18.9.02 methanol and mix.

Tumble on rotator 15 min at ca 30 rpm, and filter,

AOAC Official Method 983.28 if necessary. Pipet accurately measured aliquot (A) of this test
Amitriptyline in Drug Tablets solution, equivalent to 2 mg amitriptyline·HCl, into 50 mL
and Injectables volumetric flask, add 5.0 mL internal standard solution, dilute to
Liquid Chromatographic Method volume with methanol, and mix.
First Action 1983 (c) Injectables.—Ac cu rately pipet vol ume of in ject able,
Final Action 1986 equivalent to 10 mg amitriptyline·HCl, into 50 mL volumetric flask,
A. Principle add 25.0 mL internal standard solution, dilute to volume with
methanol, and mix. Dilute accurately measured volume of this
Amitriptyline content of tablets and injectables is determined by
solution with methanol to ca 0.04 mg/mL.
liquid chromatography, using trifluoperazine as internal standard
and UV detection at 239 nm. D. Determination

B. Apparatus and Reagents Equilibrate system with mobile phase at 1.33 mL/min, until
baseline is steady. Inject measured volume of standard solution into
(a) Liquid chromatograph.—With pump, variable wavelength
chromatograph by microsyringe or sampling valve. Adjust injection
detector, recorder, and 20 µL loop injector. Operating conditions:
volume and operating conditions so amitriptyline·HCl in standard
column temperature, ambient; solvent flow rate, 1.33 mL/min;
solution injection gives peak height ca 60% full scale and retention
detector wavelength, 239 nm; attenuation, 16 AUFS; recorder,
time ca 7 min. Under these conditions, 5 replicate injections of
1 mV; chart speed, 1 in./4 min.
standard solution should give relative standard deviation of ≤3%
(b) Chromatographic column.—Stainless steel, 300 × 3.9 mm id,
and resolution factor (R) between the 2 main peaks ≥1. Make
packed with 10 µm reverse phase cyanobonded column, or equivalent.
alternate injections of equal volumes of test sample and standard
(c) Methanol.—AR grade.
solutions. Measure peak heights for amitriptyline·HCl and internal
(d) Mobile phase.—Methanol–0.005M ammonium acetate
standard in test and standard solutions, and determine response
(38.5 mg/100 mL) (90 + 10).
ratios.
(e) Culture tubes.—95 × 25 mm with screw cap .
(f) Internal standard solution.—Accurately prepare ca 0.5 mg E. Calculations
USP Trifluoperazine·HCl Reference Standard/mL methanol.
(g) Standard solution.—0.04 mg/mL. Accurately weigh ca Tablets: mg/tablet = RR/RR′ × C × T/W
10 mg USP Amitriptyline·HCl Reference Standard and transfer to
Single tablet: mg/tablet = RR/RR′ × C × 5/A
250 mL volumetric flask. Dissolve in 1 mL methanol, add 25.0 mL
internal standard solution, dilute with methanol, and mix.
Injectables: mg/mL = RR/RR′ × C/V
C. Preparation of Test Solution
(a) Tablets.—Weigh and finely powder ≥20 tablets. Accurately where RR and RR′ = ratio of amitriptyline·HCl peak height to
weigh and trans fer amount of powder equiv alent to 10 mg internal standard peak height for test and standard solutions,
amitriptyline HCl into screw-cap culture tube and add 25.0 mL respectively; C = mg amitriptyline·HCl in 250 mL standard
internal standard solution. Tumble on rotator 15 min at ca 30 rpm, solutions; T = average tablet weight, g; W = weight test portion
and filter, if necessary. Dilute accurately measured volume of test taken, g; A = aliquot taken, mL; V = volume injectable taken, mL.
solution with methanol to ca 0.04 mg/mL. Reference: JAOAC 66, 1196(1983).
(b) Single tablet.—Place one tablet in 95 × 25 mm screw-cap
culture tube and crush to fine powder with glass rod. Add 25.0 mL CAS-549-18-8 (amitriptyline hydrochloride)

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