AMI. them. 1992, 84,3024-3028

9024

Chiral Separation of Leucovorin with Bovine Serum Albumin Using Affinity Capillary Electrophoresis Geoffrey E. Barker, Paul RUSSO, and Richard A. Hartwick' Department of Chemistry, State University of New York at Binghamton, Binghamton, New York 13902

A method for the detenninatlon of the (6R)- and (6s)stereokomen of leucovorin wlng d.ctrokhtIc chromatography (EKC) ln the affldty mode has beon developed. Bovine wrum albumin ( S A ) k used as a run buffer additive to incorporateensolectlvtty into the system. ProteinwclWInt.nrctknraremWmlz.dbyudngapoly(.thykmglycol) (PEQ) coated caplllary. Chkai resolutbn Is obtained In 12.5 mln wtth effkkndm greater than 200 000 t b r e t k a i plates using BsA as an additive, whlle no resolution k obtained in the absenceof BSA. a generalequation k derlvd to calculate the free energy of Interadon between the leucovorinhomers and the BSA md.cuk. Thk method r.pre8ents a new means of obtalning thermodynamic data for rubstrate Mndlng Interactknr and for the general study of drug crorcreactlonr and interadlonr of drug8 wtth serum and other protdns.

INTRODUCTION Leucovorin (LV) is a reduced folate that is used clinically inconjunctionwith methotrexatetherapyfor cancerpatienta.' LV has two chiral carbons; one is located in the glutamate portion of the molecule, and the other is located in the pteridine ring at the 6-poeition (Figure 1).LV is commercially available as a 1:l mixture of (6R)- and (6S)-isomers since L-glutamate is used in the synthesis. The (6S)-isomer has been determined to be the more active isomer in the treatment with methotrexak2 The ability to effectively separate the stereoisomers is crucial to the description of the clinical pharmokinetics of LV. The first chiral separation of LV reported in the literature involves a high-performanceliquid chromatographic (HPLC)method that utilizes an immobilized BSA stationary phase as a means of chiral recognition.2 Since then two more HPLC methods have been developed for the chiral separation of LV.3*4 An alternative method to HPLC is capillary electrophoresis (CE).ss This method is capable of separating ionic species based on charge to mass ratios, approaching efficiencies of 1OOO OOO theoretical plates. Electrokinetic chromatography (EKC) is a technique that can be considered to be a branch of CE and is capable of separating both neutral and charged analytes? Here analytes are separated by their differential distribution into an electrically migrating phase and the surrounding aqueous phase, where the two phases have different migration characteristics. A number of approaches have been investigated that use EKC to obtain optical (1) Djeraesi, I.; Abir, E.; Roger, G. L., Jr. Clin. Pediatr. 1966.5, 502. (2) Choi, K.E.; Schilsky, R. L. Anal. Biochem. 1988,168,398. (3)Wainer, I. W.; Stiffii, R. M. J. Chromatogr. 1988,424, 158. (4)Wainer,I. W.;Silan,L.; Jaduad,P.;Whitfield,L. R. J.Chromatogr. 1990,532,236. (5)Hjerten, S. Chromatogr. Rev. 1967,9,122. (6)Mikkers, F. E. P.; Everaerb, F. M.; Verheggen, T. P. E. M. J. Chromatogr. 1979,169,11. (7) Jorgenson, J. W.; Lukaar, K. Anal. Chem. 1981,53,1298. (8) Hjerten, S.J. Chromtogr. 1983,264,l. (9)Terabe, S.;Otauka,K.; Ichikawa, K.; Tauchiya, A.; Ando, T. Anal. Chem. 1984,56,111.

< ( c,- NII- 5H - c I I 2- c I~ 2- c 00 I I 011 C l l O

Flguo 1. Mdecular structure of leucovorin (LV). Asterisks lndhte a chlrai center. There are potentially four different dla~ter-6, although onlytwo isomers(thoselnvdvlngtheBpoeltlononthepteddne rlng) are In the sample analyzed in thls paper.

resolution. Moet of these involve the addition of stereospecific run buffer modifiers to the EKC system. Zare et al. were among the f i s t to show that optical resolution could be obtained using EKC.'OJ1 Their work demonstrated the racemic separation of a number of derivatized amino acids via diastereomeric interaction between the amino acid and a CULL-X complex (where X histidine or aspartame) present in the supporting electrolyte. Optical resolution hae also been successful in micellar EKC which employe an ionic micelle as a runbuffer modifier. Methodsinclude wing chiral s ~ r f a c t a n t aand ' ~ ~the ~ combination of sodium dodecyl sulfate (SDS)and a chiral additive.lSJs Success has also been found in employing cyclodextrin derivatives as substitutes for micelles which are chiral and ionic under the chosen conditions." A recent paper shows the EKC chiral separation of 2,3,4,6-tetra-0-acetyl-~-D-glucopyranosy~ isothiocyanate (GITC) derivatived DL-amino acids using a SDS solution.1s BSA has been used successfully in HPLC as a chiral stationary phase.24J+22 An EKC method was developed that utilizes BSA as the stereospecific run buffer modifier. In this work BSA is used as the chiral pseudostationary phase. The LV isomers and BSA are both negatively chargd, however they have different electrophoretic mobilities. Thus the net velocities of the isomers will differ depending on their extent of interaction with the BSA phase. The influence of pH and BSA concentration on the separation characteristics is examined. The use of poly(ethy1ene glycol) (PEG) coated capillaries was found to improve migration time reproducibility as well as column lifetime. Finally, an equation is derived to determine the free energy of interaction between the isomers of LV and BSA. (10)Gawmann, E.;Kuo, J. E.; Zare, R. N. Science (Wa.shington,D.C.) 1986,230,813. (11)G o d , P.;Gaseman, E.; Michelsen, H.; Zare, R. N. Anal. Chem. 1987,59,44. (12)Terabe, S.;Shibata, M.; Miyashita, Y . J. Chromatogr. 1989,480, 403. (13)Nishi, H.: Fukuyama, T.; Matauo, M.; Terabe, S. J.Microcolumn Sep. 1989,1,234. (14)Dobashi. A.; Ono, T.; Hara, S.;Yamarmchi, . J. Anal. Chem. 1989. 61,1986. (15)Coehn, A. S.;Paulus, A.; Karger, B. L. Chromatographia 1987,24, 15. (16)Oteuka, K.; Terabe, S. J. Chromatogr. 1990,515,221. (17)Terabe, S. Trends Anal. Chem. 1989,8,129. (18)Nishi, H.;Fukuyama, T.; Matauo, M. J.Microcolumn Sep. 1990, 2,234. (19)Stewart, K. K.; Doherty, R. F. Proc. Natl. Acad. Sci. U.S.A. 1978, 70,2850. (20)Reed, R. G.; Gates, T.; Peters, T., Jr. Anal. Biochem. 1976,69, 361. (21)Allenmark, 5.;Bomgren, B. J. Chromatogr. 1982,237,473. (22) Allenmark, S.; Bomgren, B. J. Chromatogr. 1983,264, 63.

0003-2700/92/03644024$03.00/0 0 1992 American Chemlcal Soclety

9NALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1002

SO28

0.0070.006-

-1 1 8

0.005

0.004-

3

0.003

Q

I Ill

1

0.002 0 001

u

10

15

20

Time (min.)

Flguro 2. Electropherogram obtained for LV using affinity EKC on a bare capillary. Condltlons: caplllary dimensions, 75-pm 1.d. X 360pm 0.d.; electric fleld strength, 325 V/cm; pH 7.0; 20 mM phosphate; BSA concentration, 1 mg/mL; detection wavelength, 280 nm.

EXPERIMENTAL SECTION Reagents and Materials. Calcium leucovorin was obtained from Lederle Laboratories,American Cyanamid Co. (Pearl River, NY). Bovine serum albumin (BSA) and sodium dodecyl sulfate (SDS)were obtained from Sigma Chemical Co. (St.Louis, MO). Sodium hydroxide, methylene chloride, and reagent grade methanol were purchased from Fisher Scientific (Fair Lawn, NJ). Reagent grade potassium phosphate was purchased from J. T. Baker Chemical Co. (Phillipsburg,NJ). The poly(ethy1eneglycol) (PEG) 8M-10was supplied by Innophase Corp. (Portland, CT). Apparatus. The EKC experimentswere performed infusedsilica capillaries of 75-pm i.d. X 360-pm 0.d. (Polymicro Technologies, Phoenix, AZ). Typical lengths were 95 cm. A spectra phoresisModel lo00 instrument (SpectraPhysics Corp.,Fremont, CA) was used for portions of the work. Other portions were performed on a laboratory-constructedinstrument using a Model PS/MK30P02.5 30-kV power supply (Glassman High Voltage, Whitehouse Station, NJ) as the voltage source. The detector was a Spectra Physics Model 100and was connected to a Spectra Physics 4400 integrator. A Varian Model 3700gas chromatograph (Varian Instrument Group, Sunnyvale, CA) was used in the coating of the capillaries. Procedures. The capillaries were coated by the following procedures: (1)the capillary was pretreated with 0.1 M NaOH at 80 "C for 4 h, (2) the capillary was rinsed with HzO and then methanol and dried at 130 O C for 6 h, (3) a solution of PEG was physically pushed through the capillary, and (4) the capillary was heated at 225 "C for 24 h. The external coating on the capillary was dissolved using hot H2S04to prepare a window in the desired position on the capillary. New capillaries were conditioned with the running buffer for at least 6 h before use. The capillary was cleaned frequently during runs by washing with methanol, 15 mM SDS, and methanol, in that order. The detection wavelength was 280 nm where the difference between LV absorption and BSA absorption is large. Electroinjection was used for the qualitative work while hydrodynamic injection was used for the rest of the study.

RESULTS AND DISCUSSION Separations Using Bare Fused Capillaries. It is possible to achieve an enantiomeric separation of LV on a bare silica capillary using a phosphate buffer (pH 7.0) that contains BSA at a concentration of 1 mg/mL, as shown in Figure 2. The same experiment in the absence of BSA (all other conditions being identical) yields only a single peak with a migration time of 12.5 min, indicating that in the presence of BSA both isomers of LV interact with BSA to some extent and the separation is based on the differential interaction. Three pK, values have been reported for LV,

Flgurs 3. Electropherogram shown for LV using bioafflnlty EKC on a PE&coated caplllary. Note the peak reversal In comparison wlth the bare capillary. Conditions: capillary dlmenslons, 75-pm 1.d. X 360pm o.d., modifled by 20% PEG 8M-10; electrlc tleld strength, 285 V/cm; pH 7.2; 20 mM phosphate; BSA concentration, 1 mg/mL; detection wavelength, 280 nm.

3.1,4.8, and 10.4, as determined by electrometric titration.23 As a result, LV is anionic at a pH value of 7 and has an electrophoretic mobility (pep)in the direction of the anode in the presence of an electric field (E). The BSA molecule consists of a single polypeptide chain made up of 582 amino acid residues and has an isoelectric point of 4.7." At a pH value of 7 BSA is also anionic and has an pepin the direction of the anode in the presence of an E. The magnitude of pep for LV is larger than that for BSA, as determined experimentally by measuring the migration time of each molecule, and therefore the "free" LV moves with a greater pepthan the LV-BSA-bound complex. Injection is made at the anode, and the (6R)-isomer of LV has a greater affinity for BSA than the (6S)-isomer2and therefore has a greater net velocity in the direction of the cathode, since the electroosmotic velocity (reo) of the aqueous phase is much greater than the electrophoretic velocities in the opposite direction. An important consideration in performing this EKC method involvea the problem of protein-wall adsorption. This phenomena could conceivably lead to changes in the pbo,as well as the pep, of LV by acting as a secondary Separation mechanism where the LV isomera are retained by the adsorbed BSA. Migration time reproducibility for the LV isomers on bare silica capillaries averaged 4.6% RSD over the course of 10injections. Migration times increased as a capillary became aged. A more obvious effect of protein-wall adsorption involves the stability of the capillary itself (measured by the number of runs the capillary could endure). Most underivatized capillaries lasted for only 10-15 runs and were replaced after that. A key advantage of the present method over affinity chromatographic methods is that the protein (or other agent) is in free solution and, if desired, near physiological conditions. Thus, extremes of pH or separate buffer additives are undesirable. A number of strategies have been developed to prevent protein-wall adsorption, including covalent attachment of specific coatings to the capillary wall and in situ methods that achieve the same effect. We chose permanent wall modification using poly(ethy1ene glyco1).26 Derivatized Capillaries. Figure 3 represents the enantiomeric separation of LV with over 200 OOO theoretical plates on a capillary coated with a 20% solution of PEG. It has been shown previously that capillaries derivatized by PEG minimize BSA-wall adsorption.26 Resolution is obtained in (23)Flynn, E.H.;Bond, T. J.; Bard-, T. J.; Shive, W. J.Am. Chem.

SOC.1951; 73, 1979.

(24)Petera,T., Jr. In Advances i n h o t e i n Chemistry;A n f i n , C . B., Edsall,J. T., Richards, F. M.,. Eds.; . Academic Press: New York, 1985;

voi. 37,pp i6i-245. (25)Hjerten, S. J. Chromatogr. 1985, 347, 191. (26) Wang,T.; Hartwick, R. A. J. Chromatogr. 1992,594, 325.

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992

hl

/

.r

1

'

6

Fbwe 4. Electropherogram showing 1.6 X g of LV injected. The conditions are the same as those of Flgure 3 except the electrlc Held strength is 225 V/cm and the LV concentration is -0.1 mg/mL. The MLOD is 5 X

g.

20r' 6.8

7

6.9

7.1

7.2 PH

7.3

7.4

7.5

;6

Flguro 8. Effect of pH on efflclency for the (SS)-isomer with varlous run buffers (differlng by BSA concentration). Condltlons are the same as those in Figure 5.

j/mL BSA 1 ...

I

2.5

s

9

21 2 mg/mL BSA

1.5:. 174 6.6

6.9

7

7.1

7.2

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Flgure 5. Influence of pH on migration time for the (BS).lsomer of LV with various run buffers(differing by BSA concentration). Conditions: capillary dimensions, 75-pm i.d. X 360-pm o.d., modlfied by 20% PEG 8M-10; electric field strength, 250 V/cm.

only 13 min, an improvement over the uncoated capillary. The p, is much smaller than the corresponding pep values, resulting in injection at the cathode. Since the (6R)-isomer has the greater affinity for BSA, it has a smaller net velocity in the direction of the anode and elutes after the (6S)-isomer (a peak reversal in comparison to the uncoated capillary). Migration time reproducibility for the LV isomers exhibited a 0.59% RSD (short term, over -6 h), and the capillary stability increased, allowing for more than 100 runs before the capillary was replaced. The mass limit of detection g corresponding to a (MLOD) was found to be 5.0 X M for concentration limit of detection (CLOD) of 3.0 X a 5-nL injection volume. An electropherogram representing 1.6 X lo4 g injected is shown in Figure 4. Optimization of Electrolyte Conditions. The effect of changes in the composition of the run buffer on the chromatographic behavior and hence the binding properties of the BSA was investigated. This study was carried out by measuring chromatographic behavior for various BSA concentrations and pH values using a 20 mM phosphate buffer and a PEG-coated capillary. The effect of pH on migration time for various run buffers (different BSA concentrations) at a field strength of 250 V/cm is shown in Figure 5. The reason for migration time variation is not clearly understood, but it may be due to changes in the pm and the pepof the BSA or changes in the binding characteristics of BSA or a combination of both. These plots suggest an optimum pH value of 7.2 for the 1and 2 mg/mL ([BSA])solutions over the range studied. Electrolyte pH values less than 6.8 produced poor reproducibility, while pH values greater than 7.6 showed poor resolution. Figure 6 shows the effect of pH on efficiency for the various run buffers at a field strength of 250 V/cm.

1II

0.5-

1 mg/mL BSA

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6.8

6'9

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Figwo 7. Effect of pH on resolution for the LV isomers with various run buffers (differing by BSA concentration). Conditions are the same as in Flgure 5.

A maximum occurs at a pH value of 7.2 for the three different BSA concentrations. Shown in Figure 7 are plots of resolution vs pH at a field strength of 250 V/cm for the various run buffers. There is a general trend for resolution to decrease as pH increases, and this phenomena is primarily due to the combination of migration times and efficiencies. Figures 5 and 6 suggest an improvement in chromatographicbehavior for lower concentrations of BSA. Furthermore, studies showed that lower BSA concentrations improved migration time reproducibility and column stability, at the expense of sample capacity. Thermodynamic Measurements. The use of HPLC to obtain accurate and realistic thermodynamic measurements for the interaction of the LV isomers and BSA is hindered by the need to modify the BSA by covalently bonding it to the stationary phase. In this system the BSAis in free solution and a general equation for the calculation of the free energy (AC)of interaction between the LV isomers and BSA can be derived. The observed migration time (tab) of a species can be determined by the distance it travels from injection to detection (1) and ita net velocity (v,.J as shown in eq 1. to,

=kiet

(1)

The vnet for LV is the sum of three velocity components, and the tObfor LV is given by eq 2 where 1 is the distance from the point of injection to the window on the capillary, R is the fraction of time the LV spends in the run buffer, VBSA and

ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, MCEMBER 1, 1992

are the electrophoretic velocity componenta for BSA and LV, respectively,and vo is the electroosmoticvelocity. The velocity of any component is given by ita electrophoretic mobility, pep,times the field strength, E, or Y = pe&. When the sign of the velocity terms is determined, an arbitrary frame of reference is used, where movement from injection to detection is given a positive sign. If the LV does not interact with BSA then R = 1, the UBSA term drops out, and the vnet for LV is the sum of the and YO, as in CE. If the LV interacts with BSA 100% of the time, R = 0 and drops out and vnet = m~+ YO for LV. Rearrangement of eq 2 gives R in terms of 1, to,, and the velocity components of the species involved. 1 --

R=

to,

Yo - VBSA

vL - %SA

(3)

Equation 3 is an important equation is that the value R for LV can be determined by five experimental parameters, four of which are constant within a given buffer system: 1 is measured from the capillary, to, is the migration time of LV, vo is determined by injecting a compound that is neutral and does not interact with BSA (acetophenone in this case), VBSA is determined by injecting a concentrated plug of BSA, and a is determined by injecting LV in the absence of BSA (all other conditions the same). The value R can also be related to the capacity factor (k'), aa described in eq 4.21 (4) Combining eqs 3 and 4, the equation k' = K*, where K is the equilibrium constant and @ is the phase ratio, and the equation AG = -R,T In K, where R, is the gas constant and T is the internal temperature of the capillary, the following equation can be used to calculate the free energy of interaction between the LV isomers and BSA

(5) Equation 5 was used to estimate the free energy of interaction between the LV isomers and BSA at pH 7.2 and 1mg/mL BSA concentration. This equation can be expressed in terms of the binding constant (K') using the equation K' = (K - 1)V, where V is the partial specific volume.28 The internal temperature was calculated by the method of Burgi et al.%to be 310 K at 800 mW of power. The total volume of the BSA in the system was used to estimate the phase ratio. Using the molecular dimensions 42 X 141A for BSAW corresponding to a phase ratio of 7.1 X (assuming all of the volume of the BSA molecule is utilized), the free energy resulta obtained for the (6R)- and (6S)-isomerswere -2665 and -2744 cal/mol, respectively. It is of c o m e not realistic to assume that the entire BSA molecule participates in the retention of the LV isomers. Overestimation of the phase ratio would have the effect of underestimating AG when eq 5 is used. For example, if the LV isomers penetrated the BSA molecule approximately a monolayer thick correspond(27) Giddings,J. C.DynamicsofChromotography;Dekker:NewYork, 1965; Chapter 2. (28) Berezin, I. V.; Martinek, K.;Yatsimirskii, A.K.R u m Chem. Rev. (Engl. Transl.)1973,42, 781. (29) Burgi, D. S.; Salomon, K.; Chien, R.-L. J.Liq. Chromutogr. 1991, 14, 847. (30)Gilpin, R.K.;Ehtesham,S. E.; Gregory, R. B. Anal. Chem. 1991, 63, 2825.

SO27

ing to a phase ratio of 1.9 X lC3,the (65')- and (6R)-isomers would have free energy values of -3369 and -3448 cal/mol, respectively. Knowledge of the interaction mechanism would appear to be a prerequisite to accurate absolute energy determinations by this technique. However, relative free energy difference between the LV isomers is given by eq 6. The subscripta 1

1

and 2 correspond to the (6s)- and (6R)-isomers of LV, respectively. Since the phase ratio term cancels, this eliminates the need to understand the retention mechanism. At conditions pH 7.2 and 1mg/mL BSA concentration,the A(AG) for the LV isomers was found to be 80 cal/mol.

CONCLUSION The resulta show that by using a biological species with high selectivity (BSA in this case) as a buffer modifier, chiral separations are feasible. This EKC method offers an advantage over some of the other chromatographic methods mentioned earlier in ita simplicity: The biological species is simply added to the run buffer, without the need for timeconsuming covalent derivatization. In addition, potentially any soluble protein, DNA, or other species can be used an the selective pseudophase, either by itaelf or in conjunctionwith other carrier molecules, thus making this technique very general. Recently, binding constants have been determined for compounds interacting with vancomycin using a method similar to ours.31 Protein/DNA interactions, drug/protein interactions, and similar measurements can be made without the need to covalently link the substrates. Errors due to deformation of the substrates upon binding to a surface are minimized. Another advantage of this method is the determination of binding interactions using only nanogram amounts of ligand. These data are typically obtained by several methods,all of which utilize quantitation of free ligand. An important drawback to these methods involves the local removal of free ligand from the presence of bound ligand which causes a shift in the equilibrium between the macromolecule and ligand and can lead to dissociation.32 Limitations of this EKC method include the need to minimize protein-wall adsorption. This is accomplished by coating the wall permanently with PEG or other polymers. The development of new coatings that are reproducible and exhibit little or no protein-wall adsorption remains a general problem for CE technology, although much progress is being made in the area The determination of thermodynamicdata for substrate binding interactions is limited by the estimation of the phase ratio, although relative data are feasible. There is also a requirement that the ligand, the run buffer modifier, or both be charged for acceptable analysis time and have optical windows with minimal overlap. Nevertheless, we believe that CE offers a unique tool by which to study the effect of competitive binding of other drugs with circulating serum proteins and to examine potential pharmacological activity in new molecules33by examining the interaction of such agents with proteins, DNA, or specific binding sites carried in the electrolyte. ~

(31)Chu, Y.;Whitesides, G. M. J. Org. Chem. 1992,57,3624. (32)Kurz, H.;Trunk, H.; Weitz, B. Arzneim.-Forsch. 1977,27,1373. (33)Kaliszan, R. Anal. Chem. 1992,64,61BA.

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ANALYTICAL CHEMISTRY, VOL. 64, NO. 23, DECEMBER 1, 1992

ACKNOWLEDGMENT This research was supported by Spectra Physics Analytical Instruments and by the New York Science Foundation through the Center for Biotechnology of SUNY at Stony Brook. We thank Dr. Robert Gaydosh and Richard Heiserodt (Lederle Laboratories) for their assistance and Professor

Roman Kaliszan of the Medical Academy, Gdansk, Poland, for his helpful discussions of this research.

RECE~VED for review June 8, 1992. Accepted September 1, 1992.