Protein precipitation, basic proteins organic solvent

Determinative and confirmatory methods of analysis for PIR residue in bovine milk and liver have been developed, based on HPLC-TS-MS (209). Milk sample preparation consisted of precipitating the milk proteins with acidified MeCN followed by partitioning with a mixture of -butylchloride and hexane, LLE of PIR from aqueous phase into methylene chloride, and SPE cleanup. The dry residue after methylene chloride extraction was dissolved in ammonium hydroxide, and this basic solution was transferred to the top of Cl8 SPE column. The PIR elution was accomplished with TEA in MeOH. For liver, the samples were extracted with trifluoroacetic acid (TFA) in MeCN. The aqueous component was released from the organic solvent with n-butyl chloride. The aqueous solution was reduced in volume by evaporation, basified with ammonium hydroxide, and then extracted with methylene chloride. The organic solvent was evaporated to dryness, and the residue was dissolved in ammonium acetate. The overall recovery of PIR in milk was 94.5%, RSD of 8.7%, for liver 97.6%, RSD of 5.1 %. A chromatographically resolved stereoisomer of PIR with TS-MS response characteristics identical to PIR was used as an internal standard for the quantitative analysis of the ratio of peak areas of PIR and internal standard in the pro-tonated molecular-ion chromatogram at m/z 411.2. The mass spectrometer was set for an 8 min SIM-MS acquisition. Six samples can be processed and analyzed in approximately 3 hours. 

It is easy to destroy micellar systems irreversibly by adding water-miscible organic solvents such as, e.g., acetone and ethanol. This technique is exceptionally effective and convenient when it is important to extract the solubilized protein (enzyme) delicately so that it retains its structure and physiological (including catalytic) activity [28]. Here, it should be emphasized that, first, the ethanol and especially acetone should be cold. Second, during precipitation with acetone in neutral and basic media, formation of Schiff bases is possible besides which, side reactions involving modification of the protein subjected to isolation (including the loss of the protein solubility in water) are also probable. 

Several extraction techniques have been reported in the literature for the analysis of sulfonamides. Because of their polar nature, sulfonamides are readily extracted by organic solvents ° ° the most commonly used are acetonitrile.Other organic solvents used for analyte extraction and protein precipitation include dichloromethane, " acetone, ethanol, chloroform, and ethyl acetate, " which are often used either alone or in conjunction with one another. Other techniques used for protein precipitation include the use of acids such as perchloric or formic and the use of basic buffers such as potassium hydrogen phosphate and ammonium sulfate. In the case of honey, the use of acids such as trichloroacetic, " " hydrochloric, and phosphoric is necessary for hydrolysis, releasing carbohydrate-bound sulfonamide residues. Other extraction techniques reported in the literature include the use of pressurized liquid extractions, " matrix solid-phase dispersion, and magnetic molec-ularly imprinted polymers. Of additional note, several authors have observed that analyte recoveries were largely... 

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