Ethanol deproteination with

Five species of common edible crabs were used. They were cooked in boiling water containing 3% NaCl for 20 minutes according to commercial practice. After cooling, the leg meat was removed from the crabs of both sexes and extracted with hot water. The extracts were then deproteinized with 80% ethanol and analyzed for free and combined amino acids, nucleotides and related compounds, quaternary ammonium bases, sugars, organic acids, and inorganic ions. 

Fig. 4 Electropherogram of (a) blank serum deproteinized with ethanol (b) serum spiked with the standard ENX solution (0.25 pmol) and IS (0.15 pmol) and (c) water spiked with the standard ENX solution (0.25 pmol). Conditions are the same as in Fig. 2.

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

Carotenoids are commonly extracted from liquid samples (plasma/serum) into lipophilic solvents such as hexane, hexane-ethyl acetate, or diethyl ether, mostly after deproteinization with ethanol or methanol, which also helps to liberate the lipidic substances from protein binding. Extracts should be protected from light and acids and antioxidants may usefully be added. The extract is either used as such or is concentrated under oxygen-free nitrogen. Solid samples, e.g., foods, are either extracted with a solvent miscible with water (acetone, methanol) or, after dehydration of the sample, with a water immiscible solvent. Cleanup of the extract and fractionation of the pigments may involve saponification and/or open-column chromatography. 

In pharmaceutical preparations and fruit juices, ascorbic acid is readily separated from other compounds by TLC on silica gel and quantified directly by absorption at 254 nm. Serum and plasma may be deproteinized with twice its volume of methanol or ethanol. Various ascorbic acid compounds in plant extracts and foods have been separated on cellulose layers and detected by spraying with 2,S dichlorophenol indophenol (36). Heulandite, a natural zeolite (particle size 45 p) has successfully been employed as an adsorbent and ascorbic acid and other hydrophilic vitamins have separated within 5 cm by ascending chromatography in dimethylformamide (37). HPTLC and OPLC methods have been developed to improve the separation of ascorbic acid from other water soluble vitamins, with mixed success (11). 

Column-switching techniques for the determination of dmgs in biological fluids usually involve on-line solid-phase extraction on reversed-phase columns coated with albumin or modified with peptides and enzymes. To determine a-tocopherol and retinol in serum, Japanese investigators developed a unique sample preparation involving treatment of the serum with SDS and ethanol, deproteinization and trapping of the analytes on a BSA-80TS precolumn, followed by stepwise... 

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. 

Blood should be deproteinized by some technique which leaves no extra salt, acid, or alkali in the supernatant Some suitable techniques are with tungstic acid, with ethanol (BIO), or with zinc sulphate and barium hydroxide (S21). The supernatant is desalted in the same way as urine and, if necessary, concentrated before applying to the paper. Subsequent technique is as for urine. 

Homocarnosine is a dipeptide of GABA and L-histidine. After deproteinizing the sample with ethanol, the mixtures are centrifuged. The clear supernatant is evaporated to dryness and derivatized with butanol. The sample is evaporated to dryness and redissolved in the mobile phase. The homocarnosine-butyl derivatives (Fig. 2.3.4) are quantified using liquid chromatography mass spectrometry/mass spectrometry (LC-MS/MS) operating in the positive mode. With multiple reaction monitoring (MRM), the transitions of m/z 297.0 to m/z 212.0 for homocarnosine and m/z 299.0 to m/z 212.0 for 2H2-L-homocarnosine are quantified. 

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). 

Carbadox, olaquindox, and their monoxy- and desoxy- metabolites are amenable to extraction from tissues and eggs with polar organic solvents. Sample extraction/deproteinization is usually accomplished with ethanol (413), acetonitrile (414, 415), and acetonitrile/methanol (416-418). To reduce interferences and concentrate the analyte(s), the primary sample extract is further subjected to... 

Bcekawa et al. [285] successfully separated thirteen amino acids on a 4-m column coated with 1.5% of SE-30 with temperature programming (170—230°C). Only Thr and Ser were not separated on this column they can, however, be separated on 1.5% of XE-61. Prior to the analysis, DNP derivatives of these amino acids were silylated by using HMDS-TMCS in pyridine (2 1 5) at room temperature for 20 min. The method was applied to the analysis of amino acids in serum, as follows. To 0.5 ml of serum, 1.5 ml of ethanol was added in order to deproteinate it. The mixture was centrifuged, the liquid layer drained off and the residue washed with 0.5 ml of water and 1.5 ml of ethanol. Combined extracts were adjusted to pH 3 and washed twice with 5 ml of chloroform. To the aqueous layer, 2 ml of phosphate buffer (pH 8.8), 1 ml of 5% dinitrofluorobenzene in ethanol and 3 ml of ethanol were added and the mixture was shaken at 40°C for 2 h, then extracted three times with 30 ml of diethyl ether. The ethereal layer was washed with the buffer solution and the washings were added to the aqueous layer, which was then acidified with 15 ml of 10% HC1 and extracted with three 20-ml portions of ethyl acetate. 

In HPLC, alcohols and especially acetonitrile are often added to the sample to remove serum proteins. In CE, in addition to removing proteins, the presence of acetonitrile in the sample leads to stacking and indirectly improves precision of quantification because of the ability to increase the sample volume. We have analyzed several drugs and other natural compounds by CE after acetonitrile deproteinization. Protein removal can also be accomplished by alcohols such as ethanol and by acids such as perchloric. Precipitation with acids is less desirable in CE than with organic solvents because it increases the salt load. Following precipitation, proteins could also be dissolved in the appropriate buffers and assayed by CE. 

Procedure. Deproteinize about 10 ml. of blood, containing more than 100 /Lig of morphine, with acetone or ethanol. Centrifuge off the precipitate, distil off the acetone and dissolve the residue in 0-3 ml. of 0-1 n hydrochlorie acid. Place an aliquot portion of this solution on a paper strip, and prepare a one-dimensional chromatogram using a butanol-acetic acid mixture. Detect the position and area of the spots from the fluorescence after irradiation by a u.v. lamp. Cut a square of paper, about 2 cm broad, around the spot and place it in a test tube. Elute the paper with 2 ml. of 1 N hydrochloric acid for 12 hr, then with another 2 ml. of 1 N hydrochloric acid for 2 hr and finally with 1 ml. of 1 N hydrochloric acid for 1 hr. Nitrate and carry out the determination in the joint eluates by the procedure given on p. 118. 

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