Stereoselective Determination of Flecainide in Human Plasma by High-Performance Liquid Chromatography with Fluorescence Detection

Februa 1990 Volume 9, Number 2

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JOURNAL OF PHARMACEUTICAL SCIENCES ~

~~

~

A publication of the American Pharmaceutical Association

ARTICLES

Stereoselective Determination of Flecainide in Human Plasma by High-Performance Liquid Chromatography with FIuorescence Detection JACQUES TURGEON, HEYOK. KROEMER, CHANDRA PRAKASH, IAN A. BLAIR, AND DANM. RODEN' Received March 8, 1989, from the Departments of Pharmacology and Medicine, Vanderbilt University, Nashville, TN 37232. Accepted for publication June 2, 1989. Abetracl 0 Enantiomers of a drug may differ in their pharmacological activities or their disposition constants. We now describe a stereoselective analytical method for the determination of the antiarrhythmic agent flecainide in plasma. The resolution of the enantiomers is achieved by high-performance liquid chromatography (HPLC) on a normal phase silica wlumn following derivatization with the optically active reagent (-)-menthy1chloroformate.The eluting diastereoisomers are monitored by fluorescence detection at an exitation wavelength of 305 nm and an emission wavelength of 340 nm. The limit of sensitlvity for the assay is as low as 2.5 ng/mL for each enantiomer using 1 mL of plasma. A new liquid-liquid extraction procedure with high recovery (>95%) and high selectivity is also reported. The intra- and interassay coefficient of variation for replicated analysis of spiked plasma samples is less than 4.0% and 7.0%, respectively. The method is suitable for single and multiple dose pharmacokinetic studies in healthy volunteers or in patients.

1

2

flgure 1-Structures of flecainide (1) and its internal standard (2). Chiral centers in these molecules are indicated by an asterisk.

similar or slightly different electrophysiological properties.8921.22 However, a recent study suggests that flecainide disposition may cosegregate with the genetically determined polymorphic metabolism of debrisoquine.29 The influence of this factor on the stereoselective disposition of flecainide needs to be evaluated and requires the development of a . stereoselective and quantitative assay. Methods for chromatographic resolution of optical isomers include the use of an optically active stationary phase, an Flecainide, N-(+)-(2-piperidylmethyl)-2,5-bis-(2,2,2-trifluo- optically active mobile phase, or conversion of the optical roeth0xy)benzamide monoacetate, is a sodium channel isomers to diastereoisomers by reaction with an optically blocker which is clinically used for the suppression of cardiac active reagent.24 The latter approach was adopted to develop arrhythmias.14 As observed with many antiarrhythmic a stereoselective analysis method for flecainide exploiting its agents, correlations can be derived between plasma concenfreely accessible secondary amine. We selected (- )-menthy1 trations of flecainide and its pharmacological effects.s.6 The chloroformate as the derivatizating reagent since this product incidence of some of its side effects appears t o be forms stable diastereoisomeric carbamates with chiral dose-dependent7and often associated with plasma concentraamines.26 Resolution can then be achieved by normal- or tions greater than 1000 nglmL.4 reversed-phase HPLC.26.27 Flecainide possesses a single chiral center adjacent to the piperidine nitrogen (Figure 1)and the drug is marketed as the Experimental Section racemic acetate.8 Monitoring plasma concentrations of Chemicals and ReagentgThe acetate salt of racernic flecainide, flecainide isomers may be of clinical interest since stereothe base form of R-(-)-flecainide and S-(+)-flecainide (>98% pure), selective differences in the disposition, the electrophysiologand the non-fluorinated analogue of flecainide uaed as the internal ical effects, or the toxicity of other antiarrhythmic agents, standard, 2,5-diethoxy-N-(2-piperidylmethyl)benzamidehydrochloincluding disopyramide,O.'o quinidine," mexiletine,'2.13 toride (S-15277),were obtained from Riker Laboratories(St. Paul, MN). cainide,"e16 propafenone,'s ~0tal01,17.18and [email protected] Butyl chloride, 2-propanol,triethylamine, toluene, hexane, and ethyl have been reported. Preliminary in vitro and in vivo results acetate, all HPLC grade, and tris(hydroxymethy1)aminomethane hydrochloride and phosphoric acid were purchased from Fisher suggest that the enantiomers of flecainide may either have OO22-3549/90/0ZOO-OO9 1$0 l.OO/O 0 1990, American Pharmaceutical Association

Journal of Pharmaceutical Sciences I 91 Vol. 79,No. 2,February 1990

Scientific (Fair Lawn,NJ). The optically active reagent, (-)-menthy1 chloroformate was purchased from Aldrich Chemical (Milwaukee, WI). All other chemicals and reagents used were of analytical grade and obtained from usual commercial sources. Standard S o l u t i o n d t o c k standard solutions of flecainide and the internal standard were prepared by dissolving 10 mg of the racemic salts in 1000 mL of distilled water to a final concentration of 10 pg/mL. Working standard solutions (final concentration of 1 pg/mL) were obtained by a further 1:lO dilution of the stock solutions. Extraction P r o c e d u r e T h e following were added to 1.0 mL of plasma in a 15 mL Pyrex tube: 50 p L of the internal standard solution (50 ng), 1mL of Tris HC12.0 M (pH 8.5)and an excess (3 g) of sodium chloride. Following maximal dissolution of the NaCl, the aqueous mixture was extracted by vortex mixing with 5 mL of butyl chloride:2-propanol(95:5,vlv) for 30 sec. After centrifugation at room temperature for 10 min at 3000 x g in a IEC-Centra 8 centrifuge (International Equipment, Needham Heights, MA), the organic phase was transferred into another 15 mL Pyrex tube. The extraction proceciure was repeated with another 5 mL of the organic mixture. Then, 1.0 mL of a 0.17 M phosphoric acid solution (1.04 mL of 85% phosphoric acid in 100 mL of water) followed by 1.0 mL of water was added to the combined organic extracts. Vortex mixing of the mixture was performed for 30 sec before and after the addition of water. The final mixture was then centrifuged a t 3000 x g and the organic layer was aspirated and discarded. After addition of 1.0 mL of Tris HCl(2.0 M, pH 8.5) and saturation with NaCl, the residual aqueous layer was reextracted with the organic mixture of butyl chloride:2-propanol as described above. The combined organic extracts were then evaporated to dryness in a Buchler Vortex-Evaporator a t 45 "C. Derivatization-After evaporation, the residue was dissolved in 50 4 of toluene. Then, 10 pL of a 10%solution of triethylamine and 20 p L of a 10% solution of (-)-menthy1 chloroformate, both in toluene, were added to this mixture. Prior to injection, the reaction mixture was evaporated with a stream of nitrogen and 100 CJ. of the mobile phase was added. After vortex mixing and centrifugation at 2000 x g for 5 min, the supernatant was injected on the column. Instrumentation-The chromatographic system used consisted of a Model M6000A pump, a U6K injector fitted with a 100 pL loop (Maasachusetts Waters Chromatography Division, Milford, MA), a Hitachi D-2000 integrator (GK Instruments, Montevallo, AL) and a variable-wavelength RF-535 fluorescence detector (Shimadzu Scientific, Columbia, MD) with the excitation and emission wavelengths Bet at 305 nm and 340 nm, respectively. Separation was performed on

an Ultrasphere silica column (25 cm x 4.6 mm i.d.; 5 jm particle size; Rainin Instrument, Woburn, MA) using a mobile phase consisting of hexane:ethyl acetate:triethylamine (54:16:0.1, vm). The flow rate was 1.0 mumin. Positive fast atom bombardment maas spectrometry (FABIMS)analysis of the diastereoisomers formed between flecainide enantiomers and (-)-menthy1 chloroformate was carried out on a VG 701250 double focusing mass spectrometer using a 3-nitrobenzyl alcohol matrix.= Calibration Curves-Aliquots of the flecainide working solution were added to 1.0 mL of blank plasma. The range of concentratiom was 10.9 to 327.5 ng of free base per mL of plasma for each enantiomer. Samples were extracted and analyzed as described above. Calibration curves baaed on the peak height ratios of each enantiomer to the internal standard were constructed using 10 different concentrations analyzed in duplicate. The data were then subjected to linear regression analysis to give calibration factors for each enantiomer. Extraction Efficiency Evaluation-To evaluate the percentage of extraction of flecainide from 1 mL of plasma, 10 mg of ( 2 ) flecainide-base and 5 mg of the internal standard were dissolved separately in 100 mL of ethanol. The flecainide stock solution was further diluted 1:lOO with ethanol to give a working solution of a final concentration of 1 WmL; the internal standard stock solution was diluted 1 : l O with ethanol to give a final concentration of 5 pg/mL. Blank plasma samples were spiked with either 25 pL (25 ng), 100 pL (100 ng) or 400 p L (400 ng) of the flecainide working solution (three for each concentration) and then extracted as described above. The same volumes of flecainide working solutions were also added to nine conical test tubes and evaporated to dryness. Prior to derivatization, 10 pL (50 ng) of the internal standard working solution in ethanol was added to each tube, both those processed with and those without the extraction step. The samples were then derivatized and analyzed 88 desrribedabove. The percentage of extraction efficiency (EE%) for a given concentration and a given enantiomer was calculated as follows: EE% = (Peak Height Rati%dnidhmd With ExtractionPeakHeight Rationdfid, I n t a d standard Without Extraction) x 100

Results and Discussion Chromatograms o f extracts from blank plasma, blank plasma spiked with 32.8 ng and 349.4 ng of (a)-flecainide, and a patient sample, all processed in the presence of the internal

100

E

C

;3 ;

D IS

R

IS

L.

0

n

---

1 1 1 1 1 1

0

10

20

0

10

20

0

10

20

0

10

20

Tim. Iminl

Flgure 24hromatograms obtained after extraction of 1.O mL of blank plasma (A); 1 .O mL of blank plasma spiked with 32.8 ng ( 8 )and 349. 4 ng of (?)-flecainide (C) and of a patient's sample (1 .O mL) containing an estimated 468 ng/mL of R(-)-flecainide and 380 ng/mL of S(+)-flecainide (D). All these plasma samples were spiked with 50 ng of the internal standard (IS) prior to extraction. 92 / Journal of Pharmaceutical Sciences Vol. 79, No. 2, February 7990

standard, are shown in Figure 2.Retention times of the peaks of interest were 12.5and 13.3min (firstand second peak of the internal standard), 21.0min [R-(-)-fleainidel and 22.5 min IS-(+ bflecainidel. Good separation with almost complete baseline resolution (a = 1.08) was obtained between flecainide enantiomers and no interference from endogenous compounds was observed. As can be seen in the extract of the blank plasma used, a small peak sometimes interferes with the first internal standard peak such that the second one was used for better precision. The elution time of the derivative corresponding to each flecainide enantiomer was determined by separate analysis of optically pure I?-(-)- and S ( +)-flecainide after derivatization with (- )-menthy1 chloroformate (Figure 3).No significant racemization of the derivatives was observed as judged by a single major peak for each enantiomer. The HPLC eluant fractions corresponding to the retention time of the two diastereoisomers were collected for FAB/MS analysis. The proposed chemical structure and positive FAB spectra obtained for these products are shown in Figure 3.The fragments observed at mlz 597 (M + 1) and d z 619(M+ 23)suggested a monoderivative while the fragments observed a t mlz 415 and mlz 301 were characteristic of the flecainide structure.26 Interestingly, as with underivatized flecainide, the menthy1 chloroformate derivative formed exhibited fluorescent properties in the mobile phase medium which increased the selectivity and the sensitivity of detection. With the excitation wavelength set a t 305 nm and the emission wavelength set at 340 nm, the limit of quantification was 2.5 ng/mL and the limit of detection 1ng/mL (as calculated by the method of Knoll)29 for each enantiomer. The assay could also be performed with a variable wavelength UV-detector set a t 298 nm. With such a detection mode and the use of 2mL of plasma,

the limit of sensitivity of the assay was 40 ng/mL of plasma for each enantiomer. Numerous optically active reagents are available for derivatization with amines. They may differ in their specificity for this functional group, their coefficientof extinction (which may be used to improve the sensitivity of the assay), and in the complexity and the time involved in the derivatization step. As shown in Figure 4,the reaction between (-)-menthy1 chloroformate and flecainide was rapid. This characteristic was advantageous to minimize the analytical time. No significant change in peak areas was observed with increasing derivatization time or temperature, suggesting a completed and rapid formation of the derivatives. The detector response demonstrates slightly higher values (-5%) for the R(-)-flecainide derivative. This may represent a modest preferential reaction between the reagent and the R4-1enantiomer or a greater extinction coefficient for this derivative. However, Figure 4 shows that this small difference was constant and therefore should not influence the quantitative characteristics of the assay. We have also observed that the derivatives formed were stable (no racemization and no significant loss in the detector signal response) for a t least 48 h in the reaction mixture. This stability is desirable since it allows one to process multiple samples in one run. Regression lines for the calibration curves over the range 10.9-327.5 ng/mL were as follows: Concentration = 0.004275(peak height ratio) + 0.0013 (r = 0.999)for the R - ( -)-flecainide enantiomer a n d Concentration = 0.003793(peak height ratio) + 0.0035 (r = 0.998)for the S-(+ )-flecainideenantiomer. h t r a - and interassay variations at three different concentrations are shown in Table I. The percentage coefficientof variation (CV)for intra- and interassay analysis of&(-)- and S-(+)-flecainidewere less than 4.0

R-(-)-Flecainidc 1

0

10

m

Time (min) 41s

loo

I

S-(+)-Flecainide

R-(-)-Flccainide

'i mh

Flgure >Proposed chemical structureand HPLC analysis of the derivativesformed between (- )-menthy1chloroformateand the optically pure R(-)-

and S(+)-flecainidestandards and the respective FAWMS spectra of the eluant fractions corresponding to both diastereoisomers.

Journal of Pharmaceutical SciencesI 93 Vol. 79, NO. 2, February 1990

25%

80'C

H

100 1

250

1a

30 0

0

30

60 90 120 30 60 DERlVATlZATlON TIME (min.)

90

of the complete derivatization time between (-)-menthy1 chloroformate and flecainide enantiomers at 25 "C and 80 "C. The dotted line represents the mean detector respanse value calculated for each enantiomer for the various times of derivatiratlon. Table Hntra- and Interassay Variations In the Slmultaneous Analysis of R(-)-Flecalnlde and S-(+)-Flecalnlde In Human Plasma

Amount of R-(-)- and S(+)-Flecalnide Added to 1 mL of plasma 10.9 ng 43.71 ng 174.7 ng

cv, Yo

11.3 2 0.4' 3.1

44.2 = 0.5 1.1

172.0 2 3.9 2.3

cv, Yo

11.3 2 0.5 4.0

43.6 2 0.9 2.0

171.2 2 3.1 1.8

cv, %

10.8 2 0.7 6.9

46.0 2 3.4 7.0

177.8 f 4.8 2.5

cv, %

10.6 2 0.7 6.1

46.4 2 2.1 4.5

178.2 2 4.4 2.5

S(+)-Flecainide Recovery Interassay variationC R(-)-Flecainide Recovery

S(+)-Flecainide Recovery a

n = 6. b Values are mean

?

SD. n = 5.

and 7.0%, respectively. A number of procedures have been reported for the extraction of flecainide from plasma.30-37 However, in all but one of these methods,36 the final medium is not appropriate for subsequent derivatization because of the difficulty in rendering it anhydrous. With the remaining met)lod,36 the extraction recovery was low (60-70%), and therefore caused a loss in sensitivity and precision. In view of these problems, a new extraction procedure based on the use of butyl chloride:2propanol and the salting-out technique was developed. In fact, the combination of mildly basic conditions [pH near flecainide PK, (9.3)1, the salting-out technique, a very polar organic phase (butyl chloride:2-propanol), and back-extraction in aqueous acidic medium, provides some selectivity and effi: ciency comparable to other previously reported methods.3036 Moreover, the extraction procedure for flecainide described herein provides cleaner samples which eases derivatization and decreases the possibility of interference from endogenous compounds. However, because of the relatively polar solvent 94 Journal of Pharmaceutical Sciences Vol. 79, No. 2, February 1990

I

I

I

1

24

36

48

60

TIME (h)

120

Figure +Estimation

Intra-assay variation' R(-)-Flecainide Recovery

1

12

Figure +Plasma concentration-timeprofile of R-(-)-flecainide (0)and S (+)-flecainide (0) observed after discontinuation of chronic oral therapy with this drug (100 mg twice a day) in a patient.

used for extraction and because of the type of column used (normal-phase), polar endogenous compounds may be extracted and may elute at long retention time (memory peak). Due to the selectivity of the fluorescence detection, only one significant peak was observed a t 28 min. To avoid possible interference with subsequent analysis, a spacing time of about 30 min should therefore be used between injections. Although this procedure may appear time-consuming, recoveries were excellent over a wide range of concentrations (Table 11). This may account for the better precision observed with this assay compared with those described previously. Moreover, since many samples can be processed at the same time, the relative time per analysis and extraction is acceptable, particularly for research purposes. The analytical method described here was applied to the enantioselective analysis of flecainide in the plasma of a patient who had been receiving chronic therapy with 100 mg of the drug twice a day and in whom it was necessary to discontinue treatment because of inefficacy. Figure 5 shows the concentration-time profile for R-( -)- and S-(+Mecainide obtained in this patient for 72 h following discontinuation of therapy. The elimination half-lives for the R4-1- and S(+ knantiomer, as klculated from the slope of the terminal linear portion of the log plasma concentration-time curves, were 29.2 and 26.7 h, respectively. Studies have addressed the possibility of stereoselective flecainide disposition.21 Single dose pharmacokinetic or in vitro drug metabolism studies can be readily performed using this method. Table ICEfflclency Evaluation of the Extraction Procedure Used In the Determination of R(-)-Flecalnlde and S(+)-Flecalnlde In Plasma

Extraction Efficiency'

Amount of R(-)- and S(+)-Flecainide Added to 1 mL of Plasma 10.9 ng 43.7 ng 174.7 ng

R-( -)-Flecainide

Amount recovered, ng Percent recovered S(+)-Flecainide Amount recovered, ng Percent recovered n

= 3.

10.5 2 0.3' 96.6 2 5.4

43.2 5 2.6 98.0 f 6.0

164.6 2 11.4 94.2 2 6.5

10.4 f 0.5 95.2 2 4.4

43.2 f 3.5 98.8 f 8.2

167.2 2 10.3 95.7 2 5.9

'Value are mean rt SD.

Conclusion The stereoselective assay described here allows quantitative determination of concentration of flecainide enantiomers in human plasma. Good chromatographic resolution of the diastereoisomers formed after a simple and fast derivatization step with (-)-menthy1 chloroformate is obtained. The selective extraction procedure decreases the likelihood of interference from endogenous compounds. The method uses the internal standard quantitation technique, is highly reproducible, very sensitive, and is performed on a readily available normal-phase HPLC system with either fluorescence or UV detection.

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A. J. J.; Eichelbaum, M.;Woosley, R. L.; Roden, D. M. Circulation 1989,79,1068-1076. 17. Johnston, G. D.; Finch, M. B.; McNeil, J. A.; Shanks, R. G. Br. J . Clin. Phnrmacol. 1985,20,507410. 18. Lynch, J . J.; Wilber, D. J.; Montgomery, D. G.; Hsieh, T. M.; Patterson, E.; Lucchesi, B. R. J. Cardiovasc. Phunnacol. 1984,6, 1132-1141. 19. Vogel esang, B: Echizen, H.; Schmidt, E.; Eichelbaum, M. Br. J. Clin. harmacot. 1984.18.733-740. 20. Echizen, H.; Vo elges&g,'B.; Eichelbaum, M. Clin. Phurmacol. Ther. 1985,38.%-76. 21. Kroemer, H. K.;Turgeon, J.; Thomas, T.; Roden, D. M. Clin. Phnrmacol. Ther. 1989,45,179. 22. Hill, R. J.;Duff, H. J.; Sheldon, R. S. Mol. Pharmacol. 1988,34, 659-663. 23. Beckmann, J.; Hertrampf, R.; Gundert-Remy, U.; Mikus, G.; Gross, A. S.; Eichelbaum, M. Brit. Med. J. 1988,297,1316. 24. Testa. B. Xenobiotica 1986.16.265-279. 25. Westiey, J. W.; Halpen, B: J. Org. Chem. 1968,33,3978-3980. 26. Prakash, C.; Kosha 1, R. P.; Wood, A. J. J.; Blair, I. A. J.Phurm. Sci., 1989,78,771- 75. 27. Prakash, C.; Jejoo, H. K.; Blair, I. A. J. Chromutogr. 1989,493, 325-335. ___ ___ 28. Sweetman, B. J.; Blair, I. A. Biomed. Mass Spectrom. 1988,17, 337-340. 29. Knoll. J. E. J. Chromatom. Sci. 1985.23.422-425. 30. Johmon, J. D.;Carleon, G.L.; Fox, J, M.; Miller, A. M.; Chang, S. F.: Conard, G. J. J. Phrrrm. Sci. 1984,73,1469-1471. 31. Plom T. A.; Boom, H. T.; Maw, R. A. A. J.Anal. Toxicol. 1986, 10,lh-106. Shenfield, G. M. J.Lq.Chromutogr. 32. Boutagy, J.; Rumble, F. M.; 1984,7,2579-2590. 33. Grgurinovich, N.J. Anal. Toxicol.1988,12,38-41. 34. Chang, S.F:; Miller, A. M.; Jernberg, M. J.; Ober,R. E.; Conard, G. J. Anneim-Forsch. 1983,33,251-253. 35. Bhamra, R. K.; Flanagan, R. J.; Holt, D. W. J . Chromutogr. 1984, 307,439-444. 36. Chang, S.F.; Welscher, T. M.; Miller, A. M.; Ober, R. E. J. Chromutogr. 1983,272,341950. 37. Chan%S. F.;Miller, A.M.; Fox, J. M.; Welscher, T. M. Ther. Drug omtoring 1984,6,105-111.

7'

Acknowledgments This work was s~pportedin art b a grant from the United States Public Health Service (GM 3804).facques Turgeon is the recipient of a Medical Research Council of Canada Fellowship. Heyo K. Kroemer was sup rted by a Fellowship of the Scientific C o m r m t F of NATO receivfihrou h the Deutache Akademische Austauschdienst, FRG. The authors t i a n k Dr. Brian J. Sweetman for running the FABMS samples and Mrs. Holly T. Gray and Mrs.Tina Thomas for technical mistance.

Journal of Pharmaceutical Sciences I 95 Vol. 79, No. 2, Februaty 7990