Document ExwJBJd5mgx1jY3zLqawO0e2N

M ate- 33or 487 DuPont Haskell Laboratory for Health and Environmental Sciences TO: FROM: MR FILI POCKET H-24921 J. C. Maslanka O ctober 30, 2003 Quality Assurance E. M ylchreest TEST SUBSTANCE STABILITY OF H-24921 M edical Research Project Number: Haskell Sample Number: Haskell Number (Analytical Reference): Analytical Test Code: Analytical Report Number: Notebook References: 2492 .2 4 9 2 1 pm i v DuPont-13708 Attached is the analytical report to satisfy protocol requirements for studies with H-24921 but may also be used for other purposes. not contain TSCA CB1 Company Sanitized. Goes TEST SUBSTANCE STABILITY OF H-24921 Medical Research Project Number: Haskell Sample Number: Analytical Report Number: DuPont-13708 A sample of H-24921 was received August 21, 2003, and an: fffh ^ ^ M p ta g e o f active ingredient (a.i.) was measured to Bominal for replicate analyses (n=3). The spon. " "when the sample was submitted. on Se tember 4, 200 rith a r; reported; jurity o ACKNOW LEGM ENTS Sample preparation by J. C. Maslanka (Senior Staff Chemist) and sample analysis by T. Ryan (Staff Chemist). SIGNATURES Report by: Janet C. Maslanka Senior Staff Chemist Zaay D ate Date Issued: 3o DuPont-13708 page 2 of 3 METHODS Analysis of H-24921 was by high-perform ance liquid chrom atography/tandem mass spectrom etry Negative-ion electrospray is used to generate negatively charged ions o f l H H i h e ions are selected w ith die firstM S quadrupole, collisionalh ndissociated using argmT, and a fragment ion is monitorec acid is used as an internal standard. SAMPLE PREPARATION & ANALYSIS Aliquots (0.0233, 0.0270, and 0.0252 grams) of H-24921 were dissolved in Nanopure water (250 mL) to the nominal concentrations of 93.2,108, and 101 ppm. These were further diluted to 0.0326, 0.0378, and 0.0354 ppm with the appropriate amount of internal standard added and analyzed according to the following method. CHROMATOGRAPHIC CONDITIONS I.C param eters Instrument: Column: Flow Rate: Injection Volume: Column Temperature: Column Switch: Mobile Phase: Gradient: Time (min.) 0.0 0.9 1.0 6.0 6.1 7.0 MS param eters Instrument: Ionization mode: Capillary voltage: Cone Voltage: Source Temperature: Desolvation Temperature: Scan function: Hewlett Packard Model 1100 HPLC Zorbax RX-C8, 2.1 mm x 150 mm, 5pm 0.4 mL/min 5 pi 30C 4.0 minutes 0.15% Acetic Acid/Acetonitrile % Acetonitrile 5 5 80 80 5 5 Micromass Quattro Micro Tandem MS Electrospray (ESI), negative ion -2.7 kV 15 V 120 350 4j3 m/z tarent) to 369 m/z (daughter) 1415 m/z (parent) to 369 m/z (daughter} Retention Time ol ant approximately 4.9 min (5 pL injections) DuPont-13708 page 3 of 3 CALIBRATION & QUANTITATION A stock solution o f the H-24921 (separate sample used as analytical reference) was made in Nanopure water. Appropriate aliquots of the stock were diluted with Nanopure water to make calibration standards that bracketed the target concentration of the diluted sample solutions. Before tiies^ fo u fots were brough t to volume, an appropriate amount of internal s t a n d a i n l H f l H ^ ^ ^ H H M H H R y a s added. Analysis of H-24921 was by high-performance liquid chromatography/tandem mass spectrometryAC-MS/MS). Negative-ion electrospray is used to generate negatively charged ions o f lp |H th e ions are selected with the f c s ^ l^ u a d r u p ^ e ^ d li^ m allv dissociated using ^ o n ,a n d a fragment ion is m o n ito r e d f lH H H H |H H p j[ ||K M H used as an internal standard. Triplicate injection of the i^ p I^ o lu u o n ^ id cah b ratio lR u w lard s solutions were made and peak areas were calculated electronically. The calibration curve was generated by regression analysis using the peak area ratio from the H-24921 and the internal standard. Data for test solutions were compared to the calibration curves to evaluate the concentrations of the H-24921. RESULTS H-24921 eluted from the HPLC column as a resolved peak with a retention time o approximately 4.9 minutes for the negative ion. The sponsor reported a purity o:^ P ^ t h e n the sample was submitted. Table 1. The percent of a.i. in the H-24921 sample analyzed September 4,2003. ppm H-24921 Percent Aliquot Targeted Measured Nominal 1 0.0326 0.0321 98.5 2 0.0378 0.0366 96.8 3 0.0354 0.0334 94.4 Average Percent Nominal S tandard Deviation Coefficient of V ariation 96.6 2 .1 2% Company Sanliizad. Does not contain TSCA CBS