Indole in Shrimp

35.1.35 solvent delivery system (Waters Associates, Inc.
), or equivalent Valco
AOAC Official Method 981.07 7000 psi injection system (Valco Instruments Co., Inc., PO Box 55603,
Indole in Shrimp Houston, TX 77255, USA; www.vici.com), or equivalent.
Liquid Chromatographic Fluorometric Method (b) LC columns.—Analytical column.—30 cm × 4 mm id, packed
First Action 1981 with 10 µm ODS, reverse phase, or equivalent. Use column and
Final Action 1982 chromatographic conditions that will produce resolution between
Caution: See Appendix B, safety notes on blenders and peaks ≥1.2. Guard column.—7 cm × 4 mm id, packed with 30 µm
methanol. Co:Pell ODS (Whatman, Inc.), or equivalent. Use replaceable 2 µm frit
on outlet. At mobile phase flow rate of 1.0 mL/min, representative
A. Principle retention times are indole 8.6 min, 2-methylindole 12.1 min. Flow rate
Test sample is blended with methanol and 2-methylindole internal may be varied from 0.7 to 1.3 mL/min to obtain best separation.
standard. Filtered extract is analyzed by LC on ODS reversed-phase D. Calibration
column using methanol–H2O (60 + 40) and spectrofluorometric
Pre pare cal i bra tion so lu tions in di cated in Ta ble 981.07. Pipet
detection.
in di cated vol umes into sep a rate 100 mL vol u met ric flasks and
B. Reagents di lute to vol ume with meth a nol. Store in re frig er a tor.
(a) Purified water.—Distilled water treated by Milli-Q purification Adjust instrument to give ≥50% full scale recorder deflection for
system (Millipore Corp., 290 Concord Rd, Billerica, MA 01821, USA; 720 pg indole (20 µL of calibration solution No. 4). Inject 20 µL
www.millipore.com), or equivalent. Use throughout. aliquots of indole calibration solutions, measure peak heights, and
(b) Methanol.—Distilled from glass, or equivalent. calculate peak ratio R = height indole peak/height internal standard
(c) Indole standard solutions.—Solution A.—1.20 mg/mL. peak. Plot R against µg indole (W) in calibration solution.
Dissolve 120 mg indole in methanol in 100 mL volumetric flask and
E. Determination
dilute to volume with methanol. Solution B.—12 µg/mL. Pipet 1.0 mL
Solution A into 100 mL volumetric flask and dilute to volume with Weigh 10.0 g test portion into blender and add 50 mL methanol. Pipet
methanol. Solution C.—1.2 µg/mL. Pipet 5.0 mL Solution B into 1.00 mL internal standard solution C (6.25 µg) into blender by letting
50 mL volumetric flask and dilute to volume with methanol. pipet drain onto side wall above liquid level. Rinse blender wall with ca
5 mL methanol. Blend slowly 1 min to ensure even distribution of
(d) 2-Methylindole internal s t a n d a rd
internal standard. Avoid splattering onto blender walls. Place Al
so lu tion.—So lu tion A.—1.25 mg/mL. Dis solve 125 mg
foil-lined cap on blender and operate at high speed 3 min. Rinse walls of
2-methylindole in methanol in 100 mL volumetric flask and dilute to
blender bowl with ca 25 mL methanol and blend combined liquids (ca
volume with methanol. Solution B.—62.5 µg/mL. Pipet 5.0 mL
80 mL) 1 min at high speed. Filter extract and inject ca 20–30 µL filtrate
Solution A into 100 mL volumetric flask and dilute to volume with
into liquid chromatograph. Measure peak heights, and calculate R as
methanol. Solution C.—6.25 µg/mL. Pipet 10.0 mL Solution B into
above. Determine W (µg indole) from calibration curve.
100 mL volumetric flask and dilute to volume with methanol.
(e) Mobile phase.—Dilute 600 mL methanol to 1.0 L with H2O.
µg Indole/100 g test portion = 10W
C. Apparatus
Reference: JAOAC 64, 592(1981).
(a) Liquid chromatograph.—Model 203 spectrofluorometer
equipped with flow cell (Perkin-Elmer Corp.), or equivalent; excitation CAS-120-72-9 (indole)
wavelength 280 nm and emission wavelength 330 nm. Model 6000 Revised: March 1996
a
Table 981.07. Calibration solutions for LC fluorometric determination of indole in shrimp
Indole standard solution C (1.2 µg/mL) 2-Methylindole internal standard solution C

Calibration solution mL µg H2O mL (6.25 µg/mL), mL
1 0.5 0.6 10 1.0
2 1.0 1.2 10 1.0
3 2.0 2.4 10 1.0
4 3.0 3.6 10 1.0
5 4.0 4.8 10 1.0
6 6.0 7.2 10 1.0
a
Final volume 100 mL in methanol.
 2005 AOAC INTERNATIONAL

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35.1.35 solvent delivery system (Waters Associates, Inc.

Indole standard solution C (1.2 µg/mL) 2-Methylindole internal standard solution C

 2005 AOAC INTERNATIONAL

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