» Flumazenil Injection is a sterile solution of Flumazenil. It contains not less than 90.0 percent and not more than 110.0 percent of the labeled amount of flumazenil (C15H14FN3O3).
Packaging and storage Preserve in tight, single-dose containers, preferably of Type I glass, and store at controlled room temperature.
USP Reference standards 11
USP Flumazenil Related Compound A RS
A: Thin-Layer Chromatographic Identification Test 201
Adsorbent: 0.25-mm layer of chromatographic silica gel mixture containing a fluorescent indicator.
Test solution Dilute, if necessary, a volume of injection with water to obtain a solution containing 0.1 mg of Flumazenil per mL.
Standard solution Prepare a solution in methanol, and dilute, step wise if necessary, with water to obtain a solution containing about 0.1 mg of USP Flumazenil RS per mL in a mixture of water and methanol (9:1).
Developing solvent system: a mixture of ethyl acetate and methanol (9:1).
B: The retention time of the major peak in the chromatogram of the Assay preparation corresponds to that in the chromatogram of the Standard preparation, as obtained in the Assay.
Bacterial endotoxins 85: not more than 100 USP Endotoxin Units per mg of flumazenil.
pH 791: between 3.4 and 4.6.
Particulate matter 788: meets the requirements for small-volume injections.
Diluent, Mobile phase, System suitability solution, and Chromatographic system Proceed as directed in the Assay.
Standard solution Use the Standard preparation as directed in the Assay.
Test solution Use the Assay preparation.
Procedure Separately inject equal volumes (about 20 µL) of the Standard solution and the Test solution into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the percentage of each impurity in the portion of Injection taken by the formula:
100(V/D)(F)(C/L)(ri / rS)in which V is the volume, in mL, of the Test Solution; C is the concentration, in mg per mL, of USP Flumazenil RS in the Standard solution; L is the dose, in mg, of Flumazenil per mL obtained in the Assay; D is the volume, in mL, of Injection taken to prepare the Test solution; F is the relative response factor as described in the table below; ri is the peak response for each impurity in the Test solution; and rS is the peak response of flumazenil obtained from the Standard solution: meets the requirements given in the table below.
Other requirements It meets the requirements under Injections 1.
Diluent Prepare a mixture of water, tetrahydrofuran, and methanol (75:20:5).
0.02 M Phosphate buffer Add 0.02 M phosphoric acid to 0.02 M monobasic potassium phosphate to obtain a solution having a pH of 2.7 ± 0.05.
Mobile phase Prepare a filtered and degassed mixture of 0.02 M Phosphate buffer, tetrahydrofuran, and methanol (75:20:5). Make adjustments if necessary (see System Suitability under Chromatography 621).
System suitability solution Dissolve appropriate quantities of USP Flumazenil RS, USP Flumazenil Related Compound A RS, and USP Flumazenil Related Compound B RS in Diluent, and dilute quantitatively, and stepwise if necessary, with Diluent to obtain a solution having known concentrations of about 0.01 mg of each per mL.
Standard preparation Dissolve an accurately weighed quantity of USP Flumazenil RS in Mobile phase, and dilute quantitatively, and stepwise if necessary, with Mobile phase to obtain a solution having a known concentration of about 0.1 mg per mL.
Assay preparation Dilute a volume of Injection, if necessary, with Diluent to obtain a solution having a known concentration of about 0.1 mg per mL of flumazenil in a known volume, V.
Chromatographic system (see Chromatography 621) The liquid chromatograph is equipped with a 230-nm detector and a 4.6-mm × 25-cm column that contains 5-µm packing L10. The flow rate is about 1.0 mL per minute. Chromatograph the System suitability solution, and record the peak areas as directed for Procedure: the relative retention times are about 0.71 for flumazenil related compound A, about 0.85 for flumazenil related compound B, and 1.0 for flumazenil; the resolution, R, between flumazenil related compound B and flumazenil is not less than 1.8; and the tailing factor for flumazenil related compound A is not more than 2.0. Chromatograph the Standard preparation, and record the peak areas as directed for Procedure: the relative standard deviation for replicate injections is not more than 2.0%.
Procedure Separately inject equal volumes (about 20 µL) of the Standard preparation and the Assay preparation into the chromatograph, record the chromatograms, and measure the peak responses. Calculate the quantity, in mg, of flumazenil (C15H14FN3O3) in the volume of Injection taken by the formula:
VC(rU / rS)in which C is the concentration, in mg per mL, of USP Flumazenil RS in the Standard preparation; V is the volume, in mL, of the Assay preparation; and rU and rS are the peak areas obtained from the Assay preparation and the Standard preparation, respectively.
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USP35NF30 Page 3219Pharmacopeial Forum: Volume No. 33(2) Page 234