Enrichment and high-performance liquid chromatography analysis of tryptophan metabolites in plasma

ANALYTICAL

BIOCHEMISTRY

Enrichment

IKWE

118,

142-146 (1981)

and High-Pe~orma~ce Liquid Chromatography Tryptophan Metabolites in Plasma

~ORITA,T~UTOMU MASUJIMA, HISANOBUYOSHIDA,AND

InsUtures of Pharmaceutical

Sciences, School of Medicine, Hiroshima Minami-ku, Hiroshima 734. Japan

Analysis of

HIDEO

IMAI

University, I-2-3 Kasumi,

Received March 16, 1981 Tryptophan metabolites with an indole ring are enriched by adsorption either as an ion pair a trichloroacetic acid anion or as its undissociated form on porous polystyrene polymer (TSK 2000 S) from strongly acidic piasma deproteinized by trichloroacetic acid, and after washing with water, they are efuted with a 90% methanol solution. Following the removal of the solvent, the residue is dissolved in a small amount of water and then subjected to highperformance liquid chromatography (hplc) analysis. Using 0.2 ml of adsorbent, the recovery of the 500 pmol added for each of the tryptophan metabolites into 1.5 ml of deproteinized plasma is above 70%. This method is used for the analysis of normal rabbit and rat plasma. The hplc analysis, with native fluorescence detection, shows several peaks corresponding to tryptophan, $hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, indole-3-acetic acid, and indole-3-propionic acid. Peak identification and cross reactivity were checked by the retention time with two hpic systems,Buorometric characterization, and electrochemical characterization. This method is easy and is simple enough for routine analysis. with

The importance of tryptophan and its m~tabolites in both normal and pathological states has been reported (l-3). Tryptophan is metabolized by two major pathways, either through kynurenine or through a series of indoles. Many methods have been reported to analyze these compounds. Recently, high-performance liquid chromatography (hplc)’ with either native fluorescence detection (4) or electr~hemi~al detection (5) has been reported to be useful for the analysis of indoles in biological samples. Tryptophan metabolites (Trp-metabolites) with an indole ring include basic compounds (serotonin, tryptamine, etc.), zwitterion compounds (%hydroxytryptophan, tryptophan, etc.), neutral compounds (melatonin, etc.), and acidic com~unds (5-hydroxyindole-3-acetic acid, indole-3-acetic acid, etc.). A simple enrichment method for ’ Abbreviations used: hplc, high-~rfo~an~ liquid chromatography; DNP, 2,4-dinitrophenyl-; TCA, trichloroacetic acid; ODS-, octadecyl silanized silica gel. ~3-2697/81/1?0142-05~02.~/0 Copyright 0 1981 by Academic Press. Inc. All rights of reproduction in any form reserved.

Trp-metabolites should be of great importance for detection of minor metabolites. For their enrichment only the use of Sephadex (6) or DNP-Sephadex (7) has been reported, but the elution is tedious for the few tested metabolites. This paper describes a simple enrichment method for Trp-metabolites with an indole ring on porous polystyrene resin by ion pair adsorption. EXPERIMENTAL

Chemicals. Trp-metabolites were obtained from Sigma, St Louis, Missouri; all other chemicals including solvents were obtained from Yoneyama Industry Ltd, Osaka, Japan. All reagents were of guaranteed grade and were used without further purification. Resins were obtained from Toyo Soda Ltd, Tokyo, Japan (TSK series). Preparation of deproteinized P~a~~u. Freshly drawn blood from the carotid artery was collected in a plastic tube containing 142

ENRICHMENT

OF Trp-METABOLITES

sodium heparin (final concentration 0.1 mg/ ml) and then centrifuged at 15OOg for 10 min at 4°C. Plasma was deproteinized by mixing with an equal volume of 10% trichloroacetic acid (TCA) and was centrifuged at 1500g for 20 min at 4°C. Enrichment

of Trp-metabolites

with TSK

2000 S. TSK 2000 S (53 pm, porous polystyrene polymer), 0.2 ml, was packed in a l-ml capacity disposable syringe. The sample, either 1.5 ml of deproteinized plasma or deproteinized plasma containing 500 pmol of each standard Trp-metabolite was passed through the resin bed by gravity and washed twice with 1 ml of water to remove salts, TCA, and hydrophilic compounds. The adsorbed materials were eluted with 1 ml of 90% methanol containing 0.01% ammonium formate. Both the water for washing and the 90% methanol for elution contained 0.1% sodium thiosulfate as an antioxidant for the Trp-metabolites. After evaporation of the eluate by a vacuum centrifuge (1 torr 55°C 20 min), the solid residue was dissolved in 100 ~1 of water and 40-80 ~1 of this solution was subjected to hplc analysis in order to determine the content of the recovery. The used resin was regenerated by successive washing with a fivefold volume each of chloroform, tetrahydrofuran, acetonitrile, methanol, and water and could be used many times without changing its characteristics. High-performance liquid chromatography analysis of Trp-metabolites. An hplc

apparatus with a single plunger-type pump, Model HU 45 (Kyowa Seimitsu Ltd, Tokyo, Japan), and a Model 7125 sample injector (Rheodyne, Berkeley, Calif.) was assembled in our laboratory. The column temperature was controlled by immersing the column in a circulating water bath. Detection was either by native fluorescence (excitation 287 nm, emission 340 nm) with a Model RF-SOOLC (Shimadzu, Kyoto, Japan) or electrochemical measurement with Model EC-8 (Toyo Soda Ltd, Tokyo, Japan) of which the working electrode was glassy carbon and the reference electrode was Ag/

IN

PLASMA

143

AgCl. Applied voltage was 0.6 V for 5-hydroxyindoles such as serotonin and 1.0 V for indoles such as tryptophan. Two separation systems were used. One was reverse-phase chromatography with a 7.5 X 75-mm column of TSK GEL LS 410 (5 pm, ODS-type resin) using a stepyise elution method. The initial solvent, 10% acetonitrile in 0.1 M phosphate buffer, pH 3.0, was used for 13 min and afterward the second solvent, 30% acetonitrile in 0.1 M phosphate buffer, pH 3.0, was used. Flow rate was 1.3 ml/min at 20°C. The other system used was ion-exchange chromatography with a 4 X 300-mm column of TSK IEX 530 (5 pm, a weak cation exchanger of silica gel core) using an isocratic elution with 15% acetonitrile in 0.1 M phosphate buffer, pH 3.5 (8). The flow rate was 0.7 ml/min at 20°C. RESULTS

AND DISCUSSION

Adsorption of Trp-Metabolites with Indole Ring to Porous Polystyrene Polymer

Neutral (e.g., melatonin) or acidic metabolites (e.g., 5-hydroxyindole-3-acetic acid, indole-3-acetic acid) were easily adsorbed from an acidic aqueous solutin onto a hydrophobic resin such as porous polystyrene polymer or ODS-type resins. But basic (e.g., serotonin, tryptamine) or zwitterion metabolites (e.g., 5-hydroxytryptophan, tryptophan) were not adsorbed tightly. When the sample solution was acidified by TCA or HC104, however, basic or zwitterion metabolites were also adsorbed onto the hydrophobic adsorbent possibly due to an ion pair formation between the basic portion of the metabolites and a strong Lewis acid such as the TCA or HC104 anion. Among the various adsorbents tested, only porous polystyrene resin showed a favorable recovery. Moreover, porous polystyrene resin was stable in an acidic aqueous solutio’n and, after regeneration with organic solvents, could be used many times (see Experimen-

144

MORITA

1 ml of Heparin

plasma

1 Addition

Residue

(IS

500 pmol)

of 10% TCA

(I ml)

Supernatant Resin

(0.2 ml)

1Washing

twice with

IElution

with

1 ml of H20

1 ml of 90% MeOH

Hz0 1 Eluate

1

Dryness

in 100 ~1 of H,O

I hplc Analysis method MeOH,

for enrichment methanol,

of Trp-me-

tal). In comparison of XAD-2 with TSK 2000 S (both were a porous polystyrene resin) TSK 2000 S had two advantages over XAD-2. First, recovery of hydrophobic metabolites such as melatonin was better by the elution of 90% methanol (suitable elution for hydrophilic metabolites such as serotonin). Second, it was easier to handle for the batchwise adsorption and elution by gravity using the commercially available resin with a 53pm particle diameter. For an acidifying and ion-pairing reagent, a final concentration of 5% TCA was used. By using this reagent almost no oxidation of Trp-metabolites was observed with good deproteinization. The deproteinization regenerated the resin and might protect the hplc column from deterioration. Loading

tested for the recovery of the added 500 pmol for each of the 10 Trp-metabolites by hplc analysis. With an increase in sample quantity, there seemed to be a decrease in the amount recovered, and up to 2.4 ml (12-fold volume of resin) of the sample, the recovery of the added Trp-metabolites was above 70%. When the loading sample was greater than 4.8 ml (24-fold volume of resin), the recovery of hydrophilic compounds such as 5hydroxytryptophan was decreased below 70% but such a trend was not observed for hydrophobic compounds such as melatonin. Effect of Washing Volume

Dissolve

FIG. 1. Standard tabolites in plasma.

ET AL.

Capacity

Using 0.2 ml of TSK 2000 S, the loading capacity of deproteinized rabbit plasma was

After loading the 1.5-ml sample onto a 0.2-ml adsorbent bed, it was washed with various amounts of water containing 0.1% sodium thiosulfate as an antioxidant for Trpmetabolites in order to remove salts, TCA, and hydrophilic compounds. After washing with 1 ml of water, the pH TABLE RECOVERY

OF ADDED

IN DEPROTEINIZED STANDARD

Compounds Tryptophanb Serotonin 5-Hydroxytryptophan 5-Hydroxyindole-3-acetic acid N-Acetylserotonin 5-Methoxytryptamine Tryptamine Melatonin Indole-3-acetic acid Indole-3-propionic acid

1 TRP-METABOLITES PLASMA METHOD’

BY THE

Recovery (%) 91 98 87 79

5.4 1.1 3.6 4.2

82 82 93 90 78 83 76

3.1 2.0 2.1 2.9 1.2 1.9 4.1

a To 1.5 ml of deproteinized plasma was added 500 pmol of each Trp-metabolite; n = 10. * Tryptophan in native samples was much higher than 500 pmol, so that the data of recovery and SD were estimated by setting the highest peak among the 10 runs of the experiment as 100%.

ENRICHMENT

OF Trp-~~TA~LITES

Retention (a)

time

IN PLASMA

145

(rain)

(b)

FIG. 2. Chromatogram of (a) standard Trp-metabolites (100 pmol) and (b) rat plasma (-300 p1 equivalent) reverse-phase chromatography. Peaks are identified as follows: (1) 5-hydroxytryptophan; (2) serotonin; (3) tryptophan; (4) 3-indoleacetaldehyde; (5) 5-hydroxyindole-3-acetic acid; (6) N-acetyl serotonin; (7) tryptamine; (8) 5-methoxytryptamine; (9) melatonin; (10) indole-3-acetic acid; and (11) indole-3-propionic acid. For chromatographic conditions, see Experimental.

of the eluate was below 3, indicating that the TCA was not completely removed, and moreover, that the residual solid, obtained by elution of the Trp-metabolites and removal of the elution solvent, was not soluble in a small amount of water. After washing with more than 2 ml of water, the pH of the eluate was almost neutral and the insolubility of the residue was avoided. Among the tested com~unds, 5hydroxytryptophan was the most sensitive to the washing procedure, but its recovery was constant at least up to a 3-ml water wash ( 15fold volume of resin). A 2-ml volume of water was settled upon for washing (lo-fold volume of resin). Effect of E&ion

electrochemical detection. Therefore, sodium thiosulfate was added to both the washing water and the eluent. In view of solubilizing and eluting sodium thiosulfate, 90% methanol containing 0.1% sodium thiosulfate and 0.01% ammonium formate was used as the eluent. Other volatile organic solvents such as acetone or acetonitrile showed poor solubilization of sodium thiosulfate. Ammonium formate was added as a volatile salt to increase the solubililty of hydrophilic compounds. At least 0.8 ml of eluent was necessary for the 0.2 ml of adsorbent to secure a recovery greater than 70%. For ease of removal of the eluent, an elution volume of 1 ml was settled upon (five-fold volume of resin).

Volume

Addition of antioxidant to both the washing water and the eluent was very important to secure a good recovery when handling picomole quantities of Trp-metabolites. Mercaptoethanol or sodium thiosulfate was effective, but the former interfered with the

Standard Method

From the results obtained above, the standard enrichment method for Trp-metabolites with an indole ring in deproteinized plasma which was settled upon is shown in Fig. 1. The recovery by this method of the

146

MORITA

4 a

5 vJ-L IO Retentton (0)

7

9 j 20

L.-

time (mm) (b)

FIG. 3. Chromatogram of (a) standard Trp-metabolites (100 pmol) and (b) rabbit plasma (-300 ~1 equivalent) by ion-exchange chromatography. Peaks are identified as follows: (1) 3-indoleacetaldehyde; (2) Shydroxytryptophan; (3) tryptophan; (4) N-acetyl serotonin; (5) S-hydroxyindote-3-acetic acid; (6) melatonin; (7) indole-3-acetic acid; (8) serotonin; (9) indole-3-propionic acid; ( 10) S-methoxytryptamine; and ( 11) tryptamine. For chromatographic conditions, see Experimental.

500 pmol added for each of the standard compounds using the same rabbit plasma is shown in Table 1. The use of an internal standard seems to be useful for correcting the data. Either melatonin or N-acetylserotonin was selected for the internal standard because these compounds when added are eluted with definite peaks in the two hplc systems, and their concentration in the plasma is far below the detection limit of the present method. However, their existence has been confirmed as metabolites in the animal kingdom. Therefore a more suitable internal standard is under investigation, Analysis of Rabbit and Rat Plasma

After enrichment by the standard method, the dried samples were dissolved in 100 ~1

ET AL.

of water, and a 40 to 80-~1 aliquot was analyzed by hplc to see the major metabolites in the plasma. As shown in Figs. 2 and 3, peaks with retention times corresponding to 5-hydroxytryptophan, serotonin, 5-hydroxyindole-3-acetic acid, indole-3-acetic acid, and indole-3-propionic acid in addition to tryptophan were observed in the hplc chromatogram by native fluorescence detection. Peak identification was done by comparing the retention time in two different hplc systems, by using fluorescent characterization (by measurement of the excitation spectrum, f%,w near 280 nm, and the emission spectrum, Emmax near 340 nm, with a stoppedflow method) and by using electrochemical characterization (by comparison of peak height when applied voltage was 0.6, 0.8, and 1.0 V). The amount detected in the peaks by both native fluorescent and electrochemical (voltammetry) methods was almost the same. The above results indicated that the peaks shown in Figs. 2 and 3 were of Trp-metabolites. ACKNOWLEDGMENTS The authors wish to thank Dr. T. Hashimoto and Dr. H. Nakamura (Toyo Soda Ltd., Tokyo, Japan) for supplying TSK resin. This research was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education in Japan.

REFERENCES 1. Barchas, J., and Usdin, E., eds. (I 973) in Serotonin and Behavior, Academic Press, New York. 2. Boullin, D. J., ed. (1978) in Serotonin in Mental Abnormalities, Wiley, New York. 3. Weil-Malherbe, H. (1972) in Handbook of Neurochemistry (Lajtha, A., ed.), Vol. 7, pp. 371416, Plenum, New York. 4. Krstulovic, A. M., and Powell, A. M., (1979) J. Chromatogr. 171, 345-356. 5. Mefford, I. N., and Barchas, J. D. (1980) J. Chromalogr. 181, 187-193. 6. Fornstedt, N. (1980) J. Chromatogr. 181,456-462. 7. Fornstedt, N. (1978) Anal. Chem. So, 1342-1346. 8. Umino, M., Komiya, K., and Watanabe, H. (1980) Bmseki kirgaku ZQ,670-674.