Reserpine Injection
»Reserpine Injection is a sterile solution of Reserpine in Water for Injection,prepared with the aid of a suitable acid.It contains not less than 90.0percent and not more than 110.0percent of the labeled amount of C33H40N2O9.It contains suitable antioxidants.
Packaging and storage— Preserve in single-dose (or,if stabilizers are present,in multiple-dose),light-resistant containers,preferably of Type Iglass.
Identification— It responds to the Identificationtest under Reserpine Elixir (Oral Solution,Official June 1,2005).
Bacterial endotoxins á85ñ— It contains not more than 71.5USP Endotoxin Units per mg of reserpine.
pHá791ñ: between 3.0and 4.0.
Other alkaloids— [NOTE—Conduct this test promptly after preparation of the test and standard solutions.]Pipet 10mLeach of the citric acid solution of the Injection,and of Solution 1used in preparing the Standard preparation,respectively,obtained as directed in the Assay,into two separators.To the Injection solution add 100mLof saturated sodium bicarbonate solution,and to Solution 1add 10mLof water,10drops of saturated sodium bicarbonate solution,and 90mLof water,and extract both of the resulting solutions with 50mLof ether.Transfer the aqueous phase to another separator,extract with a second 50-mLportion of ether,and discard the aqueous layers.Wash the ether layers in succession with two 25-mLportions of water,and discard the washings.Extract the combined ether layers with three 15-mLportions of 2Nsulfuric acid,collect the extracts in a 50-mLvolumetric flask,add 2Nsulfuric acid to volume,and mix.The absorption spectrum of the solution from the Injection,in the range of 255to 350nm,measured in a 1-cm cell,2Nsulfuric acid being used as the blank,exhibits maxima and minima only at the same wavelengths as that of the solution from the Standard preparation,concomitantly measured.Calculate the quantity,in mg,of total alkaloids in each mLof the Injection taken by the formula:
10(I/SV),
in which Iis the absorbance of the solution from the Injection at the wavelength of maximum absorbance at about 268nm;Sis that of the solution from the Standard preparation;and Vis the volume,in mL,of Injection taken.The content of total alkaloids is not more than 114.0%of the labeled amount of C33H40N2O9,and does not differ by more than 10.0%from the amount of C33H40N2O9determined in the Assay.
Other requirements— It meets the requirements under Injections á1ñ.
Assay—
Standard preparation— Dissolve 25.0mg of USP Reserpine RS,accurately weighed,in 0.25mLof chloroform,mix with about 30mLof methanol previously warmed to 50,transfer the mixture to a 250-mLvolumetric flask with the aid of warm methanol,cool the solution to room temperature,dilute with methanol to volume,and mix (Solution 1).Protect the solution from light.Just prior to use in the Assay,pipet 10mLof Solution 1into a 50-mLvolumetric flask,add 36mLof chloroform,and dilute with methanol to volume (Standard preparation).
Assay preparation— Transfer to a 100-mLvolumetric flask an accurately measured volume of Injection,equivalent to about 10mg of reserpine,and dilute with citric acid solution (1in 50)to volume.
Procedure— Pipet 10mLof the Assay preparationinto a separator,or a suitably stoppered,50-mLcentrifuge tube,add 10mLof chloroform,and shake for 2minutes.Separate and withdraw the chloroform.Wash the citric acid solution with two 10-mLportions of chloroform,adding the washings to the main chloroform solution.To the combined chloroform solutions add 10mLof sodium bicarbonate solution (1in 100),shake for 2minutes,and separate.Withdraw the chloroform,filtering it through a pledget of cotton,into a 50-mLvolumetric flask containing 14.0mLof methanol.Extract the aqueous bicarbonate layer in the extraction vessel with two 2-mLportions of chloroform,passing each portion successively through the filter into the volumetric flask.Add chloroform to volume,and mix.
Pipet duplicate 5-mLaliquots of the chloroform-methanol solution and of the Standard preparationinto separate,10-mLvolumetric flasks.Add 2.0mLof a 1in 10solution of hydrochloric acid in methanol to each flask.To one flask of each pair of duplicates (representing the Standard preparationand the extracted Assay preparation)add 1.0mLof a 3in 1000solution of sodium nitrite in dilute methanol (1in 2).To the second flask of each pair (constituting the blanks)add 1mLof dilute methanol (1in 2).Mix,and allow to stand for 30minutes.Add 0.5mLof ammonium sulfamate solution (1in 20)to each flask,add methanol to volume,mix,and allow to stand for 10minutes.Determine the absorbance of each solution in a 1-cm cell at the wavelength of maximum absorbance at about 390nm,with a suitable spectrophotometer,using a mixture of methanol,chloroform,and water (5.4:3.6:1)as the blank.
Calculate the quantity,in mg,of C33H40N2O9in each mLof the Injection taken by the formula:
10(A-A0)U/V(A-A0)S,
in which Vis the volume,in mL,of Injection taken,and the parenthetic expressions are the differences in absorbances of the nitrite-treated and blank solutions,respectively,from the Assay preparation(U)and the Standard preparation(S).
Auxiliary Information— Staff Liaison:Andrzej Wilk,Ph.D.,Senior Scientific Associate
Expert Committee:(PA5)Pharmaceutical Analysis 5
USP28–NF23Page 1713
Phone Number:1-301-816-8305