AOAC Official Method 2012.22 E.
  Preparation of Standards
Vitamin C in Infant Formula
and Adult/Pediatric Nutritional Formula
Ultra-Performance Liquid Chromatography (UPLC®)
with Ultraviolet Detection
First Action 2012
[Applicable to the determination of vitamin C (L-ascorbic acid)
in infant formula and adult/pediatric nutritional formula by ultra-
performance LC (UPLC®)-UV.]
Caution: Refer to Material Safety Data Sheets prior to use
of chemicals. Use appropriate personal protective
equipment when performing testing.
A. Principle
Ascorbic acid is extracted from the sample using trichloroacetic
acid (TCA) in the presence of Tris[2-carboxyethyl]phosphine
(TCEP) as a reducing agent. Ascorbic acid is then determined by
UPLC-UV at 265 nm. Separation takes place on a C18 column with
sodium acetate (pH 5.4) as the eluent, combined with TCEP and
decylamine as an ion-pairing agent.
B. Apparatus
(a)  Balances.—With readability of 0.1 mg and 0.01 g.
(b)  pH meter.—Metrohm 691 (Herisau, Switzerland), or
equivalent.
(c)  LC column.—Waters (Milford, MA) Acquity UPLC ethylene
bridged hybrid (BEH) C18 column, 1.75 µm, 2.1 × 100 mm, or
equivalent.
(d) UPLC system.—Waters Acquity UPLC system equipped
with a Waters Acquity UPLC photodiode array detector, or
equivalent.
C.  Reagents and Standards
(a)  Acetonitrile.—HPLC grade (Merck, Geneva, Switzerland, or
equivalent).
(b)  Ascorbic acid.—Fluka (Buchs, Switzerland), or equivalent.
(c)  Decylamine.—Fluka, or equivalent.
(d)  Phosphoric acid.—85% (Merck, or equivalent).
(e)  Purified water.—Millipore (Le Mont-sur-Lausanne,
Switzerland), or equivalent.
(f)  Sodium acetate trihydrate.—Merck, or equivalent.
(g)  TCA.—Merck, or equivalent.
(h)  TCEP.—Fluka, or equivalent.
D.  Preparation of Solutions
(a)  Sodium acetate solution (pH 5.4).—
34.0 g Sodium acetate trihydrate × 1 mol/136.08 g
= 0.25 mol sodium acetate trihydrate into 500 mL
= 0.5 mol/L = 500 mmol/L
(b)  TCA (15%).—Into a 500 mL volumetric flask, weigh 75.0 g
TCA, dissolve, and make up to volume with water.
(c)  TCEP (250 µg/mL).—Into a 500 mL volumetric flask, weigh
125 mg TCEP-HCl, dissolve, and make up to volume with water.
(d)  Mobile phase for UPLC.—Into a 250 mL flask, mix 0.4 g
decylamine, 2.5 mL acetonitrile, 25 mL sodium acetate solution
500 mmol/L (pH 5.4), and 205 mL water. Add 10 mg TCEP. No
making up to volume is preferred.
(Note: Vitamin C is sensitive to light and oxygen. Conduct
operations under subdued light, or use amber glassware. Keep all
solutions away from direct light.)
(a)  Ascorbic acid stock solution (500 µg/mL).—Into a 25 mL
amber glass volumetric flask, weigh 12.5 mg ascorbic acid.
Dissolve and make up to volume with TCEP solution. This solution
can be kept for 3 months if stored at 4°C away from light.
(b)  Ascorbic acid intermediate standard solution (50 µg/mL).—
Into a 10 mL amber glass volumetric flask, pipet 1 mL stock
solution. Make up to volume with TCEP solution. This solution
can be used for 1 month if stored at 4°C away from light.
(c)  Ascorbic acid calibration standard solutions (0.5, 1.0, 2.0,
3.0, 5.0, 7.5, and 10.0 µg/mL).—Into 10 mL amber glass volumetric
flasks, pipet 0.1, 0.2, 0.4, 0.6, 1.0, 1.5, and 2.0 mL intermediate
standard solution. Make up to volume with mobile phase to prepare
the respective concentrations given above.
F.  Preparation of Samples
(a)  Reconstitution of powder samples.—
(1)  Weigh 25 g powder into a brown glass 250 mL beaker. Add
10 mg TCEP.
(2)  Add 200 g warm water (40°C) and mix well until complete
dissolution. Make sure there are no lumps.
(b)  Extraction.—
(1)  Weigh 2 g liquid or reconstituted sample into a 10 mL amber
glass volumetric flask.
(2)  Add 4 mL TCEP solution and 2 mL TCA 15%.
(3)  Bring up to volume with water.
(4)  Filter solution through folded grade 597½ or greater filter
paper (Schleicher & Schuell, Dassel, Germany).
(5)  Transfer 1 mL filtrate into a 10 mL amber glass volumetric
flask containing 1 mL acetate solution (pH 5.4). Bring up to volume
with mobile phase.
(6) Filter about 2 mL through a 0.22 µm membrane into an
HPLC vial.
G. Analysis
(a)  Chromatographic conditions.—
(1)  Injection volume.—5 µL.
(2)  Autosampler temperature.—10°C.
(3)  Column temperature.—25°C.
(4)  Flow rate.—0.35 mL/min.
(5)  Run time.—4.0 min.
(6)  Mobile phase for UPLC.—Sodium acetate (50 mmol/L,
pH 5.4), decylamine (1.6 g/L), acetonitrile (1%), and TCEP
(40 mg/L).
Note: At the end of each analytical series, rinse the column with
acetonitrile–water (1 + 1, v/v) for 10 min at 0.4 mL/min.
(b)  System suitability test.—Equilibrate the chromatographic
system for ≥0.5 h. Inject a working standard solution at least six
times and check peak retention times and responses (peak height or
area). Inject working standard solutions on a regular basis within a
series of analyses.
(c)  Calibration.—Make single injections of working standard
solutions at least at the beginning and the end of each analytical
series. Establish the calibration curve (seven points) by plotting
peak response (height or area) versus ascorbic acid concentration.
Perform linear regression. Calculate the slope (S) and intercept (I)
of the calibration curve.
(d)  Analysis.—Make single injections of sample solutions.
© 2013 AOAC INTERNATIONAL
 (e)  Identification.—Identify the ascorbic acid peak in the Note: If results expressed in powder sample are needed, use the
chromatograms of the sample solutions by comparison with the reconstitution rate to calculate (c × 225/25).
retention time and UV spectrum of the corresponding peak in the References: J. AOAC Int. 96, 1065(2013)
standard solution. DOI: 10.5740/jaoacint.13-141
H. Calculations
AOAC SMPR 2012.012
Calculate the concentration of vitamin C, in mg ascorbic J. AOAC Int. 96, 491(2013)
acid/100 g expressed in product “as is” for RTF (ready-to-feed) DOI: 10.5740/jaoac.int.SMPR2012.012
products or as “reconstituted powder” for powder samples, as
follows: Corrected November 2013 [to correct concentrations of mobile
phase in D(a), D(d), and G(a)(6)]
( A - I ) x V1 x V3 x 100
C=
S x m x V2 x 1000
where A = response (height or area) of the ascorbic acid peak
obtained for the sample solution, m = weight of the test portion in
g (2.0 g), V1 = volume of the test solution (volume used to dissolve
the test portion) in mL (10 mL), V2 = volume used in the sample
dilution (1.0 mL), and V3 = volume of the final sample dilution
(10 mL).

© 2013 AOAC INTERNATIONAL