Colicine in Drugs

20.6.
01 replicate 20 µL volumes of same solution and compare peak

AOAC Official Method 985.49 response values (peak height or area). Relative standard deviation
Colchicine in Drugs should be <2%.
Liquid Chromatographic Method (b) Retention.—Retention time for colchicine should be between
First Action 1985 5.5 and 9.5 min (depending on brand and age of column).
Final Action 1987 (c) Column efficiency.—Number of theoreti cal plates (N)
Caution: Colchicine is extremely poisonous. determined from colchicine response is ≥4500 when calculated by
following expression:
A. Principle
Colchicine is diluted or extracted with methanol–H2O (1 + 1) and N = 5.54(tR/W0.5)2
determined directly by LC with UV detection at 254 nm.
B. Apparatus
where tR and W0.5 are retention time and peak width at 50% height
measured in mm, respectively. Use chart speed of 2–4 cm/min to
(a) Liq uid chromatograph.—LC pump, a UV de tec tor, an
facilitate accurate peak width measurement. (Note: If small
injection valve with 20.0 µL sam ple loop, and re port ing impurity peak elutes just before colchicine response, it should be
in te gra tor or equiv a lent sys tem with strip chart re corder. completely resolved from major peak.)
Op er ating con di tions: mo bile phase flow rate 1.0 mL/min,
de tec tor set at 254 nm, sen si tiv ity 0.005 AUFS, tem per a ture E. Preparation of Test Solution
am bi ent, chart speed 0.5 cm/min. Carry out all extractions and dilutions with methanol–H2O (1 + 1)
(b) LC column.—Reversed-phase, Octyl (C8), 5 µm particle in low-actinic glassware.
size, 4.6 mm id × 25 cm stainless steel or equivalent column packed (a) Tablets.—Weigh and finely powder, to pass No. 60 sieve, ≥20
with 5–6 µm octylsilane bonded spherical particles that meets tab lets. Trans fer ac cu rately weighed por tion of com pos ite
chromatographic system suitability requirements. Wash column equivalent to 0.6 mg colchicine into 100 mL volumetric flask, add ca
after use with methanol–H2O (1 + 1), and then with methanol. 50 mL methanol–H2O (1 + 1) and mechanically shake for total of
(c) Filters.—Cellulose triacetate membrane filter, pore size 15 min, rinsing down walls of flask at ca 8 min. Dilute to volume and
0.45 µm. filter portion through 0.45 µm membrane filter.
(d) Glassware.—Use low-actinic volumetric flasks for all test (b) Injections.—Transfer accurately measured volume (V) of
sample and standard solutions. liquid equivalent to 1 mg colchicine into 50 mL volumetric flask and
dilute to volume. Transfer 30.0 mL of this solution into 100 mL
C. Reagents
volumetric flask and dilute to volume.
(a) Methanol.—UV or LC grade or equivalent. (c) Bulk drug.—Accurately weigh ca 60 mg well-mixed test
(b) Extraction and dilution solvent.—Methanol–H2O (1 + 1). portion into 500 mL volumetric flask, dissolve, and dilute to volume.
Mix equal volumes of methanol and H2O, cool to room temperature. Transfer 5.0 mL of this solution to 100 mL volumetric flask and
(c) Potassium phosphate (monobasic) buffer.—0.5M. Dissolve dilute to volume.
68.0 g KH2PO4 (LC grade) in H2O and dilute to 1 L.
(d) Phosphoric acid.—0.5M. Dilute 3.4 mL H3PO4 (AR grade, F. Determination
85%) to 100 mL with H2O. Immediately after extraction and dilution of test portion, inject
(e) LC mobile phase.—Methanol–0.05M KH2PO4 (55 + 45), duplicate 20 µL volumes of colchicine standard solution and test
pH 5.5. Dilute 45 mL 0.5M KH2PO4 buffer, (c), to 450 mL with solution in alternating sequence. Calculate results by using average
H2O, then further dilute to ca 1 L (e.g., 980 mL) with methanol. Cool peak response values as follows:
to room temperature and adjust volume to 1 L with methanol. Adjust
pH to 5.5 (±0.05) with 4–10 drops 0.5M H3PO4, (d). Filter through Tablets: mg colchicine/tablet = (R/R′) × C × 100 × (T/W)
0.45 µm membrane filter.
(f) Colchicine standard solution.—0.006 mg/mL. Prepare all Injections: mg colchicine/mL = (R/R′) × C × (166.7/V)
dilutions with methanol–H2O (1 + 1). Transfer 30 mg accurately
weighed (3 mg if using microbalance) USP Colchicine Reference Bulk drug: percent colchicine (as-is basis) =
Standard, previously dried 3 h at 105°C, to 50 mL volumetric flask; (R/R′) × C × 10 000 × (100/W)
dissolve and dilute to volume. Dilute this solution quantitatively to
0.006 mg/mL. Solution is stable ≥4 months when stored tightly where R and R′ = peak response values of test and standard solutions,
stoppered and in dark. respectively; C = concentration of colchicine standard solution,
mg/mL; T = average tablet weight, mg; W = test portion weight, mg;
D. Chromatographic System Suitability Test
V = volume injection taken for analysis, mL.
(a) Repeatability.—Let chromatographic system equilibrate
with mobile phase ≥45 min. Inject 20 µL colchicine standard Reference: JAOAC 68, 1051(1985).
solution and adjust sensitivity to provide 50–70% FSD. Inject 5 CAS-64-86-8 (colchicine)
 2005 AOAC INTERNATIONAL

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20.6.

01 replicate 20 µL volumes of same solution and compare peak

 2005 AOAC INTERNATIONAL

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