Adsorption of catecholamines

Earlier fluorometric methods for analysis of urinary free catecholamines have been replaced by HPLC methods that allow selective quantitation of epinephrine, norepinephrine, and dopamine. Preliminary extraction of urine is stid required and numerous preanalytical cleanup techniques are available. An alumina extraction procedure is typically coupled with ion-exchange or adsorption chromatography. Alumina pretreatment usually involves a batch extraction technique in which catechols are first adsorbed at pH 8.6 and then eluted with boric acid, which forms a complex with cis-diol groups. Purification on boric acid affinity gels provides an alternative procedure for selective adsorption of catecholamines. 

All reagents used for extraction and the mobile phases should be of high purity and prepared in double-distilled deionized water. Small (2-5 ml) stoppered polypropylene tubes are used for the aluminum oxide extraction/adsorption of catecholamines, and stoppered tubes of a similar size for organic solvent extractions. All glassware washing should incor-... 

Electrochemical detectors, which are based on the electrochemical oxidation or reduction of the analyte, can be applied to the analysis of selected compounds such as phenols. It is physically simple, but is very sensitive for catecholamines. However, the adsorption of reacted molecules on the surface of the electrodes can reduce the conductivity. To overcome this problem a pulsed voltage is applied, which cleans the electrode surface between measurements. This pulsed amperometric detection is also sensitive for carbohydrates. 

Solid phase extraction. With the availability of pre-prepared cartridges of silica-based adsorbents, the use of solid phase extraction has increased in the last few years although the technique has been in use for many years for the isolation of many biochemicals, e.g. amino acids, catecholamines. In essence it is a version of chromatography conditions for the selective adsorption of the analytes (column, solvent, pH, etc.) are chosen, the sample is applied to a column, washed and the analytes selectively eluted with appropriate solvents. Since the columns are disposable there is no need to worry about protein contamination or infection. The adsorbents available cover an even wider range than HPLC materials since they are not required to withstand high back pressures. It is possible... 

Eiquid- or solid-phase extraction methods have been adopted for the isolation of catecholamines and their metabolites from urine samples. The liquid extraction system is ordinarily based on the formation of a complex, in alkaline medium, between diphenylborate and the diol group in the catecholamines. However, the liquid extraction methods reported in the literature are relatively tedious and often involved multiple extraction steps.For the more widely used solid-phase extraction methods, catecholamines may be selectively isolated from the urine sample by adsorption with activated alumina," " phenylboronic acid or cation-exchange resins. All the specimen preparative procedures are specific for the free catecholamines, i.e. the extracted catecholamines do not include the conjugated fraction. 

The procedure for extraction of catecholamines with activated alumina was developed by Anton and Sayre, and has subsequently been used in a number of studies. Alumina extraction has not been popular, although automated purification with alumina microcolumns was studied closely by Tsuchiya et al. The sample preparation scheme includes increasing the pH of the alumina to >8.5 and vigorous shaking of the sample with the alumina, resulting in adsorption of the catecholamines by attraction of the hydroxyl groups of the catechol nucleus. The alumina can then be washed with water or buffer, and finally the catecholamines are eluted with acid, such as 0.3 m acetic acid. Since catecholamines are... 

Recent applications of dimethylalkylsilylation include the use of DMNPS ethers for the analysis of catecholamines [377]. The DMNPS derivatives showed enhanced stability during adsorption chromatography (silica gel) compared with the corresponding TMS derivatives. In GC-MS analyses employing ammonia Cl with SIM of the [M-l-NH4l ion (base peak), the detection limit for... 

Ion pairing agents in liquid-liquid systems in reversed-phase mode have included dihydrogenphos-phate for separation of tricyclic amines, octyl sulfate for catecholamines, and tetrabutylammonium for aromatic carboxylates and anions of sulfonamides, to exemplify some of the comparatively few applications. Liquid stationary phases coated on the alkyl-bonded phase include 1-pentanol, butyronitrile, and tributylphosphate. In normal-phase liquid-liquid ion pair chromatography aqueous perchlorate solution has been coated on to silica particles for ion pair separation of catecholamines and related compovmds and tetrabutylammonium ion at neutral pH for carboxylates and anions of sulfonamides. The organic mobile phase often contained dichloromethane and butanol. In the normal-phase mode on silica alternative separation systems have been described with aqueous perchloric acid in methanol added to dichloromethane as mobile phase for separation of amines such as drug substances. This is not an extensively utilized, but quite useful, kind of separation, which has been named ion pair adsorption chromatography. 

HPLC measurements of plasma catechols are usually limited to dopamine, norepinephrine, and epinephrine. However, with an alumina adsorption extraction procedure it is also possible to simultaneously measure several other catechols by HPLC or microchip electrophoresis. These catechols include DHPG, the deaminated metabolite of norepinephrine and epinephrine DOPAC, the deaminated metaboHte of dopamine and 3,4-dihydroxyphenylalanine (r-dopa), the immediate precursor of dopamine. All are present in plasma at concentrations many fold higher than the catecholamines, making their detection relatively simple once appropriate chromatographic separation is achieved. 

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