.5 mL) to CFU
cartridge.
(6)  Centrifuge CFU cartridge at 3500 rpm (rcf 2328 g) for
15 min.
(7)  Transfer constant quantity of eluted solution (clear
solution: filtered volume between 0.1 to 0.5 mL) to plastic tube
(e.g., Eppendorf sample tube 1.5 mL).
(8)  Dilute transferred sample solution (between 0.1 to
0.4 mL) by 50% using methanol and then vortex-mix for 1 min.
(9)  Centrifuge solution at 13 000 rpm for 5 min.
(10)  Inject solutions to HILIC-MS/MS system (injection
volume: 10 mL).
F.  Calculations
Generate the calibration curves using the analyte-to-stable
isotope internal standard’s peak area ratios by weighted
(1/x2) least-squares linear regression on consecutive days.
Concentration levels are 0.04, 0.1, 0.2, 0.4, 1, 2, and 5 mg/mL
each nucleotides corrected using each stable isotopes. For IMP,
this standard was corrected by GMP-13C10, 15N5. Calculate
the sample concentration (mg/g) by “Detection values: mg/mL
by calibration curve” × 100 for range of 4 to 500 mg/g (ppm;
Table  2011.21B). Calculate liquid sample (e.g., adjustable
infant formula) concentration by “Sample concentration”/
labeled adjusting water volume (how to make infant formula
using water volume).
PA2 × 100
C=
PA1 × F
where C = concentration in mg/g of nucleotides in the sample;
PA2 = peak area of analyte in the sample chromatogram; PA1 =
peak area of the internal standard in the sample chromatogram;
F = response factor; 100 = conversion of concentration of
analyte to mg/g.
Reference: J. AOAC Int. 95, xxx(2012)
Results
The purpose of this method was to provide a simplified
method that would prove accurate and useful in quantifying the
five nucleotides (AMP, GMP, UMP, CMP, and IMP) in infant
formula and adult nutritionals. The results showed that the LOD
values were 0.005 (AMP, GMP, CMP, and IMP) and 0.01 mg/mL
(UMP), and LOQ values were 0.01 (AMP, CMP), 0.02 (UMP),
0.025 (GMP), and 0.03 (IMP) mg/mL, respectively. Seven-point
calibration was performed daily for all analytes with internal
2
standards and showed good correlation values (r  = 0.999), and
calibration errors to be <5.0% in three independent experiments.
Recovery data were generated by using spiked samples for QC
from nucleotide-free powder soy-based infant formula. Six
replicates at three concentration levels (5, 25, and 250 µg/g)
were tested. The determinations were conducted for intraday
and interday testing. The intraday test was assessed by the
recovery test of 6 times/1 day. The interday test was assessed by
the recovery test of 2 times/1 day for 3 days. Intraday recovery
ranged from 99.0 to 108.9%, and interday recovery ranged
from 98.1 to 108.9%. The precision of the intraday study was
determined to be 1.1–4.5%, while the interday was determined
to be 0.7–5.4% (2). These samples were analyzed for ranges of
the five nucleotides. Detected concentrations varied from none
detected (nd) to 278  µg/g. The total nucleotide level ranged
from nd to 600.2 µg/g in powder infant formula. For additional
information regarding the study, see Inoue et al. (2).
References
  (1) Sullivan, D. (2012) J. AOAC Int. 95, 287–290. http://dx.doi.
org/10.5740/jaoacint.Sullivan_Intro
  (2) Inoue, K., Obara, R., Hino, T., & Oka, H. (2010) J. Agric. Food
Chem. (2010) 58, 9918–9924