18.9.07
—Accurately weigh ca 80 mg
AOAC Official Method 990.14
Clioquinol in Creams and Ointments
Liquid Chromatographic Method
First Action 1990
Final Action 1992
Results of the interlaboratory study supporting acceptance of the
method: Synthetic ointment formulated at 3.24% clioquinol
(average rec. 99.1%): sr = 0.0266; sR = 0.0887; RSDr = 0.83%; RSDR
= 2.76%
Synthetic cream formulated at 2.75% clioquinol (average rec.
101.1%): sr = 0.0534; sR = 0.0993; RSDr = 1.92%; RSDR = 3.58%
Commercial 3% clioquinol ointment (average rec. 96.7%): sr =
0.0663; sR = 0.1175; RSDr = 2.29%; RSDR = 4.06%
Commercial 3% clioquinol cream (average rec. 99.7%): sr =
0.0293; sR = 0.1165; RSDr = 0.98%; RSDR = 3.89%
A. Principle
Clioquinol is de ter mined as its nickel(II) com plex by
reversed-phase liquid chromatography on phenyl-bonded column
us ing mo bile phase of 0.001M nickel chlo ride and 0.01M
ammonium acetate in acetonitrile–methanol–water (27 + 18 + 55)
and UV detection at 273 nm. Diphenylamine is added as internal
standard.
B. Apparatus
(a) Liquid chromatograph.—Isocratic pump system, injector,
photometric detector capable of monitoring absorbance at 273 nm,
and recorder.
( b ) C h ro m a t o g r a p h i c c o l u m n . — R e v e r s e d - p h a s e ,
phenyl-bonded, 300 ´ 3.9 mm id, 10 mm or equivalent column that
meets system suitability requirements). Operating conditions;
flow rate 1.2–1.5 mL/min; temperature ambient. Clioquinol elutes
at 7–9 min; diphenylamine elutes at 11–15 min.
C. Reagents
(a) Clioquinol.—USP Reference Standard.
(b) Diphenylamine.—ACS reagent grade.
(c) Tetrahydrofuran.—UV quality, LC grade.
(d) Mobile phase.—Solvent mixture; UV quality
acetonitrile–methanol–water (27 + 18 + 55, v/v/v). Prepare
solution containing 0.001 mole nickel chloride (238 mg/L) and
0.01 mole ammonium acetate (771 mg/L)/L solvent mixture.
(e) Internal standard solution.
diphenylamine (DPA) into 200 mL volumetric flask. Dissolve and
dilute to volume with methanol.
(f) Nickel(II) chloride solution.—Prepare solution of 100 mg
nickel(II) chloride hexahydrate/mL methanol.
(g) Clioquinol standard solution.—Accurately weigh 30 mg
clioquinol standard into 100 mL volumetric flask. Dissolve and
dilute to volume with tetrahydrofuran.
D. Preparation of Test and Standard Solutions
Accurately weigh test portion of ointment or cream equivalent to
30 mg clioquinol into beaker. Add ca 50 mL tetrahydrofuran and
warm beaker on steam bath to dissolve. Quantitatively transfer
solution to 100 mL volumetric flask and dilute to volume with
tetrahydrofuran.
Transfer 5 mL aliquot of clioquinol standard solution, C(g), and test
solution to separate 50 mL volumetric flasks. Add 1 mL methanolic
nickel chloride solution, C(f), to each flask and mix. Pipet 5 mL DPA
internal standard solution, C(e), into each flask, dilute solutions to
volume with methanol, and mix each solution. Filter test solution
through fluoropolymer or Nylon filter before injection.
E. System Suitability Test
Equilibrate column overnight with mobile phase at flow rate of
0.2 mL/min. Chromatograph minimum of three 10–20 mL injections
of final clioquinol standard solution at flow rate of 1.2–1.5 mL/min.
Relative standard deviation for ratio of peak responses should be
£2.0% and tailing factor should be £2.0 for either peak.
F. Liquid Chromatography
Make duplicate 10–20 mL injections of final test solutions.
Calculate percent clioquinol in test portion as follows:
(R / R ¢) ´ (C / G )
Clioquinol, % =
10
where R and R¢ are peak response ratios of clioquinol to internal
standard for assay and standard solutions, respectively; C = mg
standard weighed; and G = weight of test portion, g.
Reference: JAOAC 72, 562(1989).
CAS-130-26-7 (clioquinol)
ã 2005 AOAC INTERNATIONAL