Tissue deproteinization

Owing to the complexity of multi-residue methods for products of animal origin, it is not possible to outline a simple scheme however, readers should refer to methods described in two references for detailed guidance (Analytical Methods for Pesticides in Foodstuffs, Dutch method collection and European Norm EN 1528. ) There is no multi-method specifically designed for body fluids and tissues. The latter matrix can be partly covered by methods for products of animal origin. However, an approach published by Frenzel et al may be helpful (method principle whole blood is hemolyzed and then deproteinized. After extraction of the supernatant, the a.i. is determined by GC/MS. The LOQ is in the range 30-200 ag depending on the a.i.). 

Polymer Labs. PLRP-S 0.01M oxalic acid-ACN (75 25, v/v) UV 360 nm Animal tissues Extraction with oxalic buffer followed by chelation and deproteination, cleanup with styrene-divinylbenzene cartridge Rec 76-87% [77]... 

In each of these methods, undiluted serum, urine, acid-deproteinized milk, or a buffered saline extract of muscle was mixed with sulfamethazine-horseradish peroxidase and added to antibody-coated wells of a microtiter plate. A sulfamethazine-bovine serum albumin conjugate prepared by the glutaraldehyde procedure (56) was used for antibody production. Results showed that screening of serum was of value since sulfamethazine concentrations in serum directly correlated with those in swine tissues. Thus, for example, a level of 100 ppb of sulfamethazine in... 

For efficient extraction of macrolide and lincosamide residues from edible animal products, bound residues should be rendered soluble, most if not all of the proteins should be removed, and high recoveries for all analytes should be provided. Since tliese antibiotics do not strongly bind to proteins, many effective extraction methods have been reported. Sample extraction/deproteinization is usually accomplished by vortexing liquid samples or homogenizing semisolid samples with acetonitrile (136—139), acidified (136,140-142) orbasified acetonitrile (143), methanol (14, 144, 145), acidified (145-147) or basified methanol (148), chloroform (149-151), or dichloromethane under alkaline conditions (152). However, for extraction of sedecamycin, a neutral macrolide antibiotic, from swine tissues, use of ethyl acetate at acidic conditions has been suggested (153), while for lincomycin analysis in fish tissues, acidic buffer extraction followed by sodium tungstate deproteinization has been proposed (154). 

Alternative sample extraction techniques include an approach that combines the deproteinizing efficiency of dichloromethane with the ion-pairing ability of phenylbutazone for isolating tetracyclines from eggs (308). Another approach that was employed for extracting oxytetracycline from milk (285) or swine tissues (309), and tetracycline, oxytetracycline, and chlortetracycline from milk (284), was based on ultrafiltration. With ultrafiltration, however, not all low molecular-mass proteins are retained in the cut-off filters while interfering substances pass through the filter. 

Extraction/deproteinization has been performed by either vortexing liquid samples or homogenizing semisolid samples with acetonitrile (227, 382, 383, 386-392), methanol (14, 393-395), methanol/water mixtures (396-401), ethyl acetate (384,402-406), dichloromethane (379,380,407), and acetone (408,409). Nonpolar organic solvents, such as isooctane (410, 411) and toluene (407), have also been reported to work extremely well for extracting salinomycin and dimetridazole from chicken tissues, respectively. Sample extraction with these nonpolar solvents yields a cleaner extract and an easier workup than extraction with commonly used polar solvents. However, selecting an extraction solvent is critical in establishing an analytical method because it is closely related to the cleanup systems. 

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... 

Sample extraction and deproteinization is usually accomplished with nonpolar organic solvents at a specified pH. Organic solvents such as chloroform for the determination of cortisol in milk (529) dichloromethane/hexane (4 1) for the determination of free cortisol and its 21-acetate in milk (530) ethyl acetate for the determination of prednisolone, fluoroprednisolone, triamcinolone, and betamethasone in animal tissues (531) methylprednisolone in milk (532) fluoro-prednisolone in milk (533) and dexamethasone in milk (534) ethyl acetate in... 

A multiresidue procedure for /3-lactams in milk (67) was adapted for the determination of free CEF metabolites—DFC dimer and DFC cysteine conjugate. The extraction and deprotein-ization procedure was modified for tissue samples. For LC analysis, an ion-pair chromatographic system gave satisfactory separation from the interference in the LC fractions. The recoveries of this procedure were 54-61% with CV < 10% (71). 

Electrophoretic patterns of highly supercoiled or partially supercoiled DNA. Strip A represents a sample of circular duplex DNA obtained by deproteinization of the animal virus SV40. In strips B and C the DNA has been exposed for increasing times to an enzyme (topoisomerase) that catalyzes relaxation. Adjacent bands differ by 1 in linking number. (From W. Keller, Characterization of purified DNA-relaxing enzyme from human tissue culture cells, Proc. Natl. Acad. Sci. USA 72 2553, 1975.)... 

Although there have been numerous attempts to show the presence of other minerals (brushite, whitlockite and octacalcium phosphate ) as primary constituents of normal bones and teeth, there is no straightforward evidence that such tissues contain any mineral other than dahllite (McConnell, 1973a). This statement applies also to possible precursors within these tissues, and also to a so-called amorphous calcium phosphate, which has been assumed to be present on the basis of spurious, indirect evidence even in the case of nascent dental enamel. An electron diffraction pattern of non-deproteinized bone is shown as Fig. 3.1.12. 

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. 

Provided the tubes of deproteinizing solution are prepared in advance of the biopsy, then the reaction mixtures, and last the homogenates, it is possible to measure all 5 enzymes, including ornithine transcarbamylase at two different pH s, in a tissue specimen in 1 hour. The homogenates are kept in ice until they are used, and the last incubation is begun about 30 minutes after the homogenates are made. At least 50 mg of liver is required. 

The pink azo dye formed is completely soluble in alcoholic borate solution the color was remarkably stable even up to 24 hours at room temperature. The method is so sensitive that a fraction of a microgram of phenol can be estimated with ease. It is very economical of enzyme, requiring only 0.1 ml of properly diluted serum or tissue preparations, and the tedious deproteinization step becomes unnecessary. 

Poor tissue morphology can, among other things, be due to excessive deproteination during the pretreatments or the formation of ice crystals during freezing. Clearly, the use of nonradioactive systems is particularly helpful for a rapid optimization of ISH. 

Digestion and deproteinization of PAH-treated skin tissue extraction and precipitation of DNA hydrolysis with 1.2 M HCl... 

Urine samples are unstable, and calcium phosphate precipitates out, entrapping metal ions or other substances of interest. Precipitation can be prevented by keeping the urine acidic (pH 4.5), usually by adding 1 or 2 mL glacial acetic acid per 100-mL sample. Store under refrigeration. Urine, as well as whole blood, serum, plasma, and tissue samples, can also be frozen for prolonged storage. Deproteinized blood samples are more stable than untreated samples. 

Termine JD, Eanes ED, Greenfield DJ, Nylen MU, Harper RA (1973) Hydrazine-deproteinated bone mineral Physical and chemical properties. Calcif Tissue Res 12 73-90 Termine JD, Posner AS (1967) Amorphous/crystalline interrelationships in bone mineral. Calcif Tissue Res 1 8-23... 

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. 

A high-throughput method that combines on-line extraction and determination by LC-MS/MS has been developed for the screening of 13 multi-class antibacterials (macrolides, fluoroquinolones, lincosamides, and trimethoprim) in different animal muscle tissues. After sample deproteinization with acetontirile, the extracts were directly loaded onto the SPE cartridge, packed with an Oasis HLB... 

Samples were deproteinized with 0.2 M HC104- Before reaction with 0PA-thiol, a known amount of 3-3 -diaminodipropylamine was added to the tissue homogenates as an internal standard and perchloric acid extracts were adjusted to pH 9.5 by addition of borate buffer. For quantitation, polyamines were compared to the internal standard. The peak height ratios of polyamine to internal standard for a known concentration of polyamine was presented in Table 1. 

For the preparation of analytical samples the following procedure is recommended [419]. The deproteinized biological sample (serum or tissue extract) (0.5 rql) is mixed with 1.5 ml of a 0.13% solution of o-phenylenediamine (1.33 mg ml in 3 N HCl prepared daily), 5 /il of mer-captoethanol is added, and the volume is adjusted to 3 ml with water. The mixture is heated for 30 min in boiling water. In order to terminate the reaction, the tubes are cooled on ice and 0.5 g of anhydrous sodium sulphate is added. The quinoxalinols are extracted with three 3 ml portions of ethyl acetate. The pooled extracts are dried over anhydrous sodium sulphate and evaporated to dryness. The residue is dissolved in 0.2 ml of methanol and the solution is centrifuged and filtered through a 0.45 fan pore size filter. The filtrate is ready for HPLC using a reversed phase column. 

T IS ds-T Rat brain (100 mg) extraction (methanol-acetic acid) RP-SPE preparative NP-HPLC derivatization with 2-hydrazino-l-methylpyridine Rat serum (50 p,l) deproteinization (methanol-acetic acid) RP-SPE derivatization with 2-hydrazino-l-methylpyridine J sphere ODS H-80 (150 X 2.0 mm I.D., YMC), acetonitrile-methanol-10 mM ammonium formate and 0.2 ml/min API 2000 (Applied Biosystems), positive ESI, SRM ([M]+ residual [M]+) LOQ 0.06 ng/g tissue (brain) and 0.06 ng/ml (serum) [37]... 

Direct injection of supernatant The weighed tissue is sonicated or homogenized in 10 volumes of 0.1 to 0.2 M perchloric acid (e.g., Bennett et aL, 1981a Reinhard et al., 1980 Mefford and Barchas, 1980 Mefford, 1981). Supernatants from release studies can also be deproteinized by addition of perchloric acid (20 jjlI 0.1 M per 200 jxl sample) (Dayton et al, 1979 Bennett et al, 1981a Marsden et al., 1981). In both situations the samples are then centrifuged at 5,000-15,000 rpm for 10 min and 20-100 )xl of clear supernatant injected onto the column. The amounts of... 

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