Mass Spectrometry Quantitation

Sample Preparation

for

PK//MS Analysis

Samples are usually supplied in plasma or serum. Plasma is blood after the blood cells have been removed usually by centrifugation. Serum is the fluid that is left after blood has been clotted and the solids have been removed. Clotting removes blood cells and clotting factors. Serum is easier to handle than plasma since the clotting factors have been removed. When plasma is stored it is not uncommon to find fluffy clotted material which makes the samples difficult (goopy) to pipette. Serum or Plasma must then be prepared further before it can be loaded onto a reverse phase column for analysis. There are a number of methods that can be used to prepare plasma or serum for PK/MS analysis and several are discussed below.

Plasma Crash

Plasma crash is the method most commonly used since it does not generally require special instrumentation. With the plasma crash method acetonitrile or methanol is used to precipitate out the abundant serum proteins. A typical method is described below.

The Plasma Crash Method:

1.) Place 50 µls of plasma or serum in a 0.5 ml tube.
2.) Add 10 µls of internal standard.
3.) Add 140 µls of cold (refrigerated) organic (ACN or MeOH) and vortex.
4.) Refrigerate sample for 2 hrs.
5.) Micro-Centrifuge the sample to pellet out the precipitated proteins.
6.) Remove 150 µls of the supernatant to a clean tube and add 150 µls of the HPLC A solvent (aqueous solvent).
(at this point the organic content of the sample is approximately 38% organic. If your compound requires a lower organic content to stick to the column raise the aqueous addition in step six to lower the organic)
7.) Inject an appropriate amount of the solution from step six for PK/LC/MS/MS analysis.

Download the plasma crash method in MS Word format by right clicking on the icon and choose "save target as" to save the file to your computer.

Solid Phase Extraction

Solid phase extraction is preferred by many. In SPE, plasma or serum is added directly to a disposable hydrophobic solid support, usually a small C18 cartridge. After serum or plasma is added to the column organic solvent is passed through the column to elute the small molecule of interest. Solid phase extraction can be performed manually or with a small manifold. Performing this technique manually limits sample through-put. The technique is amenable to automation with solid phase supports available in 96 well formats. Some find the plate formats expensive with a single 96 well plate costing close to one hundred US dollars, however the cost should be balanced against ease of use and superiority of the final product. Some find that the final eluant for PK/LC/MS/MS analysis is superior in quality finding that reverse phase column fouling and roll-off of MS signal occurs at a slower rate.

Liquid-Liquid Extraction

Admittedly we have little experience with this technique. Here is what we know. Liquid-Liquid extraction only works for those compounds that partition into the organic phase. It can be a very efficient method for sample clean up. However, the method is difficult to automate.

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