AOAC Official Method 974.42 Neostigmine in Drugs
Chromatographic Method
First Action 1974
Final Action 1976
A. Apparatus
(a) Spectrophotometer.—Suitable for measurement in range 265–400 nm.
(b) Chromatographic tube and tamping rod.—See 967.31A (see 19.1.02).
B. Reagents
(a) Phosphate buffer.—pH 5.8. Mix 1 volume 1M K2HPO4 (17.4 g/100 mL) with 4 volumes 1M KH2PO4 (13.6 g/100 mL). Adjust pH, using pH meter, to 5.80 ± 0.05 with either component.
(b) Washed chloroform.—Shake equal volumes CHCl3 and H2O in separator. Let layers separate 5 min and discard upper layer.
(c) Washed ether.—Shake equal volumes ether and H2O in separator.Let layers separate 5 min and discard lower layer.
(d) Bis(2-ethylhexyl) hydrogen phosphate (DEHP) solution.—2.5%. Mix 2.5mLDEHPwith 97.5mLH2O-washed CHCl3.
(e) Diatomaceous earth.—See 960.53B (see 18.1.01).
(f) Neostigmine standard solution.—Dry USP neostigmine bromide Reference Standard 3 h in 105°C oven. Accurately weigh ca 5 mg dry standard, using microbalance, and transfer to 150 mL beaker. Add 2.0 mL pH 5.8 phosphate buffer, mix by swirling gently, and proceed as in D.
