Continuous Separation of Pantoprazole Enantiomers by Biphasic

Article pubs.acs.org/OPRD

Continuous Separation of Pantoprazole Enantiomers by Biphasic Recognition Chiral Extraction in Centrifugal Contactor Separators Yaqiong Wang,† Kewen Tang,*,‡ Panliang Zhang,‡ Jicheng Zhou,† Yan Huang,‡ Ping Wen,† and Genlin Sun§ †

College of Chemical Engineering, Xiangtan University, Xiangtan 411105, Hunan, China College of Chemistry and Chemical Engineering, Hunan Institute of Science and Technology, Yueyang 414006, Hunan, China § College of Chemistry and Chemical Engineering, Hunan University, Changsha 410083, Hunan, China ‡

ABSTRACT: Continuous separation of pantoprazole enantiomers by biphasic recognition chiral extraction (BRCE) in a cascade of centrifugal contactor separators was studied. The BRCE system consists of isobutyl D-tartrate (D-IBTA) in the organic phase and hydrophilic-β-cyclodextrin (HP-β-CD) in the aqueous phase. The effect of W/O ratio, extractant D-IBTA concentration, and number of stages on the purity and yield of the product was investigated. A higher D-IBTA concentration and a smaller W/O ratio result in a higher purity in the raffinate phase and lower in the extract phase. More stages are required to obtain higher purities in both product streams. The desired enantiomer can be obtained by asymmetric separation, i.e., by varying the phase ratio, without the need of increasing the number of stages. The optimal conditions for symmetric separation involves a W/O ratio of 5 and 0.1 mol/L D-IBTA, which can make the eeeq up to 48% and Yeq to 68%. The technology is potentially applicable to scale production.

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INTRODUCTION Many of the drugs currently used in medical practice are mixtures of enantiomers. Many times, the two enantiomers differ in their pharmacokinetic and pharmacodynamic properties, which causes the demand for enantiopure drugs to grow rapidly and has stimulated the development of new competitive technologies to obtain single enantiomers in the past few decades. Several technologies such as crystallization,1 capillary electrophoresis,2 supercritical fluid chromatography (SFC),3 liquid membrane,3,4 simulated moving bed chromatography (SMB),5,6 enzymatic resolution or biocatalysis, and so on, are being developed widely. However, these technologies are not always applicable. For example, classical crystallization is limited by its low versatility and excessive solid handling. Compared with others, enantioselective liquid−liquid extraction (ELLE) is considered as a potential attractive technique because it is cheaper and easier to scale up to commercial scale and has a larger application range.8,9 ELLE needs an enantioselective extractant dissolved in the extract phase, which reacts with the enantiomers in the feed. As is well-known, the chiral selector plays an important role in chiral solvent extraction with the distribution ratio (k) and enantioselectivity (α) depending on the chiral selector to a great extent.10 In our previous work,11,12 tartaric acid derivatives were used as extractants for the chiral separation of drug enantiomers. Recently, biphasic recognition chiral extraction has been explored for separation of aromatic acid enantiomers and excellent separation efficiency has been achieved.13 Pantoprazole,5-(difluoromethoxy)-2-[(3,4-dimethoxy-2pyridyl)methylsulfinyl]-1H-benzimidazole (Figure 1), is an administered racemic mixture, and an irreversible proton pump inhibitors, essentially used to the prevention and treatment of excessive gastric acid secretion caused by a peptic ulcer and related diseases, such as duodenal ulcer, gastric ulcer, © XXXX American Chemical Society

Figure 1. Structures of S-pantoprazole and R-pantoprazole enantiomers.

acute gastric mucosal lesion, and compound gastric ulcer.14,15 However, like other chiral drugs, pharmacological studies have shown that the metabolism of S-pantoprazole (S-PAN) is more efficient than R-pantoprazole (R-PAN) and R,S-pantoprazole in inhibiting the gastric acid secretion and in reducing the gastric and duodenal ulcers.16−18 According to the International Special Issue: Engineering Contributions to Process Chemistry Received: March 17, 2014

A

dx.doi.org/10.1021/op500090m | Org. Process Res. Dev. XXXX, XXX, XXX−XXX

Organic Process Research & Development

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Conference on Harmonisation (ICH) guidelines,19 chiral identity, enantiomeric impurity, and chiral assay tests may be needed in drug substance and product specifications. At present, the racemate of pantoprazole is the clinical drug. Specifically, it is necessary to develop a method for evaluating the purity of S-pantoprazole. Studies trying to separate pantoprazole enantiomers have been reported in recent years, which are regularly achieved by using supercritical fluid chromatography,20 HPLC techniques,21,22 and capillary electrophoresis,23,24 which can only be applied to a small amount of preparation and analysis. 25 The partition behavior of pantoprazole enantiomers was studied in a biphasic recognition chiral extraction system by our team, and the operational separation factor of 1.34 was achieved using 0.1 mol/L D-IBTA in the organic phase and 0.1 mol/L HP-β-CD in the the aqueous phase,26 which indicates that pure pantoprazole enantiomers are promising to be obtained by multistage extraction. Centrifugal contactor separators (CCS) have not only excellent throughput/hold-up ratio, but also good mass transfer characteristics due to fast mixing and separation,27−29 which is expected to achieve full separation of pantoprazole enantiomers using a number of CCS in series. In this work, centrifugal contactors are used for the continuous separation of pantoprazole enantiomers by biphasic recognition chiral extraction (Figure 2). The biphasic

Figure 4. Structures of HP-β-CD.

Similarly, the ee in the aqueous stream is defined as eeaq =

[AR ]allforms − [A S]allforms aq aq [AR ]allforms + [A S]allforms aq aq

(2)

The yield of the R-PAN is defined as the fraction of the RPAN in the feed that ends up in the outlet of aqueous stream: yield R,aq =

[AR ]allforms (W + F ) [mol] aq [AR ]F [mol]

(3)

Similarly, the yield of the S-PAN is defined as Figure 2. Flow diagram of the multistage centrifugal countercurrent extraction of PAN enantiomers.

yieldS,org =

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recognition chiral extraction system is established by adding D-IBTA (see Figure 3) in the organic phase and HP-β-CD (see

Figure 4) in the aqueous phase, which preferentially recognize S-PAN and R-PAN, respectively. The study on a biphasic recognition chiral extraction system in CCS has not been reported. According to our previous study of single stage extraction for PAN enantiomers, factors that define the extraction mechanism in multistage ELLE were analyzed, namely, the influence of the W/O ratio, the concentrations of the two extractants, and the number of stages.12,26,28 The enantiomeric excess (ee) is used as a measure of the optical purity of the described enantiomer for the system studied in this paper. The ee in the organic stream is defined as [A S]allforms − [AR ]allforms org org allforms allforms [A S]org + [AR ]org

(4)

RESULT AND DISCUSSION Exploration of Equilibrium Time. The racemic feed was added to the cascade at the middle stage (see Figure 2). To explore the equilibrium time, the function of concentration of pantoprazole enantiomers in an aqueous outlet with time (Figure 5) has been investigated. From Figure 5, it can be found that the concentration of both enantiomers increases with time in the first 180 min, and then fluctuates within a narrow range appears over the next 120 min, which shows the time that needs to reach steady state is 3 h at least. Influence of W/O Ratio. In multistage ELLE, the W/O ratio has a large influence on the purity of products in both streams. What can be observed from Figure 6 is that there is a significant relationship between the W/O ratio and ee and yield of each exiting stream. As shown in Figure 6a, a larger W/O ratio increases the purity of enantiomer in the extract phase (organic phase) while decreases the purity of the raffinate phase (aqueous phase). It can also be seen from Figure 4b that the yield extract phase in aqueous stream increases with W/O, but the yield in raffinate phase decreases. These can be explained by the fact that more ARC and ASC, which are the complexes of RPAN and HP-β-CD, S-PAN and HP-β-CD, respectively, are formed in the aqueous phase, and in the wash section more enantiomers are washed back subsequently from organic stream with the increase of aqueous stream. There is only one

Figure 3. Structures of D-IBTA.

eeorg =

[A S]allforms O [mol] org [A S]F [mol]

(1) B



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Influence of Isobutyl D-Tartrate Concentration. Our previous study on single-stage BRCE of PAN enantiomers indicated that the concentration of the hydrophobic extractant D-IBTA has a great influence on enantioselectivity. The concentration of hydrophilic extractant HP-β-CD has a strong influence on the distribution ratio but hardly has any influence on enantioselectivity. Pantoprazole enantiomers can hardly dissolved in 1,2-dichloroethane without D-IBTA, which indicates that the solubility of enantiomers in the organic phase is due to the formation of the two complexes between enantiomers and D-IBTA. Here, the change of ee and Y in both phases with the increase of D-IBTA concentration from 0.05 to 0.25 mol/L (Figure 7) is studied. As shown in Figure 7, the

Figure 5. Concentration of pantoprazole enantiomers in an aqueous outlet.

Figure 7. Influence of isobutyl D-tartrate concentration on ee and yield for separation of PAN enantiomers. W/O = 5, O/F = 4.0, pH = 8.5, [AR, S] = 0.005 mol/L, [HP-β-CD] = 0.1 mol/L, T = 278 K, N = 10, feed in the middle stage. (a) Influence on ee and (b) influence on yield.

Figure 6. Influence of the W/O ratio on ee and yield for separation of PAN enantiomers O/F = 4.0, pH = 8.5, [AR,S] = 0.005 mol/L, [DIBTA] = 0.1 mol/L, [HP-β-CD] = 0.1 mol/L, T = 278 K, N = 10, feed in the middle stage. (a) Influence on ee and (b) influence on yield.

purity of the product in extract phase increases quickly with the increase of D-IBTA concentration, while the purity of the product in raffinate phase decreases rapidly. From Figure 7b the effect of D-IBTA concentration on the yields in the two phases displays an opposite trend. The reason is that more complexes are formed between D-IBTA and enantiomers in the organic phase, resulting in transfer of more enantiomers into the organic phase.

crosspoint where ee in the aqueous phases is equal to that in organic phase, which is defined as eeeq and yield in aqueous phase is also equal to that in organic phase, which is defined as Yeq. The crosspoint is selected as the operation point for symmetric separation where eeeq and Yeq can be achieved. C



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Influence of the Number of Stages. The number of stages has a clear influence on ee and Y in both outlets in multistage ELLE system. Some 10 and 20 stages of CCS were used to separate PAN enantiomers at a W/O of 5 for symmetric separation, respectively (Table 1). From Table 1, it is obvious Table 1. Experimental date for influence of the number of stagesa N

W/O

eeaq

eeorg

Yaq

Yorg

10 20 10 20

3 3 5 5

0.46 0.74 0.23 0.47

0.07 0.01 0.24 0.48

0.19 0.03 0.66 0.75

0.93 0.99 0.56 0.68

a O/F = 4, aqueous phase: [HP-β-CD] = 0.1 mol/L; organic phase: [DIBTA] = 0.1 mol/L; feeding phase: [racemic PAN] = 0.005 mol/L, T = 278 K, pH = 8.5, feed in the middle stage.

Figure 8. Chromatogram of racemic pantoprazole: 1. S-pantoprazole; 2. R-pantoprazole.

that the purities at 20 stages are double to those at 10 stages under the same concentration and flow ratios. In terms of industrial production, if CCS is enough, the full separation of enantiomers can be accomplished. The experimental result of BRCE showed the operational separation factor is 1.34.26 According to the literature30,31 and the Fenske equation (eq 5), we predict that for a symmetrical separation of PAN enantiomers for >99% eeeq, about 50 CCSs are enough.

(

ln Nmin =

xR / (1 − x R ) xS / (1 − xS)

)

ln αop

(5)

where xR and xS are the mole fractions of R- and S-PAN in an aqueous phase outlet, respectively, and α is the operational separation factor. In addition, the asymmetric separation of PAN enantiomers was carried out by changing the W/O ratio at 10 and 20 stages CCS, respectively (Table 1). Asymmetric separation means ee in the extract phase is not equal to that in the raffinate phase. This is useful when only one enantiomer is the desired product. Increasing the W/O ratio can increase ee in extract phase and decrease ee in raffinate phase, which are shown in Figure 6a. It can be explained by the fact that with the increase of raffinate stream, more ARC and ASC complexes are formed in the wash section, and in the stripping section more enantiomers are washed back from the extract, which results in the increase of ee in extract phase with increasing of the W/O ratio. In this paper, S-PAN is the desired product and is concentrated in the extract phase. Therefore, we can obtain S-PAN with the desired purity by increasing the W/O ratio without the need of increasing the number of stages. As seen from Table 1, eeorg decreases; however, eeaq is nearly doubled, by changing the W/O ratio from 5 to 3 under the stages of 10 or 20, respectively, by which the asymmetric separation can be further verified. Chromatograms of the racemic pantoprazole plus the sample in aqueous and organic outlets under optimal conditions are shown in Figures 8−10.

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Figure 9. Chromatogram of the sample in an aqueous outlet under optimal conditions (W/O = 3, O/F = 4, aqueous phase: [HP-β-CD] = 0.1 mol/L, pH = 8.5; organic phase (1,2-dichloroethane): [D-IBTA] = 0.1 mol/L; feeding phase: [racemic PAN] = 0.005 mol/L, T = 278 K, pH = 8.5, N = 20, feed in the middle stage). 1. S-pantoprazole; 2. Rpantoprazole.

CONCLUSION AND OUTLOOK

Figure 10. Chromatogram of the sample in an organic outlet under optimal conditions (W/O = 6, O/F = 4, aqueous phase: [HP-β-CD] = 0.1 mol/L, pH = 8.5; organic phase (1,2-dichloroethane): [D-IBTA] = 0.1 mol/L; feeding phase: [racemic PAN] = 0.005 mol/L, T = 278 K, pH = 8.5, N = 20, feed in the middle stage). 1. S-pantoprazole; 2. Rpantoprazole.

The PAN racemate can be separated by a cascade of centrifugal contactor separators in a biphasic recognition chiral extraction system, which contains two different extractants, HP-β-CD in aqueous solution and D-IBTA in organic solution. The purity and yield can be improved by changing the W/O ratio, D-IBTA D



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concentration, or number of stages. The increase of the W/O ratio results in an increase of the ee in the organic stream and a decrease in the aqueous stream. The influence of the W/O ratio on Yaq and Yorg is contrary to that on eeaq and eeorg. With the increasing D-IBTA concentration, eeaq and Yorg increase. About 50 CCSs are enough for symmetrical separation of PAN enantiomers for >99% eeeq. One desired enantiomer can be obtained by asymmetrical separation, i.e., by changing the W/O ratio, but not increasing the number of stages.

solution (pH = 5.5)−acetonitrile. The flow rate was set at 1.0 mL/min.26 The pH of the aqueous phase was measured with a pH electrode and a pH meter (Orion, model 720A).

EXPERIMENT SECTION Chemicals. Racemic PAN enantiomers (purity >99% (w/ w)) were obtained from Changsha University of Science & Technology. Hydroxypropyl-β-cyclodextrin (HP-β-CD) (purity >99% (w/w)) was supplied by Qianhui Fine Chemicals (Shandong, China). D-tartaric acid (purity >99% (w/w)) was bought from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Isobutyl D-tartrate was synthesized in this laboratory. 1,2-Dichloroethane (purity >99% (w/w)) was purchased from Shanpu Co. Inc. (Shanghai, China). The solvent for chromatography was of high-performance liquid chromatography (HPLC) grade. All other reagents used in this work were of analytical grade and bought from different suppliers. Experiment Setup. All experiments were carried out in centrifugal contactor separators that were connected in turn by plastic tubes. The rotational speed was set at 2500 rpm. Both liquids were transferred to CCS using constant flow pumps. Glass containers were used to supply the solution and receive the outlet solution. Two aluminum tanks connected with a water-filled pentrough were used to keep the jacket a constant temperature of 278 K. Experimental Procedure. A biphasic recognition chiral extraction system is established by adding isobutyl D-tartrate in the organic phase and hydroxypropyl-β-cyclodextrin in the aqueous phase. The aqueous phase was prepared by dissolving HP-β-CD in 0.1 mol/L Na2HPO4/NaH2PO4 buffer solution, and the organic phase was D-IBTA dissolved in 1,2-dichloroethane. Racemic PAN was dissolved in the aqueous phase to obtain the feeding phase. Before extraction experiments began, the CCSs were filled with the heavier of the two phases; in this case it was the organic phase. When the heavy phase flowed out of the outlet, it was time to open the pump of the aqueous phase. When the aqueous phase outlet was little sticky, the feed pump was turned on. After 2 h, samples for analysis were taken from the outlet of aqueous stream every 15 min. The concentrations of R-PAN and S-PAN in aqueous phase outlet were detected by HPLC. The concentration in the organic phase was analyzed through back-extraction. A 10:1 (v/v) mixture of the aqueous phase and outlet stream of organic phase was shaken sufficiently (5 h) before being kept in a water bath at a constant temperature to reach equilibrium. After phase separation, the concentrations of PAN enantiomers in the aqueous phase were analyzed by HPLC. The efficiency of back-extraction is up to 99% according to mass balance. Analytical Method. The quantification of PAN enantiomers in the aqueous outlet is determined by the HPLC relative area % (Waters e2695 separation module) and a UV detector (Waters 2998 photodiode array detector) at the UV wavelength of 290 nm was used. The column was CHIRALCEL OJ-RH (150 mm × 4.6 mm id., 5 μm) (Daicel Chemical Industries Ltd., Japan). The mobile phase was a 76:24 (v/v) mixture of 0.1 mol/L Na2HPO4/NaH2PO4 aqueous

ACKNOWLEDGMENTS This work was supported by the Natural Science Foundation of China (No. 21176062), Hunan Provincial Natural Science Foundation of China (No. 12JJ2007), Scientific Research Fund of Hunan Provincial Education Department (12B053), Science and Technology Planning Project of Hunan Province (2012GK3107), and Aid Program for Science and Technology Innovative Research Team in Higher Educational Institutions of Hunan Province, the Planned Science and Technology Project of Hunan Province, China (No. 2013GK3094).

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

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The authors declare no competing financial interest.

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NOTATION ARC = complex of R-PAN with HP-β-CD ASC = complex of S-PAN with HP-β-CD D-IBTA = isobutyl D-tartrate D(R) = complex of R-PAN with D-IBTA D(S) = complex of S-PAN with D-IBTA ee = enantiomeric excess F = flow of feeding phase (mL·min−1) HP-β-CD = hydroxypropyl-b-cyclodextrin k = distribution ratio N = number of stages O = flow of organic phase (mL·min−1) PAN = pantoprazole T = temperature (K) W = flow of aqueous phase (mL·min−1) x = mole fraction Y = yield α = enantioselectivity [] = concentration (mol·L−1)

Subscripts

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aq = aqueous phase eq = equilibrium j = stage index min = minimization op = operational org = organic phase R = R-PAN S = S-PAN

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Continuous Separation of Pantoprazole Enantiomers by Biphasic

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