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HPLC A Praactical User'S Guide Part 9

Tham khảo tài liệu 'hplc a praactical user's guide part 9', kỹ thuật - công nghệ, cơ khí - chế tạo máy phục vụ nhu cầu học tập, nghiên cứu và làm việc hiệu quả | METHODS DEVELOPMENT 155 Heat Plasma Blank C18 70 MeOH H2O 1 AcCH 5 10 15 20 60 SFE Window SFE Windowed Plasma Standards C18 70 MeOH H2O 1 AcCH Figure 12.4 Systematic methods development samples. a Heat plasma blank b plasma blank spiked with standards c 80 SFE window d heated plasma standards e SFE win-dowed plasma standards f SFE windowed patient sample. Plasma Blank Spike with Standards C18 70 MeOH H2O 1 AcCH Heated Plasma Standards C18 70 MeOH H2O 1 AcCH SFE Windowed Patient Sample C18 70 MeOH H20 1 AcCH 156 SAMPLE PREPARATION AND METHODS DEVELOPMENT cartridge. You elute the cartridge with 2mL each of 60 80 and 100 MeOH add 1 mL of IS to each cut dilute 100X and shoot each sample into the HPLC system. The 60 wash is contaminated with B and a trace of A. You repeat moving the window frame to the left by eluting with 55 80 and 100 MeOH. All your peaks are in the 80 window Fig. 12.4c none show up in either the 55 or 100 washes. Quantisation against the internal standard s peak height shows no loss of peaks B or D on the SPE cartridge. You remove the other half of the pooled blood sample from the refrigerator. You mix 4mL of blood with 1mL each of the four standards in acetonitrile sonicate heat in boiling water and centrifuge.You place 2mL of the super and 1 mL of IS 100X in a 100-mL volumetric flask dilute with mobile phase and inject into the HPLC. Note You are looking for loss of standards by adherence to precipitated protein. Peaks A C and D are present the last two standard peaks quantitate correctly Fig. 12.4d . You take 2mL of the remaining plasma plus standards supernatant dilute it 5-fold with water and place it on an activated SFE cartridge column. You elute with 55 80 and 100 MeOH in water containing 1 acetic acid. The 80 fraction is mixed with IS diluted and run. It shows a much narrower polar peak compound B as a shoulder on the polar peak from the plasma peak resolved peaks A C D and IS and a small amount of the latter running nonpolar peaks Fig. .