Journal o.]"Chromatography, 57 i ( 1991 ) 31 ~"..-~23 Biomedical Applications Elsevier S,~ience Publishers B.V., A m s t e r d a m CHROMBIO. 6048

Short Communication Rapid method for the determinatioD of ketanserin in rat serum by high-performance liquid chromatography with fluorimetric detection Y. W. J. W O N ( 3 and M. H. S K I N N E R *

Department of Pharmacologj', Dirision of Clinical .?harmacology, Universi O"of Texas Health Sciet:e,, Center San Antonio. 7703 Floyd Curl Drive,. San Antonio, TX 78284 ( U S A )

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(First received March 26th, 1991: revised manuscript received June 21st, 199I) ABSTRACT A simplified high-performance liquid c h r o m a t o g r a p h i c ( H P L C ) assay for the determination o f ketanserin in rat serum is described. T h e c h r o m a t o g r a p h i c m e t h o d allowed complete resolution o f ketanserin from two o f its metabolites. A protein precipitation extraction procedure was e m p l o y e d which allowed rapid sample preparation for injection ;nto the H P L C system. Both intra- and inter-assay coefficients o f variation at serum ketanserin c o n c e n t r a t i o n o f 21J0 a o d 800 ng/ml were less than 6 % and the accuracy was excellent. The assay has been applied for determining the elimination kinetics o f ketanserin in the rat.

INTRODUCTION

Ketanserin, 3-{2-[4-(4-fluo robenzoyl)- 1-piperidinyl]ethyl }-2,4( 1H,3H)-quinazolinedione, is a serotonin S2-receptor antagonist effective in the treatment o f hypertension [ 1-3]. This agent has also been used extensively as a labelled ligand to characterize in vitro and in vivo serotonin S2-rcceptors [4-6]. The major phase i metabolic pathways o f ketanserin in m a m m a l s are: (a) aromatic hydroxylation at the quinazolinedione; (b) ketone reduction, (c) oxidative N-dealkylation at the piperidine nitrogen (Fig. 1). In rats, (a) and (c) are the main pathways, (b) is the minor one. h~ humans, (b) and (c) are the main pathways, whereas (a) in only a minor one [7]. Our interests in ketanserin is the application o f this agent to the kinetic modeling of serotonin S2-receptor in rat brain. This requires a sensitive assay for ketanserin in serum and brain tissue in the presence o f its metabolites. Various methods for the determination o f ketanserin in human plasma have been described; these include a radioimmunoassay [8] and several high-performance liquid chromatographic (HPLC) methods with U V [9-11] and fluorimetric detection [12,13]. We report here a method for determining ketanserin in rat serum by H P L C 0378-4347/91/S03.50

©

1991 Elsevier Science Publishers B.V. All rights reserved

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with fluorimetric detection. The assay is extremely rapid and is as z.cn~itive as other H P L C assays when less than 100/A of sample is available for analysis. In ~u,~,uu., the assay employs a rapid protein p,-'~,.cxpl""t,,-+;'--...n sample preparation procedure which allows serum samples to be prepared for analysis immediately after collection. EXPERIMENTAL

Reagents and chemicals Ketanserin tartrate (R-41,468), ketanserinol (R-46,742) and 6-hydroxyketanserin (R-49,285) were provided as reference compounds by Janssen Pharmaceutica (Beerse, Belgium). Acetonitrile and methanol were HPLC grade (OmniSolve, MCB Manufacturing Chemists, Cincinnati, OH, USA). Ace'". acid and a m m o n i u m hydroxide in analytical-grade purity were also from MC,t Manufacturing Chemists. Instrumentation The chromatograph consisted of a Waters Model 601 pump with a U 6 K universal injector (Milford, M A , USA) and a Shoeffel Model FS 970 fluorimetric

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detector (Krato, Westwood, N J, USA). C h r o m a t o g r a p h i c peaks were recorded o n a H e w l e t t ' P a c k a r d Model 3392A integrator (Houston, TX, USA). The Vortex-Genie 2* mixer and the Fishe r M o d e l 235A microcentrifuge were from Fisher Scientific (Pittsburgh, PA, USA).

Chromatographic conditions C h r o m a t o g r a p h i c analysis was carried out at n o r m a l a m b i e n t temperature, i.e. 23"C. The mobile phase consisted of acetonitrile-acetate buffer (pH 7.0)-distilled water (31:50:19). Acetate buffer (pH 7.0) was prepared by adjusting 2% acetic acid in distilled water to pH 7.0 with a m m o n i u m hydroxide. A Waters N o v a - P a k C18 (4 p m , 15 cm x 0.39 cm) c o l u m n was used as the analytical column. The flow-rate o f the mobile phase was 1 m l / m i n . •The excitation wavelength was set at 225 n m a nd the emission was m o n i t o r e d with no emission filter.

Sample preparation Serum samples were either prepared for assay i m m e d i a t e l y after collection or stored at - 2 0 ° C until assay. Each s a m p l e was allowed to thaw at r o o m temperature a n d vortex-mixed for 30 s before an aliquot was removed. Serum (25 pl) was mixed with 50 #! of m e t h a n o l in a 1.5-ml microfuge tube. The mixture was vortexmixed tbr 30 s and centrifuged at 13 600 g for 5 min. A 25-pl aliquot of the clear s u p e r n a t a n t was injected into the H P L C system. C a l i b r a t i o n plot (peak height versus concentration) was constructed using rat serum s u p p l e m e n t e d with 80, 200, 400, 800 a n d 1600 ng/ml ketanserin.

Preparation o f calibration standards Ketanserin tartrate, equivalent to 1 mg of ketanserin, was dissolved a n d m a d e tlp to 100 ml with m e t h a n o l to give solution A. A 5-ml volume o f solution A was further diluted to 50 ml with m e t h a n o l to produce solution B c o n t a i n i n g 1 ng/pl ketanserin. These solutions were freshly prepared before use. To prepare a s t a n d a r d serum sample c o n t a i n i n g 80 ng/ml ketanserin, 16 pl o f solution'B were transferred to a 1.5-ml microfuge tube with a H a m i l t o n syringe and evaporated to dryness u n d e r a stream o f dry nitrogen. The residue was reconstituted with 2 0 0 / d of b l a n k rat serum. The procedure was repeated with 40, 80, 160 a n d 320 pl of solution B to produce serum s t a n d a r d s c o n t a i n i n g 200, 400, 800 a n d 1600 ng/ml ketanserin. RESULTS AND DISCUSSION

T h e a i m of the present study was to develop a simplified m e t h o d for the determ::nation of ketanserin in rat serum. Initial investigations showed that ketanserin a n d two o f its metabolites could be resolved at p H 7.0 on a reversedp h a s e C t s column. T he retention times o f ketanserin, ketanserinol a n d 6-

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Fig. 2. R e p r e s e n t a t i v e c h r o m a t o g r a m s o f ketanserin, ketanserinol and 6-hydroxyketanserin. (A) S t a n d a r d m i x t u r e o f 2 ng o f ketanserin, 4 ng o f 6 - h y d r o n y k e t a n s e r i n and 0.5 ng o f ketanserinol; (B) b l a n k rat s e r u m sample; (C) b l a n k rat s e r u m s a m p l e s u p p l e m e n t e d with ketanserin at 80 ng/ml; (D) s e r u m s a m p l e f r o m a rat 2 h a f t e r an i n t r a v e n o u s dose o f k e t a n s e r i n at 0.5 mg/kg. Peaks: I = ketanserin; 2 ~- 6 - h y d r o x y k e t a n s e rin; 3 = ketanserinot.

h y d r o x y k e t a n s e r i n under the described c h r o m a t o g r a p h i c conditions were 7.7, 3.2 a n d 4.5 rain, respectively (Fig. 2A). T h e acidic metabolite, which is unavailable for evaluation, is not expected to interfere; as a carboxylic acid, this c o m p o u n d would p r e d o m i n a n t l y be in the ionized form at p H 7.0 and is expected to exhibit a very short retention time on a reversed-phase c h r o m a t o g r a p h i c system. T h e sensitivity o f fluorimetric detection depends on careful selection o f excitation a n d emission wavelengths. A l t h o u g h 310 and 270 n m have been used as the wavelengths of excitation for ketanserin [12,13], the detector used in the present study offered the best sensitivity with 225 n m as the excitation wavelength wh e n the emission was m o n i t o r e d with no emission filter. W i t h these detector settings, ketanserin can be detected d o w n to 0.1 ng. M o s t H P L C m e t h o d s for ketanserin published to date include a liquid extraction m e t h o d with a solvent mixture o f pe nta ne a nd isoamyl alcohol [9-13]. In order to overcome the poor extraction efficiency, the sample is n o r m a l l y extracted twice which is inconvenient. W h e n the sa mple size available for analysis is less t h a n 100 pl, a simple and m o r e efficient protein precipitation metilod offers a suitable alternative. The use o f this precipitation m e t h o d has also allowed the assay be run w ithout an internal sta nda r d. Fig. 2 D shows a c h r o m a t o g r a m resulting from the analysis of a serum sample o bt a i n e d f r om a rat 2 h after a single 0.5 m g / k g intravenous dose o f ketanserin. A

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Fig. 3. S e r u m c o n c e n t r a t i o n - t i m e profile o f ketanserin in one rat following a single 1.0 m g / k g intravenous dose o f ketanserin.

calibration plot o? peak height v e r s u s concentration of ketanserin was linear (range 80-1600 ng/ml) with correlation coefficient (r 2) greater than 0.99. The intra- and inter-assay precision and accuracy were determined by repeated analysis of serum samples supplemented with 200 and 800 ng/ml ketanserin; the results are summarized in Table I. Since the calibration standards for each inter-assay variability study run were prepared using serum from different rats, the interassay coefficient of variation reflects the consistent recovery of ketanserin provided by the extraction method. Although the lowest concentration that can be accurately determined is about 40 ng/ml, it can be improved by injecting a larger amount of supernatant in the H P L C system because the chromatograms were essentially free from interferences (Fig. 2).

TABLE i INTRA- AND INTER-ASSAY ACCURACY AND PRECISION OF THE METHOD FOR DETERMINING KETANSERIN IN RAT SERUM Theoretical concentration (ng/ml)

Number of determinations (n)

M e a n concentration (ng/ml)

Coefficient o f variation ( % )

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The assay has been applied to determining the elimination kinetics o f ketanserin in the rat. Fig. 3 shows a sertam concentration-time profile in o~ie cat following a single 1.0 mg/kg intravenous dose o f ketanserin. The method described is developed specifically for ketanserin in rat serum and is as sensitive as other published H P L C methods with fluorimetric detection when less than 100 pl o f sample is available for analysis. The major advantage o f the method is the simple and rapid sample preparation procedure, which allows samples be processed immediately after collection. In addition, the method is particularly well suited for the analysis o f radiolabelled ketanserin in serum when coupled with a radiochemical detector; because o f reduced handling in the sample preparation process, radiochemical contamination is substantially reduced. REFERENCES 1 J . N . M . Barendregt, A. van Peer, J. G. van der H o e v e n , J. C. van Oene and Y. I. T j a n d r a , Br. J. Clin. Pharmacol., 29 (1990) 715, 2 R. Donnelly, H. L. Elliott, P. A. Meredith a:ld J. L. Reid, Br. J. Clin. Pharmacol., 24 (1987) 599. 3 B. Persson, A. Pettersson and T. Hedner, Eta'. ,L Clin. Pharmacol., 32 (1987) 259. 4 J. E. Leysen, C. J. E. Neimegeers, J. M. van N u e t e n a n d P, M. L a d u r o n , Mol. Phm'macol., 21 (1982) 301. 5 P. M. L a d u r o n , P. F. NI. Janssen and J. E. Leysen, Eur. J. Pharmacol., 8I (I982) 42. 6 D. J. M c K e n n a a n d S. J. P e r o u t k a , J. Neurosci., 9 (1989) 3482. 7 W. M e u l d e r m a n s , J. Hendriekx, W, Lauwers, E. Swysen, R. H u r k m a n s , F, Knaeps, R. Woestenborghs and J. H e y k a n t s , Drug Metab. Dispos., 12 (1984) 772. 8 B. Persson, J. H e y k a n t s and T. Hedner, Clin. Pharmacokin., 20 (1991) 263. 9 V. Simon and P. Sioman, J. Cio'omatogr., 232 (1982) 186. 10 F. Lindelauf, J, Chromatogr., 277 (1983) 396. I1 A. T. K a e p r o w i e z , P. G. Shaw, R. F. W. Moulds and R. W. Bury, J, Chromatogr., 272 (1983) 417. 12 P. O. O k o n k w o , I. W. Reimann, R. Woestenborghs and U. Klotz, J. Chromotogr., 272 (I983) 41 i. 13 M. K u r o w s k i , J. Chromatogr., 341 (I985) 208.

Rapid method for the determination of ketanserin in rat serum by high-performance liquid chromatography with fluorimetric detection.

A simplified high-performance liquid chromatographic (HPLC) assay for the determination of ketanserin in rat serum is described. The chromatographic m...
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