Improved method for the determination of kynurenic acid in rat plasma by column-switching HPLC with post-column fluorescence detection

Analytica Chimica Acta 562 (2006) 36–43

Improved method for the determination of kynurenic acid in rat plasma by column-switching HPLC with post-column fluorescence detection Shogo Mitsuhashi a , Takeshi Fukushima a,∗ , Junko Kawai a , Masayuki Tomiya a , Tomofumi Santa b , Kazuhiro Imai c , Toshimasa Toyo’oka a a

Department of Analytical Chemistry, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka, Shizuoka 422-8526, Japan b Laboratory of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan c Research Institute of Pharmaceutical Sciences, Musashino University, 1-1-20, Shinmachi, Nishitokyo-shi, Tokyo 202-8585, Japan Received 14 October 2005; received in revised form 10 January 2006; accepted 10 January 2006 Available online 20 February 2006

Abstract Kynurenic acid (KYNA), an endogenous antagonist of ionotropic glutamate and ␣7 nicotinic receptors, was fluorometrically determined by column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. The HPLC system consists of two octadecyl silica (ODS) columns, both of which are connected with an anion-exchange column (trapping column). Following sample injection onto the HPLC column, KYNA was separated on the first ODS column with a mobile phase of H2 O/acetonitrile (95/5) containing 0.1% acetic acid. The peak fraction of KYNA was trapped on the anion-exchange column by changing the position of a six-port valve and then introduced into the second ODS column. Subsequently, KYNA was detected fluorometrically as a fluorescence complex formed with zinc ion which was pumped constantly. Instrumental limit of detection was approximately 0.16 nM, which corresponded to 8.0 fmol (per 50 ␮l injection, signal to noise ratio 3), and the limit of quantification was 0.53 nM (signal to noise ratio 10). Intra- and inter-day relative standard deviations were 1.1–3.9% (n = 3) and 3.0–5.3% (n = 3), respectively. The peak of KYNA in rat plasma was clearly detected by the proposed column-switching HPLC system after a facile pretreatment procedure. Intra- and inter-day relative mean errors were −1.6–1.4% (n = 3) and −2.4 to −0.4% (n = 3), respectively, with a satisfactory precision (within 5.0%). A calibration curve for the determination of KYNA showed a good linearity (r2 > 0.999) in the range of 25–200 nM. The KYNA concentrations in the plasma of male Sprague-Dawley rats (8-week-old) were 44 ± 5.5 nM (mean ± S.E., n = 5). In ketamine-treated rats, which are animal models of schizophrenia, the plasma KYNA concentrations were significantly increased compared with those in the control rats (p < 0.05). © 2006 Elsevier B.V. All rights reserved. Keywords: Kynurenic acid; Column-switching HPLC; Fluorometric detection; Ketamine; Schizophrenia

1. Introduction Kynurenic acid (KYNA), a tryptophan metabolite, acts as an endogenous antagonist of ionotropic glutamate receptors [1]. KYNA can bind at glycine binding sites to antagonize the Nmethyl-d-aspartate (NMDA) receptor function and also shows antagonistic activity with respect to ␣7 nicotinic acetylcholine receptor [2]. Therefore, endogenous KYNA plays some crucial roles in the brain [3–6], and dysregulation of KYNA levels in the brain may be one of the factors on the etiology of schizophrenia, a psychiatric disease [7,8]. As depicted in Fig. 1, a bioprecursor of KYNA, kynurenine is metabolized to KYNA by kynurenine

∗

Corresponding author. Tel.: +81 54 264 5655/4; fax: +81 54 264 5655/4. E-mail address: [email protected] (T. Fukushima).

0003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.aca.2006.01.031

aminotransferase (KAT) I and II [9,10] and is also metabolized to 3-hydroxykynurenine by kynurenine 3-hydroxylase. Inhibition of kynurenine 3-hydroxylase by 3,4-dimethoxy-[N4-(nitrophenyl)thiazol-2-yl]-benzenesulfonamide (Ro 61-8048) or m-nitrobenzoylalanine (mNBA) in vivo may result in the augmentation of the KYNA levels in rat brain microdialysate samples [11–13] due to the facilitation of kynurenine metabolism to KYNA. Apart from psychiatric diseases, KYNA has been recently reported to have some relationship with atherosclerosis [14] and multiple sclerosis [15]; plasma KYNA has been suggested to be altered in these diseases. Therefore, plasma KYNA might be a biomarker for the progression of these diseases. To clarify the biological significances of KYNA in the mammalian body, a useful determination method for KYNA is necessary.

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Fig. 1. Tryptophan metabolism pathway.

For the determination of KYNA in biological samples such as tissues, plasma, urine, and cerebrospinal fluid (CSF) in rodents or humans, a high-performance liquid chromatography (HPLC) with fluorescence detection has frequently been employed [16–19]. Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has also been utilized for the determination of KYNA in rat brain microdialysate sample [20]. These methods have mostly utilized a fluorescence complex with zinc acetate, because zinc ion can specifically form a highly fluorescent complex with KYNA [21]. These chromatographic methods have enabled the sensitive determination of KYNA in biological specimen and have been used for KYNA determination in the pharmacological and clinical fields. These methods have mostly employed a mobile phase at pH 6.2 because the fluorescent complex of KYNA with zinc ion is preferentially formed at pH 6.2. However, the separation of KYNA on an octadecyl silica (ODS) column appeared to be insufficient because the capacity factor of KYNA under the mobile phase condition (pH 6.2) was considerably small [19] due to the carboxyl group in the chemical structure of KYNA. An efficient separation is required due to the reason that unknown compounds that affect the fluorescence intensity of the complex of KYNA with zinc ion might be present in the biological

samples. Furthermore, although 0.6 ml of plasma was used in a previous method [22], it is preferable to determine KYNA in a smaller amount of plasma. Therefore, a more sensitive method is required to determine KYNA in a small amount of sample. In light of this background, we attempted to develop an HPLC method for KYNA determination that was more sensitive and convenient than the previous methods. In the present study, a column-switching HPLC method was employed, and the separation and detection conditions of KYNA were optimized; KYNA was successfully determined in 10 ␮l plasma samples from Sprague-Dawley rats. Subsequently, plasma KYNA concentrations were compared between saline- and ketamine-treated rats, which are animal models of schizophrenia. Using the proposed HPLC method, KYNA concentrations were found to be significantly increased in the plasma of ketamine-treated rats. 2. Experimental 2.1. Chemicals Kynurenic acid (KYNA) and zinc acetate (Zn(AcO)2 ) were purchased from Sigma Co. Ltd. (St. Louis, MO, USA). Acetic acid, ammonium acetate, and ketamine hydrochloride were

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purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Methanol (MeOH) and acetonitrile (CH3 CN) from Kanto Kagaku Co. Ltd. (Tokyo, Japan) were of special grade. Distilled water was used after further purification with a MilliQ system (Nihon Millipore, Tokyo, Japan). All other chemicals were of analytical reagent grade. 2.2. Measurement of fluorescence spectra A fluorescence spectrum of 5.0 ␮M KYNA was measured in the presence of 20 mM zinc acetate dissolved in H2 O. In the case of the experiment of additives in the mobile phase, each solution contained either 50 mM ammonium acetate, 50 mM ammonium formate, or 0.05% acetic acid in H2 O (n = 3). All fluorescence spectra were measured with an F-3010 spectrophotometer (Hitachi Instruments Service Co. Ltd., Tokyo, Japan) using a 1-cm quartz cell. 2.3. HPLC apparatus The HPLC system used in this study consisted of three pumps—PU-1580 (Jasco Co. Ltd., Tokyo, Japan), PU-2080 Plus (Jasco), and L-6200 (Hitachi)—an AS-2057 autosampler (Jasco), a 655A-52 column oven (Hitachi), an L-7480 fluorescence detector (Hitachi), and an HV-992-01 six-port valve (Jasco). The fluorescence detector was set at the excitation and emission wavelengths of 251 and 398 nm, respectively. Two different ODS columns were used; the first column was a CAPCELL PAK C18 MG II (100 mm × 4.6 mm i.d., 3 ␮m, Shiseido Co. Ltd, Tokyo, Japan) with a guard column CAPCELL PAK C18 MG II cartridge (10 mm × 4.0 mm i.d.), and the second column was a TSKgel ODS-80TsQA (150 mm × 4.6 mm i.d., 5 ␮m, Tosoh Co., Tokyo, Japan). The two columns were connected with the six-port valve, HV-992-01 (Jasco). Between the two ODS columns, Inertsil NH2 (10 mm × 3.0 mm i.d., 5 ␮m, GL Sciences Inc., Tokyo, Japan) was set as the trapping column. The mobile phases for each pump were as follows—eluent 1: CH3 CN/H2 O (5/95) containing 0.1% acetic acid. Eluent 2: CH3 CN/50 mM ammonium acetate in H2 O (5/95). Eluent 3: 200 mM zinc acetate in H2 O. Eluents 2 and 3 were used after filtration with a Millicup-LH (0.45 ␮m) (Millipore Corporation, Bedford, MA, USA). The flow rates of pumps 1, 2, and 3 were maintained at 1.0, 0.8, and 0.6 ml/min, respectively. The column temperature was set at 40 ◦ C by a column oven, Hitachi 655A52 (Hitachi). In the proposed HPLC system, the eluent 2 was mixed with the eluent 3 through a three-way static mixer (Jasco) before flow into the fluorescence detector. 2.4. Sensitivity, calibration, precision, and accuracy In order to determine the instrumental limit of detection for KYNA by the present HPLC system, a standard of KYNA in H2 O was directly injected onto the HPLC. The KYNA concentration showing the signal to noise ratio (S/N) 3 was regarded as the instrumental limit of detection, and S/N 10 as limit of quantification.

A physiological concentration of KYNA in rat plasma was approximately 50 nM. To construct the calibration curve, 25, 50, 100, and 200 nM KYNA standard solutions were prepared with dilution with purified H2 O (n = 3), and the 10 ␮l of these solutions were treated in a similar manner as rat plasma described later. The calibration curve for the determination of KYNA was constructed by plotting the obtained peak area against KYNA concentration in the range of 25–200 nM (31–250 fmol KYNA per injection, n = 3). The regression analysis was made using a PC software, Microsoft® Excel 2002. Precision and accuracy were expressed as relative standard deviation (R.S.D., %) and relative mean error (R.M.E., %), respectively, at the concentrations of 50, 100, and 200 nM KYNA (n = 3). Briefly, KYNA determination was carried out by adding 10 ␮l of 0, 50, 100, and 200 nM KYNA in H2 O to 10 ␮l of rat plasma prior to deproteinization with 50 mM ammonium acetate in MeOH (n = 3). Intra- and inter-day determinations of KYNA were performed by repeated analysis of KYNA on the same day and on different days (n = 3), respectively. Intra-day assay was performed within 1 day. 2.5. Determination of KYNA in rat plasma Male Sprague-Dawley rats (7-week-old) were purchased from Charles River Japan Inc. (Kanagawa, Japan) and housed in an environmentally controlled room for at least 7 days before use. All the animals received humane care in compliance with the National Institutes of Health guidelines. For collection of plasma, the rats were anesthetized with diethyl ether in a closed container, and approximately 150 ␮l of blood was then drawn from the jugular vein with a 25-G needle attached to a syringe treated with heparin (Mochida Pharmaceutical Co. Ltd., Tokyo, Japan). After centrifuging the blood at 600 × g (4 ◦ C for 10 min), rat plasma was obtained. Ten ␮l of the rat plasma was diluted twice with adding 10 ␮l of H2 O, and subsequently, was deproteinized by vortex mixing with 80 ␮l of 50 mM ammonium acetate in MeOH, and the resultant mixture was subsequently centrifuged at 600 × g (4 ◦ C for 5 min). The supernatant (50 ␮l) was mixed with 150 ␮l of H2 O/CH3 CN (95/5) containing 0.1% acetic acid. Subsequently, 50 ␮l of the final solution was injected onto the column-switching HPLC system. The concentration of KYNA in rat plasma was determined by a calibration curve described below. 2.6. Ketamine-treated rats At the beginning of this experiment, in order to examine the plasma KYNA concentration in the normal state as a control (0 week), male Sprague-Dawley rats (8-week-old, n = 6) were anesthetized with diethyl ether, and the blood was collected in a similar manner described above. Next, ketamine hydrochloride dissolved in sterilized saline was administered intraperitoneally (i.p.) daily to the rats (30 mg ml−1 kg−1 ) for 5 consecutive days. For comparison, a saline was administered (ml kg−1 ) to rats under the same conditions (n = 5). At 4 weeks after the final i.p. administration of ketamine, blood was again collected from

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each rat in a similar manner, and KYNA was determined as described above. 2.7. Statistical analysis A statistical comparison was performed using the unpaired Student’s t-test following analysis of variance. A p value less than 0.05 was judged as a significant difference. 3. Results and discussion 3.1. Fluorescence intensity of KYNA with zinc acetate Since 20 years KYNA has been reported to exhibit strong fluorescence in the presence of zinc acetate [21]; zinc acetate has mostly been used for the fluorescence detection of KYNA [16–18]. First, we investigated the fluorescence characteristics of KYNA with zinc acetate by using a fluorescence spectrophotometer. In the batch method, excitation and emission spectra of the fluorescence complex with KYNA were measured in the presence of zinc acetate dissolved in H2 O (the ratio of Zn(AcO)2 /KYNA = 4000). In previous studies, a fluorescence intensity of 398–400 nm with an excitation wavelength of 340 nm has mostly been used for the detection [12,15]. However, as shown in Fig. 2, an excitation wavelength of 251 nm yielded a stronger fluorescence intensity of KYNA than that of 340 nm. The excitation wavelength of 251 nm yielded a fluorescence intensity that was three-fold larger than that at 340 nm. Thus, 251 nm was fixed as the excitation wavelength for the highly sensitive detection of KYNA. A strong fluorescence was observed when the molar ratio of zinc acetate (Zn(AcO)2 ) to KYNA exceeded 4000, and the fluorescence intensity at the ratio of Zn(AcO)2 /KYNA above 4000 was constant (Fig. 3). This result was consistent with previous paper [21]. Fig. 4 shows the fluorescence intensities of KYNA with zinc acetate in the presence of 50 mM ammonium acetate, 50 mM ammonium formate,

Fig. 3. Relationship between concentration ratio of Zn(AcO)2 /KYNA and change of the observed fluorescence intensity. The fluorescence intensity at 4000 (Zn(AcO)2 /KYNA) was represented as 100 (%).

or 0.05% acetic acid, which have often been used as an additive in the mobile phase of HPLC. Each pH value of 50 mM ammonium acetate, 50 mM ammonium formate and 0.05% acetic acid in H2 O was 6.8, 6.3 and 3.2, respectively. As shown in Fig. 4, the fluorescence intensity decreased with a reduction in the pH of the solution, indicating that an acidic mobile phase was unsuitable for the fluorescence detection of KYNA. In the ammonium acetate and ammonium formate solutions, the percentage of ionic form of the carboxyl group of KYNA may increase to form easily the fluorescence coordination complex with zinc ion. Therefore, among the solutions examined, the aqueous solution containing ammonium acetate was considered to be the best suited mobile phase for the fluorescence detection of KYNA. It was confirmed that the addition of EDTA 2Na in the solution of KYNA with zinc ion drastically decreased the fluorescence intensity (data not shown). This result supported that the observed fluorescence might be caused by the formation of a coordinated complex between KYNA and zinc ion. 3.2. Chromatographic separation For the separation of biological sample, a reversed-phase mode using an ODS column is generally effective, and an acidic mobile phase is preferred for separation of an acidic compound such as KYNA. However, in most previous studies, an ODS column with a mobile phase at pH 6.2 containing 4.5–16% CH3 CN has been frequently used for the separation of KYNA in biological samples due to the fluorescence detection of KYNA with zinc ion [11,12]. In the mobile phase with pH 6.2, the capacity

Fig. 2. Excitation (a) and emission spectra (b) of KYNA with zinc ion. In (b), the upper and lower curves indicate the emission spectra at excitation wavelengths of 251 and 340 nm, respectively.

Fig. 4. Effect of each solution with different pH values on the fluorescence intensity of KYNA with zinc ion.

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factor of KYNA was considerably small [19], because KYNA has a carboxyl group in its chemical structure. An efficient separation is required for KYNA determination to avoid interference by unknown compounds in biological samples, but the separation of KYNA on an ODS column appeared to be inefficient at pH 6.2 of mobile phase. Therefore, we designed a column-switching HPLC system consisting of two columns. The first column is an ODS column that uses an acidic mobile phase to separate KYNA from other biological compounds, which could interfere with the fluorescence of KYNA. The second column is for the highly sensitive detection of KYNA, using a mobile phase consisting of H2 O and ammonium acetate. In order to use the column-switching HPLC system described above, the peak fraction of KYNA eluted from the first ODS column was required to be introduced into the second ODS column. KYNA has a carboxyl group in its structure; therefore, ionic interaction may occur with an amino group on the surface. We previously succeeded in trapping a fluorescence derivative of S-lactoylglutathione, which has a carboxyl group, on an anion-exchange amino column in our previous columnswitching HPLC system [23]. Based on this observation, we investigated whether KYNA could be trapped on an anionexchange amino column. Among the anion-exchange amino columns examined, when Inertsil NH2 (150 mm × 4.6 mm i.d.) was used, KYNA could not be eluted with a mobile phase of H2 O containing 0.1% acetic acid, but it could be eluted using a mobile phase of H2 O containing 50–200 mM ammonium acetate (data not shown). Therefore, this anion-exchange amino column (Inertisil NH2 ) appeared suitable to be used as the trapping column. The precise mechanism by which the retention and elution of KYNA on the amino column is unclear yet, but KYNA might be eluted by ion-exchange mode. In acidic mobile phase (0.1% acetic acid), amino moiety on the amino column is charged to ammonium form, which can bind the carboxyl group of KYNA by ionic interaction. However, under neutral pH with ammonium acetate (AcONH4 ) in the mobile phase, ammonium ion in the mobile phase could interact with the carboxyl group of KYNA as a counter ion to elute KYNA from the amino column. Thus, a column-switching

HPLC system for the determination of KYNA was established as shown in Fig. 5. The first ODS column was CAPCELL PAK C18 MG II (100 mm × 4.6 mm, 3 ␮m), and the second ODS column was TSKgel ODS-80TsQA (150 mm × 4.6 mm, 5 ␮m), because a sharp KYNA peak was obtained when TSKgel ODS-80TsQA was used as the second ODS column. Between the two ODS columns, an anion-exchange column, Inertsil NH2 (10 mm × 3.0 mm i.d.) was connected as a trapping column. The mobile phase (eluent 1) is H2 O/CH3 CN (95/5) containing 0.1% acetic acid and (eluent 2) is H2 O/CH3 CN (95/5) containing 50 mM ammonium acetate. KYNA was eluted on the first ODS column with a mobile phase (eluent 1) from 10.5 to 12.5 min. The peak fraction eluted from 10.5 to 12.5 min was introduced onto the trapping column by changing the valve position (from A to B), thereby efficiently removing the interfering compounds from the biological sample. Subsequently, KYNA was introduced onto the second ODS column by changing back the valve position (from B to A) and was then separated on the ODS column by isocratic elution of mobile phase (eluent 2). Finally, KYNA eluted from the second ODS column was specifically detected by the fluorescence complex formation with zinc ion (eluent 3). The second ODS column also plays a role for preventing co-elution of KYNA with a large amount of acetic acid, which is unnecessary for fluorescence detection, included in the mobile phase (eluent 1). Both mobile phases contained the same ratio of CH3 CN to H2 O (95/5), thereby, a flat baseline was obtained under the mobile phase condition (Fig. 6(a)). Next, we tried to optimize the post-column formation of the fluorescence complex of KYNA with zinc acetate. The concentration of zinc acetate was set at 200 mM, because noise level of baseline gradually increased above 200 mM. The flow rate in the range of 0.5–0.7 ml/min yielded a high signal to noise ratio (data not shown); thus, the flow rate of 0.6 ml/min was selected for this experiment. By optimization of the proposed HPLC conditions as mentioned above, the KYNA peak was clearly observed in the chromatogram (Fig. 6(b)). A calibration curve for the determination of KYNA showed a good linearity (r2 > 0.999) in the range of 25–250 nM (31–250 fmol KYNA per 50 ␮l injection). Instrumental limit of detection was approximately 8.0 fmol (signal to noise ratio 3), which corresponded to 0.16 nM, and the

Fig. 5. A block diagram of the column-switching HPLC system for KYNA determination (eluent 1) 0.1% acetic acid in H2 O/CH3 CN (95/5), 1.0 ml/min. (Eluent 2) 50 mM ammonium acetate in H2 O/CH3 CN (95/5), 0.8 ml/min. (Eluent 3) 200 mM zinc acetate in H2 O, 0.6 ml/min. Column 1: CAPCELL PAK C18 MG II (100 mm × 4.6 mm., 3 ␮m i.d., Shiseido Co. Ltd.). Column 2: TSKgel ODS-80TsQA (150 mm × 4.6 mm, 5 ␮m i.d., Tosoh Co. Ltd.). Trapping column: Inertsil NH2 (10 mm × 3.0 mm i.d., GL Sciences). Solid line, valve position A; dotted line, valve position B.

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Fig. 6. Representative chromatograms of blank sample (a), a standard of KYNA (31 fmol) (b), rat plasma from control rats (c), and rat plasma from ketamine-treated rats (d).

limit of quantification was 0.53 nM. In the present study, the fluorescence intensity of KYNA was greater than that of previous methods because an excitation wavelength of 251 nm was employed. Thus, the limit of fluorescence detection was greatly improved as compared with that in the previous method [22]. With respect to the analysis times, CE-LIF method [20] is superior than LC methods, because the migration time of KYNA within 60 s was reported. However, limit of detection in our proposed HPLC method is approximately 8.0 fmol corresponding to 0.16 nM, which was lower than that (1.0 nM) in CE-LIF method [20], because injection volume is limited in CE. 3.3. Determination of KYNA in rat plasma KYNA was easily dissolved in MeOH containing 50 mM ammonium acetate. Therefore, MeOH containing 50 mM ammonium acetate was used as the extraction solvent in the separation of KYNA from the rat plasma. After denaturation and precipitation of plasma proteins by adding 50 mM ammonium acetate and mixing vigorously, the supernatant was diluted 4-fold with the mobile phase (eluent 1), and an aliquot of 50 ␮l was injected onto the column-switching HPLC system after filtration through a 0.2-␮m membrane. As a result of the chromatographic separation described above, KYNA determination in the rat plasma was accomplished without any interferences (Fig. 6(c)). In 1990, Heyes et al. also employed 254 nm of excitation wavelength for the fluorescence determination of KYNA in human cerebrospinal fluids (CSF), but there were several large peaks in their chromatograms [17]. Although human CSF has not been used yet in the present study, KYNA could be efficiently purified from the interfering biological compounds in the rat plasma by using both the ODS columns with different mobile phases. Using the proposed column-switching HPLC

system, KYNA in 10 ␮l of rat plasma was successfully determined (44 ± 5.5 nM, mean ± S.E., n = 5), while 0.6 ml of rat plasma was used in previous HPLC method [22]. Furthermore, in a previous paper [15], a tedious pretreatment procedure was performed for plasma KYNA determination, i.e. deproteinization with perchloric acid, purification of KYNA with Dowex 50w cation-exchange resin including washing with 0.1 N HCl and eluting with H2 O, followed by lyophilization under vacuum. In contrast, in our proposed HPLC method, it is possible to determine KYNA in plasma by a facile pretreatment procedure as described above. Using the proposed HPLC method, 10 ␮l of rat plasma was sufficient for KYNA determination, which is beneficial in decreasing the physiological damage to small animals such as rodents. 3.4. Validation study Using the proposed column-switching HPLC, relative standard deviations (R.S.D.) and relative mean errors (R.M.E.) of KYNA determination were depicted in Table 1. The intra- and inter-day R.S.D.s of KYNA determination without rat plasma were 1.1–3.9% (n = 3) and 3.0–5.3% (n = 3), respectively, and the intra- and inter-day R.S.D.s of KYNA determination with rat plasma were 1.3–4.4% (n = 3) and 1.7–5.0% (n = 3), respectively. The intra- and inter-day R.M.E.s of KYNA determination were −1.6–1.4% (n = 3) and −2.9 to −0.4% (n = 3), respectively. The recovery (%) was determined by calculating the slope ratio of calibration curve constructed by standard KYNA solution to the calibration curve when 10 ␮l of 50–200 nM KYNA were spiked into 10 ␮l of rat plasma (n = 3). The recovery of KYNA from rat plasma was 98% for intra-day, and 97% for inter-day assay, respectively. These data showed that the HPLC method

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Table 1 Relative standard deviation (R.S.D., %) and relative mean error (R.M.E., %) of KYNA from rat plasma by the proposed HPLC method (n = 3) KYNA concentration (nM)

0

50

100

200

Intra-day, R.S.D. (%) Inter-day, R.S.D. (%)

– –

3.9 3.0

3.3 3.7

1.1 5.3

Added KYNA concentration (nM)

0

50

100

200

Intra-day, R.S.D. (%) R.M.E. (%) Inter-day, R.S.D. (%) R.M.E. (%)

3.9 – 2.8 –

2.0 1.4 5.0 −2.2

1.3 −1.5 2.7 −0.4

4.4 −1.6 1.7 −2.9

used in the present study has an adequate reproducibility for the determination of KYNA in rat plasma. 3.5. KYNA in the plasma of saline- and ketamine-treated rats It has recently been reported that ketamine, a commercially available anesthetic drug, can be used for producing an experimental animal model of schizophrenia [24,25]. This effect may be derived from its antagonistic action on the N-methyl-daspartate receptor. In accordance with previous studies [24,25], we repeatedly administered ketamine i.p. for 5 days to rats (30 mg kg−1 day−1 ). The fluorescence peak of KYNA was also observed in the plasma sample of the rats treated with ketamine (Fig. 6(d)). Subsequently, the plasma KYNA concentrations were compared between saline- and ketamine-treated rats. As mentioned above, KYNA concentration in rat plasma was 44 ± 5.5 nM (mean ± S.E., n = 5) before administration. As shown in Fig. 7, the plasma KYNA concentrations in the ketamine-treated rats were significantly increased (59 ± 4.4 nM, n = 6) when compared with those in the saline-treated rats (45 ± 4.8 nM, p < 0.05) at 4 weeks after termination of repeated ketamine administration. When compared 0 week with 4 weeks, a significant increase was observed in the ketamine-treated rats

Fig. 7. Plasma KYNA concentrations in saline- and ketamine-treated rats at 4 weeks after termination of repeated ketamine administration. Open column indicates saline-treated rats (n = 5), and closed column indicates ketamine-treated rats (n = 6). Zero week indicates the rats before administration of saline or ketamine. The detailed experimental conditions are described in the text.

(p < 0.05), while no significant changes were observed in salinetreated rats. In these plasma samples, we observed that amino acid levels also changed significantly (unpublished data). Thus, it was concluded that KYNA concentration increased significantly in the ketamine-treated schizophrenic rats, accompanied by the alterations of plasma amino acid levels. These results may be important information in the study on the relationship between KYNA and the etiology of schizophrenia. 4. Conclusion We developed a column-switching HPLC method for the highly sensitive determination of KYNA in the rat plasma with a satisfactory precision and accuracy. Plasma KYNA was successfully determined with a facile pretreatment procedure. Thus, the proposed HPLC method is expected to be used in the clinical diagnosis of neurologic and psychiatric diseases affecting plasma KYNA levels, and will be also applicable to determine KYNA in brain tissue or microdialysate sample in the pharmacological researches. Acknowledgements This work was financially supported in part by a Grant-inAid for Scientific Research (no. 17590132) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References [1] K.J. Swartz, M.J. During, A. Freese, M.F. Beal, J. Neurosci. 10 (1990) 2965. [2] C. Hilmas, E.F.R. Pereira, M. Alkondon, A. Rassoulpour, R. Schwarcz, E.X. Albuquerque, J. Neurosci. 21 (2001) 7463. [3] S. Erhardt, G. Engberg, Acta Physiol. Scand. 175 (2002) 45. [4] S. Erhardt, L. Schwieler, C. Emanuelsson, M. Geyer, Biol. Psychiatr. 56 (2004) 255. [5] R. Carpenedo, A. Pittaluga, A. Cozzi, S. Attucci, A. Galli, M. Raiteri, F. Moroni, Eur. J. Neurosci. 13 (2001) 2141. [6] H.Q. Wu, K. Fuxe, R. Schwarcz, J. Neurochem. 90 (2004) 621. [7] R. Schwarcz, A. Rassoulpour, H.Q. Wu, D. Medoff, C.A. Tamminga, R.C. Roberts, Biol. Psychiatr. 50 (2001) 521. [8] S. Erhardt, L. Schwieler, G. Engberg, Adv. Exp. Med. Biol. 527 (2003) 155. [9] E. Luchowska, P. Luchowski, R. Paczek, A. Ziembowicz, T. Kocki, W.A. Turski, M. Wielosz, J. Lazarewicz, E.M. Urbanska, J. Neurosci. Res. 79 (2005) 375. [10] W. Rzeski, T. Kocki, A. Dybel, K. Wejksza, B. Zdzisinska, M. KandeferSzerszen, W.A. Turski, E. Okuno, J. Albrecht, J. Neurosci. Res. 80 (2005) 677. [11] R. Carpenedo, A. Chiarugi, P. Russi, G. Lombardi, V. Carla, R. Pellicciari, L. Mattoli, F. Moroni, Neuroscience 61 (1994) 237. [12] A. Cozzi, R. Carpenedo, F. Moroni, J. Cereb. Blood Flow Metab. 19 (1999) 771. [13] F. Moroni, A. Cozzi, R. Carpendo, G. Cipriani, O. Veneroni, E. Izzo, Neuropharmacology 48 (2005) 788. [14] J. Stazka, P. Luchowski, E.M. Urbanska, Eur. J. Pharmacol. 517 (2005) 217. [15] Z. Hartai, P. Klivenyi, T. Janaky, B. Penke, L. Dux, L. Vecsei, Acta Neurol. Scand. 112 (2005) 93. [16] K. Shibata, J. Chromatogr. 430 (1988) 376. [17] M.P. Heyes, B.J. Quearry, J. Chromatogr. 530 (1990) 108. [18] K. Mawatari, F. Iinuma, M. Watanabe, Anal. Biochem. 190 (1990) 88.

S. Mitsuhashi et al. / Analytica Chimica Acta 562 (2006) 36–43 [19] K.J. Swartz, W.R. Matson, U. MacGarvey, E.A. Ryan, M.F. Beal, Anal. Biochem. 185 (1990) 363. [20] D.K. Hansen, S.M. Lunte, J. Chromatogr. A 781 (1997) 81. [21] F. Iinuma, M. Tabara, K. Yashiro, M. Watanabe, Bunseki Kagaku 34 (1985) 483. [22] H. Baran, M. Gramer, D. Honack, W. Loscher, Eur. J. Pharmacol. 286 (1995) 167.

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[23] E. Uchino, T. Fukushima, M. Tsunoda, T. Santa, K. Imai, Anal. Biochem. 330 (2004) 186. [24] A. Becker, B. Peters, H. Schroeder, T. Mann, G. Huether, G. Grecksch, Prog. Neuropsychopharmacol. Biol. Psychiatr. 27 (2003) 687. [25] A. Becker, G. Grecksch, Prog. Neuropsychopharmacol. Biol. Psychiatr. 28 (2004) 1267.