Simultaneous determination of purine derivatives in urine by high ...

Journal of Animal and Feed Sciences, 5, 1996, 433-439

Simultaneous determination of purine derivatives in urine by high-performance liquid chromatography M. Czauderna and J . Kowalczyk

The Kielanowski Institute of Animal Physiology and Nutrition, Polish Academy of Sciences 05-110 Jablonna, Poland (Received 9 July 1996; accepted 13 September 1996)

ABSTRACT A high-performance liquid chromatography method for the analysis of allantoin, uric acid, hypoxanthine and xanthine in urine of sheep is described. Urine samples were analyzed directly after dilution. The combination of HPLC reversed phase C columns with monitoring the effluent at 205 nm provides a relatively rapid and simple analytical tool for studying purine derivatives in the urine of ruminants. Separation and quantification of purine derivatives was achieved using three C columns connected in a series together with a NH H,PC" - N H H P 0 and methanol (95:5) gradient. The average recoveries of standard compounds added to urine samples were satisfaction (96-103%). The low value of the intra-assay coefficient of variation and high recovery point to the satisfactory precision and accuracy of the reported method. 18

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K E Y WORDS: allantoin, uric acid, hypoxanthine, xanthine, urine, determination, HPLC

INTRODUCTION The final products of nucleic acid purine base catabolism in most mammals are allantoin, uric acid, hypoxanthine and xanthine (Chen et al., 1990 a,b, 1993, 1996; Balcells et al., 1993; Susmel et al., 1994). These purine derivatives (PD) are Financial support from CRS grant No. 5 S305 033 07 is gratefully acknowledged

434

C Z A U D E R N A M . , K O W A L C Z Y K J.

excreted i n urine, i n which allantoin constitutes the greatest p r o p o r t i o n (about 85%) o f total P D (Chen et al., 1993). The purine derivatives originate mainly from the nucleic acids o f rumen microorganisms, so measurements o f urinary P D levels provide an indication o f the amount o f microbial protein supplied to ruminants and is well correlated w i t h organic matter or digestible organic matter intake (Antoniewicz et at., 1985; Lindberg et al., 1991; Balcells et al., 1993; Chen et al., 1993). A l t h o u g h some authors have proposed allantoin excretion as an index o f the microbial protein supply to the small intestine i n ruminants, a more appropriate indicator might be the excretion o f other or total purine derivatives (Topps et a l , 1965; Z i n n et al., 1986), particularly since ruminant urine also contains considerable amounts of uric acid, hypoxanthine and xanthine (Chen et al., 1990; Puchala et al., 1992). The main advantage o f such an index is thatit does not require the use o f an invasive method to estimate microbial biomass supply to the duodenum. Several methods have been developed for measuring P D (Fujihara et al., 1987; Chen et al.,1990b, 1996; Balcells et a l , 1993; Puchala et al., 1993; Czauderna and Kowalczyk, 1995), but there are few reports that include simultaneous determination o f allantoin, uric acid, hypoxanthine and xanthine (Balcells et al., 1992). The aim o f the present paper was to present a simple procedure for P D determination i n urine w i t h o u t derivatization, using H P L C - U V detection. The application o f such a method i n the study o f purine metabolism i n ruminants might provide further evidence o f the influence o f nutritional manipulation on microbial protein supply to the small intestine.

MATERIAL AND METHODS

Reagents M e t h a n o l was purchased from P O C H (Gliwice, Poland), while allantoin, uric acid, hypoxanthine and xanthine were obtained from Sigma (St. Louis, M O , U S A ) . A l l other chemicals were o f analytical grade and purchased from P O C H (Gliwice, Poland). Water was distilled and then deionized prior to use. HPLC-grade water was prepared using a M i l l i - Q system (Millipore, T o r o n t o , Canada). The mobile phase (solvents A and B) was filtered through a 0.2 / m i membrane filter (Millipore). The solvents were degassed by 10 m i n ultrasonication prior to use.

PURINE DERIVATIVES I N URINE HPLC

435

configuration

A Waters 625 L C system (including a controller and t w o pumps) was used. The apparatus consisted o f a Waters 712 W I S P autosampler, turnable Waters M o d e l 486 absorbance detector and computer data handling system (all equipment from Waters, M i l l i p o r e , M A , U S A ) . The analytical procedure, collection and data integration methods were developed using M i l l e n n i u m 2001 software and a Pentium 5P60 computer. Separations were achieved by using two Nova-Pak C - columns (4 /tm, 150 m m x 3.9 m m I . D . , Waters, Millipore) and a Chrompack column - C (4 / m i , 100 m m x 3 m m I . D . , Chrompack, The Netherlands) connected in a series. A Waters cartridge (10 m m x 6 m m I . D . , M i l l i p o r e , M A , U S A ) containing reversed phase C (30-40 /mi) pellicular packing material was used as the precolumn. 1 8

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Analytical

solvents and gradient

composition

A binary gradient program (Waters curvilinear program) was used for the complete analysis o f allantoin, uric acid, hypoxanthine and xanthine in urine samples. The following eluents were used: ( A ) - N H H P 0 (0.0025 M ) buffered to p H 3.5 w i t h 10% phosphoric acid; ( B ) - t h e solvent A and methanol (95/5 v/v). F o r the analysis o f urinary purine metabolites, the solvent B gradient was followed by the sequential steps: 0% the solvent B at 0 m i n , 80% at 21 m i n (linearly increased from 20 m i n , line N o . 6), 0% at 30 m i n (linearly decreased from 29 m i n , line N o . 6). The total flow-rate was 0.4 m l / m i n (the system pressure was 2200 + 50 psi). After 30.1 m i n , the columns were re-equilibrated for 45 m i n i n 100% solvent A at a flow-rate o f 0.5 m l / m i n (the m a x i m u m system pressure was 2870 + 50 psi). Injection volumes were 5 or 10 /d. The detector was operating at 205 n m w i t h an attenuation o f 0.050 a.u.f.s (the absorbance unit full scale). The separation was performed at 17-27°C. 4

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Standards Appropriate amounts of purine derivatives were dissolved i n the solvent A . T o provide calibration for urine samples, sets o f purine derivative standards were used. Urine was collected from sheep in metabolic cages and as a routine method o f preservation, urine samples were acidified to a p H below 3 w i t h 1 M H S 0 . F o r analysis urine samples were mixed and then diluted 1:40 or 1:20 w i t h solvent A . A l l standards and urine samples were filtered through a 0.2 / m i filter (Cole Parmers) into an autosampler vial and analyzed directly. 2

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RESULTS AND DISCUSSION

G o o d separation o f all purine derivatives-allantoin, uric acid, hypoxanthine and xanthine in urine samples was achieved during the chromatographic r u n owing to the use the gradient program and m o n i t o r i n g the effluent at 205 n m . Urine contains several components w i t h a similar high polarity and U V absorption. Thus, a long column together w i t h a low molarity and p H o f the eluent were needed to achieve satisfactory separation of P D in urine samples, and to increase the theoretical plates so as to increase the retention time o f the interfering species. I n our procedure, the allantoin peak was eluted at 8.2 + 0.6 m i n , uric acid at 10.7 + 0.8 m i n , hypoxanthine at 14.9 + 0.9 m i n and xanthine at

PURINE DERIVATIVES I N URINE

437 TABLE 1 1

Summarized results o f recoveries ( R % , i.e. mean + S D ) o f standard purine metabolites added to sheep urine and concentrations (C) o f added purine derivatives, fiM Allantoin 187.2

c R

U r i c acid (2)

102.2 + 3.8

C

C

93.6 98.7 + 4.1

(2)

R

C

62.4

(3)

R

96.8 + 4.7

C

46.8 96.2

R

C R Pooled data

136.7

91.2

110.2 98.2±1.4

(4)

75.0 (3) 102.2 + 2.8

(3)

73.5 99.4+1.1

(7)

50.0 101.9+1.9

97.7±3.8 68.4 99.2±3.3

(2)

45.6

(3)

103.8±4.3

0)

12.5 (2) 98.3 + 5.3 98.7 + 4.7

34.2 93.8

Xanthine

(2)

104.1 ± 4 . 6

124.8 (4) 101.3 + 3.4

R

2

Hypoxanthine

55.1 (5) 100.5 + 0.9 36.7

(7)

100.7+1.8

(4)

37.5 (4) 95.1 ± 4 . 6 25.0

(4)

102.6 + 3.5

(1)

27.6 (2) 103.4 + 6.5

18.7 96.1+5.4

9.1 (2) 95.3 ± 4 . 4

7.3 (3) 103.8 + 3.7 101.1+2.8

5.0 (3) 97.1+6.1 98.7 + 4.2

98.8 + 4.1

(2)

' recovery was calculated as: (S, - S ) x 100% R (%) = _ ! °. S where S and S, are measurement before and after a d d i t i o n o f standard purine derivatives, and S is the amount o f added purine derivatives number o f replicates 0

0

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17.4 + 0.9 m i n (Figure 1). G o o d linearity was obtained for a wide range o f P D standards concentrations and the following equations and correlation coefficients were calculated from the data: - for allantoin (the range o f concentration: 37.4 - 1496 /uM): y = 2.992 • 10" • S + 0.002, R = 0.9999 7

n

- for uric acid (the range o f concentration: 27.3 - 1092 fjM): y = 8.274 • 10" • S - 0.010, R = 0.9989 8

n

- for hypoxanthine (the range o f concentration: 22.0 - 880 LIM): y = 5.565 • lO" • Sn + 0.005, R = 0.9999 8

-

for xanthine (the range o f concentration: 14.9 - 596 LIM): y = 1.104 • 10" • S + 0.001, R = 0.9993 7

n

where: S and y are the peak area and the purine concentration respectively. n

(LIM),


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TABLE 2 The within-assay coefficients o f variations (C.V.) derived from the measurements o f purine derivatives i n urine samples Allantoin

U r i c acid

H y p o x a n thine

C V % 4.7

4.2

2.8

3.4

3.4

2.6

2.9

CV 1

2

2

% 3.8

Xanthine

the within-assay C V . based on three samples repeated three or t w o times (processing and injection) the within-assay C V . based on three samples each w i t h three injection

The accuracy o f the method was assessed by examining the recovery o f k n o w n quantities o f allantoin, uric acid, hypoxanthine and xanthine added to urine samples. Recoveries (R) o f standard purine metabolites added to the urine and the day-to-day precision o f the method are presented i n Table 1. I t can be seen that P D added to urine were recovered satisfactorily (96-103%). The intra-assay coefficients o f variation ( C V ) were calculated by repeatedly processing aliquots of spiked urine sample (Table 2) while the lowest concentrations o f purine derivatives that gave a reproducible integration were: 9 for allantoin, 2 LIM for uric acid, 1.5 LIM for hypoxanthine and 3.8 LIM for xanthine. I t can be concluded that the presented H P L C method for assaying purine derivatives makes it possible to separate and quantify allantoin, uric acid, hypoxanthine and xanthine i n urine samples. The urine samples can be analyzed directly after dilution. Moreover, the method described i n this study enables simple and simultaneous determination o f all P D w i t h o u t the disadvantages inherent in pre- or post-column derivatization procedures. The purine metabolites are detected at short wavelengths, as they possess a strong chromophore in the region 206-210 n m (Balcells et a l , 1992; Chen et al., 1996). A l l purine derivatives were completely resolved i n about 19 m i n , however, the total r u n time, including column re-equilibration, was 75 m i n .

REFERENCES Antoniewicz A . , 1985. U r i n a r y allantoin excretion and rumen microbial protein synthesis in sheep i n relation to protein and energy intake. Rocz. N a u k . Z o o t . 23, 169-181 Balcells J., G u a d a J.A., Peiro J . M . , 1992. Simultaneous determination o f allantoin and o x y p u r i n e s i n biological fluids by high-performance liquid chromatography. J. Chromatogr. 575, 153-157 Balcells J., G u a d a J.A., Castrillo C , Gasa J., 1993. Rumen digestion and urinary excretion o f purine derivatives i n response to urea suplementation o f sodium-treated straw fed to sheep. Brit. J. N u t r . 69, 721-732

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IN

URINE

439

Chen X . B . , Hovell F . D . D e B . , 0 r s k o v E. R., B r o w n D . S., 1990a. Excretion o f purine derivatives by ruminants: effect o f exogenous nucleic acid supply o n purine derivative excretion by sheep. Brit. J. N u t r . 63, 131-142 Chen X . B . , Mathieson J., H o v e l F . D . D e B . , Reeds P.J., 1990b. Measurement o f purine derivatives i n urine o f ruminants using automated methods. J. Sci. F o o d A g r i c . 53, 23-33 Chen X . B . , K y l e D.J., 0 r s k o v E.R., 1993. Measurement o f allantoin i n urine and plasma by high-performance liquid chromatography w i t h pre-column derivatization. J. C h r o m a t o g r . 617, 241-247 Chen X . B . , Matuszewski W . , K o w a l c z y k J., 1996. D e t e r m i n a t i o n o f allantoin i n biological, cosmetic, and pharmaceutical samples. J. Assoc. Off. A n a l . Chem. I n t . 79, 628-635 Czauderna M . , K o w a l c z y k J., 1995. D e t e r m i n a t i o n o f allantoin in b l o o d by H P L C w i t h pre-column derivatization. J. A n i m . Feed Sci. 4, 351-358 Fujihara T., Orskov E.R., Reeds P.J., K y l e D.J., 1987. The effect o f protein infusion on u r i n a r y excretion o f purine derivatives i n ruminants nourished by intragastric n u t r i t i o n . J. A g r i c . Sci., Camb. 109, 7-12 Lindberg J.E., 1991. N i t r o g e n and purine metabolism i n preruminant and r u m i n a n t goat kids given increasing amounts o f ribonucleic acids. A n i m . Feed. Sci. Technol. 35, 213-226 Puchala R., Kulasek G . W . , 1992. Estimation o f m i c r o b i a l protein flow f r o m the rumen o f sheep using microbial nucleic acid urinary excretion o f purine derivatives. Can. J. A n i m . Sci. 72, 821-830 Puchala R., Shelford J.A., Barej W . , Kulasek G W . , Pior H . , M a k o n i N . Keyserlingk M . , 1993. U r i n a r y excretion o f pseudouridine and purine metabolites i n ruminants. J. A n i m . Physiol. A n i m . N u t r . 69, 186-193 Susmel P., Spanghero M . , Stefanon B., M i l l s C.R., Plazzotta E., 1994. Digestibility and allantoin excretion i n cow fed diets differing i n nitrogen content. Livest. P r o d . Sci. 39, 97-99 Topps J . H . , Elliott R.C., 1965. Relationship between concentration o f r u m i n a l nucleic acids and excretion o f purine derivatives by sheep. Nature ( L o n d o n ) 209, 498-499 Z i n n R . A . , Owens F . N . , 1986. A rapid procedure for purine measurement and its use for estimating net r u m i n a l protein synthesis. Can. J. A n i m . Sci. 66, 157-166

STRESZCZENIE

Jednoczesne oznaczanie pochodnych purynowych w moczu metodą H P L C Opisano m e t o d ę oznaczania allantoiny, kwasu moczowego, hipoksantyny i ksantyny w moczu owiec przy użyciu zestawu H P L C . M o c z analizowano b e z p o ś r e d n i o po doprowadzeniu do p H 3 oraz o d p o w i e d n i m rozcieńczeniu. Pochodne purynowe rozdzielono w y k o r z y s t u j ą c trzy k o l u m n y z o d w r ó c o n ą fazą ( C ) poprzez elucję g r a d i e n t o w ą i monitorowanie przy 205 n m . Z m i a n y w składzie fazy ] g

ruchomej (0,4 m l / m i n ) realizowano p r o g r a m o w o przez zmiany względnej p r ę d k o ś c i podawania d w ó c h r o z t w o r ó w - A (0,025 M N H H P 0 ) i B - ( r o z t w ó r A i metanol: 95/5 v / v ) . Odzysk 4

2

4

pochodnych p u r y n o w y c h dodanych do moczu był niemal z u p e ł n y . M a ł a w a r t o ś ć w s p ó ł c z y n n i k a z m i e n n o ś c i w o b r ę b i e p r ó b y (przygotowanie i iniekcja) oraz iniekcji tej samej p r ó b y , wysoki odzysk oznaczanych z w i ą z k ó w (96-103%) d o w o d z i , że prezentowana dokładna.

prosta metoda jest

dostatecznie