Technical Notes Anal. Chem. 1995, 67, 1903-1906
Direct Determination of Substituted Azepinoindole Enantiomers in Rat Plasma Using Silica Stationary Phase and /?=Cyclodextrin as a Mobile Phase Additive R. H. Pullen,tl* J. J. Brennan,* R. Lammers,, and 0. Patonay*lt Department of Chemistry, Georgia State University, University Plaza, Atlanta, Georgia 30303, Solvay Pharmaceuticals, Inc., 901 Sawyer Road, Marietta, Georgia 30062, and Drug Disposition Department, Solvay Duphar 6.V., C. J. Van Houtenlaan 36, 7380 AA Weesp, The Netherlands
DU 124884 is a racemic serotonin receptor agonist in an early stage of drug development. DU 124884 and its potential N-desmethylmetabolite, KC 9048, both contain a single chiral center. A direct enantioselective HPLC assay was developed and validated to quanti@DU 124884 and KC 9048 in rat plasma. The drug and metabolite enantiomers were extracted from plasma and separated using silica stationary phase with an aqueous mobile phase containing/kyclodextrin @-CD),triethylamine, and 2-methyl-2-propanol. A variable wavelength detector was used to monitor absorbance at 231 nm. The assay calibration range was from 100 to 5000 ng/mL. Quality control sample precision (59%RSD) and accuracy (f10% error) were satisfactoryfor all four analytes (n= 12). The method was used to assess drug exposure during a pilot toxicologystudy in rats. Toxicokinetic study animals were dosed subcutaneously for 15 days at 0,2.5, 10, and 40 mg of DU 124884.HCl k g ' day-'. Blood was collected on the last day of dosing between 22 min and 4 h and 13 min after the last dose. The samples showed (&)-DU 124884 isomer ratios ranging ikom 1.1 to 1.3. These data suggest that DU 124884 undergoes stereoselective metabolism in rats. Levels of the N-desmethylmetabolite enantiomers were < 100 ng/mL. A recent review cites numerous examples of the utility of cyclodextrins (CDs) as mobile phase additives in reverse phase HPLC.' However, there are few examples of bioanalytical enantioseparations using CD as a chiral mobile phase additive (CMPA).2$3A recent study in our laboratory demonstrated the direct enantioseparation of DU 124884 and KC 9048 using silica stationary phase with an aqueous mobile phase containing,&CD.4
' Georgia State University. Solvay Pharmaceuticals, Inc. B.V. (1) Husain, N.; Warner, I. M. Am. Lab. 1993,11, 80-87. (2) Walhagen, A; Edholm, L. E. Chromutogruphiu 1991,32, 215-23. (3) Rona, IC;Szabo, I. /. Chromutogr. 1992,573,173-177. (4) Pullen, R H.; Brennan, J. J.; Patonay, G.]. Chromufogr. 1995,691, 187193. 5 Solvay Duphar
0003-2700/95/0367-1903$9.00/0 0 1995 American Chemical Society
DU 124884 is a racemic serotonin receptor agonist in an early stage of drug development. DU 124884 and its potential Ndesmethyl metabolite, KC 9048, both contain a single chiral center. The technique offers good column-to-column reproducibility, maximum D-CD solubility, reduced costs for column and reagents, and unique selectivities because of multiple retention mechanism~.~ This study demonstrates the feasibility of using p-CD CMPA and silica stationary phase for bioanalysis. An assay was developed for the direct determination of DU 124884 and KC 9048 enantiomers in rat plasma in a single HPLC run. The report describes the outcome of a validation study to assess method performance. The assay was used to analyze samples from a pilot rat toxicology study with DU 124884. EXPERIMENTAL SECTION Materials. DU 124884.HC1,99.9%purity, [ (&)-3[(methylamino) methyl]-3,4,5,€~tetrahydro-f%oxo-Vazepino [5,4,%cdlindole hydrochloride] and KC 9048, 95% purity, { (&)-3-(aminomethyl)3,4,5,6tetrahydro-6-ox~LH-azepino [5,4,hdlindole}were synthesized by Solvay Pharma Deutschland Research laboratories (Hannover, Germany). Figure 1 shows the structures of these compounds. Racemic albuterol hemisulfate [a'-[ [ (1,l-dimethylethy1)aminolmethyll-4-hydroxy-1,%benzenedimethanolhemisulfate], USP grade, was obtained from Research Biochemicals International (Natick, MA). Reagent-grade p-cyclodextrin was purchased from TCI America (Portland, OR). USP-grade deionized water was obtained by reverse osmosis. Pooled, heparinized, blank rat plasma was purchased from Pel-Freeze Biologicals (Rogers, AK). All other reagents were of high-purity grade (297%) and were purchased from commercial suppliers. Assay Standards. Aqueous standard solutions of DU 124884 and KC 9048 were prepared at 1000, 100, and 10.0 pg/mL of racemic free base. The lo00pg/mL aqueous standard was stable for at least 1 month when stored at 4 "C. The 100 and 10.0 pg/ mL standard solutions were prepared fresh daily. A chromatographic internal standard (IS) solution was prepared at a concentration of 10.0 pg of albuterol hemisulfate/mL of deionized water. Rat plasma calibration standards of DU 124884 and KC 9048 were Analytical Chemistty, Vol. 67, No. 11, June 1, 1995 1903
H
DU 124884
I
H -2/-
'N I
H
H
KC 9048
I
O\-NT
/AP
N
I
b.J.
-
H H
N/:
H
Figure 1. Structures of model chiral amines DU 124884 and KC 9048.
prepared by spiking 0.5 mL of blank plasma with the appropriate volume of 100 or 10.0pg/mL aqueous standard solutions. Quality control samples were prepared at 250, 750, and 2500 ng/mL by spiking pooled blank plasma with the appropriate volume of 1000 or 100 pg/mL aqueous standards. Extraction Procedure. The extraction procedure was adapted from a method developed for the achiral determination of DU 124884 and KC 9048 in human plasma.: Screw-top polypropylene tubes, 16 x 125 mm, were used for all extraction steps (Sarstedt, Inc., Newton, NC). Plasma samples (0.5 mL) were adjusted to pH 11.5 by mixing with 0.5 mL of 0.1 M NaOH (aqueous). Andytes were extracted from plasma by adding 5 mL of methylene chloride/2-propanol (7:3 v/v) and shaking at a low speed for 10 min. The phases were separated by centrifuging the samples at 1500g for 10 min. The upper aqueous layer was drawn off using a Pasteur pipet and discarded. The organic layer was transferred to a clean tube by pouring. Samples were evaporated to dryness in a 40 "C water bath under a stream of nitrogen. The chromatographic IS solution was added (0.5 mL), and samples were mixed for 1min. The samples were poured into 2 mL glass autosampler vials and capped using Teflon-lined septa. Chromatographic Instrumentationand Conditions. The HPLC apparatus consisted of a Spectra-Physics Model SP8810 isocratic pump, a Model SP8775 autosampler, and a Spectra 100 variable wavelength detector with deuterium source lamp (San Jose, CA). A 20 p L sample loop was employed, and mobile phase was delivered at a flow rate of 1.00 mL/min. Mobile phase consisted of 10 mM sodium phosphate buffer (PH 7)/15 mM P-CD/l% 2-methyl-2-propanol/2 mM TEA. The mobile phase was filtered through a 0.45 pm nylon 66 membrane filter before use. The separation was performed at ambient temperature using a silica NewGuard cartridge guard column, 15 x 3.2 mm i.d., 7 pm particle size (Applied Biosystems, Foster City, CA) and a Supelc o d LC-Si 250 x 4.6 mm i.d., 5 pm particle size silica analytical column (Supleco, Bellefonte, PA). The chromatographic run time was 24 min. Detector output at 231 nm was analyzed using Beckman PeakPro chromatography software and an HP 1000 minicomputer (Beckman Instruments, Fullerton, CA, and Hewletthckard, Los Angeles, CA, respectively). Data were quantified using a weighted (l/x) linear model and univariant least-squares regression. (5) Raghoebar, M., Solvay Duphar B.V., personal communication, 1993. 1904 Analytical Chemistry, Vol. 67, No. 7 1, June 1, 7995
Validation Study Design. Method validation runs were conducted on three separate days. On each day, plasma calibration standards were processed in duplicate at each of six concentrations (5000, 2000, 1000, 500, 200, and 100 ng/mL). Pooled plasma quality control samples were assayed in quadruplicate at three concentrations (2500, 750, 250 ng/mL) on each day. Duplicate analytical standards that were not subjected to the extraction procedure were processed on day one of the validation study. Absolute recoveries were determined for each of the four enantiomers by peak height comparison of plasma calibration standards to analytical standards at the same concentration. Assay selectivity was assessed by assaying samples from four male rats and four female rats that had not received drug. Quality control samples were used to assess analyte stability in plasma after long-term (4months) storage at -70 "C, three freeze/ thaw cycles, and 1week at ambient conditions. Statistical analysis of the stability data was performed using the procedure described by Timm and co-workers.6 In vivo Study Design. A 2-week pilot safety toxicokinetic study was conducted in rats with racemic DU 124884. Albino Sprague-Dawley rats were randomly divided into four groups and administered 0, 2.5, 10, and 40 mg of DU 124884.HCl kg-I day-' via subcutaneous injections. There were six animals in each group for both sexes. Upon completion of the study, blood samples were collected in heparinized tubes from the abdominal aorta under light ether anesthesia. Plasma was collected after centrifugation. The order of dosing determined the order of sacrifice, blood collection, and autopsy, resulting in blood collection divided over the dosing groups between 22 min and 4 h and 13 min after the last dose. RESULTS AND DISCUSSION Validation Study. The one-step Liquid-liquid extraction represents a compromise between optimal conditions for DU 124884 and KC 9048. Mean absolute recoveries for KC 9048 enantiomers were 39%, and DU 124884 enantiomer recoveries were 56%. Absolute recoveries were constant over the entire concentration range. A suitable structural analog for use as an assay IS was not available. Albuterol was selected as a suitable chromatographic IS on the basis of its HPLC retention time and response at 231 nm. The chromatographic conditions represent a compromise to permit quantitative determination of both enantiomer pairs in a single run. Resolution of KC 9048 could have been improved by increasing k . DU 124884 peak symmetry could have been improved by using a higher TEA or buffer concentration. However, these changes would have resulted in unsatisfactory separation of the other enantiomer pair. Thus, analyte retention factors were adjusted between 2 and 10, the range recommended for optimal resolution.i The blank plasma extract profile in Figure 2 shows good selectivity at a wavelength (231 nm) where many organic species absorb radiation. This selectivity is chiefly due to the unique HPLC retention mechanisms of the silica/B-CD separation system.* Tables 1-3 show spiked rat plasma calibration standard backcalculated results and quality control results. These data satisfy recommended guidelines for assay precision (515% RSD) and (6) Timm. U.; Wall, M.; Dell, D. J. Pharm. Scz. 1985,74, 972-977. (7) Dolan, J. W. LC-GC 1994,12. 368.
1:
Table 1. KC 9048 Rat Plasma Callbratlon Standard Summary
interday precision and accuracy spiked concn (ng/mL)
2
4
6
8 10 12 14 16 18 20 22
100 200 500 1000 2000 5000
(-)-KC 9048 mean RSD error (n = 6) (%I (%)
(+)-KC 9048 mean RSD error (n = 6) (%) (%)
99.1 207 490 1000 1978 5025
98.6 205 495 1002 1982 5016
5.9 5.0 4.4 5.7 1.5 3.1
-0.9 3.4 -2.0 0.0 -1.1 0.5
5.2 3.1 4.1 5.4 1.4 3.3
-1.4 2.7 -0.9 0.2 -0.9 0.3
Tlmo (mln)
9
&
fi
g
a
a
Table 2. DU 124884 Rat Plasma Calibration Standard Summary
2.9 2.6 2.3 2.0
interday precision and accuracy (-)-DU 124884
(+)-DU 124884
mean
RSD
error
mean
RSD
(n = 6)
(%)
(%)
(n = 6)
(%I
error
1.7
spiked concn (ng/mL)
1.4 1.1 0.8 0.5 0.2
100 200 500 1000 2000 5000
105 201 482 986 1958 5067
6.3 4.6 4.9 6.1 3.3 4.9
5.2 0.6 -3.6 -1.4 -2.1 1.3
109 195 479 985 1959 5072
8.5 6.7 6.2 6.7 3.4 4.8
8.6 -2.5 -4.1 -1.5 -2.1 1.4
2
4
6
8
10 12 14 16 18 20 22
Tlmo (mln)
Table 3. Rat Plasma Quality Control Summary
interday precision and accuracy, n
spiked concn (ng/mL) mean RSD (%) error (%) mean RSD (%) error (%) mean RSD (%) error (%)
250 750 2500
2
4
6
8
(%I
= 12
(-)-KC 9048
(+)-KC 9048
(-)-DU 124884
(+)-DU
228 6.7 -8.7 704 4.5 -6.1 2450 6.8 -2.0
225 7.3 -9.8 701 5.1 -6.5 2431 7.1 -2.8
224 5.1 - 10.4 694 5.0 -7.5 2433 7.9 -2.7
225 8.7 -9.8 691 5.9 -7.8 2439 7.9 -2.4
124884
10 12 14 16 18 20 22
Tlmo (mln)
Figure 2. Representative rat plasma calibration standard chromatograms: (A) 5000 ng/mL, (B) 100 ng/mL, and (C)blank plasma. Peak identification: 1, (-)-KC9048;2,(+)-KC9048;3,IS; 4,(-)DU 124884;5, (+)-DU 124884.
5
6
4
1 3
accuracy (f15% error).* Figure 2 shows representative chromatograms of plasma calibration standards at 100 and 5000 ng/ mL. A plasma stability study showed no pharmacokinetically relevant changes in concentration after long-term (-70 "C), ambient temperature, and freezehaw stability testing. There were no stereoselective dBerences in stability under any conditions for either racemate. In Vivo Study. Figure 3 shows a chromatogram of a male rat sample from the 40 mg kg-' day-l dose group collected 47 min postdose. The chromatogram shows detectable but not quantifiable peaks for the KC 9048 enantiomers and an enantio(8) Shah, V. P.;Midha, IC IC;Dighe, S.; McGilveray, I. J.; Skelly, J. P.; Yacobi, A.; Layloff, T.;Viswanathan, C. T.; Cook, C. E.; McDowall, R. D.; Pittman, IC A; Spector, S. Pharm. Res. 1992,9,588-592.
8
2
1
0 0
2
4
6
0
10 12 14 16 10 20 22
T h e (min)
Figure 3. Plasma extract chromatogram from male rat 4203 collected 47 min postdose (40mg/kg/day). Peak identification: 1, (-)KC 9048;2, (+)-KC9048;3, IS;4,(-)-DU 124884;5,(+)-DU 124884.
meric excess of (+)-DU 124884. Figure 4 presents a composite rat plasma concentration-time profile for the study. Data from the study show evidence of stereoselective metabolism of DU 124884. The (+)/(-) enantiomer ratio for plasma concentrations ranged from 1.1 to 1.3 for both sexes and dose groups. Given the subcutaneous route of administration, it appears that stereoAnalytical Chemistty, Vol. 67,No. 7 7 , June 7, 7995
1905
3 5E
+ (-)-DU 124884,40mg/kg
I
this species. Plasma levels obtained with the 10 mg/kg dose group made it clear that additional sensitivity would be needed to assay more than a few samples from the 2.5 mg/kg dose group. As a consequence, samples from that dose group were not assayed. Analytes were not detected in four male and four female rat samples from the control dose group (0 mg kg-l day-'). ACKNOWLEDGMENT The authors would like to acknowledge Ms. Kim O'Shields for assistance in preparing the manuscript. The test compounds were kind gifts of Dr. Warner Benson of Solvay Pharma Deutschland and Dr. Tom Hulkenberg of Solvay Duphar B.V. We are grateful to Dr. Maikel Raghoebar of Solvay Duphar B.V. and Dr. Aqeel A Fatrni of Solvay Pharmaceuticals, Inc., for their helpful comments.
selectivity may be due to preferential metabolic conversion, elimination, or distribution of (-)-DU 124884. KC 9048 enantiomers were not present at quantifiable levels in any samples, indicating that after subcutaneous administration, Ndemethylation is not a primary metabolic route of conversion for DU 124884 in
1906 Analytical Chemistry, Vol. 67, No. 11, June 1, 1995
Received for review October 12, 1994. Accepted March
18, 1995.@ AC941005F @
Abstract published in Advance ACS Abstracts, May 1, 1995.
