UsP 41 Assay prepaation—Use the Assy preparation prepared as clrected fn he 807 fr potasim and sodum. this solution ‘contains about 0.042 meq of chloride and 0.096 meq of sulfate per ml Chromatographic system (See Chromatography (621))—The liquid chromatograph i equipped with «Conductivity de tector, a -mm x S-cm guard column containing packn 23, and a 4emm x 30-em analytical column maintained at 3541" containing packing L23. The flow rate is about (0.9 mL per minute. Chromatograph the Standard prepara: tion as directed for Procedure: the relative retention times are about 0.25 for chloride, 0.4 for bromide, and 1.0 for suite, the resolution, between the chloride and bromide peaks isnot less than 1.5 and between the bromide and sulfate peaks i not les than 4.5. [NoTeMaintan column backe pressure at less than 1000 pounds per square inch. Backe Pressure may be reduced by changing the in-line fiers and Fri the curs, Column econ) maybe improved by backllushing the analytical column with 50 ml of the buffer solution used to prepare the Mob phase] Procedure—[NOTE—Use peak heights where peak re sponses ate indicated Separately inject equal volumes {@bout 10 ul) ofthe Standard preparation and the Assay preparation into the chromatograph, record the chromato- ‘rams, and measure the responses forthe major peaks. Cal ulate the mq of chloride per | of constituted Oral Solution taken by the formula: (500 / 58.44)(C/ 6)(R. 18) in which $8.44 is the molecular weight of sodium chloride, Cis the concentration, in ug per ml, of sodium chloride in the Standard preparation, and R. and R; are the peak re- sponse ratios of chloride to bromide obtained from the As Say preparation and the Standard preparation, respectively Calculate the mEq of sulfate per { of constituted Oral Solu- tion taken by the formula: (500 /71.02K(C/ 6)(R./ 8) in which 71.02 is one-half ofthe molecular weight of so- dum sulfate, Cs the concentration, in ug per ml, of any: kos scum sift inthe Standard prepara, ad 8 and R. are the peak response ratios ofslfate to bromide abianed rom the Asay preparation and the Standard rep rato, respectively ‘Assay for polyethylene glycol 3350— Salt solution.-Prepare a solution in water contain 0.38 mg of sodium ehlonde, 0.18 mg of potassium chloride, 10.40 mq of sodium bicarbonate, 1.37 mg of anhydrous s0- cum sulfate, and 0.88 mg of ammonium bromide per mL Mobile phase Dilute 40.0 mk of Salt solution with water to 1000 mi. Make adjustments it necessary (se System Sur ability under Chromatography (621). Standard preparation Transfer about 360 mg of USP Pol- yethylene Glyccl 3350 RS, accurately weighed, to a $00-ml Yolumetric ask, add 20.0 mL of Sait solution and about 250ml of water, issoWe by swing, dlute with water to volume, and mix. Ths Standard preparation contains about 0.72 mg of polyethylene glycol $330 per mL ‘Assay preparation—Use the Asay preparation, prepared as directed nthe kay for potastum and sodum ‘This sokton contains about 0.72 mg 8f polyethylene glycol 3380 per ml. ropatograpi stem (see Cronategaphy (621) — [NoTE—Use peak heights where peak respanses are ind ‘ated The gud cvomatogaph is equpped vith a reac tve index detector maintained a 34:20.5, a 7.8mm x 4'5.cm guard column containing packing (28, and 2 7.8- rm 30-cm analyical column containing packing (25 and maintained at ambient temperature. The flaw rate ls about ‘TL per minute. Chromatograph the Standard preparation as directed for Procedure: the relative standard deviation for replicate injections isnot more than 15%, [NOTE—Maintain Official Monographs / Polymyxin 3347 alum backpressure at les than 1000 pounds per square inch Bacipeoure may be reduce by Ceaning te eh the guard column oF by reslacng the guard cama, Base- Ine dif may be reduced by maintaining sc control af ambient Lerpeature, by itlating the Ines, the Mabie pote reservar, andthe columns, and by increasing the {ime of equation} Procedie—Separately inject equal volumes (about 20) af the Sondard preparation and the Asay preparation ita the chromatograph record the chromatograms, and meas ure the responses forthe mar pests. Cakeat te con- tent ing of polythene dyol 3350 per of constituted Oral Stn taken by the formu 500(C /6)(r fr) in which Cis the concentration, in mg per ml, of poyethyl fe geal 30 ne nd prepare, ad an sre the polyethylene glycol 3350 peak responses obtained to he ty Pepto ana the Stands preparation, re spectively Polymyxin B Sulfate e a 3 E4 3 Fs 2 ey cI z CithaN On NU(4-Aino-1-( 25,3815 4amino-1-ox0-1 (35,85, 95,125,185,185,215)-6 8, Btrs(2-aminoethy)-15-benzyl- 3((R-1-hydroxyethyi}'disobutyt2,5,8,11,14,17, 20-heptaoxo-1,4,7,10,13,16, 19-heptaazacyclotricosan- 21-yamino)butan:2-!Jamino}-3-nydroxy--oxobutan: 2oflamine-1-oxobuta-2-@methyloctanamide (4135-119), CsHhNO Polymyxin B2; Ne{(9-4-Amino-1-{(25 38)-1-{(9)-4-amino-1-0x0-1.((35,65, 95,125,155, 185,215)-68, 18-rs(2-amnoethy)-15-bendyk- Hel yon Soph 8018 17, 20-hepiaoxo-1,4,7,10,13,16,19-heptaazacyclotricosan- 21 amine) 2a) -hy cron -oxobtan 2,jllamino}-I-oxobutan-2-y]-6-methylheptanamide (34503-87-2) Caton, N{(4-Amino-1-(25,38-1-{(9)-4amino-1-0%01.4(35,85, 95,125,155,185,215)-6 8, Btrs(2-amnoethy)-15-benzyl- 3,{(-1-hydroxyethyl-12-sobutyl2,5,8,11,14,17, ao-hepaoxo-1/47,10.13,16 1dnepiaazacjclaicosan- 21-yamino)butan:2-!Jamino}-3-nydroxy-I-oxobutan- 2aflamina-t-oxobutan2yoctanamide (7140-38) 1203.48 1189.45 1189.453348 Polymyxin / Official Monographs CsHaN.0) 1203.48 Polymyxin B1 Ne{()-4-Amino-1-[ 25,38) {(9}-4-amine-1-o10-1-(5,65, 95,125,155, 185,215)-68,18-ris(2-aminoethy)-15-benzy- 3.((R)-l-hydeoxyethyi)e134(9-seccbutyl}2,58,11,14,17, 20-heptaoxo-1,4,7,10,13,16,19-heptaazacycletncosan 21-y}amino)butan:25/]amino)-3-hydrony -oxobutan. 2 jlamino}-1-oxobutan-2-yl-6-methyloctanamide Polymyxin 8, sulfate; Pobymyxin 8 sulfate [1405-20-5). Pobymyxin B (1404-26.8} DEFINITION Polymyxin 8 Sulfate i the suifate salt of a kind of pobymyan a aubstnce produced bythe goth of Bac lus polymyxa (Prazmowski) Migula (Fam. Bacilaceae), or a mixture of two or mare such salls. IU has a potency of NUT000 FajyanBumising, alee a he ed IDENTIFICATION A. The retention times of the major peaks of the Sample Solution correspond to those of the Standard solution, as _pbtaned inthe test for Cmpasian of Poms Solution A: 10 mg/ml. of cupric sulfate Sample solution: Dissolve 2 mg in 5 mL of water ‘Analysis: To the Sample solution add 5 ml. of 2.5 N so dium hydroxide, and mix. Add § drops of Solution A, shaking after the addition of each drop. Acceptance criteria: A reddlish-violet color is produced. ‘+ C.IbenmFicaTiON TesTs—GENERAL (191), Sulfate: A 50-mg/mL solution of Polymyxin 8 Sulfate meets the requirements ASSAY. ‘ PRoceDURE ‘Analysis: Proceed with Polymyxin 8 Sulfate as drected for Antibiotes—Microbial Assays (81. Acceptance criteria: NLT 6000 PolymyainBunising ‘on the dried basis IMPURITIES ‘+ RESIDUE ON IcNrTIOn (281) ‘Analysis: Proceed as directed in the chapter, moisten. ing the charred residue with 2 ml of nitric acid and 5 drops of sulfuric acid ‘Acceptance criteria: NMT 5.0% where used for pre scription compounding “+ ORCANC IMPURITIES Buffer, Mobile phase, Diluent, Standard solution, Sample solution, Chromatographic system, and Sys ‘tem suitability: ‘Proceed as directed in te lest for Composition of Polymyains. Analysis Samples: Standard solution (the Standard solution is use to determine the dregara mt) and Sane solution Calculate the percentage of each impurity in the por- tion of Polymyxin 8 Sulfate taken: Fs o & o : & 5 = a Fa 5 Result = (fr) x 100 "= peak response ofeach imp fom the ample solution Fr = sum of the responses ofall peaks from the ‘Sample solution Acceptance criteria: Disregard any peak less than 0.007 times the peak response of the polymyxin BI peak from the Standard solution ‘Any individual impurity: NWT 3.09% Total impurities: NMT 17.0% UsP 41 Pecan TESTS vast) Sample solution: 5 malt Acceptance enter "S007 + Loss on Drvine (731) Analy Dry 100"mg in a capilly-oppeed bottle Under vacaurn a 60"for 3h Acceptance eiteia® NMI 70% «Samy Tsrs 71), Mess the requirements where the abel tates that Pobmysin 8 Sulfate erie + Pynocen Test (151) ‘Sample solution" Nominal 20> 10: Pohmyxin B cnitdm{ in pyrogente sane TS Analyt Proced a ected nthe chap Gove of mig. Acceptance citer: Mets the requirements where n= Tended for injectable dorage forms or hee the abel Sates that Pojmyn 8 Sufate must be subjected fo further procering durng the preparaton of mjecable dosage forms + Cowraition oF Porras Baers qf dm late ahyow THe i jute to2.3 win diute phosphone a (10% in) pror to final dlaton Mobile phase: Acetonitrile and Buffer (20:80) Diluent: Acetonitrile and water (20:80) Standard solution: 0.3 mgim of USP Polymyxin 8 Su fate Rn ier Sample solution: 0.5 mg/ml of PoymyxinB Sule in Oiuen Chromatographic system (Gee Chromatgrphy (621), Stem Suitabity) Mode"tc Detector UY 215 nm Columns 4mm TS; 3 534m packing 1 [NOTA 4.6m x 25m; Sum pacing LT column eas ao found tobe suitable] Column temperature: 30" Flow rate: min Injection volume" 20 Run time: 14 Ses te retenton time of pmyain a System suitability ample: Stondard luton Suitably requirements Resolton: N.1 3.0 between poymyain 2 and polmyxin BS Relative standard deviation: NMT 20% forthe pohmysn peak Analyst Sample: standard soliton and Sample soon Greta the percentage of poly BI Inthe po: on of Poljmysn 8 Slate ken , Result = (rind x (CYC) x Px 100 using a test 1 =peskresponse of pobmyain 81 from the Sample run rr -=peakesponte of pobmyainB1 from the Standard outer = concentration of USP Polymyxin 8 Sut RS inthe Standart stuton (g/m) © = concentration of Pobmyaan 8 Sulate inthe fame iin oct orn aig P= potency of pobmyxin 8 in USP Pobmyain 8 Sulfate RS (mg/mg) Calculate the percentage of polymyxin 82 in the portion of Polymyxin B Sulfate taken: Result = (7rd x (CC) x Px 100 1) = peak response of polymyxin B2 from the Sample solution