185
1. Separate C57BL/6 female mice 6 weeks old into two groups
(n = 3) and inject them intraperitoneally either with a single
dose of TMZ or a single dose of TMZ@PSC4 complex.
2. Collect the blood by cardiac puncture before treatment (day 0)
and then at 0.5, 2, and 4 h posttreatment in tubes containing
heparin.
3. Centrifuge blood samples at 2862 × g for 10 min to separate the
plasma.
4. Acidify plasma (pH < 4) with phosphoric acid 85%.
5. Store all samples at −80 °C until further processing.
6. Prepare samples for analysis by adding 200 μL IS solution and
200 μL of 10 mM pH 3.5 ammonium fοrmate buffer to 100 μL
of acidified mouse plasma and precipitate this mixture with a
100 mM 1:1 methanol:zinc sulfate solution.
7. Vortex-mix for 1 min and centrifuge at 21,885 × g for 15 min.
8. Transfer the supernatant in glass vials and quantify TMZ by
LC-MS/MS (Fig. 7).
4 Notes
1. The pH of the solution must be stable on slightly basic conditions during the reaction.
2. The solution was further sonicated for 15 min to obtain a clear
solution.
3. r
2
must be over 0.99 to have an accurate calibration curve.
4. The pH of the stock solution of TMZ should be acidified to
stabilize TMZ.
5. Further dilution of stock solutions should be made with an
appropriate solvent that is compatible with the mass
spectrometer.
6. Various factors should be examined including type of elution
(gradient or isocratic), column properties, mobile-phase ratios,
etc. to achieve reliable and consistent peak shapes for all
compounds.
7. This is important to avoid sensitivity and repeatability issues
caused by temperature fluctuations.
3.5 In Vivo
Pharmacokinetic
Analysis
Encapsulation of Small Drugs in a Supramolecule Enhances Solubility, Stability…
1. Separate C57BL/6 female mice 6 weeks old into two groups
(n = 3) and inject them intraperitoneally either with a single
dose of TMZ or a single dose of TMZ@PSC4 complex.
2. Collect the blood by cardiac puncture before treatment (day 0)
and then at 0.5, 2, and 4 h posttreatment in tubes containing
heparin.
3. Centrifuge blood samples at 2862 × g for 10 min to separate the
plasma.
4. Acidify plasma (pH < 4) with phosphoric acid 85%.
5. Store all samples at −80 °C until further processing.
6. Prepare samples for analysis by adding 200 μL IS solution and
200 μL of 10 mM pH 3.5 ammonium fοrmate buffer to 100 μL
of acidified mouse plasma and precipitate this mixture with a
100 mM 1:1 methanol:zinc sulfate solution.
7. Vortex-mix for 1 min and centrifuge at 21,885 × g for 15 min.
8. Transfer the supernatant in glass vials and quantify TMZ by
LC-MS/MS (Fig. 7).
4 Notes
1. The pH of the solution must be stable on slightly basic conditions during the reaction.
2. The solution was further sonicated for 15 min to obtain a clear
solution.
3. r
2
must be over 0.99 to have an accurate calibration curve.
4. The pH of the stock solution of TMZ should be acidified to
stabilize TMZ.
5. Further dilution of stock solutions should be made with an
appropriate solvent that is compatible with the mass
spectrometer.
6. Various factors should be examined including type of elution
(gradient or isocratic), column properties, mobile-phase ratios,
etc. to achieve reliable and consistent peak shapes for all
compounds.
7. This is important to avoid sensitivity and repeatability issues
caused by temperature fluctuations.
3.5 In Vivo
Pharmacokinetic
Analysis
Encapsulation of Small Drugs in a Supramolecule Enhances Solubility, Stability…