Edition: BP 2025 (Ph. Eur. 11.6 update)
Action and use
Cholinesterase inhibitor.
DEFINITION
Neostigmine Injection is a sterile solution of Neostigmine Metilsulfate in Water for Injections.
The injection complies with the requirements stated under Parenteral Preparations and with the following requirements.
Content of neostigmine metilsulfate, C13H22N2O6S
90.0 to 110.0% of the stated amount.
IDENTIFICATION
A. Dilute, if necessary, a volume of the injection containing 2.5 mg of Neostigmine Metilsulfate to 5 mL with water, shake with three 10-mL quantities of ether and discard the ether extracts. The light absorption of the aqueous solution, Appendix II B, in the range 230 to 350 nm exhibits two maxima, at 260 nm and 266 nm.
B. Carry out the method for thin-layer chromatography, Appendix III A, using the following solutions.
(1) Use the injection diluted, if necessary, with water to produce a solution containing 0.05% w/v of Neostigmine Metilsulfate.
(2) 0.05% w/v of neostigmine metilsulfate EPCRS.
(3) Equal volumes of solutions (1) and (2).
CHROMATOGRAPHIC CONDITIONS
(a) Use as the coating silica gel G.
(b) Use the mobile phase as described below.
(c) Apply 10 µL of each solution.
(d) Develop the plate to 15 cm.
(e) After removal of the plate, dry in air and spray with dilute potassium iodobismuthate solution.
MOBILE PHASE
5 volumes of water, 10 volumes of formic acid, 35 volumes of methanol and 50 volumes of chloroform.
CONFIRMATION
The principal spot in the chromatogram obtained with solution (1) corresponds in position and colour to that in the chromatogram obtained with solution (2).
The principal spot in the chromatogram obtained with solution (3) appears as a single, compact spot.
C. To 1 mL add 0.5 mL of 5M sodium hydroxide and evaporate to dryness on a water bath. Heat rapidly on an oil bath to about 250° and maintain at this temperature for about 30 seconds. Cool, dissolve the residue in 1 mL of water, cool in ice and add 1 mL of diazobenzenesulfonic acid solution. An orange-red colour is produced.
TESTS
Acidity
pH, 4.5 to 6.5, Appendix V L.
(3-Hydroxy)trimethylanilinium methyl sulfate
Carry out the method for liquid chromatography, Appendix III D, using the following solutions.
(1) Use the injection diluted, if necessary, with water to contain 0.05% w/v of Neostigmine Metilsulfate.
(2) Dilute 1 volume of solution (1) to 100 volumes with water.
(3) Add 0.05 mL of 5M sodium hydroxide to 1 mL of solution (1) and allow to stand for 5 minutes. Add 0.1 mL of 5M hydrochloric acid and use immediately.
CHROMATOGRAPHIC CONDITIONS
(a) Use a stainless steel column (25 cm × 4.6 mm) packed with octylsilyl silica gel for chromatography (5 µm) (Lichrospher 60 RP-select B is suitable).
(b) Use isocratic elution and the mobile phase described below.
(c) Use a flow rate of 1.1 mL per minute.
(d) Use an ambient column temperature.
(e) Use a detection wavelength of 215 nm.
(f) Inject 20 µL of each solution.
MOBILE PHASE
A 0.0015M solution of sodium heptanesulfonate in a mixture of 15 volumes of acetonitrile and 85 volumes of 0.05M potassium dihydrogen orthophosphate adjusted to pH 3.0 with orthophosphoric acid.
SYSTEM SUITABILITY
In the chromatogram obtained with solution (3) the principal peak has a retention time of about 6.8 minutes (neostigmine metilsulfate) and there is a peak with a relative retention time of about 0.5 ((3-hydroxy)trimethylanilinium methylsulfate).
LIMITS
In the chromatogram obtained with solution (1):
Identify any peak due to (3-hydroxy)trimethylanilinium methylsulfate using the chromatogram obtained with solution (3). In the chromatogram obtained with solution (1):
the area of the peak due to (3-hydroxy)trimethylanilinium methylsulfate is not greater than the area of the principal peak in the chromatogram obtained with solution (2) (1%).
ASSAY
Dilute a quantity containing 25 mg of Neostigmine Metilsulfate to 50 mL with water. Measure the absorbance of the resulting solution at the maximum at 260 nm, Appendix II B.
Calculate the content of C13H22N2O6S taking 14.35 as the value of A(1%, 1 cm) at the maximum at 260 nm.
STORAGE
Neostigmine Injection should be protected from light.
