Deproteination with trichloroacetic acid

Rabenstein and Yamashita [52] determined penicillamine and its symmetrical and mixed disulfides by HPLC in biological fluids. Plasma and urine were deproteinized with trichloroacetic acid, and HPLC was performed on a column (25 cm x 4.6 mm) or Biophase ODS (5 pm) with a mobile phase comprising 0.1 M phosphate buffer (pH 3) and 0.34 mM Na octylsulfate at 1 mL/min. Detection was with a dual Hg-Au amalgam electrode versus a Ag-AgCl reference electrode. (z>)-penicillamine and homocysteine were determined at the downstream electrode at +0.15 V, and homocystine, penicillamine-homocysteine, and penicillamine disulfides were first reduced... 

Shaw et al. [64] described a (D)-penicillamine detection method in blood samples that had been treated with EDTA, deproteinized with trichloroacetic acid, and analyzed within 1 h. Penicillamine was detected at a vitreous-carbon electrode operated at +800 mV after HPLC separation. A linear calibration graph was obtained, and the method had a limit of detection equal to 5-20 ng. The method was useful in clinical and in pharmacokinetic studies. 

Plasma Deproteinization with trichloroacetic acid bromination of supernatant and treatment with pyridine-p-phenylene diamine Spectrophotometry (thiocyanate- cyanide determination) 0.07 ppm 96 (thiocyanate) Pettigrew and Fell 1972... 

The furosemide extraction procedure was later examined for potential application in the analysis of thiazide diuretics in milk. Since this procedure could not provide sufficiently clean extracts for thiazides, additional acidic and basic extraction procedures were evaluated (557). Thus, milk was deproteinized with trichloroacetic acid, phosphoric acid, or potassium dihydrogen phosphate and centrifuged. The supernatants were extracted with ethyl acetate, evaporated to dryness, reconstituted in mobile phase, and analyzed by liquid chromatography. The recoveries in most cases were low and widely variable. Basic extraction, on the other hand, with sodium bicarbonate/potassium carbonate mixture or potassium monohydrogen phosphate followed by extraction with ethyl acetate also gave poor recoveries in most cases. It appears that a significant degradation of chlorothiazide occurred under the basic conditions. 

Stewart et al. (S8) estimated magnesium in serum and urine. Of four different methods of sample preparation (i.e., wet-ashing, deproteiniza-tion, simple dilution with water, and dilution with hydrochloric acid), deproteinization with trichloroacetic acid was found to be most satisfactory. No interference was seen from sodium, potassium, or phosphate, but sulfate produced depression. With protein a 6% decrease in the apparent magnesium concentration was seen. Calcium and sulfate were added to standards and samples to control sulfate depression. 

Deproteinate with trichloroacetic acid derivatize with vanillin in ethanol acidify with sulfuric acid. 

Schrijver et al. (25) described a reliable postcolumn derivatization HPLC method for total thiamine in whole blood. Two milliliters of whole blood was deproteinized with trichloroacetic acid, neutralized with sodium acetate buffer to a final pH of 4.5, and then treated with Taka-diastase for 2 h at 45°C. After centrifugation, the clear supernatant was used for direct HPLC analysis. A LiCh-rosorb Si-100 column (250 X 4.6 mm lOpm) was used and 240 XL of the extract was injected onto the column, eluted with a mobile phase composed of 40 vaM Na2HPO4-30 mM KH2PO4 and ethanol (87 13, v/v), at pH 6.8. 

More recent determinations of serum iron have been reported by Schmidt 57), who simply diluted with lanthanum chloride solution, and by Tavenier and Hellen-doorn58), who deproteinized samples in the latter study, iron in the protein precipitate is analyzed to correct the serum iron level. Uny etal. 59) determined serum iron, using ultrasonic nebulization of the sample to increase the sensitivity. Olson and Hamlin 6°) have determined serum iron and total iron-binding capacity. Proteins are precipitated and iron (III) is released by heating with trichloroacetic acid. 

Thus, simple deproteinization of plasma with trichloroacetic acid, perchloric acid, phosphoric acid, or acetonitrile, followed by centrifugation and direct injection of the supernatants, yielded low recoveries of malachite green and leuco-malachite green, probably due to insufficient debinding of the analytes (495). Acidification or alkalinization of plasma and subsequent extraction with ethyl acetate also resulted in poor recoveries. In contrast, protein denaturation with a mixture of either acetonitrile or methanol and citric acid could substantially improve Ute recovery of the analytes, possibly due to the pairing-ion function of Ure citrate ions. 

While flame AAS is adequate for routine determination of serum Fe, micro methods utilizing the furnace have been developed (Lewis et al., 1984) for pediatrics, etc. Of course, deproteinization is still required, usually with trichloroacetic acid. Because of furnace sensitivity, the serum sample is diluted 1 -i- 9 in a solution containing the matrix modifier and about 0.2% Triton X-100, and a 10-/other biological materials, once the sample is in solution. 

Sample preparation of meat and fish or other perishable materials involves, in general, a rapid cessation of enzymatic reactions and exclusion of protein to provide a clean supernatant. The fundamental method includes (1) deproteinization of materials by precipitation with trichloroacetic acid or perchloric acid, (2) centrifugation, and (3) neutralization of the supernatant with sodium hydroxide. However, these procedures are tedious and are not always required for all food samples, so simpler methods are ordinarily adopted. The simpler method is based on the homogenization of the food material with an appropriate amount of distilled water. The homogenate is then filtered through a membrane filter (0.2- xm pore size). The filtrate is diluted to a certain volume with distilled water and used immediately as the sample. In some cases, the filtrate is subjected to a 30-kDa cutoff membrane pass before injection. 

There is a great deal of interest in the determination of lead, particularly micromethods applicable to the analysis blood lead in children. Consequently, reports continue to appear on the atomic absorption determination of lead in blood and urine. Ninety percent of blood lead is found in the erythrocytes and, therefore, whole blood is analyzed rather than serum or plasma. Berman etal. 134) have described a procedure for determining normal lead levels in which only 250 fd of blood are taken. The blood is deproteinized with 1 ml of 10 % trichloroacetic acid and then the lead is extracted with APDC into 1 ml of MIBK, at pH 3.5. 

Tryptophan is another amino acid that tends to bind with protein it is also sensitive to the deproteinization process. The use of sulfosalicylic acid (SSA) in particular is contraindicated. Trichloroacetic acid, on the other hand, leaves tryptophan unaffected. 

Sample extraction and deproteinization is usually accomplished with organic solvents including ethyl acetate (182-187, 189-192), acetone (193-196), methanol (177,197-200), acetonitrile (201,202), and ethanol (188). To optimize the extraction efficiency, acidification of the sample has been suggested by many workers (177, 188, 192, 197-199). In acidic conditions (pH 3), quinolones, being zwitterions, are fully protonated and, therefore, are becoming less bound by the matrix and more soluble in organic extraction solvents. Extraction of quinolones from food samples can also be accomplished using water (203), phosphate buffer, pH 9 (204), or trichloroacetic acid (205). 

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. 

To analyze free amino acids in plasma or tissue homogenates, it is necessary to remove proteins and peptides present in solution. The most widely used deproteinization method is precipitation with 5-sulfosalicylic acid followed by centrifugation for separating the precipitate. In comparison to other precipitation agents such as trichloroacetic acid, perchloric acid, picrinic acid, or acetonitrile, the best results with respect to completeness of precipitation are obtained with 5-sulfosalicylic acid [39]. Other deproteinization methods comprise ultrafiltration and ultracentrifugation [40], which have only recently been considered as sample preparation methods for amino acid analysis. 

Deproteinize 2-5 ml of serum which has been diluted with an equal volume of water by adding 1 volume of 20% trichloroacetic acid. The precipitate is centrifuged and washed once with 10% TCA the combined solutions are evaporated under vacuum to dryness, the residue is taken up in 20 ml of distilled water, and 5 ml of pH 8.8 NaHCOa buffer is added. The remainder of the procedure is the same as was described above for the urine sample. 

Sample preparation Deproteinize with 12.5% trichloroacetic acid. 

Andreoleti et al. (A6) also hemolyzed blood samples with saponin followed by deproteinization in two stages, with 2.5X and 50X trichloroacetic acid, respectively. A dilution factor was determined using a cadmium tracer. 

After deproteinization of e.g., fresh blood with 10% trichloroacetic acid containing 1 mM EDTA, 20 1 of deproteinized sample, 80/il of 1 mM EDTA in water, 0.2 ml of 2.5 M borate buffer (pH 9.5) containing 4 mM EDTA and 0.1 ml of SBD-F (2 mg ml in borate buffer) are mixed and heated for Ih. An aliquot can be used immediately for separation by HPLC. 

Where protein may interfere with subsequent TLC analyses, it should be removed (deproteinization procedures). A suitable procedure for an approximate 50-pl sample of serum involves addition of 100 pi of methanol to precipitate the protein, followed by shaking and centrifugation of the mixture to obtain a clear supernatant. This technique has been used to deproteinize biological fluids prior to the determination of drugs in these fluids (Touchstone and Dobbins, 1979). Other commonly used techniques to precipitate protein in either serum, urine, or tissue homogenates involve the addition of either trichloroacetic acid, perchloric acid, or sulfosalicylic acid followed by centrifugation, and then removal of the supernatant. The supernatant may require further cleanup or be used directly for TLC. Further information on deproteinization can be found in Jain (1996). 

Other authors have used trichloroacetic acid (Katayama et al., 1993) for sample deproteination and determination of histamine by the chemiluminescence FLA method. Extraction from minced tuna fish (10 g) has been carried out first with water (15 mL) followed by extraction with 20 mL of 10% (w/v) trichloroacetic acid solution and filtration. 

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