1100 series hplc

Example 28

Synthetic fluorogenic substrate of NE, (MeOSuc)-AAPV-(pNA) (160 μM), was incubated with bioactive NE (500 nM)±the NE inhibitor, sivelestat (160 μM) or nNIF (50 nM), for 3 hours at 37° C. The reactions were quenched with 5% glacial acetic acid and centrifuged at 14,000 rpm for 5 minutes. Chromatograms were obtained using an AGILENT™ 1100 Series HPLC and a PHENOMENEX® 5 μm C18 LUNA® column (100 Å, 4.6×150 mm) over a 30 minute 10% to 90% B gradient (Buffer A 0.1% TFA in H₂O, Buffer B 0.1% TFA in ACN). Mass spectra were obtained for secondary validation of the reaction products using an API® 3500 triple quadrupole mass spectrometer. Chromatograms were offset on both the X and Y axes (by 0.5 minutes and 0.1 A₂₁₄, respectively) for greater visibility. Relative A₂₁₄ was determined by normalizing all of the data to the tallest HPLC peak displayed in each graph.

Example 26

C57/BL6 mice were pretreated in blinded fashion with CRISPP (10 mg/kg), nNIF (10 mg/kg), or CRISPP-SCR (10 mg/kg) by i.p. injection 1 hour prior to and 6 hours after inoculation with LPS (25 mg/kg, i.p. injection) Control mice were i.p. injected with saline alone. Fluid resuscitation and antibiotic treatment were not used in these experiments. Survival was assessed over 50 or 72 hour intervals. All mice were included in the final survival analysis.

Synthetic fluorogenic substrate of NE, (MeOSuc)-AAPV-(pNA) (160 μM), was incubated with bioactive NE (500 nM)±the NE inhibitor, sivelestat (160 μM) or nNIF (50 nM), for 3 hours at 37° C. The reactions were quenched with 5% glacial acetic acid and centrifuged at 14,000 rpm for 5 minutes. Chromatograms were obtained using an AGILENT™ 1100 Series HPLC and a PHENOMENEX® 5 μm C18 LUNA® column (100 Å, 4.6×150 mm) over a 30 minute 10% to 90% B gradient (Buffer A 0.1% TFA in H₂O, Buffer B 0.1% TFA in ACN). Mass spectra were obtained for secondary validation of the reaction products using an API® 3500 triple quadrupole mass spectrometer. Chromatograms were offset on both the X and Y axes (by 0.5 minutes and 0.1 A₂₁₄, respectively) for greater visibility. Relative A₂₁₄ was determined by normalizing all of the data to the tallest HPLC peak displayed in each graph.