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trans isomerization
Journal of Chromatography A, 1157 (2007) 101–107
Counter-current chromatographic estimation of hydrophobicity of Z-(cis) and E-(trans) enalapril and kinetics of cis/trans isomerization Atsushi Shoji a , Akio Yanagida a , Heisaburo Shindo a , Yoichiro Ito b , Yoichi Shibusawa a,∗ a
Division of Structural Biology and Analytical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan b Center for Biochemistry and Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8014, USA Received 3 October 2006; received in revised form 13 April 2007; accepted 13 April 2007 Available online 19 April 2007
Abstract The kinetics of Z-(cis)/E-(trans) isomerization of enalapril was investigated by reversed phase high-performance liquid chromatography (RPHPLC) using a monolith ODS column under a series of different temperature and pH conditions. At a neutral pH 7, the rate (kobs ) of Z-(cis)/E-(trans) isomerization of enalapril at 4 ◦ C (9.4 × 10−3 min−1 ) is much lower than at 23 ◦ C (1.8 × 10−1 min−1 ), while the fractional concentration of Z-(cis) isomer is always higher than that of E-(trans) isomer in the pH range 2–7. The fractional concentration of the E-(trans) isomer becomes a maximum (about 40%) in the pH range 3–6, where enalapril exists as a zwitterion. The hydrophobicity (log PO/W ) of both isomers was estimated by highspeed counter-current chromatography (HSCCC). Normal phase HSCCC separation using a tert-butyl methyl ether–acetonitrile–20 mM potassium phosphate buffer (pH 5) two-phase solvent system (2:2:3, v/v/v) at 4 ◦ C was effective in partially separating the isomers, and the partition coefficient (K) of each isomer was directly calculated from the retention volume (VR ). The log PO/W values of Z-(cis) and E-(trans) isomers were −0.46 and −0.65, respectively. © 2007 Elsevier B.V. All rights reserved. Keywords: Enalapril; cis/trans Isomerization; High-speed counter-current chromatography; log PO/W
1. Introduction Angiotensin I-converting enzyme (ACE) inhibitors that block the production of vasoconstricting Angiotensin II are generally used as effective drugs in the treatment of hypertension and heart failure [1,2]. The active parts of ACE inhibitors are peptide derivatives containing C-terminal proline residues, and the compounds populate in both cis/trans isomers due to the partial double bond character of the tertiary amide bond in the proline containing peptide bond [3,4]. Among such ACE inhibitors, enalapril (and/or its active metabolite enalaprilat) is one of the most extensively investigated compounds in terms of its cis/trans interconversion and structural characteristics. In many previous reports (e.g. [5–7]), the cis/trans nomenclature of the isomers used the protein convention which is based on the polypeptide chain entering and exiting opposite corners of the rectangle
∗
Corresponding author. Tel.: +81 42 676 4544; fax: +81 42 676 4542. E-mail address: [email protected] (Y. Shibusawa).
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.04.027
formed by the planar peptide bond atoms. However, Ledger and Stellwagen [8] recently proposed that the rotational isomers containing a proline tertiary amide bond should be described as either Z-(cis) or E-(trans) according to the Cahn–Ingold–Prelog (CIP) rules as encoded in IUPAC. In the present paper, the molecular configurations of the enalapril isomers are described as Z-(cis)/E-(trans) forms according to their proposal, as illustrated in Fig. 1. A recent X-ray crystallographic study on the ACE-inhibitor complex confirmed that the Z-(cis) isomer bonds to the catalytic Zn2+ ion of ACE [9,10], suggesting that the physiologically active form of enalapril is also of Z-(cis) configuration. Furthermore, other structural studies using X-ray analyses proved that enalapril in the solid state, such as a crystal or powder, usually exists as the Z-(cis) form [11,12]. On the other hand, enalapril in solution forms Z-(cis)/E-(trans) mixtures by undergoing reversible interconversion. The Z-(cis)/E-(trans) isomeric composition of enalapril in polar solution has been investigated by NMR [5,13,14], which indicated that the Z-(cis) isomer is predominant in aqueous solution. Also, the effects of pH,
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Fig. 1. Z-(cis)/E-(trans) isomerization of enalapril based on rotation around the alanyl–proline peptide bond. The broken arrow in the structure of Z-(cis) isomer indicates an intramolecular hydrogen bonding between the proline carboxyl group and the carbonyl group in the Z-(cis) isomer.
temperature and ionic strength on the isomeric composition of enalapril have been investigated by reversed phase (RP) HPLC and capillary electrophoresis (CE) [6–8,15–18]. Stellwagen and Ledger [7] estimated the dissociation constants of the enalapril carboxyl and amine groups of both isomers based on their elution behavior under various buffer pH conditions. They reported that the Z-(cis) isomer composition changed from 68% in cationic form (pH < 2) to 50% in isoelectric form (pH ∼4.5) and to 60% in the anionic form (pH > 7) [7,8]. Under the RP-HPLC and CE conditions, the retention behavior of enalapril Z-(cis)/E-(trans) isomers is also significantly affected by temperature, since the exchange rate of the Z-(cis)/E-(trans) isomer increases with increasing temperature. Actually, both HPLC [6,8,15–17] and CE studies [7,8,18] revealed that enalapril appeared as a nonseparable single peak at elevated temperature (80 ◦ C in HPLC or 60 ◦ C in CE) while its Z-(cis)/E-(trans) components were clearly separated with base line resolution at low temperature of 6 ◦ C in HPLC or 15 ◦ C in CE. However, there have been longstanding arguments on peak assignments of the Z-(cis)/E-(trans) enalapril (and/or enalaprilat) isomers in the separation profiles of HPLC or CE. Kocijan et al. [6] and Bouabdallah et al. [17] predicted that the E-(trans) isomer (which they termed cis) was more hydrophobic and therefore was retained longer than the Z-(cis) isomer (which they termed trans) in RP-HPLC. Their conclusion was based on the statement that E-(trans) alanylproline has a larger hydrophobic surface area than its Z-(cis) isomer. For the same reason, Trapp et al. [18] also predicted that the elution order of enalaprilat isomers in their CE experiment should be trans before cis, whereas the chemical structures illustrated as trans and cis in Trapp’s paper are those of E-(trans) and Z-(cis), respectively. Thus, the elution order of enalaprilat isomers in the CE experiment was first E(trans) isomer (which they termed trans) and next Z-(cis) isomer (which they termed cis). Furthermore, Ledger and Stellwagen [8] predicted that the slower component in the HPLC profile of enalapril is the Z-(cis) isomer. The elution order of both isomers is same as that of above Trapp’s CE experiment, but their prediction was based on the crystallographic structure of the Z(cis) isomer of enalapril maleate [11]. To solve this confusion, a greater understanding of the hydrophobicity of the Z-(cis)/E(trans) isomers is necessary. Because ACE inhibitors including enalapril are transported into intestinal epithelial cells through the H+ /oligopeptide transporter [19], accurate estimation of the hydrophobicity of the Z-(cis)/E-(trans) isomers and their iso-
meric composition in solution would be useful for understanding their high affinity with the transporter. In general, the hydrophobicity of drugs is often expressed as the logarithm of the 1-octanol–water partition coefficient or log PO/W [20]. The conventional procedure for determining log PO/W is a batch-shaking method or a shake-flask method using a 1-octanol–water two-phase solvent system. This traditional method is tedious and time consuming, and in some cases, such as for the cis/trans isomers of enalapril, it is difficult to measure log PO/W values because of its fast cis/trans interconversion. RP-HPLC or CE has recently been used to estimate log PO/W in preference to the shake-flask method [21–25]. In the chromatographic method, the retention factor (k) of a solute is calculated from its retention time. Since the log k value is linearly correlated with the log PO/W value, correlations between log k and log PO/W can be obtained for different standards. Hence, these chromatographic methods are considered to be indirect methods for the determination of log PO/W . On the other hand, high-speed counter-current chromatography (HSCCC) can directly determine the partition coefficients of solutes from their retention volume because both mobile and stationary phases are in liquid form [26–28]. In our previous HSCCC studies using a 1-octanol–50 mM potassium phosphate (1:1, v/v) two-phase system [28], log PO/W values ranging from −1.35 to +3.60 were readily determined for several aromatic compounds within 21 min. The present paper describes the separation of Z-(cis)/E-(trans) enalapril isomers and estimation of their log PO/W values by HSCCC using two-phase systems. In order to optimize temperature and pH conditions in the HSCCC experiments, the kinetics of cis/trans isomerization of enalapril in aqueous solution was also investigated by RP-HPLC. 2. Experimental 2.1. Reagents Enalapril maleate, (S)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyl]-l-alanyl]-l-proline maleate, was purchased from Sigma (St. Louis, MO, USA). Organic solvents including tert-butyl methyl ether (BME), acetonitrile (ACN) and 1-octanol (Oct) were purchased from Kanto (Tokyo, Japan). Standard compounds for log PO/W determination, theobromine, caffeine and benzoic acid, were purchased from Wako (Osaka, Japan); cresol, 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid were from Tokyo Kasei (Tokyo, Japan); and benzene, phenol, anthracene and potassium nitrate were from Kanto. Other chemicals were of reagent grade. 2.2. Equipment The HPLC system (Hitachi, Tokyo, Japan) consisted of a type L-7100 pump, a Rheodyne 7166 sample injector, a type L-7420 UV–vis detector and a type D-2500 chromatointegrator. For RP-HPLC analysis of enalapril, a Chromolith Performance RP-18e column (Merck, Tokyo, Japan) was connected to the Hitachi HPLC system. For HSCCC experiments,
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a type-J coil-planet centrifuge (J-CPC; Renesas Eastern Japan Semiconductor, Tokyo, Japan) was connected to the Hitachi HPLC instruments. The J-CPC holds a pair of column holders symmetrically on the rotary frame at a distance of 5 cm from the central axis of the centrifuge. Each multilayer coiled column was fabricated by winding a single piece of polytetrafluoroethylene (PTFE) tubing (50 m × 1.0 mm I.D., total capacity about 40 ml) directly onto the holder hub making eight coiled layers (26 turns on each layer) between a pair of flanges (β = 0.5–0.6). The revolution speed of the apparatus was regulated at 1400 rpm with a speed controller. The separation coil rotates around its own axis as it synchronously revolves around a central axis, producing an efficient mixing of the two phases while retaining a sufficient amount of the stationary phase in the coiled column.
mobile phase was eluted through the column in the head to tail (H–T) direction at various flow rates of 1 ml/min (for theobromine and caffeine), 2 ml/min (for 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid and benzoic acid) and 4 ml/min (for benzene, phenol and cresol). After hydrodynamic equilibrium between the two phases was established in the rotating column, sample solution containing 1 mg of each standard dissolved in 50 l of both phases was injected and eluted with the aqueous lower mobile phase. Under these HSCCC conditions using the Oct–PB system in a reversed phase partition mode, the PO/W values can be directly obtained from their VR values according to the following equation [26,27]:
2.3. Reversed phase chromatography
where VR is the retention volume of the solute, and Vc is the PTFE column volume from the injector to the detector. Under the above HSCCC conditions, the mobile phase volume Vm is equal to the elution volume of unretained polar potassium nitrate, and the partition coefficient K in the equation can be expressed as the PO/W . HSCCC for the BME–ACN–PB system was performed in a reversed phase mode at 4 ◦ C. The HSCCC procedure and standard sample solutions were identical to those described for the Oct–PB two-phase system except that the lower aqueous ACN–PB phase was used as the mobile phase. The reversed phase partition coefficient (KRP ) of each standard compound was calculated from the VR value according to Eq. (1). Under the above HSCCC conditions, the K in the equation can be expressed as the KRP . HSCCC separation of the Z-(cis)/E-(trans)-enalapril isomers was carried out with the BME–ACN–PB two-phase system in a normal phase partition mode (mobile phase: organic upper phase; stationary phase: aqueous lower phase) at 4 ◦ C. First, the PTFE coiled column was first entirely filled with the lower aqueous stationary phase. Then, the column was rotated at 1400 rpm while the organic upper mobile phase was eluted through the column in a tail to head direction at a flow rate of 2 ml/min. After hydrodynamic equilibrium between the two phases was reached in the rotating column, the sample solution containing enalapril maleate was injected and eluted with the organic upper mobile phase. The eluent was monitored by a UV detector at 210 nm. Before the HSCCC injection, 1 mg of enalapril maleate was dissolved in 50 l of lower phase and incubated at 4 ◦ C. After the incubation, 50 l of upper phase was added to the lower phase solution containing enalapril, and the mixture solution (total 100 l) was used as the sample solution for above HSCCC separation. The normal phase partition coefficient (KNP ) of each enalapril isomer was calculated from the VR value according to Eq. (1). Under the above normal phase HSCCC conditions, the mobile phase volume Vm is equal to the elution volume of unretained hydrophobic anthracene, and the K in the equation can be expressed as KNP . In addition, the reversed phase partition coefficient (KRP ) of each isomer in the same solvent system was calculated from the relation, KNP = 1/KRP .
Separation of Z-(cis)/E-(trans) enalapril isomers was performed by HPLC using a Chromolith Performance RP-18e column (100 mm × 4.6 mm I.D.) at 23 ◦ C (in an air-conditioned laboratory) and 4 ◦ C (in a cold room). An acetonitrile–20 mM potassium phosphate buffer (pH 7) mixture (18:82, v/v) was used for the mobile phase at a flow rate of 4 ml/min. Enalapril maleate was dissolved in the 20 mM potassium phosphate buffer (0.5 mg/ml), and 5 l of this solution was injected and isocratically eluted from the column. The effluent was monitored by a UV detector at 210 nm. Before the HPLC injection, the enalapril solution was incubated at each temperature. The incubation time was 0–40 min at 23 ◦ C and was 0–1500 min at 4 ◦ C. After HPLC analysis, a concentration of the Z-(cis) isomer ([Z]) in the enalapril solution after incubation with a certain time was calculated from the peak area of the Z-(cis) isomer on the chromatogram, and the percentage of fractional concentration of the Z-(cis) isomer was calculated from the following equation: ([Z]/[Z]0 ) × 100, where [Z]0 is the concentration of the Z isomer in the enalapril solution without incubation at each temperature. 2.4. Counter-current chromatography Two two-phase solvent systems were used for HSCCC analyses of enalapril and the organic standards: (1) a BME–ACN–20 mM potassium phosphate buffer (PB; pH 5) two-phase system (2:2:3, v/v/v) and (2) an Oct–50 mM PB (pH 5) two-phase system (1:1, v/v). To establish an appropriate correlation between the reversed phase partition coefficient (KRP ) obtained in the BME-ACNPB system and the PO/W obtained in the Oct-PB system, the partition coefficients of eight different standard compounds, as listed in Section 2.1, were measured using the following HSCCC procedure. HSCCC separation was carried out using the Oct–PB twophase system in a reversed phase partition mode (stationary phase: upper octanol phase; mobile phase: aqueous lower phase) at 4 ◦ C. The PTFE coiled column previously filled with the upper stationary phase was rotated at 1400 rpm while the lower
K=
Cs VR − V m = Cm Vc − V m
(1)
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3. Results and discussion 3.1. Kinetics of Z-(cis)/E-(trans) isomerization of enalapril For the kinetic study of the isomerization of Z-(cis)/E-(trans) enalapril in aqueous solution at neutral pH, RP-HPLC of the two isomers was performed using a PB–acetonitrile mobile phase at 23 ◦ C and 4 ◦ C. Enalapril maleate was dissolved in 20 mM PB (pH 7) and immediately analyzed by HPLC. Further, aliquots of solution were incubated for 12 min at 23 ◦ C and for 600 min at 4 ◦ C before analysis. The separation profiles of cis/trans enalapril are shown in Fig. 2. We confirmed that maleate (MA) was eluted at the void volume of the ODS column in all chromatograms. We also confirmed that the elution profile of enalapril was significantly different before and after incubation. When the enalapril solution was analyzed without incubation, enalapril was eluted as a single peak (peak 2) at about 2 min. This peak 2 was assigned to the Z-(cis) isomer as indicated in previous studies [11,12]. When the solution was analyzed after incubation (for 12 min at 23 ◦ C or for 600 min at 4 ◦ C), the enalapril was eluted as two peaks (1 and 2) indicating Z-(cis)/E-(trans) equilibration during the incubation. The new peak 1 at about 0.8 min was assigned to the E-(trans) isomer. The elution order of the two peaks indicated that the later eluting Z-(cis) isomer was more hydrophobic than the earlier eluting E-(trans) isomer. A comparison of the separation profile obtained at 23 ◦ C (Fig. 2A) and 4 ◦ C (Fig. 2B) showed a plateau between the two peaks due to on-column interconversion of the Z-(cis)/E-
Fig. 3. Change over time in fractional concentration (%) of the Z-(cis) isomer in enalapril maleate solution at 23 ◦ C or 4 ◦ C. Enalapril maleate was dissolved in 20 mM potassium phosphate buffer (pH 7), and the solution was incubated for 30 min maximum at 23 ◦ C or 1500 min maximum at 4 ◦ C before HPLC analysis. The fractional concentration (%) of the Z-(cis) isomer after each incubation phase was calculated from its peak area on the HPLC chromatogram. The inset plot at each temperature shows relationship between ln([Z] − [Z]eq ) and incubation time (t). Regression equation at 23 ◦ C: y = −0.1837 (±0.0201)x + 3.3864 (±0.2187) (n = 7, r2 = 0.9435). Regression equation at 4 ◦ C: y = −0.0094 (±0.0003)x + 3.2955 (±0.0492) (n = 13, r2 = 0.9826).
(trans) isomers. Many previous researchers have reported that chromatographic conditions, such as pH, temperature and flow rate, significantly affected the appearance of the plateau and the shape of the separated peaks [6–8,17,18]. Although the HPLC separation shown in Fig. 2A was performed within 2 min and the analysis time was shortest among all LC and/or CE separation reports of enalapril, the appearance of the plateau was not completely suppressed at 23 ◦ C. On the basis of the above results, we next examined the kinetics of Z-(cis)/E-(trans) interconversion of enalapril over various incubation times under the same RP-HPLC and pH conditions. Fig. 3 shows the change over time in the fractional concentration (%) of the Z-(cis) isomer in enalapril maleate solution at 23 and 4 ◦ C. With increasing incubation time, the Z-(cis) isomer concentration decays exponentially before reaching a constant value. Since the Z-(cis)/E-(trans) isomerization of enalapril is the first-order reversible reaction as shown in Fig. 1, the isomerization rate at a certain time is expressed by following equation (Eq. (2)), −d[E] d[Z] = = −k1 [Z] + k−1 [E] dt dt
Fig. 2. Separation profiles of Z-(cis)/E-(trans) enalapril in reversed phase HPLC using monolith ODS column at 23 and 4 ◦ C (A and B), respectively. Enalapril maleate was dissolved in 20 mM potassium phosphate buffer (pH 7) and analyzed immediately by RP-HPLC. Enalapril maleate solution was also incubated for 12 min at 23 ◦ C or 600 min at 4 ◦ C before analysis. RP-HPLC was performed using a Chromolith Performance RP-18e column (100 mm × 4.6 mm I.D., MERCK) with a mobile phase composed of 20 mM potassium phosphate buffer (pH 7) and acetonitrile (82:18, v/v) at a flow rate of 4 ml/min. UV absorbance of the effluent was monitored at 210 nm. MA: maleate. Peaks: (1) E-(trans) enalapril and (2) Z-(cis) enalapril.
(2)
where k1 is the first-order rate constant of isomerization from the Z-(cis) to E-(trans) isomer, k−1 that of the reverse reaction, [Z] the concentration of the Z-(cis) isomer and [E] is that of the E-(trans) isomer. Since enalapril exists in the Z-(cis) form at the initial time t = 0, [E] = [Z]0 − [Z], where [Z]0 is the fraction of [Z] at t = 0. Therefore, Eq. (2) can be rewritten as follows: d[Z] = −k1 [Z] + k−1 ([Z]0 − [Z]) dt
(3)
Integration of Eq. (3) gives ln([Z] − [Z]eq ) = ln([Z]0 − [Z]eq ) − (k1 + k−1 )t = ln([Z]0 − [Z]eq ) − kobs t
(4)
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Table 1 Kinetic parameters of isomerization of Z-(cis)/E-(trans) enalapril in 20 mM phosphate buffer (pH 7) at 23 and 4 ◦ C Temperature (◦ C)
K (k1 /k−1 )
k1 (min−1 ) (Z → E)
k−1 (min−1 ) (E → Z)
kobs (min−1 )a (k1 + k−1 )
23 4
3.5 × 10−1 4.6 × 10−1
4.8 × 10−2 3.0 × 10−3
1.4 × 10−1 6.4 × 10−3
1.8 × 10−1 9.4 × 10−3
a
kobs was calculated as the slope of a linear least-square fitting of relationship between ln([Z] − [Z]eq ) and t.
where [Z]eq is the fraction of [Z] at equilibrium, and kobs = k1 + k−1 . Using Eq. (4), kobs of the Z-(cis)/E-(trans) isomerization of enalapril can be calculated from the slope of linear least-square fitting of ln([Z] − [Z]eq ) as a function of incubation time (t). As listed in Table 1, along with the data for the regression equations and statistics, the kobs values for the isomerization were found to be 1.8 × 10−1 min−1 at 23 ◦ C and 9.4 × 10−3 min−1 at 4 ◦ C, respectively. Since the forward and backward reaction rates are equal at equilibrium, we have the following equation (Eq. (5)). k1 [Z]eq = k−1 [E]eq
(5)
The equilibrium constant K (K = k1 /k−1 ) is given by the following equation (Eq. (6)). K=
[E]eq k1 = k−1 [Z]eq
(6)
Using Eq. (6), the equilibrium constants K were calculated to be 3.5 × 10−1 ([E]eq /[Z]eq = 26.0/74.0) at 23 ◦ C and 4.6 × 10−1 ([E]eq /[Z]eq = 31.7/68.3) at 4 ◦ C. Furthermore, these K and kobs values yield forward and backward rate constants k1 and k−1 using Eqs. (4) and (6). All the kinetic parameters (K, k1 , k−1 , kobs ) thus obtained for Z-(cis)/E-(trans) isomerization are summarized in Table 1. These kinetics data demonstrate that the backward reaction from E-(trans) to Z-(cis) is faster than the forward reaction from Z-(cis) to E-(trans), indicating that the Z(cis) form is more stable than the E-(trans) form. The stability of the Z-(cis) isomer of enalapril in solution is attributed to hydrogen bonding between the carbonyl group and carboxyl groups in the structure of Z-(cis) isomer (Fig. 1) [5]. Recently, Ledger and Stellwagen [8] reported the kinetic study of the isomerization of enalapril maleate in water (pH 4.2) at 8 ◦ C, and the isomerization was found to occur in a single kinetic phase having a half-time (t1/2 ) of 58 min with 52% in the Z-(cis) isomer at equilibrium. According to the following equation t1/2 = ln 2/kobs , the value of kobs of the isomerization under their conditions is calculated to be 1.2 × 10−2 min−1 . Furthermore, since the percentage of fractional concentration of [Z]eq is 52%, according to the previous Eqs. (5) and (6), the values K, k1 and k−1 are calculated to be 9.2 × 10−1 , 5.8 × 10−3 min−1 and 6.3 × 10−3 min−1 , respectively. These values are close to our kinetics values measured at pH 7 and at 4 ◦ C, listed in Table 1. 3.2. pH effects on isomerization of enalapril Next, we examined changes in the equilibrium ratio of Z-(cis)/E-(trans) enalapril in phosphate buffered solution at various pHs ranging from 2 to 7 at 23 ◦ C. The pH of phosphate buffer
Fig. 4. The change in fractional concentration (%) of Z-(cis) isomer under various pH conditions. Enalapril maleate was dissolved in 20 mM potassium phosphate buffer (pH 2–7), and the solution was incubated for 60 min at 23 ◦ C before HPLC analysis.
in the sample solution and in the mobile phase was adjusted to the same value. The sample solutions were incubated for 30 min at 23 ◦ C before HPLC analysis. Fig. 4 shows the fractional change in the Z-(cis) isomer as a function of pH. The data confirmed that the Z-(cis) isomer is always more stable than the E-(trans) isomer in aqueous buffered solution between pH 2 and 7. The fractional concentration of the E-(trans) isomer was estimated to be about 40% under weakly acidic conditions (pH range from 3 to 6), whereas the fraction was markedly decreased in both acidic (pH 2) and neutral (pH 7) conditions (28% at pH 2 and 32% at pH 7, respectively). Similar results from CE analysis of enalapril have been reported by Stellwagen and Ledger [7,8]. In these reports, the dissociation constants of the carbonyl group in Z-(cis) and E(trans) enalapril, pKZ and pKE , were estimated at 3.1 and 2.6, respectively, and the pKZ and pKE of the amine group were estimated at 5.6 and 5.9 from pH titration curves of CE mobility measurements. These pK values indicate that both enalapril isomers are in a zwitterionic form in the pH range from 3 to 6. In the E-(trans) configuration especially, the two charged groups (i.e. deprotonated carboxyl and protonated amine groups) may attract one another to stabilize the E-(trans) configuration [29,30]. If this speculation is correct, the intramolecular ionic interaction in the E-(trans) configuration may affect the composition ratio of Z-(cis)/E-(trans) enalapril in buffered solution in the pH range from 3 to 6. Stabilization of Z-(cis) at a low pH of 2 may be explained as the result of hydrogen bond formation between the carbonyl and carboxyl groups as shown in Fig. 1.
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3.3. HSCCC separation of Z-(cis)/E-(trans) enalapril As mentioned in Section 3.1, the elution order of the enalapril isomers in RP-HPLC confirmed that the Z-(cis) isomer was more hydrophobic than the E-(cis) isomer. However, a difference in the log PO/W values of the Z-(cis) and E-(trans) isomers is difficult to determine by the traditional shake-flask method because of fast cis/trans intercoversion in solution. As mentioned above, a chromatographic method, such as HSCCC, can achieve separation of cis/trans forms and allows direct evaluation of the partition coefficients of each isomer. We thus tried to separate the Z-(cis)/E(trans) enalapril isomers by HSCCC using either an Oct–PB two-phase system or BME–ACN–PB two-phase system. The HSCCC using the Oct–PB system can be used to readily determine log PO/W value of the solute based on the VR value [28]. On the other hand, the BME–ACN–PB system is the most appropriate two-phase system for HSCCC separation of polar compounds [31–34]. In HSCCC operation, the normal phase partition mode (stationary phase: aqueous lower phase) was selected for efficient retention and separation of enalapril isomers because enalapril in the pH 5 buffer solution exists in a polar zwitterionic form. Before HSCCC separation of the enalapril isomers, we examined the retention of the stationary phase solvent (i.e. aqueous lower phase) in revolving HSCCC column under hydrodynamic two-phase equilibrium at different temperature conditions (4 or 23 ◦ C). In general, a higher retention of the stationary phase leads to higher separation efficiency. In the case of the Oct–PB system, 40% of the stationary phase was retained in the column at 23 ◦ C while none was retained at 4 ◦ C, presumably because of the high viscosity of the octanol phase at low temperature. For the same reason, the retention of the stationary phase in the BME–ACN–PB system was greater at 23 ◦ C than at 4 ◦ C (76.3% at 23 ◦ C; 29.2% at 4 ◦ C). The retention of the stationary phase in the BME–ACN–PB system (76.3%) was higher than that of the Oct–PB system (40.0%) because the BME-rich mobile phase had a much lower viscosity than the Oct-rich mobile phase. We initially tried to separate the enalapril isomers by normal phase HSCCC using the Oct–PB (pH 5) two-phase system at 23 ◦ C. However, the enalapril isomers could not be separated from each other with this two-phase system. Therefore, a BME–ACN–PB (pH 5) two-phase system was used for the HSCCC separation. The chromatograms of enalapril maleate in the normal phase HSCCC are shown in Fig. 5. It can be seen that the Z-(cis)/E-(trans) enalapril isomers were not separated at 23 ◦ C because the rate of Z-(cis)/E-(trans) interconversion of enalapril was too fast (Fig. 3; Table 1). Since the rate of Z-(cis)/E(trans) interconversion is markedly slower at low temperature (Fig. 3), HSCCC was carried out at 4 ◦ C. As shown in Fig. 5B, when the sample solution was injected to the column without incubation, enalapril was eluted as a single peak with a small shoulder at 24.6 min. As in the RP-HPLC results shown in Fig. 2, this single peak at 24.6 min was assigned to the Z-(cis) isomer. On the other hand, when the sample solution was injected into the HSCCC after the incubation for 360 min, enalapril appeared as broad split peaks. The second peak at 28.7 min is assigned to the E-(trans) isomer. The elution order of these peaks in the
Fig. 5. HSCCC chromatograms of Z-(cis) (Z) and E-(trans) (E) enalapril in normal phase partition mode using BME/ACN/PB (2:2:2; 50 mM pH 5 buffer) (2:2:3) two-phase system at 23 and 4 ◦ C (A and B) respectively. Enalapril maleate was dissolved in the lower phase and analyzed immediately by HSCCC. The enalapril maleate solution was also incubated for 60 min at 23 ◦ C or 360 min at 4 ◦ C before analysis. HSCCC was performed using a PTFE multilayer coiled column (50 m × 1.0 mm I.D., 40 ml capacity; operated at 1400 rpm) with a lower aqueous stationary phase and an organic upper mobile phase (flow rate of 2 ml/min) was monitored at 210 nm.
normal phase HSCCC chromatograms indicated that the earlier eluted Z-(cis) isomer was more hydrophobic than the later eluted E-(trans) isomer. 3.4. Evaluation of hydrophobicity of Z-(cis)/E-(trans) enalapril The partition coefficients, KNP and KRP , obtained for the enalapril isomers under the above HSCCC condition are listed in Table 2. The KRP values of the Z-(cis) and E-(trans) isomers were calculated to be 0.52 and 0.37, respectively. Furthermore, eight standard compounds (theobromine, caffeine, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, benzoic acid, benzene, phenol and cresol) were analyzed by reversed phase HSCCC using the Oct–PB and BME–ACN–PB two-phase systems to establish a correlation between PO/W and KRP at 4 ◦ C. As shown in Fig. 6, the log KRP value of each standard was plotted against each log PO/W value. The relationship between log KRP and log PO/W was linear over the range log KRP −0.4 to 1.7 and expressed as the following regression equation: log PO/W = 1.268 log KRP − 0.107
(n = 8; r 2 = 0.982) (7)
Using this equation, we could estimate the log PO/W values of the Z-(cis) and E-(trans) isomers from their KRP values. These are Table 2 Partition coefficients and hydrophobicity of enalapril isomers BME–ACN–PB two-phase system
Z-(cis) E-(trans)
KNP
KRP
log KRP
1.91 2.72
0.52 0.37
−0.28 −0.43
Estimated log PO/W −0.46 −0.65
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References
Fig. 6. The relationship between log PO/W and log KRP for eight different standard compounds (theobromine, caffeine, 3,4-dihydroxybenzoic acid, 4hydroxybenzoic acid, benzoic acid, benzene, phenol and cresol). log PO/W values were obtained from HSCCC measurements using Oct–PB at 4 ◦ C while KRP values were determined using BME–ACN–PB at 4 ◦ C. The Regression equation: y = 1.268 (±0.074)x − 0.107 (±0.071); (n = 8, r2 = 0.982).
listed in Table 2 as −0.46 and −0.65, respectively, for log PO/W of Z-(cis) isomer and E-(trans) isomer. 4. Conclusion The bioactive Z-(cis) isomer of enalapril was found to be the predominant species in the buffered solution, and the change in fractional content of the Z-(cis) isomer under a set of different temperature and pH conditions was similar to the results in previous literatures [7,8]. The estimation of rate constants for enalapril isomerization allows optimization of the HSCCC conditions for the determination of log PO/W values for the rapidly isomerizing Z-(cis)/E-(trans) species. The estimated log PO/W values for the Z-(cis) and E-(trans) isomers, along with the observed elution order of the both isomers in RP-HPLC and in HSCCC, confirmed that the Z-(cis) isomer was more hydrophobic than the E-(trans) isomer. The HSCCC method will be useful for the estimation of log PO/W values for other related ACE inhibitors that exist as cis/trans isomers undergoing fastreversible interconversion.
[1] G. Simon, S. Morioka, D.K. Snyder, J.N. Cohon, Clin. Pharmacol. Ther. 34 (1983) 459. [2] C.R. Benedict, Curr. Hypertens. Rep. 1 (1999) 305. [3] W.A. Thomas, M.K. Williams, J. C. S. Chem. Commun. 17 (1972) 994. [4] C. Grathwohl, W. Kurt, Biopolymers 20 (1981) 2623. [5] Y. Sakamoto, Y. Sakamoto, I. Oonishi, T. Ohmoto, J. Mol. Struct. 238 (1990) 325. [6] A. Kocijan, R. Grahek, D. Kocjan, L. Zupancic-Kralj, J. Chromatogr. B 755 (2001) 229. [7] E. Stellwagen, R. Ledger, Anal. Biochem. 321 (2003) 167. [8] R. Ledger, E. Stellwagen, J. Pharm. Pharmacol. 57 (2005) 845. [9] R. Natesh, S.L.U. Schwager, E.D. Sturrock, K.R. Acharya, Nature 421 (2003) 551. [10] R. Natesh, S.L.U. Schwager, H.R. Evans, E.D. Sturrock, K.R. Acharya, Biochemistry 43 (2004) 8718. [11] Y. In, M. Shibata, M. Doi, T. Ishida, M. Inoue, Y. Sasaki, S. Morimoto, J. Chem. Soc. Commun. 6 (1986) 437. [12] Y.-H. Kiang, A. Huq, P.W. Stephens, W. Xu, J. Pharm. Sci. 92 (2003) 1844. [13] A. Skogl¨of, I. Nilsson, S. Gustafsson, J. Deinum, P.O. G¨othe, Biochim. Biophys. Acta 1041 (1990) 22. [14] D.V.S. Green, I.H. Hillier, G.A. Morris, N. Gensmantel, D.W. Payling, D.H. Robinson, J. Mol. Struct. (Theochem) 251 (1991) 173. [15] F. Barbato, P. Morrica, F. Quaglia, IL Farmaco. 49 (1994) 457. [16] H. Trabelsi, S. Bouabdallah, S. Sabbah, F. Raouafi, K. Bouzouita, J. Chromatogr. A 871 (2000) 189. [17] S. Bouabdallah, H. Trabelsi, T.B. Dhia, S. Sabbah, K. Bouzouita, R. Khaddar, J. Pharm. Biomed. Anal. 31 (2003) 731. [18] O. Trapp, G. Trapp, V. Schurig, Electrophoresis 25 (2004) 318. [19] S. Kitagawa, J. Takeda, S. Sato, Biol. Pharm. Bull. 22 (1999) 721. [20] J. Sangster, Octanol–Water Partition Coefficients; Fundamentals and Physical Chemistry, Wiley, New York, 1997 (Chapter 1). [21] A. Bechalany, A. Tsantili-Kakoulidou, E.I. Tayar, N.B. Testa, J. Chromatogr. 541 (1991) 221. [22] Y. Ishihama, Y. Oda, K. Uchikawa, N. Asakawa, Anal. Chem. 67 (1995) 1588. [23] F. Lombardo, Y.S. Marina, A.T. Karl, F.E. Gao, J. Med. Chem. 44 (2001) 2490. [24] S.K. Poole, C.F. Poole, J. Chromatogr. B 797 (2003) 3. [25] S.K. Poole, S. Patel, K. Dehring, H. Workman, J. Dong, J. Chromatogr. B 793 (2003) 265. [26] Y. Ito, Adv. Chromatogr. 24 (1984) 181. [27] Y. Ito, J. Chromatogr. A 1065 (2005) 145. [28] Y. Shibusawa, A. Shoji, A. Yanagida, H. Shindo, M. Tagashira, M. Ikeda, Y. Ito, J. Liq. Chromatogr. Related Technol. 28 (2005) 2819. [29] C. Grathwohl, K. Wuethrich, Biopolymers 15 (1976) 2043. [30] M. Christl, J.D. Roberts, J. Am. Chem. Soc. 94 (1972) 4565. [31] K. Shinomiya, Y. Ito, J. Liq. Chromatogr. Related Technol. 29 (2006) 733. [32] A. Yanagida, A. Shoji, Y. Shibusawa, H. Shindo, M. Tagashira, M. Ikeda, Y. Ito, J. Chromatogr. A 1112 (2006) 195. [33] Y. Shibusawa, A. Yanagida, H. Shindo, Y. Ito, J. Liq. Chromatogr. Related Technol. 26 (2003) 1609. [34] Y. Shibusawa, A. Yanagida, M. Isozaki, H. Shindo, Y. Ito, J. Chromatogr. A 915 (2001) 253.
Counter-current chromatographic estimation of hydrophobicity of Z-(cis) and E-(trans) enalapril and kinetics of cis/trans isomerization Atsushi Shoji a , Akio Yanagida a , Heisaburo Shindo a , Yoichiro Ito b , Yoichi Shibusawa a,∗ a
Division of Structural Biology and Analytical Science, School of Pharmacy, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-0392, Japan b Center for Biochemistry and Biophysics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892-8014, USA Received 3 October 2006; received in revised form 13 April 2007; accepted 13 April 2007 Available online 19 April 2007
Abstract The kinetics of Z-(cis)/E-(trans) isomerization of enalapril was investigated by reversed phase high-performance liquid chromatography (RPHPLC) using a monolith ODS column under a series of different temperature and pH conditions. At a neutral pH 7, the rate (kobs ) of Z-(cis)/E-(trans) isomerization of enalapril at 4 ◦ C (9.4 × 10−3 min−1 ) is much lower than at 23 ◦ C (1.8 × 10−1 min−1 ), while the fractional concentration of Z-(cis) isomer is always higher than that of E-(trans) isomer in the pH range 2–7. The fractional concentration of the E-(trans) isomer becomes a maximum (about 40%) in the pH range 3–6, where enalapril exists as a zwitterion. The hydrophobicity (log PO/W ) of both isomers was estimated by highspeed counter-current chromatography (HSCCC). Normal phase HSCCC separation using a tert-butyl methyl ether–acetonitrile–20 mM potassium phosphate buffer (pH 5) two-phase solvent system (2:2:3, v/v/v) at 4 ◦ C was effective in partially separating the isomers, and the partition coefficient (K) of each isomer was directly calculated from the retention volume (VR ). The log PO/W values of Z-(cis) and E-(trans) isomers were −0.46 and −0.65, respectively. © 2007 Elsevier B.V. All rights reserved. Keywords: Enalapril; cis/trans Isomerization; High-speed counter-current chromatography; log PO/W
1. Introduction Angiotensin I-converting enzyme (ACE) inhibitors that block the production of vasoconstricting Angiotensin II are generally used as effective drugs in the treatment of hypertension and heart failure [1,2]. The active parts of ACE inhibitors are peptide derivatives containing C-terminal proline residues, and the compounds populate in both cis/trans isomers due to the partial double bond character of the tertiary amide bond in the proline containing peptide bond [3,4]. Among such ACE inhibitors, enalapril (and/or its active metabolite enalaprilat) is one of the most extensively investigated compounds in terms of its cis/trans interconversion and structural characteristics. In many previous reports (e.g. [5–7]), the cis/trans nomenclature of the isomers used the protein convention which is based on the polypeptide chain entering and exiting opposite corners of the rectangle
∗
Corresponding author. Tel.: +81 42 676 4544; fax: +81 42 676 4542. E-mail address: [email protected] (Y. Shibusawa).
0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.04.027
formed by the planar peptide bond atoms. However, Ledger and Stellwagen [8] recently proposed that the rotational isomers containing a proline tertiary amide bond should be described as either Z-(cis) or E-(trans) according to the Cahn–Ingold–Prelog (CIP) rules as encoded in IUPAC. In the present paper, the molecular configurations of the enalapril isomers are described as Z-(cis)/E-(trans) forms according to their proposal, as illustrated in Fig. 1. A recent X-ray crystallographic study on the ACE-inhibitor complex confirmed that the Z-(cis) isomer bonds to the catalytic Zn2+ ion of ACE [9,10], suggesting that the physiologically active form of enalapril is also of Z-(cis) configuration. Furthermore, other structural studies using X-ray analyses proved that enalapril in the solid state, such as a crystal or powder, usually exists as the Z-(cis) form [11,12]. On the other hand, enalapril in solution forms Z-(cis)/E-(trans) mixtures by undergoing reversible interconversion. The Z-(cis)/E-(trans) isomeric composition of enalapril in polar solution has been investigated by NMR [5,13,14], which indicated that the Z-(cis) isomer is predominant in aqueous solution. Also, the effects of pH,
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Fig. 1. Z-(cis)/E-(trans) isomerization of enalapril based on rotation around the alanyl–proline peptide bond. The broken arrow in the structure of Z-(cis) isomer indicates an intramolecular hydrogen bonding between the proline carboxyl group and the carbonyl group in the Z-(cis) isomer.
temperature and ionic strength on the isomeric composition of enalapril have been investigated by reversed phase (RP) HPLC and capillary electrophoresis (CE) [6–8,15–18]. Stellwagen and Ledger [7] estimated the dissociation constants of the enalapril carboxyl and amine groups of both isomers based on their elution behavior under various buffer pH conditions. They reported that the Z-(cis) isomer composition changed from 68% in cationic form (pH < 2) to 50% in isoelectric form (pH ∼4.5) and to 60% in the anionic form (pH > 7) [7,8]. Under the RP-HPLC and CE conditions, the retention behavior of enalapril Z-(cis)/E-(trans) isomers is also significantly affected by temperature, since the exchange rate of the Z-(cis)/E-(trans) isomer increases with increasing temperature. Actually, both HPLC [6,8,15–17] and CE studies [7,8,18] revealed that enalapril appeared as a nonseparable single peak at elevated temperature (80 ◦ C in HPLC or 60 ◦ C in CE) while its Z-(cis)/E-(trans) components were clearly separated with base line resolution at low temperature of 6 ◦ C in HPLC or 15 ◦ C in CE. However, there have been longstanding arguments on peak assignments of the Z-(cis)/E-(trans) enalapril (and/or enalaprilat) isomers in the separation profiles of HPLC or CE. Kocijan et al. [6] and Bouabdallah et al. [17] predicted that the E-(trans) isomer (which they termed cis) was more hydrophobic and therefore was retained longer than the Z-(cis) isomer (which they termed trans) in RP-HPLC. Their conclusion was based on the statement that E-(trans) alanylproline has a larger hydrophobic surface area than its Z-(cis) isomer. For the same reason, Trapp et al. [18] also predicted that the elution order of enalaprilat isomers in their CE experiment should be trans before cis, whereas the chemical structures illustrated as trans and cis in Trapp’s paper are those of E-(trans) and Z-(cis), respectively. Thus, the elution order of enalaprilat isomers in the CE experiment was first E(trans) isomer (which they termed trans) and next Z-(cis) isomer (which they termed cis). Furthermore, Ledger and Stellwagen [8] predicted that the slower component in the HPLC profile of enalapril is the Z-(cis) isomer. The elution order of both isomers is same as that of above Trapp’s CE experiment, but their prediction was based on the crystallographic structure of the Z(cis) isomer of enalapril maleate [11]. To solve this confusion, a greater understanding of the hydrophobicity of the Z-(cis)/E(trans) isomers is necessary. Because ACE inhibitors including enalapril are transported into intestinal epithelial cells through the H+ /oligopeptide transporter [19], accurate estimation of the hydrophobicity of the Z-(cis)/E-(trans) isomers and their iso-
meric composition in solution would be useful for understanding their high affinity with the transporter. In general, the hydrophobicity of drugs is often expressed as the logarithm of the 1-octanol–water partition coefficient or log PO/W [20]. The conventional procedure for determining log PO/W is a batch-shaking method or a shake-flask method using a 1-octanol–water two-phase solvent system. This traditional method is tedious and time consuming, and in some cases, such as for the cis/trans isomers of enalapril, it is difficult to measure log PO/W values because of its fast cis/trans interconversion. RP-HPLC or CE has recently been used to estimate log PO/W in preference to the shake-flask method [21–25]. In the chromatographic method, the retention factor (k) of a solute is calculated from its retention time. Since the log k value is linearly correlated with the log PO/W value, correlations between log k and log PO/W can be obtained for different standards. Hence, these chromatographic methods are considered to be indirect methods for the determination of log PO/W . On the other hand, high-speed counter-current chromatography (HSCCC) can directly determine the partition coefficients of solutes from their retention volume because both mobile and stationary phases are in liquid form [26–28]. In our previous HSCCC studies using a 1-octanol–50 mM potassium phosphate (1:1, v/v) two-phase system [28], log PO/W values ranging from −1.35 to +3.60 were readily determined for several aromatic compounds within 21 min. The present paper describes the separation of Z-(cis)/E-(trans) enalapril isomers and estimation of their log PO/W values by HSCCC using two-phase systems. In order to optimize temperature and pH conditions in the HSCCC experiments, the kinetics of cis/trans isomerization of enalapril in aqueous solution was also investigated by RP-HPLC. 2. Experimental 2.1. Reagents Enalapril maleate, (S)-1-[N-[1-(ethoxycarbonyl)-3-phenylpropyl]-l-alanyl]-l-proline maleate, was purchased from Sigma (St. Louis, MO, USA). Organic solvents including tert-butyl methyl ether (BME), acetonitrile (ACN) and 1-octanol (Oct) were purchased from Kanto (Tokyo, Japan). Standard compounds for log PO/W determination, theobromine, caffeine and benzoic acid, were purchased from Wako (Osaka, Japan); cresol, 3,4-dihydroxybenzoic acid and 4-hydroxybenzoic acid were from Tokyo Kasei (Tokyo, Japan); and benzene, phenol, anthracene and potassium nitrate were from Kanto. Other chemicals were of reagent grade. 2.2. Equipment The HPLC system (Hitachi, Tokyo, Japan) consisted of a type L-7100 pump, a Rheodyne 7166 sample injector, a type L-7420 UV–vis detector and a type D-2500 chromatointegrator. For RP-HPLC analysis of enalapril, a Chromolith Performance RP-18e column (Merck, Tokyo, Japan) was connected to the Hitachi HPLC system. For HSCCC experiments,
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a type-J coil-planet centrifuge (J-CPC; Renesas Eastern Japan Semiconductor, Tokyo, Japan) was connected to the Hitachi HPLC instruments. The J-CPC holds a pair of column holders symmetrically on the rotary frame at a distance of 5 cm from the central axis of the centrifuge. Each multilayer coiled column was fabricated by winding a single piece of polytetrafluoroethylene (PTFE) tubing (50 m × 1.0 mm I.D., total capacity about 40 ml) directly onto the holder hub making eight coiled layers (26 turns on each layer) between a pair of flanges (β = 0.5–0.6). The revolution speed of the apparatus was regulated at 1400 rpm with a speed controller. The separation coil rotates around its own axis as it synchronously revolves around a central axis, producing an efficient mixing of the two phases while retaining a sufficient amount of the stationary phase in the coiled column.
mobile phase was eluted through the column in the head to tail (H–T) direction at various flow rates of 1 ml/min (for theobromine and caffeine), 2 ml/min (for 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid and benzoic acid) and 4 ml/min (for benzene, phenol and cresol). After hydrodynamic equilibrium between the two phases was established in the rotating column, sample solution containing 1 mg of each standard dissolved in 50 l of both phases was injected and eluted with the aqueous lower mobile phase. Under these HSCCC conditions using the Oct–PB system in a reversed phase partition mode, the PO/W values can be directly obtained from their VR values according to the following equation [26,27]:
2.3. Reversed phase chromatography
where VR is the retention volume of the solute, and Vc is the PTFE column volume from the injector to the detector. Under the above HSCCC conditions, the mobile phase volume Vm is equal to the elution volume of unretained polar potassium nitrate, and the partition coefficient K in the equation can be expressed as the PO/W . HSCCC for the BME–ACN–PB system was performed in a reversed phase mode at 4 ◦ C. The HSCCC procedure and standard sample solutions were identical to those described for the Oct–PB two-phase system except that the lower aqueous ACN–PB phase was used as the mobile phase. The reversed phase partition coefficient (KRP ) of each standard compound was calculated from the VR value according to Eq. (1). Under the above HSCCC conditions, the K in the equation can be expressed as the KRP . HSCCC separation of the Z-(cis)/E-(trans)-enalapril isomers was carried out with the BME–ACN–PB two-phase system in a normal phase partition mode (mobile phase: organic upper phase; stationary phase: aqueous lower phase) at 4 ◦ C. First, the PTFE coiled column was first entirely filled with the lower aqueous stationary phase. Then, the column was rotated at 1400 rpm while the organic upper mobile phase was eluted through the column in a tail to head direction at a flow rate of 2 ml/min. After hydrodynamic equilibrium between the two phases was reached in the rotating column, the sample solution containing enalapril maleate was injected and eluted with the organic upper mobile phase. The eluent was monitored by a UV detector at 210 nm. Before the HSCCC injection, 1 mg of enalapril maleate was dissolved in 50 l of lower phase and incubated at 4 ◦ C. After the incubation, 50 l of upper phase was added to the lower phase solution containing enalapril, and the mixture solution (total 100 l) was used as the sample solution for above HSCCC separation. The normal phase partition coefficient (KNP ) of each enalapril isomer was calculated from the VR value according to Eq. (1). Under the above normal phase HSCCC conditions, the mobile phase volume Vm is equal to the elution volume of unretained hydrophobic anthracene, and the K in the equation can be expressed as KNP . In addition, the reversed phase partition coefficient (KRP ) of each isomer in the same solvent system was calculated from the relation, KNP = 1/KRP .
Separation of Z-(cis)/E-(trans) enalapril isomers was performed by HPLC using a Chromolith Performance RP-18e column (100 mm × 4.6 mm I.D.) at 23 ◦ C (in an air-conditioned laboratory) and 4 ◦ C (in a cold room). An acetonitrile–20 mM potassium phosphate buffer (pH 7) mixture (18:82, v/v) was used for the mobile phase at a flow rate of 4 ml/min. Enalapril maleate was dissolved in the 20 mM potassium phosphate buffer (0.5 mg/ml), and 5 l of this solution was injected and isocratically eluted from the column. The effluent was monitored by a UV detector at 210 nm. Before the HPLC injection, the enalapril solution was incubated at each temperature. The incubation time was 0–40 min at 23 ◦ C and was 0–1500 min at 4 ◦ C. After HPLC analysis, a concentration of the Z-(cis) isomer ([Z]) in the enalapril solution after incubation with a certain time was calculated from the peak area of the Z-(cis) isomer on the chromatogram, and the percentage of fractional concentration of the Z-(cis) isomer was calculated from the following equation: ([Z]/[Z]0 ) × 100, where [Z]0 is the concentration of the Z isomer in the enalapril solution without incubation at each temperature. 2.4. Counter-current chromatography Two two-phase solvent systems were used for HSCCC analyses of enalapril and the organic standards: (1) a BME–ACN–20 mM potassium phosphate buffer (PB; pH 5) two-phase system (2:2:3, v/v/v) and (2) an Oct–50 mM PB (pH 5) two-phase system (1:1, v/v). To establish an appropriate correlation between the reversed phase partition coefficient (KRP ) obtained in the BME-ACNPB system and the PO/W obtained in the Oct-PB system, the partition coefficients of eight different standard compounds, as listed in Section 2.1, were measured using the following HSCCC procedure. HSCCC separation was carried out using the Oct–PB twophase system in a reversed phase partition mode (stationary phase: upper octanol phase; mobile phase: aqueous lower phase) at 4 ◦ C. The PTFE coiled column previously filled with the upper stationary phase was rotated at 1400 rpm while the lower
K=
Cs VR − V m = Cm Vc − V m
(1)
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3. Results and discussion 3.1. Kinetics of Z-(cis)/E-(trans) isomerization of enalapril For the kinetic study of the isomerization of Z-(cis)/E-(trans) enalapril in aqueous solution at neutral pH, RP-HPLC of the two isomers was performed using a PB–acetonitrile mobile phase at 23 ◦ C and 4 ◦ C. Enalapril maleate was dissolved in 20 mM PB (pH 7) and immediately analyzed by HPLC. Further, aliquots of solution were incubated for 12 min at 23 ◦ C and for 600 min at 4 ◦ C before analysis. The separation profiles of cis/trans enalapril are shown in Fig. 2. We confirmed that maleate (MA) was eluted at the void volume of the ODS column in all chromatograms. We also confirmed that the elution profile of enalapril was significantly different before and after incubation. When the enalapril solution was analyzed without incubation, enalapril was eluted as a single peak (peak 2) at about 2 min. This peak 2 was assigned to the Z-(cis) isomer as indicated in previous studies [11,12]. When the solution was analyzed after incubation (for 12 min at 23 ◦ C or for 600 min at 4 ◦ C), the enalapril was eluted as two peaks (1 and 2) indicating Z-(cis)/E-(trans) equilibration during the incubation. The new peak 1 at about 0.8 min was assigned to the E-(trans) isomer. The elution order of the two peaks indicated that the later eluting Z-(cis) isomer was more hydrophobic than the earlier eluting E-(trans) isomer. A comparison of the separation profile obtained at 23 ◦ C (Fig. 2A) and 4 ◦ C (Fig. 2B) showed a plateau between the two peaks due to on-column interconversion of the Z-(cis)/E-
Fig. 3. Change over time in fractional concentration (%) of the Z-(cis) isomer in enalapril maleate solution at 23 ◦ C or 4 ◦ C. Enalapril maleate was dissolved in 20 mM potassium phosphate buffer (pH 7), and the solution was incubated for 30 min maximum at 23 ◦ C or 1500 min maximum at 4 ◦ C before HPLC analysis. The fractional concentration (%) of the Z-(cis) isomer after each incubation phase was calculated from its peak area on the HPLC chromatogram. The inset plot at each temperature shows relationship between ln([Z] − [Z]eq ) and incubation time (t). Regression equation at 23 ◦ C: y = −0.1837 (±0.0201)x + 3.3864 (±0.2187) (n = 7, r2 = 0.9435). Regression equation at 4 ◦ C: y = −0.0094 (±0.0003)x + 3.2955 (±0.0492) (n = 13, r2 = 0.9826).
(trans) isomers. Many previous researchers have reported that chromatographic conditions, such as pH, temperature and flow rate, significantly affected the appearance of the plateau and the shape of the separated peaks [6–8,17,18]. Although the HPLC separation shown in Fig. 2A was performed within 2 min and the analysis time was shortest among all LC and/or CE separation reports of enalapril, the appearance of the plateau was not completely suppressed at 23 ◦ C. On the basis of the above results, we next examined the kinetics of Z-(cis)/E-(trans) interconversion of enalapril over various incubation times under the same RP-HPLC and pH conditions. Fig. 3 shows the change over time in the fractional concentration (%) of the Z-(cis) isomer in enalapril maleate solution at 23 and 4 ◦ C. With increasing incubation time, the Z-(cis) isomer concentration decays exponentially before reaching a constant value. Since the Z-(cis)/E-(trans) isomerization of enalapril is the first-order reversible reaction as shown in Fig. 1, the isomerization rate at a certain time is expressed by following equation (Eq. (2)), −d[E] d[Z] = = −k1 [Z] + k−1 [E] dt dt
Fig. 2. Separation profiles of Z-(cis)/E-(trans) enalapril in reversed phase HPLC using monolith ODS column at 23 and 4 ◦ C (A and B), respectively. Enalapril maleate was dissolved in 20 mM potassium phosphate buffer (pH 7) and analyzed immediately by RP-HPLC. Enalapril maleate solution was also incubated for 12 min at 23 ◦ C or 600 min at 4 ◦ C before analysis. RP-HPLC was performed using a Chromolith Performance RP-18e column (100 mm × 4.6 mm I.D., MERCK) with a mobile phase composed of 20 mM potassium phosphate buffer (pH 7) and acetonitrile (82:18, v/v) at a flow rate of 4 ml/min. UV absorbance of the effluent was monitored at 210 nm. MA: maleate. Peaks: (1) E-(trans) enalapril and (2) Z-(cis) enalapril.
(2)
where k1 is the first-order rate constant of isomerization from the Z-(cis) to E-(trans) isomer, k−1 that of the reverse reaction, [Z] the concentration of the Z-(cis) isomer and [E] is that of the E-(trans) isomer. Since enalapril exists in the Z-(cis) form at the initial time t = 0, [E] = [Z]0 − [Z], where [Z]0 is the fraction of [Z] at t = 0. Therefore, Eq. (2) can be rewritten as follows: d[Z] = −k1 [Z] + k−1 ([Z]0 − [Z]) dt
(3)
Integration of Eq. (3) gives ln([Z] − [Z]eq ) = ln([Z]0 − [Z]eq ) − (k1 + k−1 )t = ln([Z]0 − [Z]eq ) − kobs t
(4)
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Table 1 Kinetic parameters of isomerization of Z-(cis)/E-(trans) enalapril in 20 mM phosphate buffer (pH 7) at 23 and 4 ◦ C Temperature (◦ C)
K (k1 /k−1 )
k1 (min−1 ) (Z → E)
k−1 (min−1 ) (E → Z)
kobs (min−1 )a (k1 + k−1 )
23 4
3.5 × 10−1 4.6 × 10−1
4.8 × 10−2 3.0 × 10−3
1.4 × 10−1 6.4 × 10−3
1.8 × 10−1 9.4 × 10−3
a
kobs was calculated as the slope of a linear least-square fitting of relationship between ln([Z] − [Z]eq ) and t.
where [Z]eq is the fraction of [Z] at equilibrium, and kobs = k1 + k−1 . Using Eq. (4), kobs of the Z-(cis)/E-(trans) isomerization of enalapril can be calculated from the slope of linear least-square fitting of ln([Z] − [Z]eq ) as a function of incubation time (t). As listed in Table 1, along with the data for the regression equations and statistics, the kobs values for the isomerization were found to be 1.8 × 10−1 min−1 at 23 ◦ C and 9.4 × 10−3 min−1 at 4 ◦ C, respectively. Since the forward and backward reaction rates are equal at equilibrium, we have the following equation (Eq. (5)). k1 [Z]eq = k−1 [E]eq
(5)
The equilibrium constant K (K = k1 /k−1 ) is given by the following equation (Eq. (6)). K=
[E]eq k1 = k−1 [Z]eq
(6)
Using Eq. (6), the equilibrium constants K were calculated to be 3.5 × 10−1 ([E]eq /[Z]eq = 26.0/74.0) at 23 ◦ C and 4.6 × 10−1 ([E]eq /[Z]eq = 31.7/68.3) at 4 ◦ C. Furthermore, these K and kobs values yield forward and backward rate constants k1 and k−1 using Eqs. (4) and (6). All the kinetic parameters (K, k1 , k−1 , kobs ) thus obtained for Z-(cis)/E-(trans) isomerization are summarized in Table 1. These kinetics data demonstrate that the backward reaction from E-(trans) to Z-(cis) is faster than the forward reaction from Z-(cis) to E-(trans), indicating that the Z(cis) form is more stable than the E-(trans) form. The stability of the Z-(cis) isomer of enalapril in solution is attributed to hydrogen bonding between the carbonyl group and carboxyl groups in the structure of Z-(cis) isomer (Fig. 1) [5]. Recently, Ledger and Stellwagen [8] reported the kinetic study of the isomerization of enalapril maleate in water (pH 4.2) at 8 ◦ C, and the isomerization was found to occur in a single kinetic phase having a half-time (t1/2 ) of 58 min with 52% in the Z-(cis) isomer at equilibrium. According to the following equation t1/2 = ln 2/kobs , the value of kobs of the isomerization under their conditions is calculated to be 1.2 × 10−2 min−1 . Furthermore, since the percentage of fractional concentration of [Z]eq is 52%, according to the previous Eqs. (5) and (6), the values K, k1 and k−1 are calculated to be 9.2 × 10−1 , 5.8 × 10−3 min−1 and 6.3 × 10−3 min−1 , respectively. These values are close to our kinetics values measured at pH 7 and at 4 ◦ C, listed in Table 1. 3.2. pH effects on isomerization of enalapril Next, we examined changes in the equilibrium ratio of Z-(cis)/E-(trans) enalapril in phosphate buffered solution at various pHs ranging from 2 to 7 at 23 ◦ C. The pH of phosphate buffer
Fig. 4. The change in fractional concentration (%) of Z-(cis) isomer under various pH conditions. Enalapril maleate was dissolved in 20 mM potassium phosphate buffer (pH 2–7), and the solution was incubated for 60 min at 23 ◦ C before HPLC analysis.
in the sample solution and in the mobile phase was adjusted to the same value. The sample solutions were incubated for 30 min at 23 ◦ C before HPLC analysis. Fig. 4 shows the fractional change in the Z-(cis) isomer as a function of pH. The data confirmed that the Z-(cis) isomer is always more stable than the E-(trans) isomer in aqueous buffered solution between pH 2 and 7. The fractional concentration of the E-(trans) isomer was estimated to be about 40% under weakly acidic conditions (pH range from 3 to 6), whereas the fraction was markedly decreased in both acidic (pH 2) and neutral (pH 7) conditions (28% at pH 2 and 32% at pH 7, respectively). Similar results from CE analysis of enalapril have been reported by Stellwagen and Ledger [7,8]. In these reports, the dissociation constants of the carbonyl group in Z-(cis) and E(trans) enalapril, pKZ and pKE , were estimated at 3.1 and 2.6, respectively, and the pKZ and pKE of the amine group were estimated at 5.6 and 5.9 from pH titration curves of CE mobility measurements. These pK values indicate that both enalapril isomers are in a zwitterionic form in the pH range from 3 to 6. In the E-(trans) configuration especially, the two charged groups (i.e. deprotonated carboxyl and protonated amine groups) may attract one another to stabilize the E-(trans) configuration [29,30]. If this speculation is correct, the intramolecular ionic interaction in the E-(trans) configuration may affect the composition ratio of Z-(cis)/E-(trans) enalapril in buffered solution in the pH range from 3 to 6. Stabilization of Z-(cis) at a low pH of 2 may be explained as the result of hydrogen bond formation between the carbonyl and carboxyl groups as shown in Fig. 1.
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3.3. HSCCC separation of Z-(cis)/E-(trans) enalapril As mentioned in Section 3.1, the elution order of the enalapril isomers in RP-HPLC confirmed that the Z-(cis) isomer was more hydrophobic than the E-(cis) isomer. However, a difference in the log PO/W values of the Z-(cis) and E-(trans) isomers is difficult to determine by the traditional shake-flask method because of fast cis/trans intercoversion in solution. As mentioned above, a chromatographic method, such as HSCCC, can achieve separation of cis/trans forms and allows direct evaluation of the partition coefficients of each isomer. We thus tried to separate the Z-(cis)/E(trans) enalapril isomers by HSCCC using either an Oct–PB two-phase system or BME–ACN–PB two-phase system. The HSCCC using the Oct–PB system can be used to readily determine log PO/W value of the solute based on the VR value [28]. On the other hand, the BME–ACN–PB system is the most appropriate two-phase system for HSCCC separation of polar compounds [31–34]. In HSCCC operation, the normal phase partition mode (stationary phase: aqueous lower phase) was selected for efficient retention and separation of enalapril isomers because enalapril in the pH 5 buffer solution exists in a polar zwitterionic form. Before HSCCC separation of the enalapril isomers, we examined the retention of the stationary phase solvent (i.e. aqueous lower phase) in revolving HSCCC column under hydrodynamic two-phase equilibrium at different temperature conditions (4 or 23 ◦ C). In general, a higher retention of the stationary phase leads to higher separation efficiency. In the case of the Oct–PB system, 40% of the stationary phase was retained in the column at 23 ◦ C while none was retained at 4 ◦ C, presumably because of the high viscosity of the octanol phase at low temperature. For the same reason, the retention of the stationary phase in the BME–ACN–PB system was greater at 23 ◦ C than at 4 ◦ C (76.3% at 23 ◦ C; 29.2% at 4 ◦ C). The retention of the stationary phase in the BME–ACN–PB system (76.3%) was higher than that of the Oct–PB system (40.0%) because the BME-rich mobile phase had a much lower viscosity than the Oct-rich mobile phase. We initially tried to separate the enalapril isomers by normal phase HSCCC using the Oct–PB (pH 5) two-phase system at 23 ◦ C. However, the enalapril isomers could not be separated from each other with this two-phase system. Therefore, a BME–ACN–PB (pH 5) two-phase system was used for the HSCCC separation. The chromatograms of enalapril maleate in the normal phase HSCCC are shown in Fig. 5. It can be seen that the Z-(cis)/E-(trans) enalapril isomers were not separated at 23 ◦ C because the rate of Z-(cis)/E-(trans) interconversion of enalapril was too fast (Fig. 3; Table 1). Since the rate of Z-(cis)/E(trans) interconversion is markedly slower at low temperature (Fig. 3), HSCCC was carried out at 4 ◦ C. As shown in Fig. 5B, when the sample solution was injected to the column without incubation, enalapril was eluted as a single peak with a small shoulder at 24.6 min. As in the RP-HPLC results shown in Fig. 2, this single peak at 24.6 min was assigned to the Z-(cis) isomer. On the other hand, when the sample solution was injected into the HSCCC after the incubation for 360 min, enalapril appeared as broad split peaks. The second peak at 28.7 min is assigned to the E-(trans) isomer. The elution order of these peaks in the
Fig. 5. HSCCC chromatograms of Z-(cis) (Z) and E-(trans) (E) enalapril in normal phase partition mode using BME/ACN/PB (2:2:2; 50 mM pH 5 buffer) (2:2:3) two-phase system at 23 and 4 ◦ C (A and B) respectively. Enalapril maleate was dissolved in the lower phase and analyzed immediately by HSCCC. The enalapril maleate solution was also incubated for 60 min at 23 ◦ C or 360 min at 4 ◦ C before analysis. HSCCC was performed using a PTFE multilayer coiled column (50 m × 1.0 mm I.D., 40 ml capacity; operated at 1400 rpm) with a lower aqueous stationary phase and an organic upper mobile phase (flow rate of 2 ml/min) was monitored at 210 nm.
normal phase HSCCC chromatograms indicated that the earlier eluted Z-(cis) isomer was more hydrophobic than the later eluted E-(trans) isomer. 3.4. Evaluation of hydrophobicity of Z-(cis)/E-(trans) enalapril The partition coefficients, KNP and KRP , obtained for the enalapril isomers under the above HSCCC condition are listed in Table 2. The KRP values of the Z-(cis) and E-(trans) isomers were calculated to be 0.52 and 0.37, respectively. Furthermore, eight standard compounds (theobromine, caffeine, 3,4-dihydroxybenzoic acid, 4-hydroxybenzoic acid, benzoic acid, benzene, phenol and cresol) were analyzed by reversed phase HSCCC using the Oct–PB and BME–ACN–PB two-phase systems to establish a correlation between PO/W and KRP at 4 ◦ C. As shown in Fig. 6, the log KRP value of each standard was plotted against each log PO/W value. The relationship between log KRP and log PO/W was linear over the range log KRP −0.4 to 1.7 and expressed as the following regression equation: log PO/W = 1.268 log KRP − 0.107
(n = 8; r 2 = 0.982) (7)
Using this equation, we could estimate the log PO/W values of the Z-(cis) and E-(trans) isomers from their KRP values. These are Table 2 Partition coefficients and hydrophobicity of enalapril isomers BME–ACN–PB two-phase system
Z-(cis) E-(trans)
KNP
KRP
log KRP
1.91 2.72
0.52 0.37
−0.28 −0.43
Estimated log PO/W −0.46 −0.65
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References
Fig. 6. The relationship between log PO/W and log KRP for eight different standard compounds (theobromine, caffeine, 3,4-dihydroxybenzoic acid, 4hydroxybenzoic acid, benzoic acid, benzene, phenol and cresol). log PO/W values were obtained from HSCCC measurements using Oct–PB at 4 ◦ C while KRP values were determined using BME–ACN–PB at 4 ◦ C. The Regression equation: y = 1.268 (±0.074)x − 0.107 (±0.071); (n = 8, r2 = 0.982).
listed in Table 2 as −0.46 and −0.65, respectively, for log PO/W of Z-(cis) isomer and E-(trans) isomer. 4. Conclusion The bioactive Z-(cis) isomer of enalapril was found to be the predominant species in the buffered solution, and the change in fractional content of the Z-(cis) isomer under a set of different temperature and pH conditions was similar to the results in previous literatures [7,8]. The estimation of rate constants for enalapril isomerization allows optimization of the HSCCC conditions for the determination of log PO/W values for the rapidly isomerizing Z-(cis)/E-(trans) species. The estimated log PO/W values for the Z-(cis) and E-(trans) isomers, along with the observed elution order of the both isomers in RP-HPLC and in HSCCC, confirmed that the Z-(cis) isomer was more hydrophobic than the E-(trans) isomer. The HSCCC method will be useful for the estimation of log PO/W values for other related ACE inhibitors that exist as cis/trans isomers undergoing fastreversible interconversion.
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