AOAC Official Method
Quinic, Malic, and Citric Acids
in Cranberry Juice Cocktail and Apple Juice
Liquid Chromatographic Method
First Action 1986
Final Action 1989
A. Principle
Juice is eluted through disposable cartridge to remove interfer-
ences, filtered, and injected into liquid chromatographic system.
Quinic, malic, and citric acids are separated by using 2 reverse phase
LC columns in tandem with UV detection at 214 nm. Compounds
are quantitated by comparison with external standards.
B. Apparatus and Reagents
(a) Liquid chromatograph.—System equipped with Model 7725i in-
jector, Model 2487 variable wavelength detector operable at 214 nm,
AUFS (Waters Associates, Inc.) and computing integrator
(Hewlett-PackardIntegrator3390[],orequivalent).
(b) Analytical columns:—(1) Supelcosil LC-18, or equivalent,
5 m m particle size, 25 cm · mm, in tandem with and followed by
(2) Radial-Pak C18 cartridge (Waters Associates, Inc.), 5 m m particle size,
10 cm long, used with Radial Compression Module. Radial-Pak C18 car-
tridge can be substituted by any standard 25 or 30 cm stainless steel re-
verse phase C18 column with 10 m m particle size. Connect Bio-Rad
reverse phase micro-guard column (ODS-10) ahead of column 1. Mo-
bile phase: phosphate buffer at mL/min; sensitivity AUFS.
(c) Disposable cartridges.—Sep-Pak C18 (Waters Associates,
Inc).
(d) Chemicals.—Potassium phosphate monobasic (KH2PO4),
85% H3PO4, methanol, CH3CN. Filter all solvents through aqueous
or organic m m filter.
(e) LC mobile phase.—Phosphate buffer, KH2PO4, pH .
Weigh g KH2PO4 in beaker. Add H2O to 950 mL. Using pH me-
ter and H3PO4, adjust to pH . Pour into 1 L graduate and adjust to
volume with H2O; filter.
C. Preparation of Test Sample and Standards
(a) Working standard solutions.—Accurately weigh, to nearest
mg, g ACS grade quinic, malic, and citric acids. Dilute
combined acids to 100 mL in volumetric flask with H2O; filter.
(b) Test sample solutions.—Condition cartridge by eluting
10 mL CH3CN–H2O (50 + 50) through 10 mL Luer-Lok syringe. Re-
move syringe and pass 10 mL air through cartridge. Elute 10 mL
sample through conditioned cartridge. Discard first 4–5 mL and col-
lect next 4–5 mL. Filter test sample for LC analysis.
D. Determination
Condition system with 100% methanol (or methanol–H2O [70 +
30]) followed by H2O and then phosphate buffer. Reverse order at
end of working day; never let methyl alcohol come into contact with
phosphate buffer. Operating conditions: flow rate mL/min;
214 nm detector; temperature ambient; sensitivity AUFS. Col-
umn system is satisfactory when baseline separation is achieved be-
tween sugar front peak and quinic acid peak in cranberry juice
cocktail and/or apple juice. Inject 5–20 m L standard solution after
each 2 test sample injections to check linearity. Inject 5 m L test sam-
ple solution. Use average of 2 injections for standard and test sample
responses. Elution pattern: quinic, malic, citric, fumaric.
E. Calculations
Quinic, malic, citric, or fumaric acid, % =
(PA/PA¢ ) · (V¢ /V) · C
where PA and PA¢ = peak area of test sample and standard, respec-
tively; V and V¢ = volume of test sample and standard, respectively;
and C = concentration of standard, %.
[Note: Malic standards (L-malic or D-L-malic) will yield 2 peaks,
the second of which is fumaric acid. Amount of fumaric acid present
in malic acid standard is so small that standard is ‡ % fumaric
acid-free. Impurity does not affect analysis. Cranberry juice cock-
tail, fortified with vitamin C, will exhibit large peak appearing after
malic. (This is due to coelution of ascorbic acid + shikimic acid and
is not quantified.) For details about this separation see Fig-
ure .]
Reference: JAOAC 69, 594(1986).
CAS-77-92-9 (citric acid)
CAS-6915-15-7 (malic acid)
CAS-77-95-2 (quinic acid)
Revised: March 1996
Figure —LC chromatogram of cranberry juice
cocktail sample: (1) solvent and sugar front; (2) quinic
acid; (3) malic acid; (4) ascorbic acid and some
shikimic acid; and (5) citric acid.
