Determination of selected pyrimidines, purines and their metabolites in serum and urine by reversed-phase ion-pair chromatography

foumd E&via

of Chromatography, 199 f1980) 345-354 Scienfitic Publishing Cbnpany, Amsterdam -

tinted

in The Netherlands

cJxROM..f3,1u4

DETERMINATION OF SELECTED PYRIMIDINES, PURINES AND THEIR METABOLITES IN SERUM AND URINE BY REVERSED-PHASE ION-PAIR CEPROMATOGRAFHY

WOLFGANG Institut

fir

VOELTER*

Organkcke Chemie. Auf&r

Morgenstelle 18, D-7400 Tiibingen (G.F.R.)

and KARL

ZECH, PETER ARNOLD

and GERHARD

LUDWIG

Byk Gulden Lomberg, Forschungsabteilwrg, Byk-Gulden-Strasse 2, D-7750 Konstanz (G.F.R.)

SUMMARY

The qualitative and quantitative determination of selected pyrimidines, purines and azapurines and their metabolites by reversed-phase ion-pair high-performance liquid chromatography, using tetraburylammonium hydroxide as pairing ion and isocratic chromatographic conditions, is described. The method provides a sefective and sensitive assay for these classes of compounds examined in complex biological fluids.

INTRODUCTION

The analysis of pyrimidine and purine derivatives and their biotransformation products is important from a clinical point of view. This applies to disorders of purine and pyrimidine metabolism (gout) and subsequent treatment with azapurine’~* and inter-individual studies of the bioavailability and biotransformation oftheophylline3*+. Several workers described the determination of selected pukes and pyrimidines and their derivatives and metabolites in body fluids by high-performance liquid chromatography (HPLC) 5-8. However, the separation conditions used in these methods are only suitable for a limited number of compounds. Ion-exchange HPLC is used for rhe determination of uric acidLo in biological fluids and also caffeine, theopylline and hypoxanthine are separated by the same principle”*12. For the. separation of the azapurines ahopurinol and oxipurinol, three dierent HPLC methods are described: reversed-phase HPLC’, ion-cxchange HPLC and ion-exchange HPLC in combination with puritktion on CheIex-100 rcsi&. Recently Brown et aL8 described a reversed-phase paired-ion chromatographic system for the simultaneous determination of-hypoxanthine, xanthine, allopurinol and uric acid. Sodium acetate (pPH4.08~l-heptane sulfone acid was used as the mobile phase. However, under these l

Dedicatedto Praksor Dr. Dr. G. Weitzel on the occasion of his 65th birthday_

tl021-9673!so/ocXnrtXxlo/$02.25

0 1980 Ekevier Scieaitk PublishingCompany

346

W. VOELTER

et a!.

conditions it was not possible to separate hypoxanthine, xanthine, uric acid; allopurinol and oxipurinol. For the studies of reactions with the flavoprotein xanthine oxidase the simti: taneous determination of ah these compounds was essential. Therefore a more efficient reversed-phase paired-ion liquid chromatographic system was developed in our laboratories and will be discussedhere. The separation method is also suitable to determine substituted xanthine and their metabolites simultaneously in body fluids and appears to be an excellent tool for the study of the biotransformation of theophylline. EXPERIMENTAL

Apparatus

A Hewlett-Packard (HP) Model 1084B liquid chromatograph, equipped with an HP 798775A UV detector, was used. A ready-to-use column (250 x 4.6 mm I.D.; Knauer, Berlin, G.F.R.), filled with RP-18 (10 pm), was employed. Analytical procedures

Separation of purines, pyrimidines and their metabolites was achieved by means of ion-pair HPLC. Tetrabutylammonium hydroxide was added to the mobile phase as counter-ion. All separations were performed in the isocratic mode after addition of different amounts of acetonitrile to the aqueous mobile phase. Equilibration of the column was .usualIy obtained with 50 ml of mobile phase. Chemicals and compounds investigated

The purines, pyrimidines and their derivatives were obtained from commercial sources (Serva, Heidelberg, G.F.R.; Ferak, Berlin, G.F.R.). Acetonitrile (LiChro-

sol@) was purchased from Merck (Darmstadt, G.F.R.) and tetrabutylammonium hydroxide from Fluka (Buchs, Switzeriandj. The structures of the compounds investigated are given in Table 1. Sample preparation Urine_ An ahquot of a urine sample was adjusted to pH 7.5 and diluted with

the eluent in the ratio of 1:2. A 1%I5-~1 volume of this solution was injected directly into the separation system. Senrm. A OS-I&ml volume of a serum sample was centrifuged for 20 min at ca. 15Og using core-shaped filter inserts (Centriflo type 2200 CF 50) and a 4crlOO-~l aliquot of the ultrafiltrate obtained was injected directlyU-15. RESULTS AND DISCUSSION

Chromatography KFIorder to

obtain optimal selectivity and resolution for the analysis of purines and pyrimidines, the dependence of the capacity ratio (k’) on the column temperature, molar&y of counter-ion, pH and concentration of acetonitrile in the mobile phase were investigated.

HPLC OF PYREMIDINW,

PURINES

AND THEIR METABOLITES

341

TABLE I STRUCTURES

OF -mGATED

PURINES

PYRXMIDINES Name

Rl

Formu la Furhles

AND

HO HO H

J$J H

HO HO

HO

HO H H

Uric acid Xanthine Hypoxanthine

H CH3 CH3 H H

Theophylline Thcobromine Caffeine 1-Methylxanthine 3-Methykanthine I-Methyluric acid 1,fDimethyluric acid

N

Substituted xanthikles

CHs

H CHJ CHj H CH, CH2

Substituted uric acids

‘Z%

CH3 CH3 H CH, H CHr

Orotic acid

Puke metabolite H HO HO

Azapurines

H HO

AlIopuri~~ol Oxipurinol

Infruence ofcouprter-ion concentration on the capacity r&o. The influence of the molar&y of the counter-ion and the column temperature at pH 7.5 and an acetonitrile concentration of 2.5% on k’ for compounds 1-8 is demonstrated in Tables II-IV. The values in Tables H-IV clearly demonstrate a general trend for the capacity factors (with the exception of theophylline, 7): the highest k’values are found at a TABLE II k’ VALUES

FOR 5 mM COUNTER-

ION CONCENTRATION

Column: 125 x 4.6 mm I.D. Stationary phase: RP-18 (5,~m). Flow-rate: 1 mI/min. Compounds investigated:theobromine (I), uric acid (2). 3-methykanthine (3), I-methykanthme (4). I-methyluric acid (5). caffeine (6). theophylline (7) and 1.3-dimethyluricacid (8). Compound Temper&rue(“CT)

1 2 3 4 5 6 7

3.5

50

60

70

3.47 3.47 3.47 4.58 7.43 10.63 15.12

271 3.34 3.34 4.07 6.16 8.23 9.86

2.02 2.79 3.05 3.56 5.07 7.05 1283

1.75 252 2.90 3.27 437 6.17 10.28

W:VOELTER

348

et al.

TABLE tD[ k’ VAJXJES FOR 10 mM COUNTER-ION Conditions as in Table II.

CONCENTRATION

Conq~oumi Temperature(“C) 30

iI: 4.40 5.71 8.67 11.11 1273. 23.77

TABLE

JO 2.84 3.66 4.08 5.16 7.49 9.86 9.86 19.71

so 237

3.34 3.88 4.72 6.41 8.58 7.80 16.21

60

70

2.07 3.06 3.75 4.35 5.62 7.79 6.24 13.31

1.74 2.67 3.45 3.82 4.69 7.06 4.96 10.59

IV

k’ VALUES

FOR 20 m&f COUNTER-ION

CONCENTRATXON

Conditions as in Table IL

____-

Compound Temperature(“C) 35

50

60

70

:

2.10 4.40 3.27

1.66 3.99 3.21

1.44 3.71 3.15

1.30 3.49 3.11

6 7 8

9.07 11.00 -

7.71 7.29 14.76

7.03 5.70 Il.89

6.64 4.65 10.19

1

concentration of 10 mM; at the higher and lower concentrations lower values of k are observed. InpUnce of ph’ on de &zpczcity ratio. me influence of pH and column temperature on the capacity ratios for the eight compounds is obvious from Figs. I-3. As expected, the k’ values decrease with increasing column temperature. At pH 7 (Fig. 1)5 theobromine (I), uric acid (2) and 3-methylxanthine (3) (at column temperatures of 35-5O”C), and l-methyluric acid (5) and caffeine (6), have identiwl k’values. Much better resolution can be achieved at pH 8.0; however, the k’ values of 3-meth~lyanthine (3) and I-methylxanthine (4) coincide at higher temperatures. As indicated in Fig. 2, the compounds are best separated at pH 7.5. However, the elution order may be dependent on the column temperature for purine derivatives, as is seen for caffeine (6) and theophylline (7) (Fig. 2) Inf;‘uence of acetonitrile concentration on the capacity ratio. From the above results, purine derivatives are best separated at pH 7.5, a column temperature of 35°C and a count&r-ion concentration of 10 mM. Using these parameters, the dependence of the capacity ratio on the acetonitrile concentration was studied (Fig. 4). Similar investigations were undertaken for the separation of xantbine, hypoxanthine, uric acid, the azapurine ailopurinol, its metabolite oxipurinol and erotic

HPLC OF PYRIMID INES, PURINES

k

AND THEIR ME?XBOUTES

349

25

11

70

80

50

3s

Fig. 1. infkence ofcoIumn temperature on the capacity ratio (k’) at pH 7.0. Column: 125 x 4.6 mm I.D. Stationary phase: W-18 (5 pm). Mobile phase: 10 mM tetrabutylammonium hydroxide in water-acetonitrik (97.5:25). FIgw-rate: I ml/min. Compounds investigated: theobromine (l), uric

acid (2). 3-methylxanthine (3). 1-methylxanthine (41, I-methyluric acid (5), caffeine (6). theophylline (7) and 1,3_dimethyIuric acid (8).

7 6

1%

5 4

5

% 1 ~

1

I

I

1

I

333

40

50

60

I 70

Fig. 2. Influence of coolumn temperature on the capacity ratio (k’) at pH 7.5. Separation conditions and compounds as in Fig- 1.

4%

i0

60

GE

i0 Pa

Fig. 3. Influence of ~A~uI temperature on the capacity mtio (k’) at pH 8.0. Separation conditions and compounds as in Fig. 1. 25‘r t) 2%‘-

8------_

‘yx

2

10 -

ii ---._ t

5

-

1

-

+---__

---._~ -+---__

I.

---Wr

-----_-c

Fig_ 4. Influence of acetonitrileconcentration oti the capacity ratio (k’) at a col& 35°C. Separation conditions and compounds as in Fig. 1.

temperature of

HPLC OF PYREMIDINES,

PlLlRmEs

AND

THEIR

351

ME-IXBOLFIES

acid-: The .optimaf separation con&ions were found to be as follows: tetrabutylammonium ion concentration, IO m.M; ~$3, 8.0; acetonitrile conc-entiaf3on, 0.2%; arid column temperature,.35°C.

BiomalyseS

ofpurines ani2uzapurines

Serum. Fig. 5 shows the analysis of a serum sample after addition of hypoxanthine, xantbine, erotic acid, allopurinol and oxipurinol. The uric acid identified corresponds to endogenic serum uric acid, the only peak found in the analysis of a condiblank-sample using-the above-mentioned detect&-setting and separating tions.

A

c

TIME

(MINI

Fig. 5. Chromatogram of the serum filtrate from a patient (blank serum) (A) and of the serum filtrate (identical patient) undertreatment with aUopurino1 (B). Each peak in chromatogram g COKESponds to the following quantity: hypoxanthine (retention time, ER = 3.8 min), 1.2 nmob; xantbine @It = 6.2 ruin), 0.2 more; uric acid (fn = 7.4min), 24.3 nmole; erotic acid (r~ = 9-S min),0.08nmoIe; aad oxipurinol (TV = 11.7 min), 0.8 nmole. Mobile-phase, 10 miW tetrabutylarrunonium hydroxide in water (pH S.O)_acetonitrile (99.8:O.Z). Flow-rate_ 2 ml/min. Injection volume, 40 &I.

:fG 22.24

“a= ag

f%

7.32 3.78 9.82 11.63

6.17 5.58

2.69 2.18 3.81 4.77 3.72 3.21

DFt

E2

1.59 1.76

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1:342

DROTIC

ACID

XANTHINE

URIC ACID

3:m;fz

HVPDXANTttINE ALLOPURINOL

0: 146 13.335

8”* f;;

1.307 8.105 0.564 1.752 3.618

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OF PYRIMIDTNES, PURINES ANDTHEIRMETABO~

TABLEV~

353

..

SERUM URIC ACID (REpEATEDTEsTs)

CONCENTEtATIONS

Dm

BY DIFFEECE-NT METHODS

tin-c a&i (~mto~~l)

Uricuse m&t&

HPLC

WZ-fhmd deprsieinization

Wifh deproteinization

354.5 377.7 397.4 403.9

321.4 348.8 367.0 368.0

354.7 365.3 3924 410.2

m H m’

Fii 7. Analysis of a spiked control serum sample. Each peak cm-respondsto theobromine SOng, 3-methylxanthine20 n& 1-rnethylxanthine50 ng. l-methyluric acid 100 ng, caffeine 50 ng, theophylline SOng and l&Jimethylwic acid 200 ng. Separationconditions as in Fig. 5, except water-acetonitrZe (97.525) was used.

354

sample, and the rest&s were compared with those obtained by the chromatographic method (Table V). The values obtained for uric acid in serum by the uricase method without deproteiuizationare almost identical with those obtained by HPLC. If the uricase methodwith deproteinizationis used, the values wereabout g-10 % lower, owing to a constant portion of uric acid being lost during proteinprecipitation. Bioanalyses of substituted xanthines and uric acids (theophylline and membolltes)

To demonstratethe high resolution of the proposed separationtechnique,a serum sample spiked with theobromine, 3-methylxanthine, I-methykanthine, Imethyluricacid, caffeine, theophylline and 1,3-dimethyluricacid was investigated. The resultsare shown in Fig. 7. ACKNOWLEDGEMENTS

We expressour thanks to Mrs. M. Naher and Mr. B. Cettierfor carryingout ffie enzymaticassays. We are also gratefulfor the valuableassistanceof Miss E. Wegmann in preparingthe samples. REFERENCES 1 W. Grijbaer andN. ziiker, Knin.Wuchenwhr., 53 (1975) 255. F. Mat&es and G. Berg, Deur. Med. Wuchenwhr., 99 (1974) 2464. M. H. Jacob. R. M. Senior and G. Kessler, J. Amer. Med. Ass., 235 (1976) 1983. J. W. Jenne, H. T. Nzg2s2w2 and R T. Thompson, C&z. Pharmacol. 2&r., 19 (1976) 375. R. EndeIe and G. Lettenbauer, J. Ctiotnafogr., 115 (1975) 228. 6 M. Brown and A Bye, J. Chromafogr., 143 (1977) 19.5. 7 W. G. Kramer and S. Feldman, J. Chromatogr., 162 (1979) 94. 8 N. D. Brown, J. A. RXntziosand S. E. Koetitz, J. Chromafogr_ 177 (1979) 170. 9 A. H. Germip, J. Grift, E. J. Bree-Blom, D. Ketting and S. K. Wadman, J. Chromarogr., 163 (1979) 351. 10 J. A. MiIner and E. G. Perkins, Amd. Biuchem., 88 (1978) 560. 11 H. F. Walton, G. A. Ekeand J. L. Otto, J. Chromatogr., 180 (1979) 145. 12 C. V. Manion, D. W. Shoeman and D. L. Azamoff, J. Chramatagr., 101(1974) 169. 13 R. Seufkr, W. Voelter and H. Bauer, J. Clin. Chem. Clin. Biochem., 15 (1977) 663. 14 W. Voelter and H. Bauer, Clin. Chem..21 (1975) 1882. 15 R. A. Hartwick, A. M. KrstuIovic and P. R. Brown, J. Chrumarogr., 186 (1979) 659. 2 3 4 5