Protein precipitation PPT

More information on the comparative evaluation of protein precipitation methods may be obtained from Lei and coworkers.163 An interesting comparison of protein precipitation (PPT) and solid phase extraction (SPE) methods was presented in a technical library publication from Millipore164 that describes use of its Multi-SPE-MPC extraction plate and Multiscreen deep well Solvinert filter plate for SPE and PPT, respectively (Figure 1.45). A Biohit Proline multichannel pipette was used to add 400 /iL of acetonitrile to each well of the filter plate and then, using the pipette s double aspiration program, 100 /iL of spiked serum was aspirated and 100 /iL of acetonitrile from the filter plate was aspirated to initiate protein precipitation in the pipette tip. The mixture was deposited back in the filter plate and shaken vigorously for 2 min. 

Sample Preparation/Extraction The process of separating potentially interfering components from a sample prior to LC-MS analysis for the purposes of improving sensitivity, specificity, and/or method ruggedness. Variations include solid phase extraction (SPE), liquid-liquid extraction (LLE), and protein precipitation (PPT). Extraction may be performed off-line, in which the cleanup is completely independent from the LC-MS analysis, or on-line, in which the cleanup is integrated directly into the LC-MS analysis. 

Figure 13-13. Comparison of choice of sample preparation on MRM signal intensity of an investigation compound. The injection volume was 40pL.The count per second (CPS) signifies the detector (an electron multiplier) response. Protein precipitation (PPT), hydrophilic-lipophilic balanced co-polymer-based SPE (Oasis HLB- copolymer of styrene, divinylbenzene and -vinylpirrolidone monomers the hydrophihc refers to the NVP monomer, and the lipophilic refers to the SDVB monomers), and strong anion exchange SPE (Max) (all in 96-well plate format) were used in control rat plasma (unpublished data).

The three main formats for sample preparation used in drug-discovery are protein precipitation (PPT), SPE, and LLE. Several examples of off-line sample preparation have been reported and involve SPE [37,38,46,47], LLE [38,48], and PPT [39,49]. In each of the examples cited, semi- or fully automated strategies for liquid handling were incorporated to enhance throughput. Even with the recent popularity of on-line methods, off-line techniques continue to be widely employed. The key advantage to off-line methods is that sample preparation may be independently optimized from the mass spectrometer and does not contribute overhead to the LC-MS injection duty cycle. 

Walter, R.E. Cramer, J.A Tse, F.L.S. Comparison of Manual Protein Precipitation (PPT) versus a New Small Volume PPT 96-Well Filter Plate to Decrease Sample Preparation Time, J. Pharm. Biomed. Anal. 25, 331-337 (2001). 

Briem et al. [33] also describe the use of a liquid handler to perform the sample preparation steps in an automated manner. The sample preparation consisted in taking a 25 pL aliquot of the plasma sample and adding 150 pL of acetonitrile (ACN) containing the internal standard (IS) and then following typical protein precipitation (PPT) procedure steps. The authors noted that the 1 6 ratio of plasma to ACN worked well for the liquid handler and exceeded the 1 3 criterion reported by Poison et al. [34] as the minimum ratio of plasma to ACN needed to remove at least 96% of the proteins from the plasma sample. The authors also noted that use of EDTA (instead of heparin) as the anticoagulant helped to avoid clots in plasma as has been reported previously by other authors [35-37], The authors also stated that the robotic method... 

FIGURE 1.39 Solvent-first procedure using ISOLUTE PPT Protein Precipitation Plates.159 (Reproduced with permission from Biotage AB.)... 

Direct injection of plasma or supernatant after protein precipitation on a short column with a high liquid flow rate is a common method for reducing analysis time in the pharmaceutical industry. The direct injection of a sample matrix is also known as the dilute-and-shoot (DAS) approach.62 DAS can be applied to all types of matrices and approaches and is the simplest sample preparation method with matrix dependency. Direct injection can also be approached through the extraction of eluent from PPT, SPE, and LLE onto a normal phase analytical column. The procedure is called hydrophilic interaction liquid chromatography (HILIC)70110111 and it avoids the evaporation and reconstitution steps that may cause loss of samples from heat degradation and absorption. 

Figure 1.28. Generic quantitative bioanalytical sample preparation scheme. (SPE — solid phase extraction LLE - liquid-liquid extraction PPT—protein precipitation). (Reprinted with permission from Chang et al., 2007a.)...

Protein precipitation in 96-well format is commonplace and frequently involves 96-well pipetting stations. These techniques are considered semiautomated, since manual intervention is needed for protein removal. Protein removal usually occurs hy centrifugation, although hltration has also been used [50]. Due to the sensitivity of the current-generation TQMS instruments, the amount of plasma taken for analysis is typically less than 100 /zL. In our laboratory, protein precipitation with 50 /zL of plasma is typically sufficient to achieve LLOQs in the range of 1 to 10 ng/mL. Interested readers are referred to a recent paper hy Polson and co-workers, who undertook a detailed investigation with regard to factors that affect optimization of PPT for LC-MS/MS [51]. 

Abbreviations LOD- limit of detection STX- saxitoxin NEO- neosaxitoxin GTX- gonyautoxin RT- room temperature Ppt- protein precipitation SPE- solid phase extraction APB- ammonium phosphate buffer CAN- acetonitrile NPB- sodium phosphate buffer PPB- potassium phosphate buffer rxn-reaction... 

Fig. 8.2. Urea-SDS-PAGE of soy proteins with and without phytase treatment in supernatants (super) and precipitates (ppt) (Aldin, 2004).

However, PPT cannot be considered a true sample preparation technique because it removes plasma proteins only through the addition of a precipitating solvent and subsequent homogenization and centrifugation. 

A simple PPT was developed for plasma and OFs recoveries of analytes from OF are often higher when compared to plasma, but sonication before precipitation has been proven necessary to help the analytes to release the protein binding, also in OF. Developing a multiclass method with PPT has proved to be simpler in respect to other extractive procedures, so it has been possible to obtain recoveries averaging 100 % and minimized matrix effects by the use of a deuterated ISTDs for each class of substances [93],... 

After frozen storage at several temperatures in various media. All samples were quenched in alcohol at —72° before transfer to alcohol at the stated storage temperatures, and all were thawed rapidly in a 37° water bath for assay. All samples in a given experiment were prepared, frozen, stored, thawed, and assayed simultanously. ppt = precipitated on thawing. P = protein. MEt = mercaptoethanol. 

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