Catecholamines ionization

Chan EC, Ho PC. 2000. High-performance liquid chromatog-raphy/atmospheric pressure chemical ionization mass spec-trometric method for the analysis of catecholamines and metanephrines in human urine. Rapid Commun Mass Spectrom 14 1959. 

Armstrong, J. and Barlow, R.B. (1976) The ionization of phenolic amines, including apomorphine, dopamine and catecholamines and an assessment of zwitterion constants. Br. J. Pharmacol. 57, 501-516. 

Following the adjustment of pH of the eluent to achieve, if possible, total ionization of the solutes, a counterion is added, starting with one of low hydro-phobicity, e.g., a short chain alkane sulphonate. The retention may be increased by increasing the alkane chain length or the concentration of the counterion. Fig. 4.4.12 shows a typical example of the separation of a mixture of catecholamines. It should be emphasized that the previously mentioned short equilibration times of bonded phase chromatography do not apply to ion-pair partition chromatography. The counterions used often exhibit a considerable affinity to the apolar stationary phase and also, eventually, to residual silanol groups, hence the column is able to adsorb a certain amount of counterions. Equilibrium is not established until the column is... 

Chemically, NE is classified as a catecholamine. A catechol is a 1,2-dihydroxybenzene, and NE is a p-hydroxyethylaminodihydroxybenzene. Epinephrine is the N-methyl derivative of NE, and they both have acidic and basic functional groups. Physiologically, however, they behave as a base, being more than 90% protonated at pH 7.4 (pKa = 9.6) and functioning as an ionized acid. 

Schiisler-van Hees, MTIW, Beijersbergen van Henegouwen GMJ and Driever MFJ, Ionization constants of catechols and catecholamines, Pharm. Weekblad, Sci. Edn., 5,102-108 (1983). 

Many metabolites both ionizable and nonionizable can be measured by different forms of CE such as CZE, MEKC, and chirality. Metabolites with strong UV absorption such as nucleotides, phenolic amino acids, and their metabolites are easy to measure by CE. However, some of these require concentration and clean up before the CE step [5]. This can be achieved by traditional concentrating methods, such as solid phase and solvent extraction or by concentration on the capillary (stacking). Examples of small molecules that have been analyzed by CE are nucleotides [79,80], amino acids, catecholamines [81-83], and sugars [84,85]. Below is a more detailed discussion of some of these compounds. 

Peterson, Z.D., Collins, D.C., Bowerbank, C.R., Lee, M.L., and Graves, S.W. Determination of catecholamines and metanephrines in urine by capillary electrophoresis-electrospray ionization-time-of-flightmass spectrometry. J. Chromatogr. B, 776, 221, 2002. 

In a zwitterion, each ionizing group has two constants, one for the fraction that has the other group ionized and one for the remaining fraction. These are called microscopic constants, and the macroscopic constants (as determined by spectrometry or potentiometric titration) are gross composites of these. Historically, tyrosine was one of the first zwitterions to be examined for microconstants (Edsall, Martin and Hollingworth, 1958) and the same methods are still used. See Section 12.4 for the pursuit of microconstants in the catecholamine series. 

Table 12.1 Ionization of natural catecholamines and related agonists. (Percentage, in water at pH 7.4 and 25°C)...

Attention has been centered on the haiogenated derivatives of primary amines. This development started with the introduction of electron capture detection systems. With pentafluoro-derivatives for example, detection by electron capture is over 1000 times more sensitive than by flame ionization. If hydroxyl groups are present, they are usually trimethylsilylated, which augments the electron affinity of the product. Picogram amounts of catecholamines have been separated on methylsilicone phases and detected by electron capture after derivatization with pentafluorobenzaldehyde and silylation [37,43]. Such methods can be a great aid in the ultra-trace analysis of primary amines in biological specimens. 

Ion pair LC in the reversed-phase mode is the overall dominating separation technique, not least within the biomedical field. Many compounds of interest occur in ionic form or are ionizable, and chromatographic separation can be performed by counterion-aided distribution. The range of applications is extremely wide and includes small hydrophilic aminophenols like catecholamines, carboxylic acids,... 

Delva, R Pastori, C. Began, M. Montesi, G. Lechi, A. Catecholamine-induced regulation in vitro and ex vivo of intralymphocyte ionized magnesium. J. Membr. Biol. 2004, 199, 163-171. 

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