Using a centrifuge to process and measure plasma samples

Release time:

2014-04-02

Overview:

For centrifugation, we usually use a speed of 16,000 rpm—it works quite well. We haven’t specifically examined the effect of centrifugal speed yet, but higher centrifuge speeds tend to generate more heat. If your drug is thermally unstable, you’ll want to pay close attention.

Take 200 μl of plasma, add 100 μl of 10% perchloric acid solution, vortex to mix thoroughly, and centrifuge at 10,000 r·min⁻¹ for 5 min. Take 100 μl of the supernatant, add 50 μl of NaOH solution, vortex to mix for 30 s, then add 100 μl of OPA derivatization reagent, vortex to mix again, and place in the dark for 10 min. Pipette 20 μl into the injection port, analyze under the conditions described above, record the chromatogram, and measure the peak area A of the GSH derivative.

Treatment method
For 1,200 and 100 μl, it’s generally more convenient to use a pipette. Whether you choose a domestically produced or an imported one depends on your experimental requirements and budget.

2. When preparing plasma samples, we use a 10% perchloric acid solution to precipitate proteins, and then we need to adjust the pH. Of course, you can experiment with the amount of NaOH added—afterward, you won’t need to adjust the pH for every single tube; just add a fixed amount instead.

3. For centrifugation, we usually use a speed of 16,000 rpm—it works quite well. We haven’t specifically examined the effect of centrifugal speed yet, but higher centrifuge speeds tend to generate more heat. If your drug is thermally unstable, you’ll want to pay close attention.

4. The concentration and amount of NaOH solution were already discussed in point 2.

5. After adding the derivatization solution, it should be kept in the dark—this is because the derivatization reagent or the conditions under which the derivatization reaction takes place require protection from light. This reagent should be fairly well-established by now.

In addition, “blank plasma” does not mean plasma without any derivatization reagents added; rather, aside from the absence of the drug, all other reagents and processing methods are identical to those used for drug-containing plasma. Adding a derivatization reagent allows us to determine whether the derivatization reagent itself interferes with the sample peak.

Application of Ultracentrifuges in Public Experimental Platforms

Notice of Competitive Negotiation for the Procurement of Centrifuges by Huazhong University of Science and Technology