BIOMEDICAL CHROMATOGRAPHY, VOL. 6 , 124-127 (1992)

A Sensitive Determination Method for Mexiletine Derivatized with Dansyl Chloride in Rat Plasma Utilizing a HPLC Peroxyoxalate Chemiluminescence Detection System Atsuhiko Nishitani, Susumu Kanda and Kazuhiro Imai* Branch Hospital Pharmacy, University of Tokyo, 3-28-6 Mejirodai, Bunkyo-ku, Tokyo 112, Japan

A sensitive determination method for a non-fluorescent anti-arrhythmic drug, mexiletine, in rat plasma is presented utilizing a HPLC peroxyoxalate chemiluminescence (PO-CL) detection system. After an internal standard (4-methylmexiletine, 4.35 pmol) and 0.1 N sodium hydroxide solution were added to 5 pL rat plasma, the solution was poured onto an Extrelut 1column. Both mexiletine and the internal standard were eluted with diethy ether and then the eluate was evaporated to dryness. The residue was dissolved in 0.2 M borate buffer (pH8.5) and mixed with dansyl chloride (75nmol) in acetronitrile. After standing of 90min at room temperature, 0.5 N HCl was added to the reaction mixture to stop the reaction and a 2/45 aliquot of the mixture was subjected to a HPLC PO-CL detection system using bis(4-nitro-2(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) and hydrogen peroxide. The calibration curve for mexiletine in rat plasma was linear over the range 20-100 ng/mL plasma (20.6-103 fmolhnjection). The detection limit (S/N=2)was 1.0 fmol over the whole procedure. The method was applied to the measurement of the time courses of plasma mexiletine concentration after oral administration of the drug 125 mg (115.9 pmsl)lkg] to rats.

INTRODUCTION Mexiletine (1-(2,6-dimethylphenoxy)-2-propamine, MX) is an anti-arrhythmic drug. While its structure and electrophysiological properties resemble those of lidocaine, MX has an advantage of being effective with oral administration and having a longer half-life. The therapeutically effective blood level of M X is reported to be narrow at 0.5-2.0 yg/mL. Moreover, the occurrence rate of adverse effects increases when the blood level is above 2pgImL (Talbot, 1973; Campbell and Kelly, 1978). Therefore, the monitoring of the plasma level is necessary. Mexiletine in biological fluids has been quantitated by a number of different gas-liquid chromatographic methods, using a variety of detection systemsnitrogen/phosphorus-selective detection (NPD) (Smith and Meffin, 1980), flame ionization detection (FID) (Holt et al., 1979), electron capture detection (ECD) (Willox and Singh, 1976). However, these methods are not sensitive and selective enough for the measurement of minute amounts of MX in biological specimens. The detection limits of MX in blood are 5 ng/mL, 500 ng/ mL and 20 ng/mL determined by NPD, FID and ECD, respectively. Recently, the peroxyoxalate chemiluminescence (PO-CL) reaction has been successfully applied to a detection system for fluorescent compounds and drugs using HPLC (Kobayashi and Imai, 1980a; Imai, 1986). Since this PO-CL detection system requires fluorescent drugs to generate CL without a light source, they can be quite sensitively detected at the fmol to amol range with absence of interference by a light source as in the case of fluorometric analysis (Imai et al., 1987a, 1987b). * Author to whom

correspondence should be addressed.

0269-38791921030124-04 $05.00 01992 by John Wiley & Sons, Ltd.

In a previous paper (Nishitani et al., 1991), we reported a simple, sensitive determination method for the fluorescent drugs dipyridamole and benzydamine in rat plasma samples. The method consists of deproteinization of 0.1-10 pL plasma with 13 volumes of acetonitrile and separation by HPLC with PO-CL detection. This paper describes a simple and sensitive method for the determination of the non-fluorescent drug, MX, in rat plasma after its oral administration using a HPLC PO-CL detection system, which should require only a small volume of plasma and is more convenient than the previous methods (Ueno et af.,1989; Mastropaolo et af., 1984). The conditions for extraction and fluorogenic derivatization of MX with dansyl chloride (DNS(3)are studied and the resultant fluorescent derivative is finally determined by the HPLC PO-CL detection system.

EXPERIMENTAL Chemicals.

Bis(bnitro-2-(3,6,9-trioxadecyloxycarbonyl)phenyl) oxalate (TDPO) was purchased from Wako Pure Chemical Industries Ltd (Osaka, Japan). Hydrogen peroxide (30%) was purchased from Mitsubishi Gas Kagaku (Tokyo, Japan). Imidazole (zone refined) and 5-( dimet hylamino)- 1naphthalenesulphonyl chloride (DNS-CI) were donated from Tokyo Kasei Kogyo Co. Ltd (Tokyo, Japan). Distilled water (HPLC grade), acetonitrile (HPLC grade), and ethyl acetate (HPLC grade) were from Wako. Mexiletine and 4methylmexiletine (internal standard) were donated from Nippon Boehringer Ingelheim Ltd (Tokyo, Japan). All other chemicals were of reagent grade. Extrelut 1 was purchased from Merck. Received I9 July 1991 Accepted 16 October 1991

HPLC- PO-CL DETECTION OF MEXILETlNE Eluent

Il-I

column oven

Figure 1. Flow diagram for chemiluminescence reaction detection system for HPLC. PI and P2: pumps; C, and Cz: d u m m y columns for dampers; I: injector; TDPO: bis(4-nitro-2-(3,6,9trioxadecy1oxycarbonyl)p henyl) oxalate; H,O,: hydrogen peroxide; MD: mixing device; R; recorder; Det: chemiluminescence monitor.

HPLC chemiluminescencedetection system. The flow diagram for this system is shown in Fig. 1. The pumps used were a KHP-011 (Kyowa Seimitsu Co., Tokyo, Japan) and an LC-6A (Shimadzu Seisakusho Co., Tokyo, Japan). The injection valve (Rheodyne, Cotati, CA, USA) with a 20 pL loop, an analytical column (TSK ODS 80Tm 150 x 4 mm i.d., 5 pm) and a dummy column (TSK ODS 120A, 100 x 4.6 mm i.d., 5 pm) were used. A chemiluminescence monitor, 825-CL (Jasco, Tokyo, Japan) and a recorder (T-626DS Nippon Denki Kagaku Co. Ltd, Tokyo, Japan) were used. A 25 pL rotating flow mixing device (Kyowa Seimitsu Co., Tokyo, Japan) (Kobayashi and Imai, 1980b) was heated at 40 "C in a column oven (655A-52, Hitachi Seisakusho Co. Ltd, Tokyo, Japan). The flow line (PTFE tubing, 50 X 0.5 mm i.d. and 50 x 0.8 mm i.d.) was connected to the detector next to the mixing device. The eluent consisted of 5 0 m ~imidazole buffer(pH 6.0) :acetonitrile (35: 65, v/v), the flow rate of which was 1.0 mL/min (Imai et al., 1990). The chemiluminogenic reagent solution was a mixture of 0.25 mM TDPO and 12.5 mM hydrogen peroxide in acetonitrile : ethyl acetate (1 :1, v/v). The flow rate of the solution was 1.6 mL/min (Nishitani et al., 1991). Both solutions were mixed just prior to use. Investigation of the fluorogenic reaction. To 10pL of the aqueous solution of MX (0.6 p ~ in) a test-tube, 50 pL internal standard (Cmethylmexiletine), 40 pL 0.2 M borate buffer (pH 7.5, 8.0, 8.5 and 9.0) and 150 pL 500 p~ DNS-Cl in acetonitrile were added successively. After certain time intervals at room temperature in the dark, the tube was taken and 150 yL 0.5 N HCI was added. The aliquot of the reaction mixture was subjected to HPLC with the same separation conditions as for the CL detection system. The fluorescence intensities of the column eluate were measured with emission at 510 nm (excitation at 350 nm). Sample preparation. The sample preparation procedure (Fig. 2.) was performed according to the previous paper with slight modifications (Takanaka et al., 1987). Five pL intact rat plasma was added with 10 pL of the internal standard [lo0 ng/ mL (435.3 pmol/rnL), 4-methylmexiletine], 70 pL water and 50 pL 0.1 N sodium hydroxide solution. The mixture was stirred with a Vortex mixer for 10 s and then poured onto an Extrelut 1 column. Mexiletine and 4-methylmexiletine were eluted with 5 mL diethyl ether and the eluate was evaporated to dryness. The residue was dissolved in 150 pL 0.2 M borate buffer (pH 8.5) with the successive addition of 150 pL 500 p~ DNS-Cl acetonitrile solution, and reacted at room temperature in the dark for 90min. The solution was mixed with

125

150 pL 0.5 N HCl and then a 20 pL aliquot of the mixture was subjected to HPLC analysis. The calibration curve was obtained by adding 5 p L MX (92.7-463.5 n M ) , 10 pL internal standard (435.3 nM), 65 pL water and 50 pL 0.1 N sodium hydroxide to 5 pL rat plasma. The mixture was further treated as described above.

The plasma concentration of MX in rats after oral administration. The aqueous solution of MX hydrochloride was administered at once to female Wistar rats (10 weeks old, 250260g) in a dose of 25 mg (115.9 pmol)/kg into the stomach with a tube. After administration, 100 pL blood was drawn from the jugular vein at the following times: 30 min, I , 2 , 3 , 4 , 5 , 6, 8 and 10h. The collected blood was centrifuged at 2500 x g for 5 min. Five pL plasma sample was transferred into a heparinized 2 m L Eppendorf tube and treated as described above.

~~~

RESULTS AND DISCUSSION

In a previous paper (Nishitani et al., 1991), we reported a simple method for t h e determination of the fluorescent drugs, dipyridamole (2,6-bis(diethanolamino)4,8-dipiperidinopyrimido-[5,4-d]-pyrimidine,platelet inhibitor) and benzydamine hydrochloride (1-benzyl-3[3-(dimethylamino-propoxy]-lH-indazole hydrochloride, anti-inflammatory drug) with HPLC PO-CL detection. T h e detection limits of dipyridamole and benzydamine hydrochloride were 34.5 amol and 147 fmol on injection, respectively (SIN= 2). Recently, non-fluorescent compounds and drugs have been detected by a HPLC PO-CL system after conversion to fluorescent compounds. Nitrated pyrenes were detected after on-line electrochemical reduction (Imaizumi et al., 1990). Amphetamine-related compounds were detected after derivatization with DNS-Cl (Hayakawa et al., 1989). Bile acids in urine were plasma 5 , u l t internal standard (100 ng/ml); 10 ,uL1 t 0.1 N NaOH 50 f i l + H20 70 ~1

I

Extre lut

\I/

+

ether

ether extract

\1

evaporate t o dryness

dissolve i n 150 u l o f 0 . 2 M borate buffer (pH 8.5) and then add 150 u1 of 500 ,uM DNS-Cl

\1

standing for 90 min

add 150 , u l of 0.5N HCI

20 ,u1 Injection t o HPLC Figure 2. Sample preparation procedure for mexiletine determi-

nation in plasma.

ATSUHIKO NISHITANI ET AL.

126

DNS-C I

kxiletine hydrochloride

DNS-Mexiletine

Scheme 1. Reaction of mexiletine with dansyl chloride [5-(dimethylamino)-l-naphthalenesulphonylchloride]

detected after derivatization with dansylhydrazine (Imai et af., 1989; Higashidate et af., 1990). Ketocorticosteroid in blood plasma was detected after derivatization with dansylhydrazine (Koziol et al., 1984). In this experiment, we investigated the use of the HPLC with PO-CL detection system for the determination of non-fluorescent drugs in plasma in order to monitor the level of drugs in blood using a minimum amount of blood. Mexiletine has an amino group for the fluorogenic reaction with DNS-CI (Scheme 1). The fluorogenic reaction of DNS-CI was affected by the concentration and pH of the buffer. Therefore the conditions for derivatization were first examined before the analytical technique was developed.

As shown in Fig. 3, the largest peak height was obtained when MX in 0.2 M borate buffer (pH 8.5) was mixed with DNS-CI in acetonitrile and reacted for more than 60 min. Also the optimum concentration of borate was 0 . 2 when ~ the pH of the borate buffer was 8.5 (data not shown). Thus, for the derivatization of the plasma MX, we decided to use 0 . 2 ~borate buffer (pH 8.5) and react for 90 min at room temperature. The calibration curve for MX obtained by addition of the drug to rat plasma, derivatized and isolated by the above procedure and determined by the proposed method was linear over the range 20-100 ng/mL plasma (20.6-103 fmol/injection). The regression equation was as follows: Y = 0.024X- 0.008(r = 0.99),

In (u

c a In

a(u: I

k

.s

'c)

0 W

e:

5

10

15

20

15

20

Retention Time (min)

B

i-

m

In C In a

m

@=

k 'c)

0

e0 W : -

0

30

60

90

120

Time ( m i n t

Figure 3. Effect of pH on the apparent time course of the peak height of dansylated mexiletine. The experimental details are described in the text. e: 0.2 M borate buffer (pH 7.5); A: 0.2 M borate buffer (pH8.0); m: 0.2 M borate buffer (pH 8.5); 0: 0.2 M borate fuffer (pH 9.0).

I

1 1- ~ - . _ 5

- .10 ~

Retention Time (min)

Figure 4. Chromatogram of dansyl mexiletine in rat plasma after oral administration of obtained at 0 min (A) and 30 min (6) mexiletine hydrochloride 125 mg (115.9 pmol)/kg]. The experimental details are described in the text.

HPLC- PO-CL DETECTION OF MEXILETINE

rl X

E"

0.01 0

1

2

4

6

8

10

Time (hour) Figure 5. Plasma concentration-time curve of mexiletine following oral administration of mexiletine hydrochloride [25 m g (115.9 ~ m o l ) / k g to l Rats (n=3). The experimental details are described in the text.

where Y is the peak height ratio of the drug to the internal standard, X is the amount of MX in injected samples (20 pL) and r is the coefficient of correlation. The advantage of this highly sensitive detection method is that only 5 pL plasma was required to achieve the measurements, while in a recent report, where FK-506, a macrolide antibiotic in rat serum and lymph was detected after derivatization with dansylhydrazine, 100 pL serum and lymph was required (Takada et al., 1990). In the case of a fluorescent drug, dipyridamole, only 0.1 pL plasma was needed (Nishitani et al., 1991). This difference may be due to the difference in chemiluminescence intensity of the drugs.

127

Dipyridamole shows very strong chemiluminescence intensity (Imai et al., 1987b) and so a very small volume of plasma is required. Also, in the presence of plasma, about 10% loss of MX in the sample was observed through the extraction procedure. Figure 4(B) shows the chromatogram of the plasma MX obtained at 30 min after the oral administration to rats and derivatized with DNS-Cl. Although a large peak was observed in both (A) and (B) before the peak of dansylated MX, it did not interfere the measurement of MX. Figure 5 shows the time course of the plasma concentration of MX administered orally to rats [25 mg (115.9 pmol)lkg]. As shown in the figure, the elimination half-life of MX in rats was about 2 h. Yoshida et al. (1983) reported that the administration of MX orally to rats showed biphase elimination. The a-half-life was 0.5 h and the b-half-life was 10 h. This discrepancy might be ascribed to the difference in the determination method of MX. They administered radioactive MX and measured total radioactivity in plasma, whereas we measured unchanged MX by HPLC with the administration of non-radioactive MX hydrochloride. By using the HPLC PO-CL detection system, we were able to detect a fmol level of MX in blood plasma. The detection limit of MX was extremely low compared to the previous papers (Breithaupt and Wilfling, 1982; McErlane et al., 1987; Katagiri et al., 1989). These results suggest that the plasma concentration of nonfluorescent drugs having amino residues which can be labelled with dansyl chloride may be determined by this method.

Acknowledgements The authors express their thanks to Tosoh Co. for the generous gift of TSK ODS columns. A part of the work was supported by a Grant-in-Aid from the Tokyo Biochemical Research Foundation.

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Kobayashi, S. and Imai, K. (1980a). Anal. Chem. 52,424. Kobayashi, S. and Imai, K. (1980b). Anal. Chem. 52, 1548. Koziol, T., Grayeski, M. L. and Weinberger, R. (1984). J. Chromafogr. 317, 355. Mastropaola, W., Holmes, D. R., Osborn, M. J., Rooke, J. and Moyer, T. P. (1984). Clin. Chem. 30, 319. McErlane, K. M., Igwemezie, L. and Kerr, C. R. (1987). J. Chromatogr. 415, 335. Nishitani, A., Kanda, S. and Imai, K. (1991). Anal. Chim. Acfa, 251, 247. Smith, K. J. and Meffin P. J. (1980). J. Chrornatogr. 181, 469. Talbot, R. G. (1973). Lancet2, 399. Takada, K., Oh-hashi, M., Yoshikawa, H., Muranishi, S., Nishiyama, H., Yoshida, H., Hata, T. and Tanaka, H. (1990). J. Chromafogr. 530. 212. Takanaka, A., Furusho, M., Matsunaga, M., Koyasu, M., Hattori, M., Ito, A.. Sugawara, K. and Miyamura, E. (1987). Jpn J. Pharm. 13, 132. Ueno, K., Miyai, K. and Kawaguchi, Y. (1989). Jpn Pharm. Ther. 17, 4367. Willox, S. and Singh, B. N. (1976). J. Chromatogr. 128, 196. Yoshida. T., Kobayashi, S., Matsurnura, R. and Kohei, H. (1983). Jpn Pharm. Ther. 11, 1761.