Solid-phase extraction proteins

Several strategies have been described for the preconcentration of sample components present at low concentrations. These techniques include zone sharpening,28-29 on-line packed columns,30 and transient capillary isotachophoresis (cITP).31-32 Other standard laboratory techniques are often used, including solid-phase extraction, protein precipitation, ultrafiltration, etc. Two important points to keep in mind when selecting a concentration protocol are the sample requirements of the method and the potential selectivity on relative concentrations of sample components. The latter point applies to purity and concentration analysis. 

Extraction efficiency. Recovery of the analyte from biological matrix after sample pretreatment (i.e., liquid-liquid extraction, solid-phase extraction, protein precipitation, etc.) to remove endogenous substances. 

The liquid chromatography - tandem mass spectrometry (LC/MS/MS) technique was proposed for the determination of corticosteroids in plasma and cerebrospinal fluid (CSF, liquor) of children with leucosis. Preliminai y sample prepai ation included the sedimentation of proteins, spinning and solid-phase extraction. MS detection was performed by scanning selected ions, with three chai acteristic ions for every corticosteroids. The limit of detection was found 80 pg/ml of plasma. 

D. Eigeys, A. Dua et and R. Aebersold, Identification of proteins by capillary electrophoresis-tandem mass specti ometiy . Evaluation of an on-line solid-phase extraction device , J. Chromatogr. A 763 295-306 (1997). 

Exudate collection in trap solutions usually requires subsequent concentration steps (vacuum evaporation, lyophilization) due to the low concentration of exudate compounds. Depending on the composition of the trap solution, the reduction of sample volume can lead to high salt concentrations, which may interfere with subsequent analysis or may even cause irreversible precipitation of certain exudate compounds (e.g., Ca-citrate, Ca-oxalate, proteins). Therefore, if possible, removal of interfering salts by use of ion exchange resins prior to sample concentration is recommended. Alternatively, solid-phase extraction techniques may be employed for enrichment of exudate compounds from the diluted trap solution (11,22). High-molecular-weight compounds may be concentrated by precipitation with organic solvents [methanol, ethanol, acetone 80% (v/v) for polysaccharides and proteins] or acidification [trichloroacetic acid 10% (w/v), per-... 

Extraction of lyophilized protein powder by w/o-ME solution (solid-phase extraction method, SPE). 

FIG. 3 Time course of the solid-phase extraction of bovine serum albumin by an AOT-based w/o-Me solution. Conditions 500 mg of solid-phase protein 20 mL of iso-octane containing 25 mM AOT and 1000 mM water (w = 40). (Reprinted with permission from Ref. 58.)... 

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. 

Conditions for Automated Solid Phase Extraction of Vitamin D Metabolite from Plasma after Protein Precipitation... 

The study concluded that Once wash steps are optimized, samples prepared by solid phase extraction are cleaner than those prepared by protein precipitation. Samples prepared by extraction with a Multi-SPE plate resulted in lower LOQs than samples prepared by solvent precipitation. Drug recoveries were acceptable (>80%) for both the SPE and the solvent precipitation methods. Well-to-well reproducibility of samples was slightly better with extraction with a Multi-SPE plate. Evaporation and reconstitution, while more time-consuming, yield better chromatographic performance, allow analysis of lower concentration samples, and require optimization for good analyte recovery. 

The most common (off-line) sample preparation procedures after protein precipitation are solid phase extraction and liquid-liquid extraction. Multiple vendors and available chemistries utilize 96-well plates for solid phase extraction systems and liquid-liquid extraction procedures. Both extraction process can prepare samples for HPLC/MS/MS assay. Jemal et al.110 compared liquid-liquid extraction in a 96-well plate to semi-automated solid phase extraction in a 96-well plate for a carboxylic acid containing analyte in a human plasma matrix and reported that both clean-up procedures worked well. Yang et al.111 112 described two validated methods for compounds in plasma using semi-automated 96-well plate solid phase extraction procedures. Zimmer et al.113 compared solid phase extraction and liquid-liquid extraction to a turbulent flow chromatography clean-up for two test compounds in plasma all three clean-up approaches led to HPLC/MS/MS assays that met GLP requirements. 

As a general rule, APCI is less likely to demonstrate matrix effects and ESI is more likely to be affected by matrix effects. Sample clean-up is another important factor—protein precipitation is more likely to result in matrix effects than is solid phase extraction. Matrix effects may be caused by sample constituents that are not parts of the biological matrix. Mei et al.126 129 showed that certain brands of sample tube containers can produce matrix effects. They also demonstrated that Li-heparin, a common anticoagulant for plasma samples, can produce significant matrix effects... 

The biological applications of NMR include the study of the structure of macromolecules such as proteins and nucleic acids and the study of membranes, and enzymic reactions. Newer methods and instruments have overcome, to a large extent, the technical difficulties encountered with aqueous samples and the analysis of body fluids is possible, permitting the determination of both the content and concentration of many metabolites in urine and plasma. NMR is not a very sensitive technique and it is often necessary to concentrate the sample either by freeze drying and dissolving in a smaller volume cm- by solid phase extraction methods. 

Early assays for steroids involved liquid-hquid extraction (LEE) techniques, which are still widely used. However, the popularity of LEE has declined as more efficient techniques such as solid-phase extraction (SEE) have been introduced. In LEE techniques, the polarity of the steroids and their ability to bind to proteins are important aspects when selecting various solvents for their extraction from biological matrices. LEE is a nonselective procedure and nonsteroidal hpids are likely to be coextracted and interfere with the analysis. 

Before compounds in biological matrices can be analyzed by LC/MS/MS, the samples must undergo a preparation procedure. There are a variety of techniques available for sample preparation including offline sample preparation techniques (liquid-liquid extraction, protein precipitation, and solid phase extraction) and on-line sample preparation... 

Krause, A.A. and Niemczyk, H.D. Gas-liquid chromatographic analysis of chlorthal-dimethyl herbicide and its degradates in turfgrass thatch and soil using a solid-phase extraction technique, J. Environ. Sci Health, B25(5) 587-606, 1990. Kresheck, G.C., Schneider, H., and Scheraga, H.A. The effect of DzO on the thermal stability of proteins. Thermod3mamic parameters for the transfer of model compounds from HzO to DzO, J. Phys. Chem., 69(9) 3132-3144, 1965. 

Surface enhanced laser desorption/ionization (SELDI) is a distinctive form of laser desorption ionization where the target plays an active role in the sample preparation procedure and ionization process [49]. Depending on the chemical or biochemical treatment, the SELDI surface acts as solid phase extraction or an affinity probe. Chromatographic surface is used for sample fractionation and purification of biological samples prior to direct analysis by laser desorption/ ionization. SELDI is mainly applied for protein profiling and in biomarker discovery by comparing protein profiles from control and patient groups. 

Unlike clenbuterol, salbutamol is a difficult compound to analyze due to its particular chemical attributes. It is a basic compound subjected to protein binding poor recoveries are obtained especially when protein precipitation techniques are used to prepare the extracts (145). In addition, salbutamol is charged at all pH values and does not readily lend itself to simple, specific back-extracting procedures. This severely restricts the options of sample cleanup. However, a Subtilisin protease digestion step followed by acid clarification and solid-phase extraction has been suggested (146) as an adequate extraction and cleanup procedure prior to the end-point determination of salbutamol by an enzyme immunoassay (139) based on the cross-reactivity of anticlenbuterol antibodies. 

If necessary, select a sample cleanup method. Cleanup could be required if resolution is poor, if the salt content is high, or if the capillary fouls. Cleanup might involve solid-phase extraction (Section 28-3), protein precipitation, or dialysis (Demonstration 27-1). 

Figure 28-17 compares chromatograms of the drug naproxen in blood serum with or without sample cleanup by solid-phase extraction. Without cleanup, serum proteins overlap and obscure the signal from naproxen. Solid-phase extraction removes most of the protein. 

MN Vaghela, A Kilara. A rapid method for extraction of total lipids from whey protein concentrates and separation of lipid classes with solid phase extraction. J Am Oil Chem Soc 72 1117-1121, 1995. 

C protein precipitation with trichloroacetic acid at 100°C for 10 min C,8 solid-phase extraction/cleanup. method (r = 0.987). 

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