Vous êtes sur la page 1sur 2


AOAC Official Method 993.22
p-Toluenesulfonamide Residues in Ice Cream
Combined Continuous Flow and
Liquid Chromatographic Method
First Action 1993

(Applicable to determination of 0.55.0 mg/kg p-toluenesulfonamide in ice cream.)

(Caution: See Appendix B, safety notes for handling of organic solvents.)
Method Performance:
Ice cream, 0.5 mg p-toluenesulfonamide/kg
Mean recovery = 78.8%
sr = 0.02; sR = 0.04; RSDr = 3.67%; RSDR = 7.79%
Ice cream, 1.0 mg p-toluenesulfonamide/kg
Mean recovery = 78.5%
sr = 0.02; sR = 0.12; RSDr = 2.08%; RSDR = 11.68%
Ice cream, 5.0 mg p-toluenesulfonamide/kg
Mean recovery = 76.1%
sr = 0.15; sR = 0.46; RSDr = 3.34%; RSDR = 10.30%
A. Principle

Samples are homogenized and dialyzed to remove interferences

(i.e., fats, proteins) in the continuous flow (CF) system. p-Toluenesulfonamide (p-TSA) is separated by on-line liquid chromatography
(LC) and detected fluorometrically at 230 nm (excitation) and 295
nm (emission).

Figure 993.22Flow diagram for combination of

continuous flow and on-line liquid chromatographic
system for determination of p-toluenesulfonamide in
ice cream

B. Apparatus

(a) Gravimetric diluter.Capable of mass dilutions (1 + 4 m/m).

(Spiral Systems Inc, Cincinnati, OH, is suitable source).
(b) Homogenizer.Rotor/stator-type; do not use blender (Ultra
Turrax, Janke & Kunkel, Staufen, Germany, and various models
available from Fisher Scientific, Pittsburgh, PA, are suitable).
(c) Continuous flow (CF) system.Equipped with: sampler
with external remote control of 6-port valve, dialyzer, tubing
pump, p-TSA cartridge with 700 mm flat-plate dialyzer, and
debubbler (Skalar USA, Inc., Buford, GA, or Skalar Analytical
BV, Breda, The Netherlands; Technicon Instruments Corp./Bran
& Luebbe Analyzing Technologies, Inc, Elmsford, NY, or
Maarssen, The Netherlands, are suitable sources). Connected to
LC system, see Figure 993.22.
Operating conditions: sample time, 280 s; wash time, 270 s; air
bubble time, 4 s. (Note: Flow time needed to transport sample from
sampler to injection loop shall not be longer than wash time. Total
of sample and wash time, 550 s, is necessary for LC separation.)
(d) Valve.Automatic 6-port valve with 500 L injection loop.
(e) LC system.Equipped with high-pressure pump, fluorescence detector capable of wavelength selections for excitation at 230
nm and emission at 295 nm, electronic integrator, and recorder.
Operating conditions: LC mobile phase flow rate, 0.5 mL/min.
(f) LC column.Reversed-phase C18, 5 m particle size, 3.0 mm
id 100 mm stainless steel (ss) to meet system suitability requirements in E(a). (Lichrospher or Lichrocart, E. Merck, Darmstadt,
Germany; Lichrosorb, Chrompack Int, BV, Middelburg, The Netherlands, are suitable sources).
(g) Guard column.Reversed-phase C18, 5 m particle size, 3.0
mm id 10 mm ss (Lichrosorb is suitable).

C. Reagents

(a) Methanol.
(b) Phosphate buffer.pH 7.00, containing 3.522 g potassium
dihydrogen phosphate (KH2PO4) and 7.265 g disodium hydrogen
phosphate dihydrate (Na2HPO42H2O) in 1 L H2O (commercial
buffer solutions are suitable).
(c) CF buffer solution.Dissolve 2 g sodium chloride in 1 L
phosphate buffer, (b).
(d) Sampler wash solution.Add 1 mL polyoxyethylene lauryl
ether (Brij 35, 30% [volume/volume] in H2O, Atlas Chemical Ind.,
Wilmington, DE, and E. Merck are suitable sources) to 1 L H2O.
(e) LC mobile phase.Methanolwater (1 + 3). Dilute 250 mL
methanol with 750 mL H2O. Filter mobile phase through 0.45 m
(f) p-TSA stock solution.1.000 mg p-TSA/L. Dissolve 100 mg
p-TSA in 10 mL 95% (volume/volume) ethanol and dilute to 100
mL with H2O. Solution is stable for at least 4 months at 4.
(g) p-TSA intermediate and standard solutions.(1) Intermediate solution.Pipet 1 mL p-TSA stock solution, (f), into 100 mL
volumetric flask and dilute to volume with H2O. (2) Standard
solutions.0.1, 0.2, 0.3, 0.4, and 0.5 mg p-TSA/L. Pipet 1, 2, 3, 4,
and 5 mL of intermediate solution into separate 100 mL volumetric
flasks and dilute to volume with H2O. Standard solutions are stable
for at least 4 weeks at 4.
(h) Vanillin stock solution.1 mg/mL. Dissolve 100 mg vanilla
(4-hydroxy-3-methoxy-benzaldehyde) in 100 mL H2O. Solution is
stable for 4 months at 4.


(i) Vanillin standard solution.20 mg/L. Pipet 1 mL vanilla

stock solution, (h), into 50 mL volumetric flask and dilute to volume
with H2O. Prepare on day of use.
D. Preparation of Test Solutions

Dilute melted ice cream samples (ca 510 g) 1:4 (by weight) with
H2O, using balance or gravimetric diluter, B(a). Homogenize mixture ca 1 min. Remove and discard upper, fat and/or foam, layer by
E. Procedure

(a) Startup.Place all lines in their respective solutions. Let CF

system equilibrate for 30 min. Equilibrate LC system until baseline
is stable. To set fluorescence detector sensitivity, pump 0.1 mg
p-TSA/L standard solution, C(g)(2), through instrument and adjust
recorder. Signal-to-noise ratio of 10:1 is recommended.
After system equilibration, determine resolution, R, between 0.1
mg p-TSA standard solution and vanillin standard solution, C(i). R,
as calculated below, should be 5.0.
R = [2(t2 t1)]/(W1 + W2)
where t1 and t2 are elution times of p-TSA and vanillin, respectively,
and W1 and W2 are peak widths of vanillin and p-TSA, respectively.
Proceed when above requirements are fullfilled.
(b) Determination.Fill sample cups of sampler in following
order: 5 cups with 0.10.5 mg p-TSA/L standard solutions, C(g)(2);
5 cups with test solutions, D; 1 cup with 0.3 mg p-TSA/L standard
solution; 5 cups with test solutions; etc. Run H2O sample at end of
each series.
Start sampler. After last cup has been sampled, let system continue
until steady baseline is obtained.
(c) Shutdown.After each series of analyses, pump methanol
water solution (ca 80 + 20 volume/volume) through LC column until
baseline is stable.
F. Calculation

Plot peak area vs concentration of p-TSA standard solutions.

Determine p-TSA concentrations (Cx) of test solutions by interpolation. To calculate p-TSA concentration in samples, multiply Cx by 5
(dilution factor). Report results to nearest 0.01 mg p-TSA/1 kg
Reference: J. AOAC Int. 77, 672(1994).