ASPIRIN (ACETYLSALICYLIC ACID) CERTIFIED REFERENCE MATERIAL HO O O CH 3 O LOT #: BCBD0724V Fluka No. 01478 NOMINAL WT: 1 g CORRESPONDES TO RTC LOT: P500003 CERTIFICATE VALIDITY DATE: 20. October 2011 VERSION: BCBD0724V-2 Note: Certificates may be updated due to Compendial Lot changes or the availability of new data. Check our website at: www.sigma-aldrich.com for the most current version. EXPIRATION: AUG / 2013 (Proper Storage and Handling Required) STORAGE: Store at Room Temperature, keep container tightly closed. Attachment of a 20 mm aluminum crimp seal recommended for unused portions. CHEMICAL FORMULA: C 9 H 8 O 4 MW: 180.16 PHYSICAL DESCRIPTION: White powder in amber vial CAS #: 50-78-2 HAZARDS: Read MSDS before using. Always handle as if potentially hazardous using prudent laboratory techniques. INSTRUCTIONS FOR USE: The internal pressure of the container may be slightly different from the atmospheric pressure at the user s location. Open slowly and carefully to avoid dispersion of the material. This material is intended for LABORATORY USE ONLY as a Certified Reference Material, and NOT for use in humans or animals. Certificate of Analysis Version BCBD0724V-2 Page 1 of 8
TRACEABILITY ASSAY Comparative assay demonstrates direct traceability to Pharmacopeial Standards Specification: 99.5 100.5% (USP) ASSAY vs. USP REFERENCE STANDARD (as is basis) ASSAY VALUE vs. USP LOT 99.5% H Labeled Content = none Assume 100.0% ASSAY vs. EP CRS (as is basis) ASSAY VALUE vs. EP BATCH 99.5% 4.0 Labeled Content = None Assigned Content = 99.5% * METHOD: HPLC (ref.: Aspirin Tablets, USP34) Column: Ascentis C18, 4.6 x 250mm, 5µm Mobile Phase: Water/Acetonitrile (85:15), sodium 1-heptanesulfonate, ph to 3.4 w/glacial acetic acid Flow: 2 ml/min Injection: 20 ul Detector Wavelength: 280 nm *The assigned content of the EP CRS was determined by assay against the USP Reference Standard Certificate of Analysis Version BCBD0724V-2 Page 2 of 8
Representative Chromatogram from Lot: P500003 Analysis ASSAY vs. BP CRS (as is basis) ASSAY VALUE vs. BP BATCH 99.9% 2065 Labeled Content = 100.0% METHOD: HPLC (ref.: Aspirin Tablets, USP31) Column: Wakosil C18 RS, 4.6 x 250 mm, 5um Mobile Phase: Water/Acetonitrile (85:15), sodium 1-heptanesulfonate, ph to 3.4 w/glacial acetic acid Flow: 2 ml/min Injection: 10 ul Detector Wavelength: 280 nm Representative Chromatogram from Lot: P500003 Analysis Certificate of Analysis Version BCBD0724V-2 Page 3 of 8
ASSAY BY TITRATION (ref.: Aspirin USP31) Method: Dissolve about 1.5 g in 50.0 ml 0.5 N NaOH and boil for 10 minutes. Titrate to endpoint with 0.5 N H 2 SO 4. Mean of nine determinations: 99.9% PURITY DETERMINATION BY MASS BALANCE CHROMATOGRAPHIC IMPURITY ANALYSIS METHOD: HPLC (ref.: Acetylsalicylic Acid, EP 6) Column: Wakosil C18 RS, 4.6 x 250 mm, 5um Mobil Phase: Phosphoric Acid/Acetonitrile/Water, (2:400:600) Flow: 1 ml/min Injection: 10µl Detector Wavelength: 237 nm Impurities Detected Salicylic Acid: 0.005 % Impurity 1: 0.003 % Impurity 2: 0.001 % Total Impurities: 0.009 % Representative Chromatogram from Lot: P500003 Analysis Certificate of Analysis Version BCBD0724V-2 Page 4 of 8
RESIDUAL SOLVENTS Method: GC-MS Headspace (ref.: Residual Solvents <467>, USP34) Column: DB-1301 Carrier gas: He Flow: 1.2mL/min Split Ratio: 1:5 Injection/Temperature: 1µl/250 C Temperature Program: 40 C for 20min, 10 C/min to 240 C, hold 20min Solvents Detected: None LOSS ON DRYING/VOLATILES Method: Silica Gel for 5 hours followed by vacuum over P 2 O 5 for three hours Mean of three measurements, Loss = 0.08% RESIDUE ANALYSIS Method: Sulfated Ash Sample Size: ~1g Mean of three measurements, Residue = 0.02% CERTIFIED PURITY BY MASS BALANCE [100% - Impurities (normalized)] 99.9% U crm = ±0.2%, k = 2.4 (as is basis) Certificate of Analysis Version BCBD0724V-2 Page 5 of 8
IDENTIFICATION TESTS INFRARED SPECTROPHOTOMETRY (Comparative identification analysis demonstrates direct traceability to Pharmacopeial standards) Certificate of Analysis Version BCBD0724V-2 Page 6 of 8
1 H NMR (Data provided by an external laboratory; not in scope of accreditation) Consistent with structure ELEMENTAL ANALYSIS (Data provided by an external laboratory; not in scope of accreditation) Exeter Analytical 440 Elemental Analyzer Combustion method % Theoretical Result 1 Result 2 Mean C 60.00 59.95 59.89 59.92 H 4.48 4.50 4.41 4.46 MELTING POINT Specification: None, (about 143 C (EP); lit.: 135 C) Mettler Toledo FP900 Thermosystem with FP81 Measuring Cell Mean of seven measurements = 137.0 C HOMOGENEITY ASSESSMENT Between-bottle homogeneity was assessed in accordance with ISO Guide 35. Completed units were sampled over the course of the bottling operation. Samples were taken in the following manner: the units produced in the bottling operation were divided into three chronological groups, those from the Early third, the Middle third, and the Late third (Groups). A pre-determined number of sample units were then randomly selected from each group. A subset of each group was then randomly selected for chemical analysis. The results of the chemical analysis were then compared by Single Factor Analysis of Variance (ANOVA). Certificate of Analysis Version BCBD0724V-2 Page 7 of 8
ANALYTICAL METHOD: HPLC (ref.: Traceability Assay) An analytical sample was prepared from each of four bottles from each Group (Early, Middle, and Late/twelve total). Sample size: ~50 mg Replicate (triplicate) analysis was performed on each sample. The order of replicate analyses was randomized. The between bottle standard deviation was derived from the ANOVA. No significant difference between the Groups was indicated. Heterogeneity was not detected under the conditions of the ANOVA. Certified by Reviewed by Validity Date Quality Systems 20.10.2011 THIS PRODUCT WAS DESIGNED, PRODUCED AND VERIFIED BY RTC- USA FOR ACCURACY AND STABILITY IN ACCORDANCE WITH ISO 17025:2005 (ACLASS Cert AT-1467) and ISO GUIDE 34:2009 (ACLASS Cert AR-1470). Certificate of Analysis Version BCBD0724V-2 Page 8 of 8