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Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography
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Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography Wenyu Sun, Yang Jin, Chaoyue Wang, Shanshan Zhao, Xiang Wang, Meng Luo, Jizhong Yan, Shengqiang Tong∗ College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China
a r t i c l e
i n f o
Article history: Received 24 August 2019 Revised 23 December 2019 Accepted 29 December 2019 Available online xxx Keywords: Countercurrent chromatography Hydroxypropyl-β -cyclodextrin Isomeric sertraline Methyl-β -cyclodextrin Stereoselective liquid-liquid extraction
a b s t r a c t Sertraline is an antidepressant in a group of drugs called selective serotonin reuptake inhibitors. Four stereoisomeric compounds would be produced in its synthetic preparation due to two chiral carbons on its chemical structures. In the present work, stereoselective liquid-liquid extraction of isomeric sertraline with substituted cyclodextrins as stereoselective extractant was investigated. Factors affecting the distribution performance, including organic solvents, types of extractants, pH value, buffer solution of aqueous phase, concentration of extractant and temperature, were investigated. Under optimized conditions, a stereoselectivity of 1.404 was obtained for cis-sertraline and a stereoselectivity of 2.373 was obtained for trans-sertraline when hydroxypropyl-β -cyclodextrin was used as the stereoselective extractant, and a stereoselectivity of 1.685 was achieved for trans-sertraline with methyl-β -cyclodextrin as extractant. An unusual stereoselective combination was observed for trans-sertraline and cis-sertraline when sodium carbonate buffer was used. Successful stereoselective separation of trans-sertraline and cis-sertraline, (1S, 4R) and (1R, 4S)-sertraline by analytical countercurrent chromatography with methyl-β -cyclodextrin as stereoselective selector was achieved, using a biphasic solvent system composed of n-hexane : 0.1 mol L−1 citrate buffer solution with pH7.6 (1:1, v/v). © 2019 Elsevier B.V. All rights reserved.
1. Introduction With the increasing demand for chiral compounds, especially chiral drugs, the separation technology of chiral substances has become one of the most interest subjects over the past decades [1-5]. Chiral separations are mainly conducted by mechanical, chemical, biochemical, chromatographic, extractive and membranous methods. Enantioselective liquid-liquid extraction (ELLE) is considered to be a promising technology for chiral separation, which combines the concepts of solvent extraction and chiral recognition in a single technology [6]. It has the advantage of lower cost in energy consumption, easier to scale up to commercial scale, and a wide range of application. In the past decades, increasing number of papers with regarding to ELLE has been published, and almost all the published papers are about chiral separation of racemic compound with one chiral center in its chemical structure or enantioseparation of optical isomers [7-11]. Various chiral extractants have been studied in ELLE [12-16]. The main extractant classes are as followings: substituted cyclodextrins, crown ether based ∗ Corresponding author at: College of Pharmaceutical Science, Zhejiang University of Technology, Chaowang Road 18, Chaohui No. 6 District, Hangzhou, China. E-mail address: [email protected] (S. Tong).
extractants, metal complexes and metalloids, extractants based on tartrates, and other types of chiral extractants. ELLE is easy to be scaled up using a countercurrent cascade of centrifugal contactor separators or modern liquid-liquid partition chromatography for preparative chiral separation. To the best of our knowledge, few literatures were available with regarding to stereoselective liquidliquid extraction of drugs with two chiral centers in its chemical structure, yielding four stereoisomers. Sertraline hydrochloride, cis-(1S,4S)-N-methyl-4-(3, 4dichlorophenyl)−1, 2, 3, 4-tetrahydro-1-naphthalenamine hydrochloride, is a selective inhibitor of central serotonin reuptake and it is used as an antidepressant for oral administration [17]. Four stereoisomeric compounds, including (1S, 4R), (1R, 4S), (1S, 4S) and (1R, 4R)-isomer, were produced during its synthesis due to two chiral carbon centers in its chemical structure (Fig. 1). It has been proved that only (1S, 4S)-sertraline owns intended pharmacological activity due to stereospecific combination with receptor in the body [18]. In our very recent work, stereoselective separation of (1S, 4S)-sertraline from its medicinal reaction mixtures by countercurrent chromatography using hydroxypropyl-β -cyclodextrin as the stereoselective selector was investigated, in which a fabricated in-house analytical countercurrent chromatographic apparatus was used for optimization of the separation conditions, and two
https://doi.org/10.1016/j.chroma.2019.460834 0021-9673/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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2.4. Stereoselective liquid-liquid extraction procedures
Fig. 1.. Chemical structure of sertraline hydrochloride.
cis-isomers were successfully separated [19]. However, no success of stereoselective separation of trans-isomers was achieved. Meanwhile, in order to further investigate the distribution behavior and stereoselectivity of stereoisomeric sertraline with substituted β cyclodextrin as stereoselective selector, stereoselective liquid-liquid extraction of the four isomeric sertraline using substituted β cyclodextrins as extractant was investigated in our present work, in which an interesting phenomenon was found when different type of buffer solutions were used. Furthermore, stereoselective separation of trans-sertraline and cis-sertraline, (1S, 4R) and (1R, 4S)-sertraline by analytical countercurrent chromatography with methyl-β -cyclodextrin as stereoselective selector was achieved. 2. Experimental methods 2.1. Apparatus The high-performance liquid chromatography (HPLC) was a Shimadzu HPLC LC-solution system comprised of a SCL-10Avp controller, a Shimadzu LC-10Avp UV detector, a Shimadzu LC-10ATvp multi solvent delivery system and a Shimadzu LC pump. The pH value for the test was determined with a PB-10 pH meter (Sartorius, Germany). A fabricated in-house analytical type-J counter-current chromatographic apparatus with a total capacity of 15 ml was used [19]. The solvents were pumped into the column with a model TBP 5002 constant-flow pump (ShanghaiTauto Biotechnique, Shanghai, China). A model UVD-200 detector (Shanghai Jinda Biotechnology Co., Ltd., Shanghai, China) was used for continuous monitoring of the effluent and the chromatogram was recorded with a SEPU30 0 0 workstation (Hangzhou Puhui Technology, Hangzhou, China). 2.2. Materials Hydroxypropyl-β -cyclodextrin (HP-β -CD), methyl-β -cyclodextrin (Me-β -CD) and sulfobutylether-β -cyclodextrin (SBE-β CD) were purchased from Qianhui Fine Chemical, Shandong, China. Hexanol, octanol, n-Hexane, n-heptane, petroleum ether, dichloromethane, chloroform, 1, 2-dichloroethane, cyclohexane, ethyl acetate were purchased from Macklin Biochemical Co. Ltd, Shandong, China. Dibasic sodium phosphate, citric acid, sodium carbonate, sodium bicarbonate, glycine, sodium hydroxide, ammonia, ammonium chloride were purchased from Huipu Chemical, Hangzhou, China. Acetonitrile used for HPLC analysis was of chromatographic grade. Water was redistilled. 2.3. Preparation of sertraline hydrochloride The preparation of isomeric sertraline hydrochloride was according to the literature [20], and experimental section in detail could be found in our previous work [19]. The sample of isomeric sertraline was composed of equal amount of cis-crude product and trans-crude product synthesized in our lab.
HP-β -CD, Me-β -CD and SBE-β -CD were used as the extractants in aqueous phase. Aqueous phases were prepared by dissolving substituted β -CD and 1 mg mL−1 of isomeric sertraline hydrochloride in 0.10 mol L−1 buffer solution. Liquid-liquid extraction was performed in a 10 ml glass-stoppered tube. Equal volumes (each 2 ml) of the aqueous and the organic phase were placed together, and shaken sufficiently (5 min) before being kept in a water bath for 1 h at a fixed temperature to reach equilibrium. The investigated temperature was in the range of 5–40 °C. After phase separation, the concentrations of isomeric sertraline in the aqueous phase were determined by HPLC. Each experiment was duplicated under identical conditions. The concentrations of sertraline isomers in organic phase were calculated by subtractive method. The distribution ratio and stereoselectivity are defined as the followings:
D(1S,4R) =
CO,SR CW,SR
(1)
D(1R,4S ) =
CO,RS CO,RS
(2)
D(1S,4S ) =
CO,SS CW,SS
(3)
D(1R,4R) =
CO,RR CW,RR
(4)
α(trans) =
D(1S,4R ) D(1R,4S ) Or D(1R,4S ) D(1S,4R )
(5)
where D(1S, 4R) ≥ D(1R, 4S) or D(1R, 4S) ≥ D(1S, 4R)
α(cis) =
D(1S,4S ) D(1R,4R ) Or D(1R,4R ) D(1S,4S )
(6)
where D(1S, 4S) ≥ D(1R, 4R) or D(1R, 4R) ≥ D(1S, 4S) , where CO,SR and CW,SR respectively represent concentration of (1S, 4R)-sertraline in organic phase and aqueous phase; CO,RS and CW, RS respectively represent concentration of (1R, 4S)-sertraline in organic phase and aqueous phase; CO,SS and CW,SS respectively represent concentration of (1S, 4S)-sertraline in organic phase and aqueous phase; CO,RR and CW,RR respectively represent concentration of (1R, 4R)sertraline in organic phase and aqueous phase. 2.5. Countercurrent chromatographic separation Solvent systems consisting of n-hexane : 0.10 mol L−1 of disodium hydrogen phosphate-citric acid buffer pH 7.6 containing 50 mmol L−1 of Me-β -CD (1:1, v/v) were used. The aqueous buffer solution with pH 7.6 was prepared before adding 50 mmol L−1 Meβ -CD. Then the solvent mixture was mixed and thoroughly equilibrated in a separatory funnel, the saturated aqueous phase and organic phase were separated shortly and degassed by ultrasound for 30 min before use. The sample solution was prepared by dissolving 1 mg of isomeric sertraline into 0.5 mL the aqueous mobile phase for countercurrent chromatographic separation. The separation was initiated by entirely filling the column with the organic stationary phase. The aqueous mobile phase was pumped into the column at a flow rate 0.5 mL min−1 while the apparatus was rotated at 1120 rpm. Subsequently, the sample solution was injected after observing a clear elution of mobile phase at the tail outlet, which indicated hydrodynamic equilibrium was established in the column.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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W. Sun, Y. Jin and C. Wang et al. / Journal of Chromatography A xxx (xxxx) xxx Table 1 Influence of different extractant in distribution ratio and stereoselectivity. Extractant
D(1S, 4R)
D(1R, 4S)
α
HP-β -CD Me-β -CD SBE-β -CD
0.424 0.200 0.109
0.534 0.337 0.110
1.259 1.685 1.010
(trans)
D(1S, 4S)
D(1R, 4R)
α (cis)
1.314 0.980 0.173
1.705 0.989 0.192
1.298 1.010 1.110
Aqueous phase: 0.1 mol L−1 of disodium hydrogen phosphate-citric acid at pH=7.6; organic phase: n-hexane; equilibrium temperature: 10 °C; Concentration of extractant: 50 mmol L−1 of cyclodextrin in the aqueous phase; equilibrium temperature: 10 °C.
2.6. Analytical method The quantification of isomeric sertraline was performed by HPLC using a UV detector set at 225 nm with an YMC-Pack ODS-A column (150×4.6 mm i.d., 5 μm) according to reference [21]. The mobile phase was 20 mmol L−1 of HP-β -CD aqueous buffer solution (pH 3.0, adjusted with phosphoric acid) : acetonitrile (78:22, v/v) at a flow rate of 0.8 mL min−1 , the column temperature was 35 °C. 3. Results and discussion 3.1. Stereoselective liquid-liquid extractions 3.1.1. Selection of chiral extractants and organic solvent It was reported that complete separation of four isomers by liquid chromatography with HP-β -CD as mobile phase additive could be achieved [21]. Therefore, three substituted β -cyclodextrins, HPβ -CD, Me-β -CD and SBE-β -CD were investigated for stereoselective liquid-liquid extraction of the four isomeric sertraline in light of the above reported results. Substituted β -cyclodextrins were highly hydrophilic and were added in the aqueous phase. Distribution ratio and stereoselectivity for isomeric sertraline were investigated in several different liquid-liquid extraction systems containing 50 mmol L−1 substituted β -CD in aqueous phase. As shown in Table 1, very limited stereoselectivity was obtained for trans-sertraline and cis-sertraline when SBE-β -CD was used as stereoselective selector. It was found that high stereoselectivity, α =1.685, was obtained for trans-sertraline but low stereoselectivity, α =1.010, was obtained for cis-sertraline when Me-β -CD was used as stereoselective selector. Meanwhile, relatively high stereoselectivities for both trans-sertraline and cis-sertraline, α =1.259 and α =1.298, were obtained with moderate distribution ratio when HP-β -CD was used as stereoselective selector under pH7.6. As shown in Table 1, D (1R, 4S ) were found to be always larger than D(1S, 4R) , and D(1R, 4R) were always larger than D(1S, 4S) , which indicated that HP-β -CD and Me-β -CD preferentially recognized (1S, 4R)-sertraline and (1S, 4S)-sertraline in trans-sertraline and cissertraline, respectively. HP-β -CD could be chosen as the suitable
3
chiral extractant for both cis-sertraline and trans-sertraline, while Me-β -CD could be selected as the suitable chiral extractant for trans-sertraline because of the higher stereoselectivities. The influence of organic solvents on distribution behavior was investigated using HP-β -CD as extractant, as shown in Table 2. Very low stereoselectivities were found when dichloromethane, chloroform and 1,2-dichloroethane were used, while a moderate distribution ratio and a high stereoselectivity were obtained when n-hexane, cyclohexane, n-heptane and petroleum ether was used as the organic solvent, which indicated n-hexane, cyclohexane, nheptane and petroleum ether could be selected for stereoselective liquid-liquid extraction. Distribution ratio for all the stereoisomers were too large to be tested when ethyl acetate was used as the organic solvent, and so ethyl acetate was not applied in the present work. 3.1.2. Influence of pH and buffer solution 3.1.2.1. HP-β -CD. Sertraline is a kind of organic base in light of its chemical structure. So its distribution behavior in the biphasic solvent system would be greatly influenced by pH value of aqueous phase. Meanwhile, inclusion complex formed by isomeric sertraline and cyclodextrin might be greatly affected by its status in the aqueous phase. Fig. 2 shows the influence of pH on the stereoselective separation of four isomeric sertraline with HP-β -CD as extractant. As is shown in Fig. 2, both of the distribution ratio and stereoselectivity increased with the increasing of pH value, which might be caused by the fact that HP-β -CD would mainly have stereospecific affinity with molecular sertraline instead of ionic sertraline. Therefore, high pH value would be favorable for stereoselective separation for isomeric sertraline. A moderate value of distribution ratio could be obtained for both of cis and trans-isomers when pH was 9.6 and 10.6, which would be suitable for stereoselective separation of the four isomers by multistage continuous liquid-liquid separations, such as countercurrent cascade of centrifugal contactor separators and countercurrent chromatography. During the investigation of influence of pH value in distribution ratio and stereoselectivities, an unusual phenomenon was observed when different type of buffer solution at pH9.6 or pH10.6 was used. As is shown in Fig. 3, a variety of buffer solutions including borax-sodium carbonate buffer, sodium carbonate-sodium bicarbonate buffer, ammonium hydroxide-ammonium chloride buffer and glycine-sodium hydroxide buffer, each at pH9.6 and pH10.6, were tested. Very interestingly, a complete different stereoselective combination between HP-β -CD and isomeric sertraline was found when sodium carbonate was used in the buffer solution. As shown in Fig. 2, D (1S, 4R) were always smaller than D(1R, 4S) and D(1S, 4S) were always smaller than D(1R, 4R) , which indicated HPβ -CD preferentially combined with (1S, 4R)-sertraline or (1S, 4S)sertraline. However, as shown in Fig. 3, it was found that D(1S, 4R) were greater than D (1R, 4S) and D (1S, 4S) were greater than D(1R, 4R )
Table 2 Distribution ratio and stereoselectivity with different organic solvent. Organic solvent
D(1S, 4R)
D(1R, 4S)
α
n-hexane cyclohexane ethyl acetate n-heptane petroleum ether dichloromethane chloroform 1,2-dichloroethane
0.424 1.786 – 0.231 0.193 11.97 89.54 16.17
0.534 2.322 – 0.289 0.249 13.89 100.7 19.35
1.259 1.301 – 1.251 1.290 1.160 1.121 1.196
(trans)
D(1S, 4S)
D(1R, 4R)
α
1.314 5.520 – 0.735 0.652 16.67 143.7 36.50
1.705 7.221 – 0.952 0.844 17.19 178.1 45.19
1.298 1.308 – 1.295 1.292 1.031 1.234 1.238
(cis)
Aqueous phase: 0.1 mol L−1 of disodium hydrogen phosphate-citric acid at pH 7.6 dissolved with 50 mmol L−1 of HPβ -CD; organic phase: different organic solvent; equilibrium temperature: 10 °C. ‘–’ means that the distribution ratio were too large to be measured and ‘-’ indicated no stereoselectivity could be obtained.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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Fig. 2.. Influence of pH in distribution ratio and stereoselectivity. Organic phase: n-hexane; aqueous phase: buffer solution added with 50 mmol L−1 of HP-β -CD; equilibrium temperature: 10 °C. Buffer solution: 0.1 mol L−1 disodium hydrogen phosphate-citric acid pH4.6–7.6, 0.1 mol L−1 hydrogen phosphate-potassium dihydrogen phosphate pH8.6, 0.1 mol L−1 ammonium hydroxide-ammonium chloride pH9.6, 0.1 mol L−1 glycine-sodium hydroxide pH10.6 and 0.1 mol L−1 hydrogen phosphate-sodium hydroxide pH11.6.
when sodium carbonate was used in the buffer solutions, which indicated HP-β -CD preferentially combined with (1R, 4S)-sertraline or (1R, 4R)-sertraline when sodium carbonate buffer was used. This unusual phenomenon might be due to carbonate salt formation which could influence the inclusion complexes of the different enantiomers when a carbonate buffer is used. Meanwhile, different distribution ratios and stereoselectivities were obtained with different buffer types even when the same pH value was used, as indicated in Fig. 3. For instance, different distribution ratio and stereoselectivity were observed when borax-sodium carbonate pH9.6, sodium carbonate-sodium bicarbonate pH9.6 and ammonium hydroxide-ammonium chloride pH9.6 were used. Figs. 4(a), (b) and (a)’, (b)’ shows the HPLC chromatogram of stereoselective analysis of aqueous phase before and after stereoselective liquidliquid extraction of isomeric sertraline with HP-β -CD as extractant using the above different buffer. Big difference in peak area of each isomer between (1S, 4R)-sertraline and (1R, 4S)-sertraline was found. 3.1.2.2. Me-β -CD. As elucidated in Table 1, a high stereoselectivity of being 1.685 was obtained for trans-sertraline when Me-β CD was used as the extractant. So pH value of aqueous phase was
investigated for trans-sertraline using Me-β -CD as extractant. As shown in Fig. 5, pH 7.6–8.6 could be selected because a moderate distribution ratio and a high stereoselectivity for trans-sertraline could be achieved. Interestingly, when sodium carbonate-sodium bicarbonate buffer at pH 9.6 and 10.6 was used, no stereoselectivities was obtained for trans-sertraline, although a high stereoselectivity could still be obtained when glycine-sodium hydroxide buffer at pH 9.6 and 10.6 was used as is shown in Fig. 6. Figs. 7(a), (b) and (a)’, (b)’ shows the HPLC chromatogram of aqueous phase before and after stereoselective liquid-liquid extraction of trans-sertraline with Me-β -CD as extractant using different buffer. 3.1.3. Influence of concentration of cyclodextrin 3.1.3.1. HP-β -CD. HP-β -CD and isomeric sertraline could form diastereomeric complexes with different stabilities with regarding to different concentrations, which not only enhances the solubility of the stereoisomers in aqueous buffer, but also improves the stereoselectivity for isomeric sertraline. As shown in Fig. 2, high stereoselectivities for cis-sertraline were obtained when 0.10 mol L−1 glycine-sodium hydroxide pH10.6 and 0.10 mol L−1 hydrogen phosphate-sodium hydroxide pH 11.6 were used, while no much change of stereoselectivities was found for trans-sertraline, when
Fig. 3.. Influence of pH and type of buffer solution in distribution ratio and stereoselectivity. Organic phase: n-hexane; aqueous phase: buffer solution added with 50 mmol L−1 of HP-β -CD; equilibrium temperature: 10 °C.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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Fig. 4.. Chromatogram for stereoselective analyses of the aqueous phase before and after stereoselective liquid-liquid extraction of isomeric sertraline with HP-β -CD by reverse phase high performance liquid chromatography. (a) chromatogram for the aqueous phase before stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 glycine-sodium hydroxide pH10.6;(a’): chromatogram for the aqueous phase after stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 glycine-sodium hydroxide pH10.6;(b) chromatogram for the aqueous phase before stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6;(b’): chromatogram for the aqueous phase after stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6. Chromatographic condition was given in Section 2.5.
the above buffer was used. However, as shown in Fig. 3, stereoselectivity for trans-sertraline was greatly increased to 1.909 when sodium bicarbonate buffer at pH 10.6 was used with otherwise the same conditions. Therefore, high stereoselectivities for cis- and trans-sertraline were obtained when pH was over 10. So the influences of concentration of HP-β -CD in distribution ratio and stereoselectivity of cis- and trans-sertraline were investigated under pH 10.6, and it was shown in Fig. 8. With the increase of the concentration of HP-β -CD, the distribution ratios decreased greatly, which might be caused by the fact that a larger amount of inclusion complexes favorably formed in aqueous phase. Meanwhile, stereoselectivity increased to a maximum value when the concentration
reached 50 mmol L−1 . However, it was found that stereoselectivity of trans-sertraline were decreased greatly with increasing concentration of HP-β -CD. 3.1.3.2. Me-β -CD. As shown in Fig. 5, high stereoselectivity for trans-sertraline were obtained using Me-β -CD as extractant only when pH was less than 8. So the influences of concentration of Me-β -CD in distribution ratio and stereoselectivity of transsertraline were investigated when pH was 7.6. Fig. 9 shows the distribution ratio and stereoselectivity of trans-sertraline under different concentrations of Me-β -CD. With increasing concentrations of Me-β -CD, the distribution ratio significantly decreased while the
Fig. 5.. Influence of pH in distribution ratio and stereoselectivity for trans-isomer. Organic phase: n-hexane; aqueous phase: buffer solution added with 50 mmol L−1 of Meβ -CD; equilibrium temperature: 10 °C. Buffer solution: 0.1 mol/L disodium hydrogen phosphate-citric acid pH4.6–7.6, 0.1 mol L−1 hydrogen phosphate-potassium dihydrogen phosphate pH8.6, 0.1 mol L−1 ammonium hydroxide-ammonium chloride pH9.6, 0.1 mol L−1 glycine-sodium hydroxide pH10.6 and 0.1 mol L−1 hydrogen phosphate-sodium hydroxide pH11.6.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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Fig. 6.. Influence of pH and type of buffer solution in distribution ratio and stereoselectivity. Organic phase: n-hexane; aqueous phase: buffer solution added with 50 mmol L−1 of Me-β -CD; equilibrium temperature: 10 °C.
Fig. 7.. Chromatogram for stereoselective analyses of the aqueous phase before and after stereoselective liquid-liquid extraction of isomeric sertraline with Me-β -CD by reverse phase high performance liquid chromatography. (a) chromatogram for the aqueous phase before stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 glycine-sodium hydroxide pH10.6;(a’): chromatogram for the aqueous phase after stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 glycine-sodium hydroxide pH10.6;(b) chromatogram for the aqueous phase before stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6;(b’): chromatogram for the aqueous phase after stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6. Chromatographic condition was given in Section 2.5.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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Fig. 8.. Influence of concentration of HP-β -CD in distribution ratio and stereoselectivity for cis-sertraline and trans-sertraline. Organic phase: n-hexane; aqueous phase: 0.10 mol L−1 glycine-sodium hydroxide buffer at pH 10.6 for cis-sertraline and 0.10 mol L−1 sodium carbonate-sodium bicarbonate buffer at pH 10.6 for trans-sertraline; equilibrium temperature: 10 °C.
Fig. 9.. Influence of concentration of Me-β -CD in distribution ratio and stereoselectivity for trans-sertraline. Organic phase: n-hexane; aqueous phase: 0.10 mol L−1 disodium hydrogen phosphate-citric acid buffer at pH 7.6; equilibrium temperature: 10 °C.
stereoselectivity increased to a constant value when the concentration of Me-β -CD reached 50 mmol L−1 . Therefore, a concentration of 50 mmol L−1 Me-β -CD could be selected for stereoselective separation. 3.1.4. Influence of temperature 3.1.4.1. HP-β -CD. The effect of temperature on distribution ratio and stereoselectivity of trans-sertraline and cis-sertraline was shown in Table 3. An increase in distribution ratio was found with the increasing of temperature. It was noticed that no much change was found as for stereoselectivity of trans-isomer, while slight decrease was found as for that of cis-isomer when the temperature was increased from 5 °C to 40 °C. 3.1.4.2. Me-β -CD. Table 4 was the experimental results of the influence of temperature in distribution ratio and stereoselectivity for trans-sertraline with Me-β -CD as extractant. An increase of distribution ratio and a slight decrease of stereoselectivity were observed when the temperature was increased from 5 °C to 40 °C. The stereoselectivity, α , is correlated with the difference between the free energies of formation of inclusion complex between
isomeric sertraline and cyclodextrin by:
−i, j (G ) = RT ln
Di = RT ln α Dj
(7)
where i and j represent (1S, 4R) and (1R, 4S)-sertraline, or (1R, 4R) and (1S, 4S)-sertraline, respectively. R is the gas constant Table 3 Influence of temperature in distribution ratio and stereoselectivity with HP-β CD as extractant in the aqueous phase. Temperature ( °C)
D
5 10 15 20 25 30 40
3.766 4.489 5.038 6.731 7.397 8.102 10.97
(1S, 4R)
D
(1R, 4S)
1.587 1.939 2.098 2.784 3.221 3.337 4.523
α (trans)
D
2.373 2.316 2.401 2.417 2.296 2.428 2.427
18.39 18.55 20.28 22.22 22.44 24.25 31.79
(1S, 4S)
D
(1R, 4R)
25.83 26.01 27.25 30.03 30.59 33.06 44.63
α (cis) 1.404 1.402 1.343 1.351 1.363 1.363 1.404
Organic phase: n-hexane; aqueous phase: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer at pH 10.6 containing 5 mmol L−1 of HP-β -CD for transisomer and 0.10 mol L−1 glycine-sodium hydroxide buffer solution at pH 10.6 containing 50 mmol L−1 of HP-β -CD for cis-isomer.
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indicated that inclusion complex formed by Me-β -CD and transsertraline was enthalpy driven process.
Table 4 Influence of temperature in distribution ratio and stereoselectivity with Me-β -CD as extractant. Temperature( °C)
D
5 10 15 20 25 30 40
0.157 0.200 0.245 0.320 0.435 0.470 0.677
(1S, 4R)
D
(1R, 4S)
0.262 0.337 0.388 0.509 0.674 0.711 0.952
α (trans)
3.2. Stereoselective separation by countercurrent chromatography
1.669 1.685 1.616 1.592 1.548 1.513 1.406
Organic phase: n-hexane; aqueous phase: 0.10 mol L−1 disodium hydrogen phosphate-citric acid buffer at pH 7.6 containing 50 mmol L−1 of Me-β -CD.
(R = 8.3143 J K−1 mol−1 ) and T is thermodynamic temperature in K. combination of (7) with the Gibbs-Helmholz equation could give the following equation:
ln α =
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−i, j (H ) i, j (S ) + RT R
(8)
where i,j (H) and i,j (S) are the differences in enthalpy and entropy of complex formation between stereoisomers, which could be determined by plotting lnα with 1/T. A straight line could be obtained if no change was happened for the separation mechanism [22-25]. No straight line was achieved for stereoisomeric sertraline in the range of 5–40 °C when HP-β -CD was used as extractant, which indicated the combination of HP-β -CD and stereoisomeric sertraline did not fit with the Van’t Hoff model. However, a straight line could be obtained when Me-β -CD used as extractant: y = 438.67x - 1.0419, R2 =0.944. i,j (H),i,j (S) and i,j (G) were determined from the regress analysis along with Gibbs-Helmholz equation: −3198.59 J mol−1 , −7.11 J K−1 mol−1 and −1079.10 J mol−1 (T = 278.1 K). Calculation of thermodynamic parameters
The results of stereoselective liquid-liquid extraction of four isomeric sertraline provided an optimized separation conditions for trans-sertraline and cis-sertraline with high stereoselectivity. On the one hand, two biphasic solvent systems could be used for separation trans-sertraline: system (1): n-hexane : 0.10 mol L−1 of sodium carbonate-sodium bicarbonate buffer at pH 10.6 containing 5 mmol L−1 of HP-β -CD (1:1, v/v); system (2): n-hexane : 0.10 mol L−1 of disodium hydrogen phosphate-citric acid buffer at pH 7.6 containing 50 mmol L−1 of Me-β -CD (1:1, v/v). The distribution ratios of trans-sertraline, (1S, 4R) and (1R, 4S)-isomer, were 3.766 and 1.587, 0.200 and 0.337, and the stereoselectivities of transsertraline, (1R, 4S) and (1S, 4R)-isomer, reached 2.373 and 1.685 with the above two systems, respectively. On the other hand, only HP-β -CD could be selected for separation of cis-sertraline, (1S, 4S) and (1R, 4R)-isomer. A biphasic solvent system composed of nhexane : 0.10 mol L−1 of glycine-sodium hydroxide buffer at pH 10.6 containing 50 mmol L−1 of HP-β -CD (1:1, v/v) provided high stereoselectivity for (1S, 4S) and (1R, 4R)-isomer. The distribution ratios of cis-sertraline, (1S, 4S) and (1R, 4R)-isomer, were 18.30 and 25.83, and the stereoselectivity reached 1.404 using the above system. With the above selected solvent systems, an analytical countercurrent chromatographic apparatus was used to separate stereoisomeric sertraline. It was found that very long retention time of the isomeric sertraline was always involved if pH were greater than 9.0 due to large distribution ratios, even though a high stereoselectivities were obtained with high pH value. An acceptable retention time was obtained for isomeric sertraline only when the pH value was between7.0 and 8.0. Fig. 10 shows the chromatogram
Fig. 10.. Chromatogram for stereoselective separation of isomeric sertraline by analytical countercurrent chromatography. The two-phase solvent system: n-hexane : 0.10 mol L−1 of disodium hydrogen phosphate-citric acid buffer pH 7.6 containing 50 mmol L−1 of Me-β -CD (1:1, v/v); flow rate: 0.5 ml min−1 ; revolution: 1120 rpm; column temperature: 25 °C; stationary phase retention: 65.3%.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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for stereoselective separation of isomeric sertraline with system (2) by analytical countercurrent chromatography. A baseline separation of trans-sertraline and cis-sertraline could be obtained. Meanwhile, (1S, 4R) and (1R, 4S)-sertraline were completely separated with high peak resolution using Me-β -CD as stereoselective selector. The analysis of HPLC showed that (1S, 4R)-sertraline of 98.7% purity, (1R, 4S)-sertraline of 98.2% purity and cis-sertraline of 99.1% purity were obtained. Compared with our previous work [19], peak resolution for cis- and trans-sertraline was increased from 0.91 to 1.56. Meanwhile, separation for trans-isomers, (1S, 4R) and (1R, 4S)sertraline, was achieved. 4. Conclusion Stereoselective liquid-liquid extraction of four isomeric sertraline was investigated with HP-β -CD and Me-β -CD as extractant, respectively. A complete different stereoselective combination between HP-β -CD and isomeric sertraline was observed when sodium carbonate was used in the buffer solution. The stereoselectivity of trans-sertraline was greatly improved when sodium carbonate was used in the buffer solution. The stereoselectivity of trans-sertraline could reach 2.373 and 1.685, when the solvent system were composed of n-hexane and 0.10 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6 containing 5 mmol L−1 HP-β -CD and disodium hydrogen phosphate-citric acid buffer pH 7.6 containing 50 mmol L−1 of Me-β -CD, respectively. The stereoselectivity of cis-sertraline could reach 1.404 when the solvent system composed of n-hexane : 0.10 mol L-1 of glycinesodium hydroxide buffer at pH 10.6 containing 50 mmol L−1 of HP-β -CD (1:1, v/v) was used. Successful stereoselective separation of cis-sertraline and trans-sertraline by countercurrent chromatography using the above systems was only obtained when pH was in the range of 7.0–8.0 using Me-β -CD as stereoselective selector. An biphasic solvent system composed of n-hexane and 0.10 mol L−1 of disodium hydrogen phosphate-citric acid buffer at pH 7.6 containing 50 mmol L−1 of Me-β -CD (1:1, v/v) was feasible for successful separation of trans-sertraline and cis-sertraline, and separation of (1R, 4S) and (1S, 4R)-isomer. Complete separation trans-sertraline and cis-sertraline, (1R, 4S) and (1S, 4R)-sertraline by countercurrent chromatography with high peak resolution was achieved using Meβ -CD as stereoselective selector. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was financially supported by National Natural Science Foundation of China (21978266). References
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Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography Wenyu Sun, Yang Jin, Chaoyue Wang, Shanshan Zhao, Xiang Wang, Meng Luo, Jizhong Yan, Shengqiang Tong∗ College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou 310032, China
a r t i c l e
i n f o
Article history: Received 24 August 2019 Revised 23 December 2019 Accepted 29 December 2019 Available online xxx Keywords: Countercurrent chromatography Hydroxypropyl-β -cyclodextrin Isomeric sertraline Methyl-β -cyclodextrin Stereoselective liquid-liquid extraction
a b s t r a c t Sertraline is an antidepressant in a group of drugs called selective serotonin reuptake inhibitors. Four stereoisomeric compounds would be produced in its synthetic preparation due to two chiral carbons on its chemical structures. In the present work, stereoselective liquid-liquid extraction of isomeric sertraline with substituted cyclodextrins as stereoselective extractant was investigated. Factors affecting the distribution performance, including organic solvents, types of extractants, pH value, buffer solution of aqueous phase, concentration of extractant and temperature, were investigated. Under optimized conditions, a stereoselectivity of 1.404 was obtained for cis-sertraline and a stereoselectivity of 2.373 was obtained for trans-sertraline when hydroxypropyl-β -cyclodextrin was used as the stereoselective extractant, and a stereoselectivity of 1.685 was achieved for trans-sertraline with methyl-β -cyclodextrin as extractant. An unusual stereoselective combination was observed for trans-sertraline and cis-sertraline when sodium carbonate buffer was used. Successful stereoselective separation of trans-sertraline and cis-sertraline, (1S, 4R) and (1R, 4S)-sertraline by analytical countercurrent chromatography with methyl-β -cyclodextrin as stereoselective selector was achieved, using a biphasic solvent system composed of n-hexane : 0.1 mol L−1 citrate buffer solution with pH7.6 (1:1, v/v). © 2019 Elsevier B.V. All rights reserved.
1. Introduction With the increasing demand for chiral compounds, especially chiral drugs, the separation technology of chiral substances has become one of the most interest subjects over the past decades [1-5]. Chiral separations are mainly conducted by mechanical, chemical, biochemical, chromatographic, extractive and membranous methods. Enantioselective liquid-liquid extraction (ELLE) is considered to be a promising technology for chiral separation, which combines the concepts of solvent extraction and chiral recognition in a single technology [6]. It has the advantage of lower cost in energy consumption, easier to scale up to commercial scale, and a wide range of application. In the past decades, increasing number of papers with regarding to ELLE has been published, and almost all the published papers are about chiral separation of racemic compound with one chiral center in its chemical structure or enantioseparation of optical isomers [7-11]. Various chiral extractants have been studied in ELLE [12-16]. The main extractant classes are as followings: substituted cyclodextrins, crown ether based ∗ Corresponding author at: College of Pharmaceutical Science, Zhejiang University of Technology, Chaowang Road 18, Chaohui No. 6 District, Hangzhou, China. E-mail address: [email protected] (S. Tong).
extractants, metal complexes and metalloids, extractants based on tartrates, and other types of chiral extractants. ELLE is easy to be scaled up using a countercurrent cascade of centrifugal contactor separators or modern liquid-liquid partition chromatography for preparative chiral separation. To the best of our knowledge, few literatures were available with regarding to stereoselective liquidliquid extraction of drugs with two chiral centers in its chemical structure, yielding four stereoisomers. Sertraline hydrochloride, cis-(1S,4S)-N-methyl-4-(3, 4dichlorophenyl)−1, 2, 3, 4-tetrahydro-1-naphthalenamine hydrochloride, is a selective inhibitor of central serotonin reuptake and it is used as an antidepressant for oral administration [17]. Four stereoisomeric compounds, including (1S, 4R), (1R, 4S), (1S, 4S) and (1R, 4R)-isomer, were produced during its synthesis due to two chiral carbon centers in its chemical structure (Fig. 1). It has been proved that only (1S, 4S)-sertraline owns intended pharmacological activity due to stereospecific combination with receptor in the body [18]. In our very recent work, stereoselective separation of (1S, 4S)-sertraline from its medicinal reaction mixtures by countercurrent chromatography using hydroxypropyl-β -cyclodextrin as the stereoselective selector was investigated, in which a fabricated in-house analytical countercurrent chromatographic apparatus was used for optimization of the separation conditions, and two
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Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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2.4. Stereoselective liquid-liquid extraction procedures
Fig. 1.. Chemical structure of sertraline hydrochloride.
cis-isomers were successfully separated [19]. However, no success of stereoselective separation of trans-isomers was achieved. Meanwhile, in order to further investigate the distribution behavior and stereoselectivity of stereoisomeric sertraline with substituted β cyclodextrin as stereoselective selector, stereoselective liquid-liquid extraction of the four isomeric sertraline using substituted β cyclodextrins as extractant was investigated in our present work, in which an interesting phenomenon was found when different type of buffer solutions were used. Furthermore, stereoselective separation of trans-sertraline and cis-sertraline, (1S, 4R) and (1R, 4S)-sertraline by analytical countercurrent chromatography with methyl-β -cyclodextrin as stereoselective selector was achieved. 2. Experimental methods 2.1. Apparatus The high-performance liquid chromatography (HPLC) was a Shimadzu HPLC LC-solution system comprised of a SCL-10Avp controller, a Shimadzu LC-10Avp UV detector, a Shimadzu LC-10ATvp multi solvent delivery system and a Shimadzu LC pump. The pH value for the test was determined with a PB-10 pH meter (Sartorius, Germany). A fabricated in-house analytical type-J counter-current chromatographic apparatus with a total capacity of 15 ml was used [19]. The solvents were pumped into the column with a model TBP 5002 constant-flow pump (ShanghaiTauto Biotechnique, Shanghai, China). A model UVD-200 detector (Shanghai Jinda Biotechnology Co., Ltd., Shanghai, China) was used for continuous monitoring of the effluent and the chromatogram was recorded with a SEPU30 0 0 workstation (Hangzhou Puhui Technology, Hangzhou, China). 2.2. Materials Hydroxypropyl-β -cyclodextrin (HP-β -CD), methyl-β -cyclodextrin (Me-β -CD) and sulfobutylether-β -cyclodextrin (SBE-β CD) were purchased from Qianhui Fine Chemical, Shandong, China. Hexanol, octanol, n-Hexane, n-heptane, petroleum ether, dichloromethane, chloroform, 1, 2-dichloroethane, cyclohexane, ethyl acetate were purchased from Macklin Biochemical Co. Ltd, Shandong, China. Dibasic sodium phosphate, citric acid, sodium carbonate, sodium bicarbonate, glycine, sodium hydroxide, ammonia, ammonium chloride were purchased from Huipu Chemical, Hangzhou, China. Acetonitrile used for HPLC analysis was of chromatographic grade. Water was redistilled. 2.3. Preparation of sertraline hydrochloride The preparation of isomeric sertraline hydrochloride was according to the literature [20], and experimental section in detail could be found in our previous work [19]. The sample of isomeric sertraline was composed of equal amount of cis-crude product and trans-crude product synthesized in our lab.
HP-β -CD, Me-β -CD and SBE-β -CD were used as the extractants in aqueous phase. Aqueous phases were prepared by dissolving substituted β -CD and 1 mg mL−1 of isomeric sertraline hydrochloride in 0.10 mol L−1 buffer solution. Liquid-liquid extraction was performed in a 10 ml glass-stoppered tube. Equal volumes (each 2 ml) of the aqueous and the organic phase were placed together, and shaken sufficiently (5 min) before being kept in a water bath for 1 h at a fixed temperature to reach equilibrium. The investigated temperature was in the range of 5–40 °C. After phase separation, the concentrations of isomeric sertraline in the aqueous phase were determined by HPLC. Each experiment was duplicated under identical conditions. The concentrations of sertraline isomers in organic phase were calculated by subtractive method. The distribution ratio and stereoselectivity are defined as the followings:
D(1S,4R) =
CO,SR CW,SR
(1)
D(1R,4S ) =
CO,RS CO,RS
(2)
D(1S,4S ) =
CO,SS CW,SS
(3)
D(1R,4R) =
CO,RR CW,RR
(4)
α(trans) =
D(1S,4R ) D(1R,4S ) Or D(1R,4S ) D(1S,4R )
(5)
where D(1S, 4R) ≥ D(1R, 4S) or D(1R, 4S) ≥ D(1S, 4R)
α(cis) =
D(1S,4S ) D(1R,4R ) Or D(1R,4R ) D(1S,4S )
(6)
where D(1S, 4S) ≥ D(1R, 4R) or D(1R, 4R) ≥ D(1S, 4S) , where CO,SR and CW,SR respectively represent concentration of (1S, 4R)-sertraline in organic phase and aqueous phase; CO,RS and CW, RS respectively represent concentration of (1R, 4S)-sertraline in organic phase and aqueous phase; CO,SS and CW,SS respectively represent concentration of (1S, 4S)-sertraline in organic phase and aqueous phase; CO,RR and CW,RR respectively represent concentration of (1R, 4R)sertraline in organic phase and aqueous phase. 2.5. Countercurrent chromatographic separation Solvent systems consisting of n-hexane : 0.10 mol L−1 of disodium hydrogen phosphate-citric acid buffer pH 7.6 containing 50 mmol L−1 of Me-β -CD (1:1, v/v) were used. The aqueous buffer solution with pH 7.6 was prepared before adding 50 mmol L−1 Meβ -CD. Then the solvent mixture was mixed and thoroughly equilibrated in a separatory funnel, the saturated aqueous phase and organic phase were separated shortly and degassed by ultrasound for 30 min before use. The sample solution was prepared by dissolving 1 mg of isomeric sertraline into 0.5 mL the aqueous mobile phase for countercurrent chromatographic separation. The separation was initiated by entirely filling the column with the organic stationary phase. The aqueous mobile phase was pumped into the column at a flow rate 0.5 mL min−1 while the apparatus was rotated at 1120 rpm. Subsequently, the sample solution was injected after observing a clear elution of mobile phase at the tail outlet, which indicated hydrodynamic equilibrium was established in the column.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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W. Sun, Y. Jin and C. Wang et al. / Journal of Chromatography A xxx (xxxx) xxx Table 1 Influence of different extractant in distribution ratio and stereoselectivity. Extractant
D(1S, 4R)
D(1R, 4S)
α
HP-β -CD Me-β -CD SBE-β -CD
0.424 0.200 0.109
0.534 0.337 0.110
1.259 1.685 1.010
(trans)
D(1S, 4S)
D(1R, 4R)
α (cis)
1.314 0.980 0.173
1.705 0.989 0.192
1.298 1.010 1.110
Aqueous phase: 0.1 mol L−1 of disodium hydrogen phosphate-citric acid at pH=7.6; organic phase: n-hexane; equilibrium temperature: 10 °C; Concentration of extractant: 50 mmol L−1 of cyclodextrin in the aqueous phase; equilibrium temperature: 10 °C.
2.6. Analytical method The quantification of isomeric sertraline was performed by HPLC using a UV detector set at 225 nm with an YMC-Pack ODS-A column (150×4.6 mm i.d., 5 μm) according to reference [21]. The mobile phase was 20 mmol L−1 of HP-β -CD aqueous buffer solution (pH 3.0, adjusted with phosphoric acid) : acetonitrile (78:22, v/v) at a flow rate of 0.8 mL min−1 , the column temperature was 35 °C. 3. Results and discussion 3.1. Stereoselective liquid-liquid extractions 3.1.1. Selection of chiral extractants and organic solvent It was reported that complete separation of four isomers by liquid chromatography with HP-β -CD as mobile phase additive could be achieved [21]. Therefore, three substituted β -cyclodextrins, HPβ -CD, Me-β -CD and SBE-β -CD were investigated for stereoselective liquid-liquid extraction of the four isomeric sertraline in light of the above reported results. Substituted β -cyclodextrins were highly hydrophilic and were added in the aqueous phase. Distribution ratio and stereoselectivity for isomeric sertraline were investigated in several different liquid-liquid extraction systems containing 50 mmol L−1 substituted β -CD in aqueous phase. As shown in Table 1, very limited stereoselectivity was obtained for trans-sertraline and cis-sertraline when SBE-β -CD was used as stereoselective selector. It was found that high stereoselectivity, α =1.685, was obtained for trans-sertraline but low stereoselectivity, α =1.010, was obtained for cis-sertraline when Me-β -CD was used as stereoselective selector. Meanwhile, relatively high stereoselectivities for both trans-sertraline and cis-sertraline, α =1.259 and α =1.298, were obtained with moderate distribution ratio when HP-β -CD was used as stereoselective selector under pH7.6. As shown in Table 1, D (1R, 4S ) were found to be always larger than D(1S, 4R) , and D(1R, 4R) were always larger than D(1S, 4S) , which indicated that HP-β -CD and Me-β -CD preferentially recognized (1S, 4R)-sertraline and (1S, 4S)-sertraline in trans-sertraline and cissertraline, respectively. HP-β -CD could be chosen as the suitable
3
chiral extractant for both cis-sertraline and trans-sertraline, while Me-β -CD could be selected as the suitable chiral extractant for trans-sertraline because of the higher stereoselectivities. The influence of organic solvents on distribution behavior was investigated using HP-β -CD as extractant, as shown in Table 2. Very low stereoselectivities were found when dichloromethane, chloroform and 1,2-dichloroethane were used, while a moderate distribution ratio and a high stereoselectivity were obtained when n-hexane, cyclohexane, n-heptane and petroleum ether was used as the organic solvent, which indicated n-hexane, cyclohexane, nheptane and petroleum ether could be selected for stereoselective liquid-liquid extraction. Distribution ratio for all the stereoisomers were too large to be tested when ethyl acetate was used as the organic solvent, and so ethyl acetate was not applied in the present work. 3.1.2. Influence of pH and buffer solution 3.1.2.1. HP-β -CD. Sertraline is a kind of organic base in light of its chemical structure. So its distribution behavior in the biphasic solvent system would be greatly influenced by pH value of aqueous phase. Meanwhile, inclusion complex formed by isomeric sertraline and cyclodextrin might be greatly affected by its status in the aqueous phase. Fig. 2 shows the influence of pH on the stereoselective separation of four isomeric sertraline with HP-β -CD as extractant. As is shown in Fig. 2, both of the distribution ratio and stereoselectivity increased with the increasing of pH value, which might be caused by the fact that HP-β -CD would mainly have stereospecific affinity with molecular sertraline instead of ionic sertraline. Therefore, high pH value would be favorable for stereoselective separation for isomeric sertraline. A moderate value of distribution ratio could be obtained for both of cis and trans-isomers when pH was 9.6 and 10.6, which would be suitable for stereoselective separation of the four isomers by multistage continuous liquid-liquid separations, such as countercurrent cascade of centrifugal contactor separators and countercurrent chromatography. During the investigation of influence of pH value in distribution ratio and stereoselectivities, an unusual phenomenon was observed when different type of buffer solution at pH9.6 or pH10.6 was used. As is shown in Fig. 3, a variety of buffer solutions including borax-sodium carbonate buffer, sodium carbonate-sodium bicarbonate buffer, ammonium hydroxide-ammonium chloride buffer and glycine-sodium hydroxide buffer, each at pH9.6 and pH10.6, were tested. Very interestingly, a complete different stereoselective combination between HP-β -CD and isomeric sertraline was found when sodium carbonate was used in the buffer solution. As shown in Fig. 2, D (1S, 4R) were always smaller than D(1R, 4S) and D(1S, 4S) were always smaller than D(1R, 4R) , which indicated HPβ -CD preferentially combined with (1S, 4R)-sertraline or (1S, 4S)sertraline. However, as shown in Fig. 3, it was found that D(1S, 4R) were greater than D (1R, 4S) and D (1S, 4S) were greater than D(1R, 4R )
Table 2 Distribution ratio and stereoselectivity with different organic solvent. Organic solvent
D(1S, 4R)
D(1R, 4S)
α
n-hexane cyclohexane ethyl acetate n-heptane petroleum ether dichloromethane chloroform 1,2-dichloroethane
0.424 1.786 – 0.231 0.193 11.97 89.54 16.17
0.534 2.322 – 0.289 0.249 13.89 100.7 19.35
1.259 1.301 – 1.251 1.290 1.160 1.121 1.196
(trans)
D(1S, 4S)
D(1R, 4R)
α
1.314 5.520 – 0.735 0.652 16.67 143.7 36.50
1.705 7.221 – 0.952 0.844 17.19 178.1 45.19
1.298 1.308 – 1.295 1.292 1.031 1.234 1.238
(cis)
Aqueous phase: 0.1 mol L−1 of disodium hydrogen phosphate-citric acid at pH 7.6 dissolved with 50 mmol L−1 of HPβ -CD; organic phase: different organic solvent; equilibrium temperature: 10 °C. ‘–’ means that the distribution ratio were too large to be measured and ‘-’ indicated no stereoselectivity could be obtained.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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Fig. 2.. Influence of pH in distribution ratio and stereoselectivity. Organic phase: n-hexane; aqueous phase: buffer solution added with 50 mmol L−1 of HP-β -CD; equilibrium temperature: 10 °C. Buffer solution: 0.1 mol L−1 disodium hydrogen phosphate-citric acid pH4.6–7.6, 0.1 mol L−1 hydrogen phosphate-potassium dihydrogen phosphate pH8.6, 0.1 mol L−1 ammonium hydroxide-ammonium chloride pH9.6, 0.1 mol L−1 glycine-sodium hydroxide pH10.6 and 0.1 mol L−1 hydrogen phosphate-sodium hydroxide pH11.6.
when sodium carbonate was used in the buffer solutions, which indicated HP-β -CD preferentially combined with (1R, 4S)-sertraline or (1R, 4R)-sertraline when sodium carbonate buffer was used. This unusual phenomenon might be due to carbonate salt formation which could influence the inclusion complexes of the different enantiomers when a carbonate buffer is used. Meanwhile, different distribution ratios and stereoselectivities were obtained with different buffer types even when the same pH value was used, as indicated in Fig. 3. For instance, different distribution ratio and stereoselectivity were observed when borax-sodium carbonate pH9.6, sodium carbonate-sodium bicarbonate pH9.6 and ammonium hydroxide-ammonium chloride pH9.6 were used. Figs. 4(a), (b) and (a)’, (b)’ shows the HPLC chromatogram of stereoselective analysis of aqueous phase before and after stereoselective liquidliquid extraction of isomeric sertraline with HP-β -CD as extractant using the above different buffer. Big difference in peak area of each isomer between (1S, 4R)-sertraline and (1R, 4S)-sertraline was found. 3.1.2.2. Me-β -CD. As elucidated in Table 1, a high stereoselectivity of being 1.685 was obtained for trans-sertraline when Me-β CD was used as the extractant. So pH value of aqueous phase was
investigated for trans-sertraline using Me-β -CD as extractant. As shown in Fig. 5, pH 7.6–8.6 could be selected because a moderate distribution ratio and a high stereoselectivity for trans-sertraline could be achieved. Interestingly, when sodium carbonate-sodium bicarbonate buffer at pH 9.6 and 10.6 was used, no stereoselectivities was obtained for trans-sertraline, although a high stereoselectivity could still be obtained when glycine-sodium hydroxide buffer at pH 9.6 and 10.6 was used as is shown in Fig. 6. Figs. 7(a), (b) and (a)’, (b)’ shows the HPLC chromatogram of aqueous phase before and after stereoselective liquid-liquid extraction of trans-sertraline with Me-β -CD as extractant using different buffer. 3.1.3. Influence of concentration of cyclodextrin 3.1.3.1. HP-β -CD. HP-β -CD and isomeric sertraline could form diastereomeric complexes with different stabilities with regarding to different concentrations, which not only enhances the solubility of the stereoisomers in aqueous buffer, but also improves the stereoselectivity for isomeric sertraline. As shown in Fig. 2, high stereoselectivities for cis-sertraline were obtained when 0.10 mol L−1 glycine-sodium hydroxide pH10.6 and 0.10 mol L−1 hydrogen phosphate-sodium hydroxide pH 11.6 were used, while no much change of stereoselectivities was found for trans-sertraline, when
Fig. 3.. Influence of pH and type of buffer solution in distribution ratio and stereoselectivity. Organic phase: n-hexane; aqueous phase: buffer solution added with 50 mmol L−1 of HP-β -CD; equilibrium temperature: 10 °C.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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Fig. 4.. Chromatogram for stereoselective analyses of the aqueous phase before and after stereoselective liquid-liquid extraction of isomeric sertraline with HP-β -CD by reverse phase high performance liquid chromatography. (a) chromatogram for the aqueous phase before stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 glycine-sodium hydroxide pH10.6;(a’): chromatogram for the aqueous phase after stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 glycine-sodium hydroxide pH10.6;(b) chromatogram for the aqueous phase before stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6;(b’): chromatogram for the aqueous phase after stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6. Chromatographic condition was given in Section 2.5.
the above buffer was used. However, as shown in Fig. 3, stereoselectivity for trans-sertraline was greatly increased to 1.909 when sodium bicarbonate buffer at pH 10.6 was used with otherwise the same conditions. Therefore, high stereoselectivities for cis- and trans-sertraline were obtained when pH was over 10. So the influences of concentration of HP-β -CD in distribution ratio and stereoselectivity of cis- and trans-sertraline were investigated under pH 10.6, and it was shown in Fig. 8. With the increase of the concentration of HP-β -CD, the distribution ratios decreased greatly, which might be caused by the fact that a larger amount of inclusion complexes favorably formed in aqueous phase. Meanwhile, stereoselectivity increased to a maximum value when the concentration
reached 50 mmol L−1 . However, it was found that stereoselectivity of trans-sertraline were decreased greatly with increasing concentration of HP-β -CD. 3.1.3.2. Me-β -CD. As shown in Fig. 5, high stereoselectivity for trans-sertraline were obtained using Me-β -CD as extractant only when pH was less than 8. So the influences of concentration of Me-β -CD in distribution ratio and stereoselectivity of transsertraline were investigated when pH was 7.6. Fig. 9 shows the distribution ratio and stereoselectivity of trans-sertraline under different concentrations of Me-β -CD. With increasing concentrations of Me-β -CD, the distribution ratio significantly decreased while the
Fig. 5.. Influence of pH in distribution ratio and stereoselectivity for trans-isomer. Organic phase: n-hexane; aqueous phase: buffer solution added with 50 mmol L−1 of Meβ -CD; equilibrium temperature: 10 °C. Buffer solution: 0.1 mol/L disodium hydrogen phosphate-citric acid pH4.6–7.6, 0.1 mol L−1 hydrogen phosphate-potassium dihydrogen phosphate pH8.6, 0.1 mol L−1 ammonium hydroxide-ammonium chloride pH9.6, 0.1 mol L−1 glycine-sodium hydroxide pH10.6 and 0.1 mol L−1 hydrogen phosphate-sodium hydroxide pH11.6.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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Fig. 6.. Influence of pH and type of buffer solution in distribution ratio and stereoselectivity. Organic phase: n-hexane; aqueous phase: buffer solution added with 50 mmol L−1 of Me-β -CD; equilibrium temperature: 10 °C.
Fig. 7.. Chromatogram for stereoselective analyses of the aqueous phase before and after stereoselective liquid-liquid extraction of isomeric sertraline with Me-β -CD by reverse phase high performance liquid chromatography. (a) chromatogram for the aqueous phase before stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 glycine-sodium hydroxide pH10.6;(a’): chromatogram for the aqueous phase after stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 glycine-sodium hydroxide pH10.6;(b) chromatogram for the aqueous phase before stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6;(b’): chromatogram for the aqueous phase after stereoselective liquid-liquid extraction, buffer solution: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6. Chromatographic condition was given in Section 2.5.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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Fig. 8.. Influence of concentration of HP-β -CD in distribution ratio and stereoselectivity for cis-sertraline and trans-sertraline. Organic phase: n-hexane; aqueous phase: 0.10 mol L−1 glycine-sodium hydroxide buffer at pH 10.6 for cis-sertraline and 0.10 mol L−1 sodium carbonate-sodium bicarbonate buffer at pH 10.6 for trans-sertraline; equilibrium temperature: 10 °C.
Fig. 9.. Influence of concentration of Me-β -CD in distribution ratio and stereoselectivity for trans-sertraline. Organic phase: n-hexane; aqueous phase: 0.10 mol L−1 disodium hydrogen phosphate-citric acid buffer at pH 7.6; equilibrium temperature: 10 °C.
stereoselectivity increased to a constant value when the concentration of Me-β -CD reached 50 mmol L−1 . Therefore, a concentration of 50 mmol L−1 Me-β -CD could be selected for stereoselective separation. 3.1.4. Influence of temperature 3.1.4.1. HP-β -CD. The effect of temperature on distribution ratio and stereoselectivity of trans-sertraline and cis-sertraline was shown in Table 3. An increase in distribution ratio was found with the increasing of temperature. It was noticed that no much change was found as for stereoselectivity of trans-isomer, while slight decrease was found as for that of cis-isomer when the temperature was increased from 5 °C to 40 °C. 3.1.4.2. Me-β -CD. Table 4 was the experimental results of the influence of temperature in distribution ratio and stereoselectivity for trans-sertraline with Me-β -CD as extractant. An increase of distribution ratio and a slight decrease of stereoselectivity were observed when the temperature was increased from 5 °C to 40 °C. The stereoselectivity, α , is correlated with the difference between the free energies of formation of inclusion complex between
isomeric sertraline and cyclodextrin by:
−i, j (G ) = RT ln
Di = RT ln α Dj
(7)
where i and j represent (1S, 4R) and (1R, 4S)-sertraline, or (1R, 4R) and (1S, 4S)-sertraline, respectively. R is the gas constant Table 3 Influence of temperature in distribution ratio and stereoselectivity with HP-β CD as extractant in the aqueous phase. Temperature ( °C)
D
5 10 15 20 25 30 40
3.766 4.489 5.038 6.731 7.397 8.102 10.97
(1S, 4R)
D
(1R, 4S)
1.587 1.939 2.098 2.784 3.221 3.337 4.523
α (trans)
D
2.373 2.316 2.401 2.417 2.296 2.428 2.427
18.39 18.55 20.28 22.22 22.44 24.25 31.79
(1S, 4S)
D
(1R, 4R)
25.83 26.01 27.25 30.03 30.59 33.06 44.63
α (cis) 1.404 1.402 1.343 1.351 1.363 1.363 1.404
Organic phase: n-hexane; aqueous phase: 0.1 mol L−1 sodium carbonate-sodium bicarbonate buffer at pH 10.6 containing 5 mmol L−1 of HP-β -CD for transisomer and 0.10 mol L−1 glycine-sodium hydroxide buffer solution at pH 10.6 containing 50 mmol L−1 of HP-β -CD for cis-isomer.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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indicated that inclusion complex formed by Me-β -CD and transsertraline was enthalpy driven process.
Table 4 Influence of temperature in distribution ratio and stereoselectivity with Me-β -CD as extractant. Temperature( °C)
D
5 10 15 20 25 30 40
0.157 0.200 0.245 0.320 0.435 0.470 0.677
(1S, 4R)
D
(1R, 4S)
0.262 0.337 0.388 0.509 0.674 0.711 0.952
α (trans)
3.2. Stereoselective separation by countercurrent chromatography
1.669 1.685 1.616 1.592 1.548 1.513 1.406
Organic phase: n-hexane; aqueous phase: 0.10 mol L−1 disodium hydrogen phosphate-citric acid buffer at pH 7.6 containing 50 mmol L−1 of Me-β -CD.
(R = 8.3143 J K−1 mol−1 ) and T is thermodynamic temperature in K. combination of (7) with the Gibbs-Helmholz equation could give the following equation:
ln α =
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−i, j (H ) i, j (S ) + RT R
(8)
where i,j (H) and i,j (S) are the differences in enthalpy and entropy of complex formation between stereoisomers, which could be determined by plotting lnα with 1/T. A straight line could be obtained if no change was happened for the separation mechanism [22-25]. No straight line was achieved for stereoisomeric sertraline in the range of 5–40 °C when HP-β -CD was used as extractant, which indicated the combination of HP-β -CD and stereoisomeric sertraline did not fit with the Van’t Hoff model. However, a straight line could be obtained when Me-β -CD used as extractant: y = 438.67x - 1.0419, R2 =0.944. i,j (H),i,j (S) and i,j (G) were determined from the regress analysis along with Gibbs-Helmholz equation: −3198.59 J mol−1 , −7.11 J K−1 mol−1 and −1079.10 J mol−1 (T = 278.1 K). Calculation of thermodynamic parameters
The results of stereoselective liquid-liquid extraction of four isomeric sertraline provided an optimized separation conditions for trans-sertraline and cis-sertraline with high stereoselectivity. On the one hand, two biphasic solvent systems could be used for separation trans-sertraline: system (1): n-hexane : 0.10 mol L−1 of sodium carbonate-sodium bicarbonate buffer at pH 10.6 containing 5 mmol L−1 of HP-β -CD (1:1, v/v); system (2): n-hexane : 0.10 mol L−1 of disodium hydrogen phosphate-citric acid buffer at pH 7.6 containing 50 mmol L−1 of Me-β -CD (1:1, v/v). The distribution ratios of trans-sertraline, (1S, 4R) and (1R, 4S)-isomer, were 3.766 and 1.587, 0.200 and 0.337, and the stereoselectivities of transsertraline, (1R, 4S) and (1S, 4R)-isomer, reached 2.373 and 1.685 with the above two systems, respectively. On the other hand, only HP-β -CD could be selected for separation of cis-sertraline, (1S, 4S) and (1R, 4R)-isomer. A biphasic solvent system composed of nhexane : 0.10 mol L−1 of glycine-sodium hydroxide buffer at pH 10.6 containing 50 mmol L−1 of HP-β -CD (1:1, v/v) provided high stereoselectivity for (1S, 4S) and (1R, 4R)-isomer. The distribution ratios of cis-sertraline, (1S, 4S) and (1R, 4R)-isomer, were 18.30 and 25.83, and the stereoselectivity reached 1.404 using the above system. With the above selected solvent systems, an analytical countercurrent chromatographic apparatus was used to separate stereoisomeric sertraline. It was found that very long retention time of the isomeric sertraline was always involved if pH were greater than 9.0 due to large distribution ratios, even though a high stereoselectivities were obtained with high pH value. An acceptable retention time was obtained for isomeric sertraline only when the pH value was between7.0 and 8.0. Fig. 10 shows the chromatogram
Fig. 10.. Chromatogram for stereoselective separation of isomeric sertraline by analytical countercurrent chromatography. The two-phase solvent system: n-hexane : 0.10 mol L−1 of disodium hydrogen phosphate-citric acid buffer pH 7.6 containing 50 mmol L−1 of Me-β -CD (1:1, v/v); flow rate: 0.5 ml min−1 ; revolution: 1120 rpm; column temperature: 25 °C; stationary phase retention: 65.3%.
Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834
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for stereoselective separation of isomeric sertraline with system (2) by analytical countercurrent chromatography. A baseline separation of trans-sertraline and cis-sertraline could be obtained. Meanwhile, (1S, 4R) and (1R, 4S)-sertraline were completely separated with high peak resolution using Me-β -CD as stereoselective selector. The analysis of HPLC showed that (1S, 4R)-sertraline of 98.7% purity, (1R, 4S)-sertraline of 98.2% purity and cis-sertraline of 99.1% purity were obtained. Compared with our previous work [19], peak resolution for cis- and trans-sertraline was increased from 0.91 to 1.56. Meanwhile, separation for trans-isomers, (1S, 4R) and (1R, 4S)sertraline, was achieved. 4. Conclusion Stereoselective liquid-liquid extraction of four isomeric sertraline was investigated with HP-β -CD and Me-β -CD as extractant, respectively. A complete different stereoselective combination between HP-β -CD and isomeric sertraline was observed when sodium carbonate was used in the buffer solution. The stereoselectivity of trans-sertraline was greatly improved when sodium carbonate was used in the buffer solution. The stereoselectivity of trans-sertraline could reach 2.373 and 1.685, when the solvent system were composed of n-hexane and 0.10 mol L−1 sodium carbonate-sodium bicarbonate buffer pH 10.6 containing 5 mmol L−1 HP-β -CD and disodium hydrogen phosphate-citric acid buffer pH 7.6 containing 50 mmol L−1 of Me-β -CD, respectively. The stereoselectivity of cis-sertraline could reach 1.404 when the solvent system composed of n-hexane : 0.10 mol L-1 of glycinesodium hydroxide buffer at pH 10.6 containing 50 mmol L−1 of HP-β -CD (1:1, v/v) was used. Successful stereoselective separation of cis-sertraline and trans-sertraline by countercurrent chromatography using the above systems was only obtained when pH was in the range of 7.0–8.0 using Me-β -CD as stereoselective selector. An biphasic solvent system composed of n-hexane and 0.10 mol L−1 of disodium hydrogen phosphate-citric acid buffer at pH 7.6 containing 50 mmol L−1 of Me-β -CD (1:1, v/v) was feasible for successful separation of trans-sertraline and cis-sertraline, and separation of (1R, 4S) and (1S, 4R)-isomer. Complete separation trans-sertraline and cis-sertraline, (1R, 4S) and (1S, 4R)-sertraline by countercurrent chromatography with high peak resolution was achieved using Meβ -CD as stereoselective selector. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This work was financially supported by National Natural Science Foundation of China (21978266). References
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Please cite this article as: W. Sun, Y. Jin and C. Wang et al., Stereoselective separation of isomeric sertraline with analytical countercurrent chromatography, Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2019.460834