RAPID COMMUNICATIONS IN MASS SPECTROMETRY, VOL. 6,547-549 (1992)
Determination of 15N Enrichment of Taurine in Cat Urine by High Resolution Fast-atom Bombardment Mass Spectrometry Andreas A. Stampfli,* Olivier Ball&vre.and Laurent B. Fay Nest16 Research Centre, Vers-chez-les-Blanc, P O Box 44,,1000 Lausanne 26, Switzerland
A method is described for measuring the stable isotopic enrichment of taurine in cat urine samples by high resolution fast-atom bombardment mass spectrometry, after ',5Nlabelled taurine was given to cats for the purpose of investigating taurine metabolism. The *'N enrichment of taurine was measured after hydrolysis and purification of taurine by aniodcation exchange chromatography. The isotopic ratio of taurine was determined by measuring the [M HI+ ion peaks in the spectra of the pnlabelled and labelled compounds under multiple ion scan conditions. The overall standard deviation of the measurement is better than 4%. This method requires no derivation and uses only 500 pL of urine samples.
+
Taurine (2-aminoethanesulfonic acid) is a conditionally essential amino acid for human and other mammal species.' Especially in the cat, a dietary taurine deficiency has been associated with retinopathy and and more recently with dilated cardiomyopathy and vascular change^.^ Cat foods must, therefore, contain the cat's daily need of taurine to avoid illness and disease. In order to elucidate the metabolic basis of taurine requirements in cats we fed '5N-labelled taurine as a bolus and measured taurine enrichment in urine. To estimate the "N enrichment of taurine in cat urine we used high resolution fast-atom bombardment mass spectrometry. The ratio of I4N/I5Nwas measured by monitoring the pseudo-molecular ions of the unlabelled and labelled compounds. This technique eliminates the need for timeconsuming clean-up procedures of urinary samples and avoids cumbersome derivatization steps necessary for capillary gas chromatography/mass spectrometry of taurine .', EXPERIMENTAL All chemicals used were of analytical grade. "N-tgurine was synthesized by Philippossian et al. with an isotopic purity of greater than 98%.' Standard solutions of 15N-taurine were prepared up to 12 mole per cent excess (MPE) in de-ionized water with a final concen. solutions were then used for tration of 1 3 m ~These calibration purposes. Urine samples (1 mL) spiked with a known amount of ''N taurine were hydrolysed by adding 1mL of 12 N hydrochloric acid. The sealed tubes were kept for 24 h at 130 "C. After evaporation to dryness under vacuum, the residue was dissolved in 2 m L of water. 1 mL of the sample was loaded on a small ion-exchange column filled with both an anionic and cationic resin (Amberlite AGlx8, 200-400 mesh, 0.5 mL and Amberlite AG50W-x8, 200-400 mesh, 1mL). Taurine was washed out with 6 mL of water. The water was again evaporated to dryness and the residue stored at 4 "C for further treatment. The quantity of taurine obtained was between 500-1000 nmol. The sample was redissolved in 20 pL of water and Author to whom correspondence should be addressed. 095 1-4198/92/090547-03 $06.50
01992 by John Wiley & Sons, Ltd
mixed for 5 min in an ultrasonic bath. After centrifuging (10 min at 8000 rpm) 5 yL of the diluted sample was thoroughly mixed with 5 pL of glycerol containing threonine (2 M, as internal standard, for lockmass). 2-3 pL of this mixture was applied to the fast-atom bombardment (FAB) probe tip and transferred into the ion chamber to measure the ratio of the unlabelled and labelled taurine. Mass spectrometry was performed on a Finnigan MAT 8430 mass spectrometer (San Jose, CA, USA) at 3 kV acceleration voltage with a resolution of 5000. For FAB ionization, the mass spectrometer was equipped with a saddle-field atom gun (Ion Tech, Teddington, UK) which was operated with 2 mA and 7-8 kV with xenon. Glycerol was the matrix. The mass spectrometer was under full data control (SS300 on a PDP-11/73). The mu1,tiple ion-scan (MIS) measurement was done over a period of 2 min to obtain more than 100 single measurements. The ratio of the [M + H]+ ion peak of taurine (rnlz 126.0225 u) and the [M + H]+ ion peak of the "N-labelled compound (mlz 127.0195 u) was then determined using 50 scans, by integrating the areas below both single-ion traces of the two pseudo-molecular ions. The lockmass for the MIS measurement was the [M + H]+ ion of threonine at mlz 120.0661 u. RESULTS The calibration for "N-enriched taurine standards in water is displayed in Fig. 1 which shows a calibration curve of observed vs theoretical MPE. The MPE measured for the samples were calculated using the formula:
MPE = 100(R, - &)/( 1- ( R , - R,)) where R,, and Rs are the ratios of the integrated areas of the peaks corresponding to ionS [ M + H ] + and [M + 1+ HI+ for a sample of natural abundance and an enriched , sample, respectively. The correlation coefficient of 0.9985 obtained by a linear regression fit of the theoretical vs measured MPE indicates that the estimation is linear over a range of 0-11 MPE. Preliminary tests with urine showed that there is a peak at mlz 127 interfering with the [M +HI+ peak of 4
Received 2 July 1992 Accepted 16 July 1992
DETERMINATION OF I5N ENRICHMENT OF TAURINE IN CAT URINE
548
i///
Table 1. Ratio of m/z327/126 for urine samples (5OOpL) spiked with the indicated amount of "N-taurine. All values were measured not less than 3 times
1I 0
/
Amount of "N (nmol)
[ M + 1]+/[M]+
CV ("A)"
MPE theoretical
MPE measured
MPE calculatedh
0.00 8.00 15.50 39.50 78.70
0.0251 0.0335 0.0396 0.0593 0.0991
2.4 1.7 5.2 5.8 3.3
0.00 0.83 1.42 3.30 6.89
0.37 1.11 1.65 3.38 6.88
0.00 0.74 1.43 3.65 6.85
Ratio
* CV = coefficient of variance.
'Using the biological level of I4N-taurine given in the text.
Figure 1. Mole percent excess of measured vs theoretical value of standard I4N- and "N-taurine in water.
labelled taurine due either to the biological background of the samples, or to the cleaning steps involved. The interfering compound was never observed with standards in pure water. In samples with a biological background, an increase of the resolution to 5000 was enough to resolve the pseudo-molecular ion of taurine from its contaminant. The resolving power of 5000 proved to be a good compromise between selectivity and sensitivity. Figure 2 shows a plot of the measured MPE of spiked urine vs the MPE calculated with the calibration curve established in pure water. The correlation coefficient of 0.9998 from a linear-regression fit indicates that the curve is linear over the investigated range. The slope of 0.95 shows that there is no matrix effect due to the biological background of the samples. The physiological level of 14N-taurinein cat urine was calculated with the following formula using the amount of I5N-taurine as internal standard: X = A / ( R , - R")
where A is the amount of 15N-taurineadded (nmol), Rs is the ratio of (M 1)/M in the enriched sample; and Ro is the ratio of (M+ 1)/M in the non-enriched sample. The amount estimated was 134.75 k 9.43 pg in 500 pL
+
ywu*olaicpl
Figure2. Mole percent excess of measured vs calculated value in urinary samples by using the linear regression obtained from standards in water for calculation.
of urine. Therefore, the amount applied to the probe tip was approximately 5 pg of taurine. The MPE, the theoretical and measured values, and values calculated using the biological level of 14N-taurinegiven above, for the spiked urine samples are listed in Table 1 together with the measured ratios Ro and Rs , respectively. The overall standard deviation of the measurements is about 4%. This method was then applied to a quantitative determination of "N-taurine enrichment in the urine of cats that had received a bolus of "N-taurine. A typical curve is displayed in Fig. 3 showing I5N-taurine enrichment vs time after bolus, in the urine of a cat over a period of 10 days. An exponential fit of this curve allows us to calculate taurine kinetics in cats using a stochastic analysis of a one-pool model.' CONCLUSION High resolution fast-atom bombardment mass spectrometry seems to be a suitable method to measure "N-enrichment of taurine in urine. The ratio of the intensities of labelled and unlabelled pseudo-molecular ions is linear over a range of 0 to 11MPE. The advantages over a combination of gas chromatography and mass spectrometry (GUMS) are that fewer clean-up steps are required and there is no need for derivatization. One drawback is that due to the high resolution, more sample is required. The amount applied to the probe tip for a single measurement was about 5 pg of total taurine. Although this amount is relatively high in
DAYS posl BOLUS
Figure3. Typical curve obtained from cat urine (mean of three animals k standard error of the means showing mole percent excess of taurine over a period of 10 days after the bolus.
D E T E R M I N A T I O N OF "N E N R I C H M E N T OF T A U R I N E IN C A T U R I N E
relation to the amount necessary for a G U M S run, the method could be applicable to biological sample fluids where taurine concentrations are relatively high (urine, blood). Another drawback compared to other techniques like G U M S is that no automation is possible. Nevertheless the technique provides an alternative method which can be.easily applied to estimate enrichments for a whole variety of non-volatile compounds without any prior derivatization steps. Acknowledgement The authors thank Dr I. Horman for revising the manuscript.
549
REFERENCES 1 . K. C. Hayes, Nutr. Sci. Reu. 1, 99 (1988). 2. A . R . Rabin, K. C. Hayes and E. I . Berson, Inuest. Ophthamol. Vis. Sci., 12, 694 (1973). 3. K. C. Hayes, R . E. Carey and S: Y. Schmidt, Science 88, 949 ( 1975). 4. P. D. Pion, M. D. Kittleson, Q. R. Rogers and J . G. Morris, Science 237, 764 (1987). 5. H . Kataoka, S. Yamamoto and M . Makita, J . Chrornafogr. 306, 61 (1984). 6. H . Kataoka, M. Ohnishi and M. Makita, J. Chrornafogr.339,370 (1985). 7. G . Philippossian, D. H. Welti, R . Fumeaux, U . Richli and K. Anantharaman, J. Labelled Comp. Rndiopharm. 27, 1267 (1989). 8. 0. Ballltvre (unpublished results, 1992).
Determination of 15N Enrichment of Taurine in Cat Urine by High Resolution Fast-atom Bombardment Mass Spectrometry Andreas A. Stampfli,* Olivier Ball&vre.and Laurent B. Fay Nest16 Research Centre, Vers-chez-les-Blanc, P O Box 44,,1000 Lausanne 26, Switzerland
A method is described for measuring the stable isotopic enrichment of taurine in cat urine samples by high resolution fast-atom bombardment mass spectrometry, after ',5Nlabelled taurine was given to cats for the purpose of investigating taurine metabolism. The *'N enrichment of taurine was measured after hydrolysis and purification of taurine by aniodcation exchange chromatography. The isotopic ratio of taurine was determined by measuring the [M HI+ ion peaks in the spectra of the pnlabelled and labelled compounds under multiple ion scan conditions. The overall standard deviation of the measurement is better than 4%. This method requires no derivation and uses only 500 pL of urine samples.
+
Taurine (2-aminoethanesulfonic acid) is a conditionally essential amino acid for human and other mammal species.' Especially in the cat, a dietary taurine deficiency has been associated with retinopathy and and more recently with dilated cardiomyopathy and vascular change^.^ Cat foods must, therefore, contain the cat's daily need of taurine to avoid illness and disease. In order to elucidate the metabolic basis of taurine requirements in cats we fed '5N-labelled taurine as a bolus and measured taurine enrichment in urine. To estimate the "N enrichment of taurine in cat urine we used high resolution fast-atom bombardment mass spectrometry. The ratio of I4N/I5Nwas measured by monitoring the pseudo-molecular ions of the unlabelled and labelled compounds. This technique eliminates the need for timeconsuming clean-up procedures of urinary samples and avoids cumbersome derivatization steps necessary for capillary gas chromatography/mass spectrometry of taurine .', EXPERIMENTAL All chemicals used were of analytical grade. "N-tgurine was synthesized by Philippossian et al. with an isotopic purity of greater than 98%.' Standard solutions of 15N-taurine were prepared up to 12 mole per cent excess (MPE) in de-ionized water with a final concen. solutions were then used for tration of 1 3 m ~These calibration purposes. Urine samples (1 mL) spiked with a known amount of ''N taurine were hydrolysed by adding 1mL of 12 N hydrochloric acid. The sealed tubes were kept for 24 h at 130 "C. After evaporation to dryness under vacuum, the residue was dissolved in 2 m L of water. 1 mL of the sample was loaded on a small ion-exchange column filled with both an anionic and cationic resin (Amberlite AGlx8, 200-400 mesh, 0.5 mL and Amberlite AG50W-x8, 200-400 mesh, 1mL). Taurine was washed out with 6 mL of water. The water was again evaporated to dryness and the residue stored at 4 "C for further treatment. The quantity of taurine obtained was between 500-1000 nmol. The sample was redissolved in 20 pL of water and Author to whom correspondence should be addressed. 095 1-4198/92/090547-03 $06.50
01992 by John Wiley & Sons, Ltd
mixed for 5 min in an ultrasonic bath. After centrifuging (10 min at 8000 rpm) 5 yL of the diluted sample was thoroughly mixed with 5 pL of glycerol containing threonine (2 M, as internal standard, for lockmass). 2-3 pL of this mixture was applied to the fast-atom bombardment (FAB) probe tip and transferred into the ion chamber to measure the ratio of the unlabelled and labelled taurine. Mass spectrometry was performed on a Finnigan MAT 8430 mass spectrometer (San Jose, CA, USA) at 3 kV acceleration voltage with a resolution of 5000. For FAB ionization, the mass spectrometer was equipped with a saddle-field atom gun (Ion Tech, Teddington, UK) which was operated with 2 mA and 7-8 kV with xenon. Glycerol was the matrix. The mass spectrometer was under full data control (SS300 on a PDP-11/73). The mu1,tiple ion-scan (MIS) measurement was done over a period of 2 min to obtain more than 100 single measurements. The ratio of the [M + H]+ ion peak of taurine (rnlz 126.0225 u) and the [M + H]+ ion peak of the "N-labelled compound (mlz 127.0195 u) was then determined using 50 scans, by integrating the areas below both single-ion traces of the two pseudo-molecular ions. The lockmass for the MIS measurement was the [M + H]+ ion of threonine at mlz 120.0661 u. RESULTS The calibration for "N-enriched taurine standards in water is displayed in Fig. 1 which shows a calibration curve of observed vs theoretical MPE. The MPE measured for the samples were calculated using the formula:
MPE = 100(R, - &)/( 1- ( R , - R,)) where R,, and Rs are the ratios of the integrated areas of the peaks corresponding to ionS [ M + H ] + and [M + 1+ HI+ for a sample of natural abundance and an enriched , sample, respectively. The correlation coefficient of 0.9985 obtained by a linear regression fit of the theoretical vs measured MPE indicates that the estimation is linear over a range of 0-11 MPE. Preliminary tests with urine showed that there is a peak at mlz 127 interfering with the [M +HI+ peak of 4
Received 2 July 1992 Accepted 16 July 1992
DETERMINATION OF I5N ENRICHMENT OF TAURINE IN CAT URINE
548
i///
Table 1. Ratio of m/z327/126 for urine samples (5OOpL) spiked with the indicated amount of "N-taurine. All values were measured not less than 3 times
1I 0
/
Amount of "N (nmol)
[ M + 1]+/[M]+
CV ("A)"
MPE theoretical
MPE measured
MPE calculatedh
0.00 8.00 15.50 39.50 78.70
0.0251 0.0335 0.0396 0.0593 0.0991
2.4 1.7 5.2 5.8 3.3
0.00 0.83 1.42 3.30 6.89
0.37 1.11 1.65 3.38 6.88
0.00 0.74 1.43 3.65 6.85
Ratio
* CV = coefficient of variance.
'Using the biological level of I4N-taurine given in the text.
Figure 1. Mole percent excess of measured vs theoretical value of standard I4N- and "N-taurine in water.
labelled taurine due either to the biological background of the samples, or to the cleaning steps involved. The interfering compound was never observed with standards in pure water. In samples with a biological background, an increase of the resolution to 5000 was enough to resolve the pseudo-molecular ion of taurine from its contaminant. The resolving power of 5000 proved to be a good compromise between selectivity and sensitivity. Figure 2 shows a plot of the measured MPE of spiked urine vs the MPE calculated with the calibration curve established in pure water. The correlation coefficient of 0.9998 from a linear-regression fit indicates that the curve is linear over the investigated range. The slope of 0.95 shows that there is no matrix effect due to the biological background of the samples. The physiological level of 14N-taurinein cat urine was calculated with the following formula using the amount of I5N-taurine as internal standard: X = A / ( R , - R")
where A is the amount of 15N-taurineadded (nmol), Rs is the ratio of (M 1)/M in the enriched sample; and Ro is the ratio of (M+ 1)/M in the non-enriched sample. The amount estimated was 134.75 k 9.43 pg in 500 pL
+
ywu*olaicpl
Figure2. Mole percent excess of measured vs calculated value in urinary samples by using the linear regression obtained from standards in water for calculation.
of urine. Therefore, the amount applied to the probe tip was approximately 5 pg of taurine. The MPE, the theoretical and measured values, and values calculated using the biological level of 14N-taurinegiven above, for the spiked urine samples are listed in Table 1 together with the measured ratios Ro and Rs , respectively. The overall standard deviation of the measurements is about 4%. This method was then applied to a quantitative determination of "N-taurine enrichment in the urine of cats that had received a bolus of "N-taurine. A typical curve is displayed in Fig. 3 showing I5N-taurine enrichment vs time after bolus, in the urine of a cat over a period of 10 days. An exponential fit of this curve allows us to calculate taurine kinetics in cats using a stochastic analysis of a one-pool model.' CONCLUSION High resolution fast-atom bombardment mass spectrometry seems to be a suitable method to measure "N-enrichment of taurine in urine. The ratio of the intensities of labelled and unlabelled pseudo-molecular ions is linear over a range of 0 to 11MPE. The advantages over a combination of gas chromatography and mass spectrometry (GUMS) are that fewer clean-up steps are required and there is no need for derivatization. One drawback is that due to the high resolution, more sample is required. The amount applied to the probe tip for a single measurement was about 5 pg of total taurine. Although this amount is relatively high in
DAYS posl BOLUS
Figure3. Typical curve obtained from cat urine (mean of three animals k standard error of the means showing mole percent excess of taurine over a period of 10 days after the bolus.
D E T E R M I N A T I O N OF "N E N R I C H M E N T OF T A U R I N E IN C A T U R I N E
relation to the amount necessary for a G U M S run, the method could be applicable to biological sample fluids where taurine concentrations are relatively high (urine, blood). Another drawback compared to other techniques like G U M S is that no automation is possible. Nevertheless the technique provides an alternative method which can be.easily applied to estimate enrichments for a whole variety of non-volatile compounds without any prior derivatization steps. Acknowledgement The authors thank Dr I. Horman for revising the manuscript.
549
REFERENCES 1 . K. C. Hayes, Nutr. Sci. Reu. 1, 99 (1988). 2. A . R . Rabin, K. C. Hayes and E. I . Berson, Inuest. Ophthamol. Vis. Sci., 12, 694 (1973). 3. K. C. Hayes, R . E. Carey and S: Y. Schmidt, Science 88, 949 ( 1975). 4. P. D. Pion, M. D. Kittleson, Q. R. Rogers and J . G. Morris, Science 237, 764 (1987). 5. H . Kataoka, S. Yamamoto and M . Makita, J . Chrornafogr. 306, 61 (1984). 6. H . Kataoka, M. Ohnishi and M. Makita, J. Chrornafogr.339,370 (1985). 7. G . Philippossian, D. H. Welti, R . Fumeaux, U . Richli and K. Anantharaman, J. Labelled Comp. Rndiopharm. 27, 1267 (1989). 8. 0. Ballltvre (unpublished results, 1992).
