Reversed phase nucleotides extraction

Juengling and Kammermeier (J2) reported the separation of purine nucleotides from rat heart extract using tetra-/t-butylammonium phosphate on a C-18 column. Although the chromatographic separation has poor resolution, they emphasize the need for a chromatographic technique for the simultaneous analysis of ionic and nonionic compounds that retains the flexibility of the reversed-phase mode. 

The purification procedure should remove potentially interfering compounds and, moreover, fractionate the entire spectrum of cytokinins into groups. It is usually not possible to analyze nucleotides and O-glucosides together with free bases, ribosides and iV-glucosides. Classical liquid-liquid partition steps [274] have been recently replaced by less labor-intensive, rapid and selective solid-liquid extraction. When applied in neutral aqueous solutions, cytokinin nucleotides are retained on weak anion-exchangers (DEA -Sephadex, DEAE-cellulose), while the other cytokinin forms are retained on reversed phase sorbents. Dobrev and Kaminek [275] used mixed-mode solid-phase extraction for step-wise... 

The relative merits of ion-exchange and reversed phase modes are discussed in a study of the nucleotide pools from mammalian tissues (Brown et al., 1982). The tissue extracts were initially purified on a Cu -loaded Chelex 100 resin to maximise both resolution and sensitivity during the main HPLC separations. It was concluded that the best routine method was on the anion-exchange column containing jaBondapak NH2 (Fig. 11.1.7), but where necessary this could be complemented with reversed phase or reversed phase ion-pair HPLC. 

PGA extracts were analyzed by HPLG using described methods (6). Purine nucleosides and bases were separated with a reversed-phase column. Purine nucleotides were separated by anion-exchange HPLG. Simultaneous UV monitoring and radioactivity detection were performed with an on-line radioactivity flow detector. 

A second advantage of the LC-ESI-MS approach is the increased mass accn-racy of the measurement, making possible the detection of small mass differences between proteins. These mass differences can be eqirivalent to single-nucleotide polymorphisms (SNP) mutations or post-translational modifications. Recent work by McFarland and coworkers (2014) best illustrates the implementation of LC-(ESI)-MS (intact protein mass) and MS/MS top-down analyses to bacteria differentiation at the strain level. Proteins extracted from bacterial samples of Salmonella typhimurium (strain LT2) and S. heidelberg (strain A3 9) were first separated by reversed-phase (RP) LC and the eluent analyzed directly by ESI-MS using Q-TOF MS system (operated in the full-scan mode or MS). Following the data processing... 

Usually, in reversed-phase systems, ascorbic acid is eluted within 3 to 10 min. However, as extracting biological samples there may be compounds that elute far after ascorbic acid. Gradient elution may be needed to shorten the column purification time between injections. Tanishima and Kita (74) used a stepwise increasing methanol concentration to remove uric acid from the column after plasma samples. Lazzarino et al. (88) separated ascorbic acid, uric acid, xanthine, hypoxanthine, malondialdehyde, and several nucleotide derivatives with ion pairing and methanol gradient in a reversed phase system. The method was used for determining those compounds in ischemic-reperfused rat heart. 

Dideoxy-adenosine, -inosine, and -cytidine have been determined in biological samples by both reversed-phase and ion-pair reversed-phase methods. An ion-pair (Bu" N ) reversed-phase analysis of purine nucleosides, bases, nucleotides, dinucleotides, and nucleoside derivatives (adenosyl-homocysteine, -succinate, and -methionine) from acid-extracted tissues was undertaken to investigate adenosine reservoirs in rats on treatment with a metabolic inhibitor which blocks adenosine transport. 

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