Protein precipitation -extracted samples

Relative extraction efficiencies of polar polymeric neutral, cation, and anion exchange sorbents (HLB, MCX, and MAX) for 11 beta antagonists and 6 beta agonists in human whole blood were probed.109 Initial characterization of MCX and MAX for acidic and basic load conditions, respectively, showed that both the agonists and antagonists were well retained on MCX, while they were recovered from MAX in the wash with either methanol or 2% ammonia in methanol (see Table 1.6). Blood samples were treated with ethanol containing 10% zinc sulfate to precipitate proteins and the supernatants loaded in 2% aqueous ammonium hydroxide onto the sorbents. After a 30% methanol and 2% aqueous ammonia wash, the analytes were eluted with methanol (HLB), 2% ammonia in methanol (MCX), or 2% formic acid in methanol (MAX). The best recoveries were observed with MCX under aqueous conditions or blood supernatant (after protein precipitation) spiked sample load conditions (see Table 1.7). Ion suppression studies by post-column infusion showed no suppression for propranolol and terbutaline with MCX, while HLB and MAX exhibited suppression (see Figure 1.6). 

Figure 4 LC/MS/MS of a mixture of gabapentin and naproxen using polarity switching. Micromass Quattro II, electrospray ionization rat plasma sample prepared by protein precipitation/extraction using a MetaChem 4.6 X 30-mm Polaris column with 3- Xm packing with a MetaChem 2000-MG2 guard column. Flow rate = 2.0 mL/min split 1 10 to MS. Injection volume 10 uL step gradient 100% 0.1% acetic acid, 0—1.0 min 70/30 0.1% acetic acid/acetoni-trile, 1.0—1.5 min 100% acetonitrile, 1.5—2.0 min. (Courtesy of Laura F. Polchinski, Ion Ebright, and Scott T. Fountain, personal communication.)...

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. 

In some cases, so called direct plasma injection techniques may be used23 83 104 108 instead of protein precipitation for loading plasma samples onto an HPLC/MS/MS system. Some direct plasma injection systems use a column switching technique in which the plasma is loaded onto an extraction column that retains the small molecules. The other plasma components are sent to waste and the flow is switched so that the small molecules are eluted onto an analytical column that connects to the MS/MS.23 83 108 One variation of the column switching method is turbulent flow chromatography commercialized by Cohesive Technologies (now part of Thermo, San Jose, CA).23... 

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... 

Koal et al. (2004) measured four immunosuppressants (cyclosporine A, tacrolimus, sirolimus, and everolimus) in whole blood samples from transplant recipients. The samples were treated first with a protein precipitation step. The supernatant was extracted with a Poros Rl/20 perfusion column (30 x 2.1 mm, 20 tm, Applied Biosystems, Darmstadt, Germany) online. A Luna phenyl hexyl column (2 x 50 mm, Phenomenex, Schaffenburg, Germany) was used for separation. The total run time was 2.5 min. The lower limit of quantitation was 10 ng/mL for cyclosporine A and 1 ng/mL for the other three analytes. 

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. 

Protein precipitation is used routinely in bioanalytical laboratories in the pharmaceutical industry. Plasma is mixed with an excess (3 to 5 times) of organic solvent (typically acetonitrile or methanol) or an acid such as formic acid. The proteins precipitate out of solution, the sample is centrifuged, and the supernatant is analyzed. While this method is relatively fast and simple, the extract contains salts and lipids that can interfere with subsequent analyses. Often, a second technique such as SPE is used for further cleanup. Table 2.4 exhibits various samples that... 

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. 

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... 

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. 

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. 

Sample extraction/deproteinization is usually accomplished with mild acidic solvents to free the noncovalently bound tetracyclines from macromolecules. Mcllvaine buffer, pH 4.0 (286, 287), Mcllvaine/EDTA buffer, pH 4.0 (283, 287-293), succinate buffer, pH 4,0 (278-281,294-296), acidic acetonitrile (297-299), and acidic methanol (14, 199, 300) have all been used successfully. Moreover, trichloroacetic acid, pH 2.0 (301, 302), metaphosphoric acid (303), acetate buffer (126, 280), citrate buffer, pH 4.0 (304), citrate buffer/ethyl acetate, pH 4-5 (305), and hydrochloric acid/glycine buffer (306, 307) have all been employed with varying success to precipitate proteins from the sample homogenates. 

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). 

The uses of constant-current coulometry for the determination of drugs in biological fluids are few, basically due to sensitivity restriction. Monforte and Purdy [46] have reported an assay for two allylic barbituric acid derivatives, sodium seconal and sodium sandoptal, with electrogenerated bromine as the titrant and biamperometry for endpoint detection. Quantitative bromination required an excess of bromine hence back titration with standard arsenite was performed. The assay required the formation of a protein-free filtrate of serum with tungstic acid, extraction into chloroform, and sample cleanup by back extraction, followed by coulometric titration with electrogenerated bromine. The protein precipitation step resulted in losses of compound due to coprecipitation. The recoveries of sodium seconal and sodium sandoptal carried through the serum assay were approximately 81 and 88%, respectively. Samples in the concentration range 7.5-50 pg/mL serum were analyzed by this procedure. 

When resuspending isoflavones in pure organic solvent, extra care should be taken to completely dissolve extracted isoflavones in the solvent. Solublization can be achieved by ultrasonification and providing sufficient time for the isoflavones to dissolve. Vortexing helps to release and recover isoflavones from precipitated proteins that may stick to the wall of glass vials and containers. All solvents must be of a high degree of purity. All extracted samples should be filtered to remove small particles. 

The fish tissue sample containing AMO residues was homogenized with phosphate buffer (pH 4.5), followed by protein precipitation with TCA and SPE on a C18 cartridge. Trace amounts of nonpolar interfering substances present in the SPE eluate were removed by LLE using ether. The final extract was reacted with formaldehyde and TCA at 100°C for 30 min. A fluorescent derivative was extracted with ether three times, and the extracts were combined, evaporated, and reconstituted in the mobile phase. No interfering peaks from the control fish extract were observed. A proposed chemical structure of the fluorescent derivative was reported and confirmed by both MS and NMR experiments (73). 

Ninhydrin forms fluorophors of high intensity with guanidino compounds in alkaline media. Dihydrostreptomycin, which has two guanidino groups, yields similar fluorophors. Milk sample was treated with TCA, to precipitate proteins, and extracted with dichloromethane and NaOH, and the supernatant cleanup was performed using a Cl8 SPE column. The analyte was eluted with formic acid in MeOH. The postcolumn derivatization was performed at 80°C. The recovery from all procedures varied from 82.6% to 82.8% (only for two concentration levels), with RSD of 0.7-1.2%. This method can also be used for the determination of STR in milk (112). 

The column-switching system was applied to the determination of STR and DIHS in pork and bovine muscle and kidney. Perchloric acid was used to precipitate proteins and extract analytes from the tissue. The clear supernatant was further cleaned up by an offline SPE on a cation-exchange SPE column. After the washing of the cartridge with water, the analytes were eluted with phosphate buffer (pH 8.0) and diluted with HSA, perchloric acid, and water. The enrichment was achieved in the online mode. After loading the sample, the enrichment precolumn was flushed with HSA at pH 3.3 for 5 min. Using the ratio of MeCN to aqueous component of 83 17... 

A procedure to determine PIR residues in bovine milk using HPLC with the derivatization step for UV detection has also been published (211). The PIR was extracted from milk after protein precipitation and a two-step LLE procedure. The extract was evaporated to dryness, dissolved in dilute base, and derivatized with 9-fluorenylmethyl chloroformate (FMOC). The de-rivatized extract was analyzed by reversed-phase HPLC. Overall recovery was 89%, with 4% for coefficient of variation. A linear regression analysis of HPLC/UV results was compared with the HPLC/MS assay (209,210). The procedure takes about 2.5 hours to complete six or eight samples. Pirlimycin is stable in milk frozen to —60°C or below for at least 3 months. 

For orange juices and vegetables juice extraction using Ana-lytichem Cl 8 solid phase (SPE). For milk samples protein precipitation with HCL, vortex mixing, centrifugation, 1-octanesulfonic acid extraction. For cola MeOH addition, vortex mixing, filtration. 

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 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.)...

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