1、USP 40 Official Monographs / Heparin 4475Antithrombin IU/mL. Dilute this solution with pH 8.4 Parallel-line assay: For each series, calculate the regres-buffer to obtain a solution having a concentration of sion of the absorbance or change in absorbance/min0.125 Antithrombin IU/mL. against log conce
2、ntrations of the Standard solutions andThrombin human solution: Reconstitute thrombin the Sample solutions, and calculate the potency of Anti-human (factor IIa) (see Reagents, Indicators, and Solu- coagulant Heparin Solution in USP Units/mL using sta-tionsReagent Specifications) in water to give 20
3、tistical methods for parallel-line assays.Thrombin IU/mL, and dilute with pH 8.4 buffer to ob- Slope ratio assay: For each series, calculate the regres-tain a solution having a concentration of 5 Thrombin sion of the log absorbance or the log change in absorb-IU/mL. NOTEThe thrombin should have a sp
4、ecific ac- ance/min against concentrations of the Standard solu-tivity of NLT 750 IU/mg. tions and the Sample solutions, and calculate theChromogenic substrate solution: Prepare a solution of potency of Anticoagulant Heparin Solution in USPa suitable chromogenic thrombin substrate for Units/mL using
5、 statistical methods for slope ratio assays.amidolytic test (see Reagents, Indicators, and Solutions Acceptance criteria: 90.0%110.0% of the potencyReagent Specifications) in water to obtain a concentra- stated on the label in terms of USP Heparin Units.tion of 1.25 mM. SODIUM CHLORIDEStopping solut
6、ion: 20% (v/v) solution of acetic acid Sample solution: Solution and potassium chromate TSStandard solutions: Reconstitute the entire contents of (5:1)an ampule of USP Heparin Sodium for Assays RS with Analysis: Titrate with 0.1 N silver nitrate VS. Each mL ofwater, and dilute with pH 8.4 buffer to
7、obtain at least 0.1 N silver nitrate is equivalent to 5.844 mg of NaCl.four dilutions in the concentration range betweenSPECIFIC TESTS0.005 and 0.03 USP Heparin Unit/mL. PH 791: Between 5.0 and 7.5Sample solutions: Proceed as directed for Standard so- BACTERIAL ENDOTOXINS TEST 85: It contains NMT 2.
8、5lutions to obtain concentrations of Anticoagulant Hepa-USP Endotoxin Units/mL.rin Solution similar to those obtained for the Standard INJECTIONS AND IMPLANTED DRUG PRODUCTS 1: Meets thesolutions.requirementsAnalysisNOTEThe procedure can also be performed using al-ADDITIONAL REQUIREMENTSternative pl
9、atforms. PACKAGING AND STORAGE: Preserve in single-dose contain-For each dilution of the Standard solutions and the Sam-ers, of colorless, transparent Type I or Type II glass, or ofple solutions, at least duplicate samples should bea suitable plastic material (see Transfusion and Infusion As-tested.
10、 Label a suitable number of tubes, dependingsemblies and Similar Medical Devices 161).on the number of replicates to be tested. For example, LABELING: Label it in terms of USP Heparin Units, and toif five blanks are to be used: B1, B2, B3, B4, and B5indicate the number of mL of Solution required per
11、for the blanks; T1, T2, T3, and T4 each at least in100 mL of whole blood.duplicate for the dilutions of the Sample solutions; and USP REFERENCE STANDARDS 11S1, S2, S3, and S4 each at least in duplicate for theUSP Endotoxin RSdilutions of the Standard solutions. Distribute theUSP Heparin Sodium for A
12、ssays RSblanks over the series in such a way that they accu-rately represent the behavior of the reagents duringthe experiments. NOTETreat the tubes in the orderB1, S1, S2, S3, S4, B2, T1, T2, T3, T4, B3, T1, T2, T3,.T4, B4, S1, S2, S3, S4, B5. Note that after each addi-Heparin Sodiumtion of a reage
13、nt, the incubation mixture should bemixed without allowing bubbles to form. Add twiceDEFINITIONthe volume (100200 L) of Antithrombin solution toHeparin Sodium is the sodium salt of sulfated glycosamino-each tube containing one volume (50100 L) of ei-glycans present as a mixture of heterogeneous mole
14、culesther the pH 8.4 buffer or an appropriate dilution of thevarying in molecular weights that retains a combination ofStandard solutions or the Sample solutions. Mix, but doactivities against different factors of the blood clottingnot allow bubbles to form. Incubate at 37 for at leastcascade. It is
15、 present in mammalian tissues and is usually1 min. Add to each tube 2550 L of Thrombin humanobtained from the intestinal mucosa or other suitable tis-solution, and incubate for at least 1 min. Addsues of domestic mammals used for food by humans. The50100 L of Chromogenic substrate solution. Pleaseso
16、urcing of heparin material must be specified in compli-note that all reagents, Standard solutions, and Sampleance with applicable regulatory requirements. The manu-solutions should be prewarmed to 37 just before use.facturing process should be validated to demonstrateTwo different types of measureme
17、nts can be recorded:clearance and inactivation of relevant infectious and ad-1. Endpoint measurement: Stop the reaction after atventitious agents (e.g., viruses, TSE agents). See Viralleast 1 min with 50100 L of Stopping solution.Safety Evaluation of Biotechnology Products Derived from CellMeasure t
18、he absorbance of each solution at 405 nmLines of Human or Animal Origin 1050 for general guid-using a suitable spectrophotometer (see Ultraviolet-ance on viral safety evaluation. The heparin manufactur-Visible Spectroscopy 857). The RSD over the blanking process should also be validated to demonstra
19、te clear-readings is less than 10%.ance of lipids. It is composed of polymers of alternating2. Kinetic measurement: Follow the change in absorb-derivatives of -D-glucosamido (N-sulfated, O-sulfated, orance for each solution over 1 min at 405 nm using aN-acetylated) and O-sulfated uronic acid (-L-idu
20、ronic acidsuitable spectrophotometer (see Ultraviolet-Visibleor -D-glucuronic acid). The component activities of theSpectroscopy 857). Calculate the change in absorb-mixture are in ratios corresponding to those shown byance/min (OD/min). The blanks for kinetic meas-USP Heparin Sodium for Assays RS.
21、Some of these compo-urement are also expressed as OD/min and shouldnents have the property of prolonging the clotting timegive the highest values because they are carried outof blood. This occurs mainly through the formation of ain the absence of heparin. The RSD over the blankcomplex of each compon
22、ent with the plasma proteinsreadings is less than 10%.antithrombin and heparin cofactor II to potentiate the in-Calculations: The statistical models for Slope ratio assayactivation of thrombin (factor IIa). Other coagulation pro-or Parallel-line assay can be used, depending on whichteases in the clo
23、tting sequence, such as activated factor Xmodel best describes the correlation between concen-(factor Xa), are also inhibited. The ratio of anti-factor Xatration and response.activity to anti-factor IIa potency is between 0.9 and 1.1.USP MonographsOfficial from nullCopyright (c) 2017 The United Stat
24、es Pharmacopeial Convention. All rights reserved.Accessed from 10.6.1.1 by brunswick20 on Tue Feb 21 02:42:58 EST 20174476 Heparin / Official Monographs USP 40The potency of Heparin Sodium, calculated on the dried Analysisbasis, is NLT 180 USP Heparin Units in each mg. Sample: Sample solutionAccepta
25、nce criteria: No unidentified signals greaterIDENTIFICATIONthan 4% of the mean of the height of signals 1 and 2 A. 1.H NMR SPECTRUMare present in the following ranges: 0.102.00,(See Nuclear Magnetic Resonance Spectroscopy 761.)2.103.20, and 5.708.00 ppm. No signals greater thanStandard solution: NLT
26、 20 mg/mL of USP Heparin So-200% of the mean of the height of signals 1 and 2 aredium Identification RS in deuterium oxide with 0.002%present in the 3.754.55 ppm for porcine heparin.(w/v) deuterated trimethylsilylpropionic (TSP) acid so- B. CHROMATOGRAPHIC IDENTITYdium saltSolution A: Dissolve 0.8 g
27、 of monobasic sodium phos-System suitability solution: Prepare 0.3% (w/w) USPphate dihydrate in 2 L of water, and adjust with phos-Oversulfated Chondroitin Sulfate RS in the Standardphoric acid to a pH of 3.0. Pass the solution through asolution.membrane filter with a 0.45-m pore size, and degasSamp
28、le solution: NLT 20 mg/mL of Heparin Sodium inbefore use.deuterium oxide with 0.002% (w/v) deuterated TSP.Solution B: Dissolve 0.8 g of monobasic sodium phos-NOTEEDTA may be added to the Sample solution tophate dihydrate and 280 g of sodium perchlorateNMT 12 g/mL. In the event that EDTA is added to
29、themonohydrate in 2 L of water, and adjust with phos-Sample solution, spectra should be recorded and com-phoric acid to a pH of 3.0. Pass the solution through apared both with and without addition of EDTA.membrane filter with a 0.45-m pore size, and degasInstrumental conditionsbefore use.(See Nuclea
30、r Magnetic Resonance Spectroscopy 761.)Mobile phase: See Table 1.Mode: NMR, pulsed (Fourier transform)Frequency: NLT 500 MHz (for 1.H)Table 1Temperature: 2030System suitability Time Solution A Solution BSamples: Standard solution and System suitability (min) (%) (%)solution0 80 20Using a pulsed (Fou
31、rier transform) NMR spectrometer30 10 90operating at NLT 500 MHz for 1.H, acquire a free in-31 80 20duction decay (FID) using NLT 16 scans using a 9045 80 20pulse, an acquisition time of NLT 2 s, and at least a10-s delay. Record the 1.H NMR spectra of the StandardStandard solution: NLT 20 mg/mL of U
32、SP Heparin So-solution and the System suitability solution at a stabledium Identification RS in watertemperature between 2030. Collect the 1.H NMRSystem suitability solution: Prepare 0.1% (w/w) USPspectrum with a spectral window of at least 10 to Oversulfated Chondroitin Sulfate RS and 0.5% (w/w)2 p
33、pm and without spinning. The number of tran-USP Dermatan Sulfate RS in the Standard solution.sients should be adjusted until the signal-to-noise ratioSample solution: NLT 20 mg/mL of Heparin Sodium inof the N-acetyl heparin signal in the Standard solutionwateris at least 1000/1 in the region near 2
34、ppm. The Stan-Chromatographic systemdard solution shall be run at least daily when Sample(See Chromatography 621, System Suitability.)solutions are being run. For all samples, the TSP methylMode: LCsignal should be set to 0.00 ppm. The chemical shiftDetector: UV 202 nmfor the N-acetyl resonance of h
35、eparin and oversulfatedColumn: 2-mm 25-cm; packing L812. chondroitin sulfate in the System suitability solutionGuard column: 2-mm 5-cm; packing L61should be observed at 2.05 0.02 and 2.16 Column temperature: Maintain columns at 400.03 ppm, respectively. Record the 1.H NMR spectrumFlow rate: 0.22 mL/
36、minof the Sample solution at a stable temperature betweenInjection volume: 20 L2030. Draw a baseline from 8.00 ppm to 0.10 ppm.System suitabilityThe ppm values for H1 of GlcNAc/GlcNS, 6S (signalSample: System suitability solution1), H1 of IdoA2S (signal 2), the H2 of GlcNS (signalNOTEThe retention t
37、imes for dermatan sulfate, hepa-3), and the methyl of GlcNAc (signal 4) of heparin arerin, and oversulfated chondroitin sulfate are about 17,present at 5.42, 5.21, 3.28 (doublet centered at22, and 30 min, respectively.3.28 ppm), and 2.05 ppm, respectively.1. The chemicalSuitability requirementsshift
38、s of these signals do not differ by more thanResolution: NLT 1.0 between the dermatan sulfate0.03 ppm. Measure the signal heights above theand heparin peaks, and NLT 1.5 between the heparinbaseline of signal 1 and signal 2, and calculate theand oversulfated chondroitin sulfate peaksmean of these sig
39、nal heights. Other signals of variableRelative standard deviation: NMT 2% for the hepa-heights and ppm values, attributable to heparin andrin peak area determined from three replicateHOD, may be seen between signal 2 and 4.55 ppm.injectionsResidual solvent signals may be observed in theAnalysis0.103
40、.75 range. Heparin Sodium must meet the re-Samples: Standard solution and Sample solutionquirements stated in Residual Solvents 467.Record the chromatograms, and measure the retentionSuitability requirementstimes for the major peaks.Number of transients: Adjust until the signal-to-Acceptance criteri
41、a: The retention time of the majornoise ratio of the N-acetyl heparin signal in the Stan-peak of the Sample solution corresponds to that of thedard solution is at least 1000/1 in the region nearStandard solution.2 ppm.2. L81A hydroxide-selective, strong anion-exchange resin consisting of aChemical s
42、hift: The TSP methyl signal should be sethighly cross-linked core of 9 m porous particles having a pore size of 2000to 0.00 ppm for all samples.A units and consisting of ethylvinylbenzene cross-linked with 55%Chemical shifts (for the N-acetyl resonance of heparindivinylbenzene with a latex coating c
43、omposed of 70 nm diameter micro-and oversulfated chondroitin sulfate in the System beads (6% crosslinked) bonded with alkanol quaternary ammonium ions (Asuitable column is Dionex IonPac AS11-HC available from www.thermofisher.suitability solution): Should be observed at 2.05 com).0.02 and 2.16 0.03
44、ppm, respectively1. GlcNAc, N-acetylated glucosamine; GlcNS, N-sulfated glucosamine; S, sul-fate; IdoA, iduronic acid; GlcN, glucosamine; GalN, galactosamine.USP MonographsOfficial from nullCopyright (c) 2017 The United States Pharmacopeial Convention. All rights reserved.Accessed from 10.6.1.1 by b
45、runswick20 on Tue Feb 21 02:42:58 EST 2017USP 40 Official Monographs / Heparin 4477 C. ANTI-FACTOR Xa TO ANTI-FACTOR IIa RATIO tion), and Calibration solution (single injection), and re-Anti-Factor Xa and Anti-Factor IIa Assays for Unfrac- cord the chromatograms for a length of time to en-tionated a
46、nd Low Molecular Weight Heparins 208, sure complete elution, including salt and solvent peaksAnti-Factor Xa and Anti-Factor IIa Assays for Unfraction- (about 50 min). NOTEThe calibrant, standard, orated Heparin sample of heparin will give a broad heparin peak be-Acceptance criteria: 0.91.1 tween abo
47、ut 20 and 40 min, followed by a later elut-ing narrow salt peak, as illustrated in the USP Certifi-cate for USP Heparin Sodium Molecular WeightChange to read:Calibrant RS.Calculations: Calculate the total area under the hepa- D. MOLECULAR WEIGHT DETERMINATIONSrin peak in the Calibration solution chr
48、omatogram, and1 M ammonium acetate solution: Accurately weighthe cumulative area at each point under the peak as a77.1 g of ammonium acetate, and dissolve in 1 L ofpercent of the total. Do not include the salt peak. Us-water.ing the Broad Standard Table provided in the USP Cer-1% sodium azide soluti
49、on: Dissolve 1 g of sodiumtificate for USP Heparin Sodium Molecular Weightazide in 100 mL of water.Calibrant RS, identify those points in the chromato-Mobile phase: Transfer 100 mL of 1 M ammonium ace-gram for which the percent cumulative area is closesttate solution to a 1-L volumetric flask, add 20 mL of 1%to the percent fractions listed in the Table, and assignsodium azide solution, and dilute with water to volume.the molecular weight (MW) in the Table to the corre-Filter using a
