Examination of guanosine and xanthosine nucleotides by HPLC and electrochemical detection

0039-9140/88 $3.00 + 0.00 Copynght 0 1988 Pergamon Press plc

Tolanto. Vol. 35, No. II. PP. 911-913, 1988 Printed in Great Britam. All rights reserved

EXAMINATION NUCLEOTIDES

OF GUANOSINE AND XANTHOSINE BY HPLC AND ELECTROCHEMICAL DETECTION

ANNA MARIA GHE, GIUSEPPE CHIAVARI and CECILIABERGAMINI Scuola di Specializzazione in Chimica Analitica, Dipartimento di Chimica “G. Ciamician”, Universita de Bologna, via Selmi, 2-40126 Bologna, Italy (Received 8 April 1988. Revised 21 May 1988. Accepted 24 August 1988)

Summary-The suitability of electrochemical detection (ECD) following HPLC separation of guanosine, xanthosine and adenosine nucleotides has been evaluated. Separation of five monophosphates of guanosine was achieved by using a reversed-phase column; di- and triphosphates have also been separated from monophosphates. Adenosine compounds are insensitive to ECD.

Putine and pyrimidine bases are important components of nucleic acids, coenzymes, and the media

of hormone action. Thus qualitative and quantitative analyses for these bases are under continuous development. Recent studies have demonstrated that in certain pathological situations there is a change in the concentration of mcleotides, nucleosides, and the corresponding purint bases in biological fluids. We can therefore think of these substances as biological markers.’ Particular importance from this point of view attaches to guanosine and its compounds, especially as neoplastic disease markers.’ A rapid and easily reproducible method for determination of guanosine and its nucleotides is desirable. Several methods are available, but it is the chromatographic techniques which are preferred when separation of components is necessary-as from a complex biological matrix-prior to determination. HPLC can be used successfully in the analysis of nucleotides and nucleosides and no derivatization is necessary.3.4 The possibility of coupling HPLC and electrochemical detection (ECD) should reduce the effect of interferences. Previous work related to HPLC/ECD examination of purine and pyrimidine bases has been reported.‘q6 We have studied’ bases and related nucleosides by HPLC/ECD, both in reversed-phase and ion-exhange chromatography, and verified that not all bases and related nucleosides are electrochemically active. The aim of this work is to complete the study, examining the nucleotides of guanosine. adenosine and xanthosine by HPLC/ECD. We have used both

reversed-phase and ion-exchange columns with the aim of establishing the optimum isocratic conditions. Use of the gradient technique with an electrochemical detector is not advised, as unacceptable noise is generated

by the changes in Polat%

and composition

of the eluents during the analysis.

EXPERIMENTAL Apparatus

A Hewlett-Packard model 1OlOA liquid isochromatograph with a Rheodyne 7120 sample injector (20 ~1 loop, Varian model 2150) was used. The detectors were a Varian model 2550 variable wavelength detector and an ESA Coulochem electrochemical detector (model 5100) equipped with a model 5010 analytical cell. The output was recorded with a Houston Gmniscribe or a Varian model 4290 integrator recorder. The columns used were an Erbasil Cl8 10 p (250 mm x 4.6 mm) and a Whatman Partisil PXS IO/25 SAX (250 mm x 4.6 mm). Reagents The eluents used were: (a) KH,PG, (0.02&f) and methanol (95:5 v/v), pH 5.8; (b) KH,PO, (0.02M). pH 7; and (c) KH,PO, (0.25M), pH 6.8. The pH was adjusted by addition of acid or base to the buffer solution and measured with an Orion Research pH-meter (model 201) with an accuracy of kO.01. Carlo Erba solvents (HPLC grade) and reagents (RPE grade) were used to prepare the eluents. The standards were 5’-guanosine monophosphate (5’-GMP), 3’-guanosine monophosphate (3’-GMP), 2’guanosine monophosphate (2’~GMP), cyclic 2’,3’-guanosine monophosphate (2’,3’-GMPc), cyclic 3’,5’-guanosine monophosphate (3’,5’-GMPc), S-guanosine diphosphate (5’GDP), 5’-guanosine triphosphate (5’-GTP), guanosine, 5’-xanthine-monosphosphate -(5’-XMP), 5’-xanthosine diphosphate (5’-XDP), S-xanthosine triohosohates (5’-XTP). 5’-adenosine monophosphate (5’-AhiP), 5’-adenosine di: phosphate (S-ADP) and 5’-adenosine triphosphate (5’-ATP), supplied by Sigma, and of the highest purity available.

The standard solutions and eluents were filtered through a 0.45-pm plastic Millipore filter. For ultraviolet measurements a wavelength of 250 nm was chosen because the substances examined give maximum absorption at this wavelength. All experiments were done at room temperature, and at least in triplicate. Voltammetric measure-

ments were made with an AMEL model 556 potentiostat equipped with a Hewlett-Packard model 704OA x-y recorder. A three.-electrode cell was used with an AMEL GC492 glassy-carbon working electrode, a platinum counter-

electrode and a saturated calomel reference electrode. 911

912

SHORT

COMMIJNlCATlONS

Table I

k = (1~- hJ/h, PXS 10125 SAX

Cl8 Nucleotides

Eluent (a)

Eluent (6)

Eluent (c)

5’-GMP 3’-GMP 2’,3’-GMPc 2’-GMP 3’,5’-GMPc 5’-GDP 5’-GTP Guanosine S-XMP 5’-XDP 5’-XTP 5’-AMP’ 5’-ADP* 5’-ATP*

0.3 0.9 1.3 2.0 2.3 0 0 5.2 0.4 0 0 1.3 0 0

25.4 25.4 12.6 30.2 12.6 -

0 0 0 0 0 1.7 26.5 0 1.4 23.2 0

pp.

0.25

-

Flow-rate 1 ml/min. Ultraviolet detection at 250 nm. *Electrochemically inactive. RESULTS AND

DISCUSSION

HPLC separation of nucleotides Nucleotides of guanosine, xanthine and adenosine were considered. Table 1 gives the capacity factors (k) of these compounds on the Erbasil Cl8 and Partisil PXS IO/25 SAX columns. It appears that an isocratic separation of the mono-, di, and triphosphates of guanosine and xanthosine is not practicable. The reversed-phase column, however, gives a good separation of the five guanosine monophosphates. With the ion-exchange column PXS lo/25 SAX and eluent (b) (Table 1) there is a predictable inversion of the retention times of the different substances, but the separation between the five guanosine monophosphates is poorer. S-GMP and 2’-GMP are co-eluted and so are the two cyclic monophosphates 2’,3’-GMPc and 3’,3’-GMPc.

V Fig. 2. Monophosphates: relationship between electrochemical detector response and applied potential. Erbasil Cl8 column, eluent (a); sample solutions 10-5M. With eluent (c) (Table 1) it is possible to separate the di- and triphosphates from the monophosphates, which all have k = 0. Figure 1 shows the separation (Erbasil Cl8 column) of guanosine from its five monophosphates. Electrochemical detection of nucleosides The change in oxidation state during ECD of nucleotides and nucleosides is related to the structure

1

min

Fig. I. Separation of guanosine monophosphates on an Erbasil Cl8 column, eluent (a). Electrochemical detection (oxidation potential + 1.0 V). I = 5’-GMP, 2 = 3’-GMP, 3 = 2’,3’-GMPc, 4 = 2’-GMP, 5 = 3’,5’-GMPc, 6 = guanosine.

1.0

1

I

I

I

0.9

0.6

0.7

0.6

V

Fig. 3. Diphosphates: relationship between electrochemical detector response and applied potential. Erbasil Cl8 column, eluent (c); sample solutions 10-5M.

913

1 0

I

5

1 10

min Fig. 4. EIe&ochemicat detection at + 1.DDV; 1,Y-GMP; 2, 5’-XMP; 3, 3’-GMP; 4, S-AMP, 5,2’,3’-GMPc; 6,2’-GMP; 7, 3’,5’-GMPc. of the corresponding base. Our previous works showed that when the purine base was deetrochemically active, the related nucfeotides were oxidizable and therefore detectable by ECD. The present study, extended to nucleotides, confirms this behaviour. The electroactivity of the test compounds was investigated to find the best wurking potentids for use of LSV (linear sweep voltammet~) methods. An anodic peak with an Ep of about + 1.0 V, showing irreversibility characteristics under the experimental conditions (20 mV/sec), was observed. Over the range of potentials examined (down to - 1.0 V) no reduction was apparent. Figure 2 shows the response (current tis. applied potential) for the guanosine and xanthosine monophosphates, and Fig. 3 gives the corresponding results for the diphosphates. The electrochemical response of the monophosphates begins at +0.7 V (with a maximum at about +0.95 V) whereas a tower potential is sufficient for the diphosphates (+0.6 V). Adenosine and related nucleotides do not give oxidation curves. Figures 4 and 5 show the ultraviolet and ECD responses for a mixture of

I 0

I

s

J 10

min

Fig. 5. Ultraviolet detection at 250 nm; identification of peaks as in Fig. 4.

monophosphat~. The detection of 2’,3’-GMPc in the presence of S-AMP is difficult with an ultraviolet detector but possibIe by ECD because the adenosine compounds do not given an ECD signal. The detection limits found for the mono- and diphosphates are around 10 pmole with either detector. The sensitivity for the triphosphates is poorer, toward 5 nmoie, with both detectors, because of the “flattening” of the peaks owing to the higher retention times of these substances. REFERENCES

A. Hartwich and F. R. Brown, J. ~~~~~r~~~ 1976, rw, 769. Chaghou Yi, J. L. Fasching and P. R. Brown, ibid,, 1985, 339, 75. J. Stadler, Anal. Biochem., 1987, 86, 477. J. B. K&l, H. Y. Cheng and T. A. Last, Anal. Chem., 1986, 58, 285. A. M. Ghe, G, Chiavari and J. Evgenidis.,~~~fa* 1985,

I. R.

2. 3. 4, 5.

33, 379.

6. R. J. Henderson and C. A. Griffin, J. Cbromatog., 1984, 298, 231.