Anal. Chem. 1995, 67,2334-2341
Enantiomeric Separation of Drugs by Mucopolysaccharide-Mediated Electrokinetic Chromatography Hiroyuki Nishi,* Kouji Nakamura, Hideo Nakai, and Tadashi Sat0 Analytical Research Laboratow, Tanabe Seiyaku Company, Lid., 16-89,Kashima 3-chome, Yodogawa-ku, Osaka 532, Japan
Chondroitin sulfate C (sodium salt) and heparin (sodium salt), which are both mucopolysaccharides and natural components, have been employed as chiral selectors in electrokinetic chromatography (EKC) for the separation of enantiomers of drugs. These additives are charged, linear, sulfated polysaccharides having large mass. Ionic and hydrophobic interactions are probably the bases for the separation. Among tested drugs that are electrically neutral or basic, trimetoquinol, diltiazem, and their related compounds were successfully enantioseparated by EKC with mucopolysaccharides, especially with chondroitin sulfate C. The choices of pH and the concentration of mucopolysaccharideswere found to be important for improvement of enantioselectivity. The acidic buffer solutions were effective for enantioseparation of the solutes in the chondroitin sulfate C system, although no migration of the basic drugs was observed in the heparin system. The results were compared with the enantiomeric separationby EKC with dextran sulfate (sodium salt). The method using chondroitin sulfate C was successfully applied to the optical purity testing of the drug substances. Capillary electrophoresis (CE) is a powerful and versatile separation technique because of its fast separation and high resolution.'-" Various separation modes such as capillary gel electrophoresis (CGE) and micellar electrokinetic chromatography (MEKC)4,5have been developed. Electrokinetic chromatography (EKC) was the first to enable the separation of electrically neutral solutes by the CE technique and expanded the applicability of the CE method. A wide variety of substances, from ions to biopolymers, now can be analyzed by choosing the suitable separation mode, according to the physicochemical properties of the analyte.1-5 Separation of enantiomers of drugs is one of the important issues because stereochemistry can have a significant effect on the biological activity of a drug and the antipode of a chiral drug is regarded as one of the impurities. Various kinds of analytical methods have been developed, but high-performance liquid chromatography (HPLC) is the most widely used for the separa(1) Grossmann, P. D.. Colbum. J. C.. Eds. Capillary Electrophoresis-Theory
and Practice. Academic Press, Inc.: New York. 1992 (2) Li, S. F. Y. Capillary Electrophoresis-Principle. Practice and Applications; Elsevier Science Publishers: New York, 1992. (3) Guzman. N. A,. Ed. Capillary Electrophoresis Technologv; Marcel Dekker. Inc.: New York, 1992. (4) Vindevogel, J.; Sandra, P. Introduction to Micellar Electrokinetic Chromatography. Huething: Heidelberg, Germany, 1992. (5)Terabe. S. Trends Anal. Chem. 1989.8, 129.
2334 Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
tion of enantiomers.".' In the HPLC method, one can use chiral stationary phase columns, which are often commercially available, to develop the chiral separation method. However, typically one column can give chiral recognition only for a limit number of compounds. That is, to separate the enantiomers, we have to try to use several different columns. Advantages of CE techniques for the separation of enantiomers are the ultrahigh separation efficiency, easy changes of separation media, extremely small volumes of the sample and the media, etc., in comparison with HPLC. In the development of a CE chiral separation method, one can easily alter the separation solution to find the optimun separation media and also can use an expensive chiral selector because of the small volume required. Nowadays most of the direct CE separations employ cyclodextrins (CDs) or proteins as chiral selectors in capillary zone electrophoresis (CZE), EKC, and CGE.8-16 These chiral selectors have been demonstrated to be effective in chiral HPLC separation and have shown wide enantioselectivity.6J Recently, polysaccharides, which had already been found to be useful as chiral stationary phases for HPLC, were applied to the CE chiral separation in two modes. One is CZE with an electrically neutral maltode~trin,~'-~~ and the other is EKC with an ionic In the latter method, we consider that the mode can be included in affinity EKC, which is used for CE chiral separation with proteins as chiral selectors.l:j-li In affinity EKC, biological components are employed as an ionic pseudophase and the mode can be applicable to electrically neutral solutes as in MEKC. In this paper, chondroitin sulfate C (sodium salt) and heparin (sodium salt), which are both mucopolysaccharides and natural components, are employed as chiral selectors in EKC for the (6) Krstulvic, A. M.. Ed. Chiral Separation by HPLC Ellis Honvood: Chichester, U.K., 1989. (7) Lindner. W. Chromatographia 1987,24, 97. (8) Snopek. J.; Jelinek, I.: Smolkova-Keulemansova, I. J. Chromatogr. 1992.609, 1.
(9) Kuhn. R.; H-Kuhn, S. Chromatographia 1992.34.505. (10) Terabe. S.; Otsuka. K.; Nishi, H. J. Chromatogr. 1994,666. 295. (11) Ward. T. J. Anal. Chem. 1994.66, 633A. (12) Novotny. M.; Soini, H.; Stefansson, M. Anal. Chem. 1994,66, 646A. (13) Barker, E. G.; Russo, P.: Hartwick, R. A. Anal. Chem. 1992,64, 3024. (14) Birnbaum. S.; Nilsson. S. Anal. Chem. 1992.64, 2872. (15) Tanaka. Y.; Matsubara. N.; Terabe, S. Electrophoresis 1994,15, 848. (16) Guttmann. A,; Cooke. N. J. Chromatogr., A 1994,685, 155. (17) D'Hulst. A.; Verbeke. N.J. Chromatogr. 1992,608, 275. (18) D'Hulst, A,; Verbeke. N. Chirality 1994,6, 225. (19) Soini. H.; Stefansson. M.; Riekkola. M.-L.: Novotny. M. V.Anal. Chem. 1994, 66. 3477. (20) Nishi, H.; Nakamura. K; Nakai, H.: Sato, T.; Terabe, S. Electrophoresis 1994, 15, 1335. (21)Stalcup. A. M.; Agyei, K. M. Anal. Chem. 1994,66, 3054. 0003-270019510367-2334$9.0010 0 1995 American Chemical Society
separation of enantiomers of cationic or electrically neutral drugs. The capability of these mucopolysaccharides as chiral selectors is investigated,and these results are discussed with those obtained from EKC with dextran sulfate. Separation of enantiomers of cationic drugs such as trimetoquinol is also tried by CZE with an electrically neutral polysaccharide to evaluate the separation mechanism. The effects of buffer pH and the concentration of chondroitin sulfate C on the enantioseparation and migration are examined. The applications of the method to the optical purity testing of drugs are also described.
Propranolol
Trimetoqulnol (S-form)
H o(=)H o
bH
EXPERIMENTAL SECTION Materials. Chondroitin sulfate C (sodium salt) and heparin (sodium salt, 177.2 unitdmg) of reagent grade were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) and used without further purification. Dextran (molecularweight -40 OOO) was obtained from Tokyo Kasei Kogyo (Tokyo, Japan). More than 20 drugs (racemates) were used as the test solutes. Diltiazem hydrochloride (calcium channel blocker), diltiazem derivatives (6 chlorodiltiazem,khlorodiltiazem, khlorodiltiazem) , denopamine (cardiotonic), trimetoquinol hydrochloride (bronchodilator), trimetoquinol isomer, sulconazole nitrate (antifungal), timepidium bromide (anticholinergic), bisoprolol fumarate @-blocker), and trimebutine maleate (antispasmodic) were obtained from Tanabe Seiyaku Co. Ltd. (Osaka, Japan). Laudanosolin, norlaudanosolin, laudanosin, primaquine (antimalarial), and warfarin (anticoagulant) were purchased from Aldrich Chemical Co. (Milwaukee, W. Benzoin, promethazine hydrochloride (antipsychotic), chlorpheniramine maleate (antihistaminic),2,2'-dihydroxy-l,l'dinaphthyl, and propranolol hydrochloride @-blocker) were purchased from Wako Pure Chemical Industries Ltd. Metanephrine hydrochloride, metoprolol tartrate @-blocker) and alprenolol hydrochloride @-blocker)were obtained from Sigma Chemical Co. (St. Louis, MO). (S)-Trimetoquinolhydrochloride, which is used as a bronchodilator (Inolin), and (2S,3S)-diltiazem,which is a calcium channel blocker (Herbesser), were obtained from Tanabe Seiyaku. (R)-Trimetoquinol hydrochloride has no effect, and (2S,3S)diltiazem is the most active among four possible enantiomers. Mesityl oxide, used as a tracer of the electroosmotic flow @OF), was obtained from Nacalai Tesque (Kyoto, Japan). The structures of diltiazem, trimetoquinol and its related compounds, and some other drugs are shown in Figure 1. HPLC grade methanol was purchased from Katayama Kagaku Kogyo (Osaka, Japan). All other reagents used were of analytical reagent grade from Katayama Kagaku Kogyo. Water was purified by Milli-RO 60 water system (Millipore Japan, Tokyo, Japan). Apparatus. The instrument used was a P/ACE system 5510 equipped with a photodiode array detector (Beckman Instruments, Inc., Fullerton, CA). Fused silica capillary columns with 75 ym i.d. and 57 (effective length 50 cm) or 47 cm total length (effective length 40 cm) were used in the system for the separation. The capillary was thermostated at 23 "C with a liquid coolant. The applied voltage was held constant at 15 or 20 kV. Detection wavelength was adjusted to 214-235 nm. The instrument control and data collections were performed with a personal computer (COMPAQ ProLinea 4/33). Procedure. Running buffer solutions were freshly prepared by dissolving each mucopolysaccharide in a 20 mM phosphateborate buffer of the specified pH. The low-pH buffer used was 20 mM NaZHP04, adjusted to the desired pH with H3P04. The pH values of the buffers were always checked before and after
Trlmetoquinol Isomer
f
H
2
G
o
Diltiazem (PS,SS-form)
H OCOCH3
HO
OH
Norlaudanosollne
9-Chiorodiltiarem
I
CI
Leudanosollne
Chlorphenlramine FH3 HNHCHCH~CH~CHZNH~
bJ
CHJO
Primaquine
Figure 1. Structures of some of the tested solutes.
each experiment. These buffer solutions were filtered through a 0.45 pm pore size membrane filter (Gelmann Science Japan, Tokyo) and degassed by sonication with a Branson Model B2200 ultrasonic cleaner (Yamato, Tokyo, Japan) prior to use. The capillary was rinsed with the running buffer solution for 1 min each before every run and washed with 0.1 N KOH solution followed by water daily, usually at the end of an experiment. The injection was performed by the pressurizing method (0.5 psi, 2-10
4. Sample Preparation. Stock solutions of each racemic sample were prepared in methanol at a concentration of -1.0 mg/mL. The sample solutions for enantioseparation were prepared at a concentration of -0.1 mg/mL by mixing each stock solution and diluting with water. The sample solutions for purity testing of trimetoquinol and diltiazem were prepared as follows: 100 mg of each standard, whose optical purity was previously checked, was exactly weighed into 20 mL volumetric flask. Then each corresponding antipode was added to the flask to give a concentration from 0.2% to 1.0%and made to volume with methanol. These solutions for purity testing were diluted to one-third for diltiazem, and one-iifth for trimetoquinol, with water. RESULTS AND DISCUSSION
EKC with Three Ionic Polysaccharides. Chondroitin sulfate C and heparin are both sulfated polysaccharides composed of unit structures of 1-4liinked glucosamine and uronic acid. These are natural components found in connective tissues or mast cell granules and are soluble in water, hence they are usable as a pseudophase in EKC. These unit structures are shown in Figure Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
2335
1 NHCOCH3
ClcUA
GlcUA
GalN
CalN
2 NHS03’ GluUA
3.
NHS03’
ClcN
r
n
GlcN
LIdoUA 1
Figure 2. Unit structure of anionic polysaccharides: (1) chondroitin sulfate C; (2) heparin; (3) dextran sulfate. Table I. Characteristics of Three Ionic Polysaccharides
polysaccharide
molecular mass (Da)
S content (%)
chondroitin sulfate C heparin dextran sulfate
30000- 50000 7000-20000 -7300
11 18
7
2,22 with those of dextran sulfate, which was the first ionic polysaccharide employed as a pseudophase in EKC.20 Among these three ionic polysaccharides, the molecular weight of chondroitin sulfate C is the largest (30 000-50 000) and that of dextran sulfate is the smallest (-7300). As for the anionic character, chondroitin sulfate C has about one ionic group (carboxyl group or sulfate group) per monosaccharide unit and heparin (molecular weight 7000-20000) one to two; dextran sulfate has almost three sulfate groups per one D-glucose unit. This means that the electrophoretic mobility of chondroitin sulfate C is the smallest and that of dextran sulfate is the largest. As previously reported, in the enantiomer separation by EKC with dextran sulfate, migration of the basic solutes was not observed within -50 min under the acidic buffer conditions @H -3) in the dextran sulfate system. This can be ascribed to the strong ionic interaction between the solute and the dextran sulfate. Preliminary investigation of the ionic character of chondroitin sulfate C and heparin, as well as their enantioselectivities, was carried out by using pH 3.0 and pH 7.0 buffer solutions containing 3% each mucopolysaccharide at 20 kV. As expected from the results of dextran sulfate,20no migration of the basic solutes was observed in EKC with heparin under the acidic conditions. However, chondroitin sulfate C was successfully employed in both acidic and neutral conditions. This can be interpreted as due to the ionic character of the chiral selectors. At pH 3.0, we assume 2336 Analytical Chemistry, Vol. 67,No. 14, July 15, 1995
monosaccharide residue ( 8 ) -170 -40 -20
pep(10-2 mm2 s-1 V-1)
calcd (rel)
obsd
1
-1.28 (1) -1.57 (1.2) -2.06 (1.6)
1.4 2.3
that carboxyl groups do not dissociate and only sulfate groups dissociate, because the reported pKa value of hyaluronic acid, which is a mucopolysaccharide having similar structure and carboxyl groups, is 3.04.23 Therefore there is 0.5 ionic groups per monosaccharide residue in chondroitin sulfate C, and 1.2 in heparin, and 3 in dextran sulfate, from the structures shown in Figure 2. The electrophoretic mobility of linear-type ionic solutes is inversely proportional to (molecular mas^)^'^ and proportional to its charge.24 The relative ratio of the electrophoretic mobility calculated approximately from [(ionic group per monosaccharide residue) (number of monosaccharide residue) / (average molecular mas^)^'^] is summarized in Table 1, with the observed electrophoretic mobilitypc,,. The electrophoretic mobilities of these ionic polysaccharides were obtained by the equation (uep(app) - ped. Apparent electrophoretic mobility of the ionic polysaccharides (uep(app)) was determined by the indirect method described in Stalcup’s work. The mobility of electroosmotic flow (ueJ was determined from the mesityl oxide peak (0.83 x mm2 V-I). There is good agreement between the calculated values and the observed results. As discussed above, the electrophoretic (22) The Japanese Society for Biochemistry, Ed. Data Book on Biochemistry; Tokyo Kagaku Dojin: Tokyo, 1979; pp 491-492. Merck Index, 11th ed.; Merck & Co. Inc.: Rahway, KJ,1983; p 427. (23) Cleland, R L.; Wang. J. L.; Detweiler, D. M. Macromolecules 1982,15,386. (24) Rickard, E. C.; Strohl, M. M.; Nielsen, R G.Anal. Biochem. 1991,197, 197.
A * C
c 8
s
15
20
30 ,
,
,
I
15
IO
Mlgratlon tlme I mln
Migratlon tlme / mln
B 8 * e
c n8
a
15
20
30
Mlgratlon tlme I mln
Figure 3. Separation of enantiomers of (A) diltiazem (SS, RR, SR, RS) and (B) trimetoquinol and its four related compounds by EKC with chondoroitin sulfate C. Solutes: (1) trimetoquinol; (2) trimetoquinol isomer; (3) norlaudanosoline; (4) laudanosoline; (5) laudanosine. Conditions: 3% chondroitin sulfate C in 20 mM phosphate-borate buffer (pH 2.4); separation tube, 75 pm i.d. x 57 cm (effective length 50 cm); applied voltage, 20 kV; detection, (A) 235, (B) 230 nm; temperature 23 "C; injection times of the sample solutions, 5 s.
mobility of chondroitin sulfate C was smallest among the three ionic polysaccharides due to its small ionic character, leading to the adaptability to acidic conditions and successful enantioseparation. Other than the ionic character, the species of the monosaccharide residue of the ionic polysaccharides may be one of the important factors affecting enantioselectivity. Chird Recognition by EKC with Three Ionic Polysaccharides. In the chondroitin sulfate C system, among the tested solutes, enantiomers of diltiazem, diltiazem derivatives, trimetoquinol, trimetoquinol isomer, laudanosine, norlaudanosoline, laudanosoline, and primaquine were successfully separated. Propranolol and sulconazole were partially enantioseparated. Acidic conditions especially were superior to basic conditions for enantioseparation. Typical separations of enantiomers of diltiazem and trimetoquinol analogues at pH 2.4 are shown in Figure 3. Four possible optical isomers of diltiazem were directly separated by the method, as shown in Figure 3A Enantiomers of trimetoquinol and its four related compounds were also successfully separated in a single run, as shown in Figure 3B. In the heparin system, where only the neutral buffers are usable, as mentioned above, partial enantiomeric separation of diltiazem (separation between SS-form and RR-form), trimetoquinol, and chlorpheniramine was achieved. Almost perfect baseline separation of enantiomerswas observed in three diltiazem derivatives. Separations of the enantiomers of Schlorodiltiazem and chlorpheniramine by EKC with heparin at pH 6.0-6.5 are
10
5 Migration tlme / mln
Figure 4. Separation of enantiomers of (A) 9-chlorodiltiazem (SS, RR) and (B) chlorpheniramine by EKC with heparin. Conditions: 3% heparin in 20 mM phosphate-borate buffer of (A) pH 6.0 and (B) pH 6.5. Other conditions are the same as in Figure 3 except the detection wavelength was 214 nm.
shown in Figure 4. Recently, successful enantioseparations of several antihistaminessuch as pheniramine, doxylamine, etc., and several antimalarial drugs such as chloroquine and primaquine by EKC with heparin, where phosphate buffers of pH 4.5 and 5 were used, were reported.2I In the dextran sulfate system, enantiomers of trimetoquinol, trimetoquinol isomer, and &hlorodiltiazem were baseline resolved. Partial separation of enantiomers was observed in diltiazem and raudanosine. The pH of buffers employed was 5-7. Typical separations of enantiomers of diltiazem, trimetoquinol, and trimetoquinol isomer at pH 5.5 and 3%dextran sulfate are shown in F i i e 5. The detailed enantiomeric separation of trimetoquinol and its isomer by EKC with dextran sulfate has been discussed elsewhere.2O The first CE direct enantiomeric separation of diltiazem was achieved with MEKC with taurodeoxycholate (STDC)F5 The resolution R, of diltiazem (SS-form and RR-form) by EKC with chondroitin sulfate C (-3.6, see Figure 3A) was much larger than that for MEKC with STDC (-1.4). The possible four optical isomers of diltiazem were successfully separated by EKC with chondroitin sulfate C. The CE enantiomeric separation of trimetoquinol-relatedcompounds was also reported by both MEKC with STDCZ4and cyclodextrin-modified CZE (CDczE).2627 Among successful CE enantiomer separations of trimetoquinol, EKC with (25) Nishi, H.;Fukuyama, T.; Matsuo, M.; Terabe, S.;J. Chromatogr. 1990,515, 233.
Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
2337
25
‘“1
? 2oi\”
t‘O -15
c
E
.-0
c
c
-10
3 v)
Q,
E 5
10
-5
20
MlglatlOn llme I mln
B
I
c
1
o s , . ., 2
3
4
5
6
7
0
PH Figure 6. Effects of the buffer pH on the separation of enantiomers of diltiazem (0,SSform; 0, RR-form; +, resolution) and trimetoquinol ( A , R-form; 0, S-form; A, resolution)). Buffer, 20 mM phosphateborate buffer containing 3% chondoroitin sulfate C. Other conditions are the same as in Figure 3.
chondroitin sulfate C gave the largest Rs as in diltiazem. Moreover, all of the enantiomers of trimetoquinol-related compounds were successfully separated in a single run, as shown in Figure 3B. Chondroitin sulfate C had the widest enantioselectivity for the tested solutes, which is probably due to its large molecular mass and small ionic character. Heparin also seems to have a relatively wide enantioselectivity judging from Stalcup’s work,21although heparin was not as good for enantioseparation of the tested solutes described in the Experimental Section. We think the most important and useful character of chondoroitin sulfate C is its small ionic character, which permits EKC analysis under acidic conditions, leading to the large enantioselectivity. In CZE with the neutral polysaccharide dextran (molecular weight 40 000) at the same concentration (3%) under the acidic conditions @H 3.0), enantiomers of trimetoquinol and 9-chlorodiltiazem were not separated and they migrated with almost the same velocity as in the CZE separation without the chiral selectors (for trimetoquinol, see Figure 8B). These results show that ionic interaction contributes to the solute migration and the enantioseparation. Recently, a high concentration (-10-20%) of dextrin, which is a neutral polysaccharide, was demonstrated to be effective for the
enatiomeric separation of warfarin, ibuprofen, etc.lg This means that some hydrophobic interaction as well as ionic interaction (that is, aflinity interaction in a wide sense) must contribute to the enantiorecognition. There may be a possibility of employing a high concentration of dextran for the enantiomeric separation of trimetoquinol or other tested solutes. In EKC with an anionic pseudophase, the difference in the apparent mobilities of cationic enantiomers (Apapp) can be described by the same equaiton as in enantiomer separation by protein-mediated EKC.15 The separation model is simple and similar to that described by Wren and Rowe for the separation of enantiomers with neutral C D S . ~The ~ equation shows the dependence of Apappon the difference in the mobilities between the free and complexed analytes. It is expected that Apappincreases with a decrease in buffer pH for the cationic solute, due to the decrease of E O F therefore, the pH value will be an important factor in improving the separation in addition to the concentration of the chiral selector. Recently Vigh et al. developed a more complex model in CD-CZE.29-31They discussed three simpliied models: (1) only the non-ionic forms of the enantiomers; (2) only the ionic forms of the enantiomers; (3) both forms of the enantiomers interacting differently with the chiral selectors (CD) . The enantiomeric separation of the cationic solutes by EKC with mucopolysaccharides seems to be included in model 2 or 3. Effect of B d e r pH on Migration Times and Enantioselectivity. The effects of buffer pH on migration times and enantioseparation were investigated, using 20 mM buffer solutions of pH 3-7 containing 3%chondroitin sulfate C. The dependence of resolution Rs and the migration times of each enantiomer of trimetoquinol on the buffer pH is shown in Figure 6, where the migration time of mesityl oxide, Le., the migration of EOF, is also plotted. Separation of the enantiomers was achieved at all pH. At pH 6.0 and 7.0, migration of the enantiomers of trimetoquinol was slower than that of the EOF. In contrast, the enantiomers
(26) Nishi. H.; Kokusenya, Y.; Miyamoto, T.; Sato, T. J. Chromatogr., A 1994, 659, 449. (27) Nishi, H.:Nakamura, K; Nakai, H.: Sato, T. J. Chromatogr., A 1994,678, 333.
(28) Wren, S. A C.: Rowe, R C. J. Chromatogr. 1992,603, 235. (29) Rawjee, Y. Y.: Staerk, D. U.; Vigh, G. J. Chromatogr. 1993,635, 291. (30) Rawjee, Y. Y.; Williams, R. L.; Vigh, G. J. Chromatogr., A 1993,652, 233. (31) Rawjee, Y. Y.: Williams, R. L.: Vigh, G. J. Chromatogr., A 1993,680,599.
,
10
,
,
,
,
r
20
-
I
25
Mlgratlon llme / mln Figure 5. Separation of enantiomers of (A) diltiazem (SS, RR) and (B) trimetoquinol and its isomer by EKC with dextran sulfate. Solutes: (1) trimetoquinoi; (2) trimetoquinol isomer. Conditions: 3% dextran sulfate in 20 mM phosphate-borate buffer (pH 5.5). Other conditions are the same as in Figure 3 except applied voltage was 15 kV and detection wavelength was 214 nm.
2338 Analytical Chemistry, Vol. 67,No. 14, July 75, 7995
Diltiazem
A
ss
1
I
!
.
.
.
,
10
5
15
20
8-Chlorodiltiazem
15
isomer
25
Migration time / min Figure 7. Separation of trimetoquinol and its related compounds by EKC with 20 mM phosphate-borate buffer (pH 2.8) containing 3% chondoroitin sulfate C. Other conditions and solute numbers are the same as in Figure 3B.
:' ,.9; 3.0 %
2.0 %
.
l!,Id
L
0.5 %
migrated faster than the EOF in the acidic buffers @H 3-5). As for the separation of the enantiomers of trimetoquinol, it was more effectively improved with an increase of migration times through change of buffer pH, i.e., through the reduction of the EOF velocity. R, was 2.8 at pH 3.0, and 0.5 at pH 7.0. Acidic conditions were more effective than neutral conditions and the pH 4.0 buffer gave the largest resolution. From the results of Figure 6 and the CD-CZE enantiomer separation of trimetoquin01,2~.~~ trimetoquinol seems to be type 2 or type 3 solute in Vghs modePo although the results are not conclusive enough to delineate this. For other trimetoquinol-related compounds, the same tendency was o b served. Additionally, selectivity in separation not only between the enantiomers but also among the solutes was affected by the pH. One example is shown in Figure 7 , where the pH 2.8 buffer was employed. It is evident that the pH 2.4 condition (see Figure 3B) is superior to the pH 2.8 condition for the enatiomeric separation of the trimetoquinol isomer, although the others gave better enantiomeric separation at pH 2.8 (Figure 7 ) . The migration order between trimetoquinol and its isomer also changed between pH 2.4 and pH 2.8. Trimetoquinol has a cationic character under the investigated pH range that increases with a decrease of pH. If ionic interaction between chondroitin sulfate C and trimetoquinol is as strong as in dextran sulfate, the migration of trimetoquinol should be slow or not be observed under the acidic buffers, because of the large electrophoreticmobility of the selector and the small EOF velocity. However, migration of trimetoquinol at pH 3 (-16 min) was faster than that of EOF (-25 min). TAis means that the ionic interaction of chondroitin sulfate C is very small compared with the other chiral selectors. But as mentioned above, even small ionic interactions delayed the migration of trimetoquinol and gave a successful enantioseparation. The migration time of trimetoquinol in the dextran system (3%,pH 3), where ionic interactions cannot exit because it is a neutral polysaccharide, was almost the same as in CZE without such chiral additives at pH 3.0 (-7 min; see Figure 8B). This shows that ionic interaction is important and it contributed to the enantiomeric separation.
As for the migration order of the enantiomer of trimetoquinol, the R-form migrated faster than the S-form in EKC with chondroitin sulfate C. This migration order of the trimetoquinol enantiomers was the same as in EKC with dextran sulfate2O and different with CZE with ,!?-type CDs.25!26This means that the magnitude of interaction between each enantiomer of trimetoquinol and the chiral selector is reversed in the two modes (CZE and EKC), where chiral selectors having a D-glucose unit as a chiral moiety were employed. The effects of buffer pH on the migration times and resolution R, examined for diltiazem, using the same conditions as with trimetoquinol, are also shown in Figure 6. The results were almost the same as with trimetoquinol except the migration was faster than EOF at all pH. Enantiomers of diltiazem were separated under the pH range investigated. However, acidic conditions were superior to the basic conditions because of the slow EOF. As for the migration order of the enantiomers, the active SS-form migrated faster than the RR-form. In EKC with chondroitin sulfate C, pH affected the migration times and the enantioseparation as we had expected, although it was not so critical as in EKC with dextran sulfate. It is recommended that the EKC enantiomeric separation with the chondroitin sulfate C system be performed under the acidic conditions. Effect of Concentration of Chondroitin Sulfate C on Migration T i e s and Enantioselectivity. The effects of chondroitin sulfate C concentration on the migration times and the Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
2339
Table 2. Reproducibility of the Mlgration Times of Trlmetoquinol in EKC with Chondroltln Sulfate Ca
av migration conditions time (min) RSD (%) expt repetition column (cm) pH R-form S-form R-form S-form 1 2 3
50 50 40
6 5 5
2.2 2.4 2.8
29.95 27.88 20.58
31.71 29.27 21.00
1.51 1.09 1.41
1.52 1.23 1.35
Buffer 20 m M phosphate-borate buffer containing 3%chondroitin sulfate C; applied voltage, 20 k V temperature, 23 "C; detection, 235 nm. (I
Table 3. Linearity and Recovery of Trimetoquinol in EKC with Chondoroitin Sulfate Ca
R-form
theor
av found
repetition
R-form (%)
R-form (%)
rec (%)
0.2 0.5 1.0
5 5 4
0.22 0.55 1.10
0.26 0.56 1.15
120 102 104
Buffer, 20 mM phosphate-borate buffer, pH 2.8, containing 3% chondroitin sulfate C; applied voltage, 20 kV; temperature, 23 "C; detection, 230 nm. (I
enantioseparation were investigated with a pH 2.8 buffer solution. The concentration range of chondroitin sulfate C was from 0% (CZE mode) to 3%. The results for the enantiomers of diltiazem and khlorodiltiazem are shown in Figure 8A and for trimetoquinol and its positional isomer in Figure 8B. In the CZE separation, cationic solutes migrated faster than the signal of methanol, Le., EOF. And naturally no enantiomeric separation was observed in the absence of chondroitin sulfate C. As for trimetoquinol and its isomer, even the separation of the two solutes was not successful in CZE. However, through an increase in the concentration of chondroitin sulfate C, migration times of the solutes gradually increased and the separation of the enantiomers was improved as shown in figures. Especially marked improvement of the enantiomeric separation was observed between 1.0% and 2.0% concentration. This means that interaction between chondroitin sulfate C and the solute occurs and it increases with an increase of the concentration of the additive. The separation mechanism in EKC with mucopolysaccharides is not clear. However, successful chiral separation by CZE using neutral polysaccharide^^^-^^ suggests that some affinity in a wide sense rather than an ion exchange process is the basis for the chiral separation with mucopolysaccharides. Application of EKC with Chondroitin Sulfate C to the Optical Purity Testing of the Drugs. The method was applied to the optical purity testing of trimetoquinol and diltiazem drug substances. One of our purposes in this work is to develop an optical purity testing method for the drugs. As mentioned in the introduction, CE chiral separation has many advantages and attractive features compared with HPLC. The important factors requested for the quality control method are simplicity, rigidity, and good reproducibility, as well as detection sensitivity or selectivity. Then method valiation such as linearity, recovery, reproducibility of migration times, etc., was investigated in EKC with chondroitin sulfate C, to judge whether this method could be adaptable or not. From the results mentioned above, the acidic buffer solution containing 3%chondroitin sulfate C was selected 2340 Analytical Chemistry, Vol. 67, No. 14, July 15, 1995
20
30
20
added (%)
30
Migration time I min Figure 9. Optical purity testing of diltiazem hydrochloride: (A) standard diltiazem hydrochloride (SSform) and (B) 0.2% RR-form spkied to standard diltiazem. Injection times of the sample solutions are 6 s. Other conditions are the same as in Figure 3A.
B
A
[I c
u
c r?
0
G ' I "I 15
20
z_j
15
20
Migration time I min Figure I O . Optical purity testing of trimetoquinol hydrochloride: (A) standard trimetoquinol hydrochloride (S-form) and (B) 0.2% Rform spkied to standard trimetoquinol. injection times of the sample solutions are 10 s. Conditions are the same as in Figure 7.
as a running buffer for the optical purity testing of the drugs. The reproducibility of the migration times of the enantiomers of trimetoquinol examined under three different conditions is summarized in Table 2 . The RSD values were 1.1%-1.5%. These values are acceptable for purity testing, although those from HPLC are much better than those from CE. The linearity and recovery were then investigated by adding the inactive enantiomer (R-form) to the standard trimetoquinol (S-form) in the range 0.2%-1.0%. The results are summarized in Table 3. The optical purity of the standard trimetoquinol was previously determined by HPLC using the derivatization method described elsewhere.32 There was no minor enantiomer in the standard (not more than 0.05%). Good results were obtained in both linearity and recovery testing. Typical chromatograms in the optical purity testing of trimetoquinol and diltiazem are shown in Figures 9 and 10, indicating the 0.1%level minor enantiomers detectable in the method at S/N (32) Nishi, H.; Fujumura, N.; Yamaguchi, 8.; Jyomori, W.; Fukuyama, T. Chromatographia 1990,30, 186.
= 3. As for the major peak shapes, trimetoquinol was more critically affected by the water content of the sample solution. It was recommended for good peak shape to prepare a water-rich sample solution. However, even when peak shapes were worse than normal, the linearity of the minor component was good enough to determine its content.
CONCLUSIONS Ionic polysaccharides were found to be effective as chiral selectors in EKC. EKC with chondoroitin sulfate C was successful for both the solute separation and each enantiomeric separation and could be performed under acidic conditions. Among the tested solutes, trimetoquinol and its related compounds and diltiazem were successfully enantioseparated. The large Rsvalues in EKC with chondroitin sulfate C enabled this method to be used as an optical purity testing method for the drugs. However, EKC with anionic polysaccharide mentioned above can be successful for the enantioseparationof electrically neutral or cationic solutes. For the anionic solutes, CZE with neutral polysaccharides or EKC with cationic pseudophases should be examined. Some successful enantioseparation of anionic solutes by CZE with the neutral polysaccharides was recently reported.17-19 Chiral separation by (33) Armstrong, D. W.; Rundlett, K L.; Chen, J.-R. Chirality 1994,6, 496.
CE with polysaccharides can be selected according to the sample’s ionic character. As in HPLC chiral separation, many polysaccharides other than those examined in this work, whether ionic or not, will be successfully applied for the CE c h d separation. Again, one of the advantages of the CE chiral separation is the applicability of the relatively expensive c h d selectors. Recently, Armstrong et al. applied the macrocyclic antibiotic vancomycin for the CE chiral separation and reported high resolution factors for a variety of c0mpounds.3~ Other than CDs, proteins, polysaccharides, and vancomycin, still there is a possibility of finding a chiral selector showing excellent enantioselectivity in the CE chiral separation. ACKNOWLEDGMENT The authors gratefully acknowledge the helpful discussions with Professor S. Terabe (Himeji Institute of Technology). Received for review December 7, 1994. Accepted April 21, 1995.@ AC9411841 @Abstractpublished in Advance ACS AQstructs,June 1, 1995.
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