Nucleotides HPLC analyses

HPLC is frequently employed in the analysis of amino acids, peptides, proteins, nucleic acids, and nucleotides. HPLC is also often used to analyze for drugs in biological samples (see Workplace Scene 16.2). Due to the complex nature of the molecules to be analyzed, these techniques tend to be more complex than HPLC applications in other areas of analytical chemistry. For example, separation of nucleotides or amino acids is more difficult than testing for caffeine in beverages, even though the same instrument and same general methods would be employed. A variety of columns and mobile phases are regularly employed. 

Most HPLC instruments monitor sample elution via ultraviolet (UV) light absorption, so the technique is most useful for molecules that absorb UV. Pure amino acids generally do not absorb UV therefore, they normally must be chemically derivatized (structurally altered) before HPLC analysis is possible. The need to derivatize increases the complexity of the methods. Examples of derivatizing agents include o-phthaldehyde, dansyl chloride, and phenylisothiocyanate. Peptides, proteins, amino acids cleaved from polypeptide chains, nucleotides, and nucleic acid fragments all absorb UV, so derivatization is not required for these molecules. 

HPLC analysis of bases and small nucleotides are best accomplished by RPC or IEC,31,39 as exemplified by the example shown in Figure 7.32. 

HPLC analysis of protein-bound nucleotides showed the absence of any nucleotides on the isolated P subunit (Fi re 1). The BSA control showed less than 200 picomoles of each of AMP, ADP and ATP. This negative result is in contrast with that seen for isolated CFi which contains a tightly bound molecule of ADP. 

Wulfson, A. N. and Yakimov, S. A., HPLC of nucleotides. II. General methods and their development for analysis and preparative separation an approach to selectivity control, J. HRC CC, 7, 442,1984. 

Figure 11.4 Analysis of in vitro synthesized RNAs. 32P-Radiolabeled RNAs (48 nucleotides) capped with m7Gp3G (A and C) or m27,3 °Gp3G (B and D) were digested with either RNase T2 (A and C) or RNase T2 plus tobacco acid pyrophosphatase (TAP) (B and D) followed by anion-exchange HPLC on a Partisil 10SAX/25 column as described in the text. Fractions of 1 ml were collected, and the Cerenkov radiation was determined. The elution times of the following standard compounds, detected by ultraviolet (UV) absorption, are indicated with arrows 3,-CMP (Cp), S UMP (Up), 37-AMP (Ap), 3 -GMP (Gp), 3, 5 -m7GDP (pm7Gp), 3, 5 -GDP (pGp), 5 -GDP (p2G), 5 -GTP (p3G), and guanosine-SCtetraphosphate (P4G).

Analysis of regulatory networks involved in the adaptation of the metabolism of microorganisms to various environmental conditions, such as starvation, revealed the particular importance of nucleotides and amino acids. These compounds function as signals for starvation, precursors in metabolic pathways, energy sources or are involved in enz3une activity regulation [10,32,33,42,44,110-112]. They are usually determined off -line by HPLC [8,10,32,33,42,44,112], as chromatographic or electrophoretic determinations allow the simultaneous determination of all compounds of... 

Interferring compounds from the sample matrix can become a major problem in the analysis of cytokinins that normally occur at trace levels. Immunoaffinity purification methods that are based on polyclonal or monoclonal antibodies enable a selective single-step clean-up and concentration of a certain group of cytokinins. These methods are usually combined with various techniques of final analysis such as HPLC-UV, HPLC-ELTSA, HPLC-MS [277-279]. However, the antibodies prepared so far do not show suitable affinity to certain metabolites (O-glucosides, N -glucosides, nucleotides). 

Another alternative is the use of postlabeling, i.e., digestion of DNA to nucleotides, depurination, HPLC separation to isolate adducts, [ C]-acetylation reaction, HPLC to collect [ CJ-labeled adducts, and AMS analysis. 

LC-separation of low molecular-weight constituents of nucleic acids and intact nucleic acids was reviewed by Zadrazil [358,359], Brown [360] described an enzyme peak shift method verifying peak identities of nucleotides, Singhal [361] reviewed separation and analysis of nucleic acids and their constituents by ion-exclusion and ion exchange column chromatography, and Brown [31,362] summarized the latest developments and state-of-art in HPLC of nucleic acid constituents. Plunkett [363] dealt with the use of HPLC in research of purine nucleoside analogs. 

The monomer constituents of biomacromolecules can be separated and identified by various chromatographic methods, which have been employed extensively in biochemical analyses. High performance liquid chromatography (HPLC) is a popular technique for the analysis of small biomolecules (Lim, 1986). An application of HPLC to analyze nucleotides, a-amino acids and monosaccharides is shown in Table 2.5. 

Assay sensitivity was therefore enhanced by concentrating and isolating the adducts by HPLC prior to RIA. Recoveries of 80% were typical. The removal of cross-reacting species maximised the assay sensitivities which were now limited only by the quantity of DNA available for analysis. The adopted procedure was based on a 5 mg DNA sample, equivalent to 9.25 X10 nucleotides. The lower limits of detection of the EdA and EdC RIAs were 3 x 10 ° molecules and 1.5 x 10 ° molecules respectively. Thus the EdA RIA was capable of detecting 1 adduct in 3 x 10 nucleotides and the EdC RIA 1 adduct in 6 x 10 nucleotides. 

Figure 5 HPLC-m.d.d. analysis of a standard mixture of nucleotides and InsPy. Separation was performed on a 15cm x 0.5cm Mono Q column, Eluent A and B contained 9 and 14nmoir YCI3, respectively, and reagent C contained 200nmoll" PAR. The flow rate was 1.2 mimin for A/B, and 0.6 ml min for C. The gradient applied is depicted. The upper monitor tracing (A) was obtained by m.d.d. at...

Fig. 1A,B. pH]-Mevalonic acid incorporation into cytokinins by tobacco crown gall tissue. A Sephadex LH-20 column chromatography of the nucleotide-derived. BuOH-soluble fraction from compactin (5 jLiM)-treated tissue B HPLC of fraction 1 obtained from the analysis shown above (column juBondapak C,. gradient elution with 10-50% MeOH (containing 1% HOAc) in 30 min. 3 ml/min)...

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