In Situ Derivatization of (RS)-Mexiletine and Enantioseparation Using

Subscriber access provided by Gothenburg University Library

Article

In-situ derivatization of (RS)-Mexiletine and Enantioseparation using Micellar liquid chromatography: a green approach Shiv Alwera ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/acssuschemeng.8b01869 • Publication Date (Web): 22 Jul 2018 Downloaded from http://pubs.acs.org on July 23, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

In-situ derivatization of (RS)-Mexiletine and Enantioseparation using Micellar liquid chromatography: a green approach Shiv Alwera* Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee – 247667, India e-mail: [email protected]; [email protected]

ABSTRACT: Developing surfactant based aqueous solvents as alternatives to organic solvents for chromatographic enantioseparation is highly desired. Surfactants are inexpensive, non-toxic, environmental friendly, and increases the solubility of organic compounds in the aqueous solutions. In this work, impact of surfactant based aqueous solvents on RP-HPLC enantioseparation of (RS)mexiletine is studied. The in-situ derivatization of (RS)-mexiletine with chirally pure moieties ((S)levofloxacin, (S)-ketoprofen, (S)-ibuprofen) was carried out/performed to reduce consumption of organic solvents and side wastes, and maintain green approach most. We report the impact of increasing concentration of surfactants on separation of diastereomeric derivatives of (RS)mexiletine on a C18 column of HPLC. Surfactant in water increases solubility of diastereomeric derivative and provided lowest elution time (tR in between 2.152-10.87 min) and minimize consumption of solvent. The use of surfactant containing aqueous mobile phases in chromatographic separations is a good alternative for replacement of organic solvents.

The

hydrolysis of the synthesized diastereomeric derivatives was carried out, and native enantiomers of mexiletine were isolated and characterised. In addition, the lowest energy optimized structures were developed using Gaussian 09 Rev. A.02 program for determination of elution order, and the method was validated for linearity, accuracy, limit of detection (LOD) and limit of quantitation (LOQ). Keywords: MLC, Enantioseparation, Mexiletine, In-situ derivatization,

INTRODUCTION The development of new chromatographic methods is always being interested because the pharmaceutical industries and academia always require a more sensitive, convenient, fast, reproducible, less expensive and environmentally friendly method for the separation. The lots of 1 ACS Paragon Plus Environment

ACS Sustainable Chemistry & Engineering 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

new green chromatographic methods have been developed1-7 for separation of a variety of organic compounds, among them, very few are reported for chiral separation.6,7 The chiral separation of racemic compounds is considered very important for the synthetic laboratories or pharmaceutical industries, which are dealing with the development of new chiral drugs/compounds, for the establishment of the chirally purity of synthesized products. It is well known, in a chiral drug, only one of the enantiomer is responsible for the desired physiological effects while the other enantiomer is less active, or inactive, or sometimes even causing adverse effects. Nearly 56% of the marketed pharmaceuticals are chiral compounds, and, amongst them, 88% are administered as racemates.8-10 Because of the different physiological activity of a pair of enantiomers, it is necessary and important to establish enantiopurity of the chiral drugs, for this purpose direct and indirect chromatographic methods have been developed. The chromatographic technique, especially HPLC, is an important, inexpensive, time consuming and effective tool for the establishing enantiopurity of chiral compounds, and allows the semi or preparative isolation/separation of single enantiomer form a mixture of enantiomers. Because of the importance of chiral separation, nowadays, the development of new chromatographic methods for enantioseparation of racemic compounds has gained a huge attention, especially green developments. Previous reports on enantioseparation revels, the huge amount of organic solvents, e.g., methanol, acetonitrile, dichloromethane, chloroform, tetrahydrofuran, acetic acid, trifloroacetic acid, etc., were used during the process of chromatographic enantioseparation, and these solvents are considered hazardous on comparing them with the green solvent list given by Pfizer3, 11 and Sanders.3 Among the 12 rules of green chemistry, Welch et al. described three “Rs” principle (Reduce, Replace and Recycle) for the development of a new green analytical methods.12 By following the three “Rs” principle our research group has reported few methods on green development in chiral chromatographic separation.6, 7 where the surfactant based aqueous eluting phase was used as a replacement of organic eluting phase. Surfactant based mobile phase (mix or single surfactant based aqueous system) in combination with organic modifier, previously, have been used in a several chromatographic techniques, for determination of different types of organic compounds and biological samples.5-7,13,14 The idea to use a non-toxic, non-volatile, inexpensive and eco-friendly water-surfactant based eluting phase in chiral chromatography, favours the green developments.

2 ACS Paragon Plus Environment

Page 2 of 22

Page 3 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


By learning from previous limitations, here, we describe a green micellar liquid chromatographic method for enantioseparation of (RS)-mexiletine (Mex; a non-selective voltagegated sodium channel blocker, IB anti-arrhythmic class drug; marketed and administered as racemate) through the direct covalent derivatization (indirect approach of enantioseparation). The literature on covalent derivatization of enantiomers showing their own advantages in the establishment of the chiral purity.6,7,15-24 The covalent derivatization of enantiomers provides sensitive chromatographic UV-Visible detection with fast elution and high resolution. Literature reveals, for the covalent derivatization, lots of chiral derivatizing reagents have been developed, e.g., CDRs based on Marfey’s reagent,15,16 Sanger reagent,16 cyanuric chloride,17,18 isothiocyanate,19,20 chloroformates,19 (S,S)-O,O’-di-p-toluoyl tartaric acid anhydride,21 divinyl dicarboxylates,22 (S)-(−)-(N)-trifluoroacetyl-prolyl chloride,23 (1S)-(−)-camphanic chloride,23 2anthroyl chloride,24 (S)-naproxen ester,16, 23 levofloxacin ester7 and (S)-ketoprofen ester6; and these provided easy derivatization with target analyte. For the present work three chirally pure moieties [(S)-levofloxacin, (S)-ketoprofen and (S)ibuprofen] were selected for the covalent derivatization of (RS)-mexiletine. To maintain green chemistry most in the experiment, here, in-situ derivatization of (RS)-mexiletine was carried out (to reduce consumption of the solvents and minimize the waste). A non-toxic, inexpensive and eco-friendly water-surfactant based eluting phase was prepared by dissolving SDS and Brij-35 in water, and used in chromatographic separation as the replacement of organic solvents (organic solvents were completely swapped form eluting mobile phase). Synthesized diastereomeric derivatives were characterized and their lowest energy optimized structures were developed using “the Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G* basis set”, and used to establish elution order of synthesized diastereomeric derivatives. In order to isolate optically pure enantiomers of mexiletine the hydrolysis of synthesized diastereomeric derivatives were performed, and the isolated enantiomers were characterised. The developed separation method was validated for linearity, accuracy, limit of detection (LOD) and limit of quantitation (LOQ). Also, the efficiency of the developed method was examined in terms of green aspects by comparing developed method with Analytical Eco-Scale method25 and Green Analytical Procedure Index method (GAPI).26 Current method could be considered as a green analytical method because the method was devised in the way that it would support green



chemistry rules (such as the replacement of hazardous organic solvents, minimization of the waste, and the derivatization conditions are faster including one step synthesis and cost-effective).

Figure 1. Showing structure of (RS)-mexiletine, (S)-levofloxacin, (S)-ketoprofen and (S)-ibuprofen

EXPERIMENTAL Chemical and Equipment The detail description of chemical and equipment used is given as supporting information. The abbreviations used for mexiletine, levofloxacin, ketoprofen and ibuprofen are, respectively, Mex, Lfx, Kpf and Ibf. In-situ derivatization (RS)-Mex The solutions of the (RS)-Mex (50 µL, 1 mM in THF), (S)-Lfx (60 µL, 1 mM in THF), DCC (80 µL, 1 mM in THF) and DMAP (40 µL, 1 mM in THF) were mixed in a vial and the resulting solutions was ultra-sonicated for 30 min. After the reaction completion, the reaction mixture was filtered using 0.45 µm filter to remove side product dicyclohexylurea form reaction. Then the reaction solution was dried under vacuum and diluted with 500 µL DCM and then work-up with 1 N HCl solution to remove DMAP from reaction solution.27 The resulting reaction solution was


Page 4 of 22

Page 5 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


dried under vacuum and then diluted with 250 µL EtOH. Similarly, diastereomeric derivatives of (RS)-Mex were prepared with (S)-Kpf and (S)-Ibf. A preparative scaled up synthesis of diastereomeric derivatives were also carried out by taking substrate in gram amount at the same ratio as described above. The characterisation data is given in results and discussion. Synthesized diastereomeric derivatives of (RS)-Mex were named as LM-1 and LM-2, KM-1 and KM-2, and IM-1 and IM-2; respectively, synthesized with (S)-Lfx, (S)-Kpf and (S)-Ibf. Preparation mobile phase and of stock solutions Surfactant based mobile phase (WMP) was prepared by dissolving 1.45 g SDS (0.005 M) and 1.78 g Brij-35 (0.0015 M) in double distilled water making a total volume of 1000 mL. The resulting solution was sonicated for half an hour to obtain a clear and homogeneous solution. The solution was filtered using 0.45 µm filter, then degassed by passing dry nitrogen gas and sonicating again for half an hour. The water-surfactant solution was then used as a component of the mobile phase in HPLC. The stock solutions of NaHCO3 (0.1 M), TEAP buffer (10 mM, pH 3.5) in distilled water, and 1 mM concentrated stock solutions of (RS)-Mex, (S)-Lfx, (S)-Kpf, (S)-Ibf and DCC in THF were prepared. Sample preparation and HPLC conditions Sample for HPLC were prepared by diluting 50 µL aliquot solution obtained from each of completed reaction mixture with 250 µL EtOH and resulting solution was filtered with 0.45 µm syringe filter, and its 20 µL solution was injected onto the C18 column. Separation conditions were optimized by varying (i) mobile phase composition as follows, WMP-TEAP buffer in an isocratic mode in the ratio of (50:50, 60:40, 70:30, and 80:20, v/v) for 60 min, and (ii) flow rates, 0.5, 0.7, and 1.0 mL min−1 for each mobile phase composition. Detection were made on 294 nm, 254 nm and 262 nm; respectively, for diastereomeric derivative synthesized with (S)-Lfx, (S)-Kpf and (S)-Ibf, using PDA detector.



Figure 2. Showing synthesis of diastereomeric derivatives of (RS)-Mex with (S)-Lfx

Preparative HPLC, hydrolysis of diastereomeric derivatives and isolation of native enantiomers The preparative HPLC was performed for the separation of diastereomeric derivatives at preparative scale and the detail processor for preparative HPLC is given in supporting information. In order to obtain isolated enantiomers of (RS)-Mex hydrolysis of prepared diastereomeric derivatives (LM, KM, IM) were carried out. The isolated diastereomeric derivatives were hydrolysis via acid catalysed reaction under microwave irradiation conditions,28 the detailed processor is given in supporting information. The preparative HPLC separation was performed to isolate native enantiomers from hydrolysate solutions (similar chromatographic conditions and method were applied as described for preparative HPLC of diastereomeric derivatives).


Page 6 of 22

Page 7 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Schematic diagram of the present work

RESULTS AND DISCUSSION The structure of analyte and chiral moieties [(RS)-Mex, (S)-Lfx, (S)-Kpf and (S)-Ibf], under study, are given in Figure-1. Structures of intermediates of the in-situ derivatization are given in FigureS1. The scheme of synthesis of diastereomeric derivatives of (RS)-Mex with (S)-Lfx (as representative) is given in Figure-2. The reaction of synthesis of diastereomeric derivatives of (RS)Mex with (S)-Kpf and (S)-Ibf are given in Figure-S2. Figure-3 is representing a schematic diagram showing the approach of separation applied at present work. In-situ derivatization of diastereomeric derivatives Diastereomeric derivatives of (RS)-Mex were prepared under in-situ derivatization condition followed by nucleophilic substitution reaction. Because of the derivatization reactions were carried out in a single step at same place at same derivatizing condition (no addition and no isolation), 7 ACS Paragon Plus Environment


reaction can be say in-situ. The formation of peptide (amide) bond takes place in between amino group of (RS)-Mex and carboxylic group of chiral derivatizing moiety (Lfx/Kpf/Ibf) in presence of DCC. DCC, a dehydrating agent, removed a water molecule (-OH from carboxylic group and -H from amino group) and resultant formation of diastereomeric derivative of (RS)-Mex was taking place. The side product of the synthesis reaction, dicyclohexylurea, was precipitated out form reaction solution and removed by filter reaction mixture solution using 0.45 µm filter. During the each derivatization reaction an unstable and reactive intermediate was formed due to reaction of DCC with Lfx/Kpf/Ibf (Figure-S1), and these were readily reacted with the amino functional group of the (RS)-Mex via a nucleophilic substitution reaction and yields the diastereomeric derivatives of (RS)-Mex. These reactions were simply completed by ultra-sonication reaction mixture for 30 min. The efficiency and reproducibility of the reaction was optimized by performing reaction at both low amount scaled up (reactants in microgram amount) and high amount scaled up (reactants in gram amount), and in both cases reaction were completed and yields > 98%. Characterisation data of diastereomeric derivatives of (RS)-Mex; Prepared with (S)-Lfx; yield, 98%; m.p. 176-178 °C, UV (λmax in MeOH, 294 nm); IR (KBr): 3425, 2935, 2504, 2001, 1723, 1619, 1541, 1472, 1395, 1340, 1292, 1266, 1238, 1199, 1134, 1026, 1089, 924, 801 and 766 cm-1. 1H NMR (400 MHZ, CDCl3-d1): δ 1.52 (d, 3H), 1.56 (d, 3H), 2.15 (s, 6H), 2.25 (s, 3H), 2.36 (t, 4H), 3.24 (m, 5H), 3.46 (m, 1H), 3.71 (m, 1H), 4.10-4.21 (m, 3H), 6.956.99 (m, 3H), 7.67 (d, 1H), 7.92 (s, 1H) and 8.54 (s, 1H); HRMS: Calcd for C29H35FN4O4: 522.2642 (M++H), found 523.2672; anal. Calcd for C29H35FN4O4: C, 66.65%; H, 6.75%; N, 10.72%; Found: C, 66.52%; H, 6.44%; N, 10.46%. Prepared with (S)-Kpf; yield, 98%; m.p. 169-172 °C, UV (λmax in MeOH, 254 nm); IR (KBr): 3119, 2938, 2589, 2504, 2001, 1727, 1685, 1653, 1595, 1483, 1395, 1341, 1283, 1198, 1083, 1026, 969, 922, 830 and 765 cm-1. 1H NMR (400 MHZ, CDCl3-d1): δ 1.34 (d, 3H), 1.52 (d, 3H), 2.18 (s, 6H), 3.54-3.82 (m, 3H), 4.34 (m, 1H), 6.93-6.98 (m, 3H), 7.48-7.71 (m, 9H) and 8.14 (s, 1H); HRMS: Calcd for C27H29NO3: 416.5315 (M++H), found 416.5324; anal. Calcd for C27H29NO3: C, 78.04%; H, 7.03%; N, 3.37%; Found: C, 78.15%; H, 6.83%; N, 3.56%. Prepared with (S)-Ibf; yield, 99%; m.p. 174-176 °C, UV (λmax in MeOH, 262 nm); IR (KBr): 3421, 3212, 2954, 2935, 2495, 2005, 1719, 1662, 1631, 1598, 1513, 1457, 1383, 1257, 1231, 1181, 8 ACS Paragon Plus Environment

Page 8 of 22

Page 9 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1168, 1108, 1047, 983, 849 and 769 cm−1. 1H NMR (400 MHZ, CDCl3-d1): δ 1.09 (d, 6H), 1.29 (d, 3H), 1.48 (d, 3H), 1.85 (m, 1H), 2.17 (s, 6H), 2.43 (d, 2H), 3.52-3.56 (m, 2H), 3.82 (m, 1H), 4.32 (m, 1H), 6.91-6.96 (m, 3H), 7.08 (d, 2H), 7.28 (d, 2H) and 8.16 (s, 1H); HRMS: Calcd for C24H33NO2: 368.2545 (M++H), found 368.2578; anal. Calcd for C24H33NO2: C, 78.43%; H, 9.05%; N, 3.81%; Found: C, 78.21%; H, 9.28%; N, 3.92%. WMP (water micellar mobile phase) system: Sodium dodecyl sulphate (SDS; anionic) and Brij-35 (non-ionic) are good surfactant for preparation of mobile phase, and according to literature5,6,7,13,14,29 their individual aqueous solution and their mixed surfactant based aqueous system with combination with organic solvents have been used as mobile phase in RP-HPLC analysis of biological and non-biological samples. Previous literature shows the mixed surfactant system have been scarcely used in liquid chromatography applications30, 31 and outside of the field of chromatography.30 The advantage of using surfactant based aqueous mobile phase for RP-HPLC analysis is to allow direct injection of biological sample without any pre-treatment and at the same time reduce the use of organic solvents (reduce hazard impact as compared to traditional liquid chromatography), also both surfactant have very low molar absorbance as compared to analyte used so they doesn’t interfere during UV-visible detection on RP-HPLC. In this current study, the mixed surfactant aqueous system was used as mobile phase. The advantage to using mixed surfactants allow to handle properties like solubilisation, detergency, emulsion, aggregation and distribution of surfactant,32 and allow to design minimal and maximally adsorbing system,32,33 also, as compared to mixed surfactants the isomerically pure surfactants are often expensive to produce and having low potential in performance.32,33 The mixed surfactant having the property to have an average CMC of the CMCs of surfactants used in the system,32 and because of that mixed surfactant system allows to modify CMC of the system accordingly as compared to the single surfactant system. Also, the CMC of mixed surfactant system influenced by other factors, e.g, the structure of surfactant, polar groups, temperature, counter-ions, electrolytes and polar organic compounds.32-37 According to literature36-40 the CMC of the individual systems of SDS is 8.4 mM and Brij-35 is 0.09 mM. In the present studies, the CMC of the surfactant system is 3.5-4.0 mM. The advantage of this method over previous methods is, here, organic solvents completely replaced by aqueous mixed surfactant based mobile phase. Also, due to hydrophilic and



hydrophobic properties of surfactant, micellar mobile phase (WMP) provides good solubility for analytes. In the WMP system SDS and Brij-35 leads to form micelles, SDS formed dodecyl apolar core containing micelles and similarly Brij-35 formed micelles but relatively more polar due to oxyethylene chain in micelles. Surfactants in water micellar mobile phase system have the property to adsorb on the surface of C18 column and resultant increases the polarity of stationary phase. The interaction between the hydrocarbon chain of Brij-35 with C18 column material, the surface of the stationary phase become more polar as compared to C18 material. Similarly, absorbed SDS on the surface of the column material increase polarity of stationary phase because of the orientation of its negatively charged sulphate group. These modification reduce the retention time of analysed compound and also indicate that there are no specific interactions such as hydrogen bonding between the hydroxyl groups of the adsorbed surfactant and the phenolic group of the analytes.5,6

Figure 4. Effect of concentration of (i) SDS and (ii) Brij-35 on retention time of the diastereomeric derivatives of (RS)-Mex [LM, KM, IM] prepared with respectively; (S)-Lfx, (S)-Kpf and (S)-Ibf.


Page 10 of 22

Page 11 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Effect of the concentration of (i) SDS and (ii) Brij-35 on resolution of the diastereomeric derivatives of (RS)-mex [LM, KM, IM] prepared with respectively; (S)-Lfx, (S)-Kpf and (S)-Ibf.

WMP system shows a partitioning behaviour. According to Arunyanart and Cline-Love,31 “

there occur two association equilibria, (i) between solute and stationary phase, and (ii) between

solute and micelle. These describe the association of a solute in bulk water with the stationary phase binding sites, and with the surfactant monomers in the micelles dissolved in the mobile phase”. According to Arunyanart and Cline-Love,31 the association equilibria in between solute, micelles and stationary phase makes possible the chromatographic separation of prepared diastereomeric derivatives of (RS)-Mex. In the WMP system, the surfactants aggregated around the diastereomeric derivatives and forms micelles, and these micelles exist in an equilibrium of formation and deformation (diffusion) of micelles.41,42,43 Because the formation and deformation of the micelles, during the chromatographic separation, allowed intermediate/interacting interactions between micelles of diastereomeric derivatives and stationary phase, and these interactions found sufficient to separate both of the diastereomeric derivatives completely. The interactions between 11 ACS Paragon Plus Environment


micelles and the stationary phase dependent on the concentration of surfactants and can be modified by manipulating the concentration of surfactants in the mobile phase and can achieve required resolution and retention time (Figure-4 and Figure-5). The micelles formation of diastereomeric derivatives of (RS)-Mex with surfactant of mobile phase is considered the reason for good chromatographic separation. The UV-visible spectrum of the first and second eluted diastereomeric derivatives were captured using PDA detector. The elution time for LM-1 and LM-2 were 2.152 and 3.754 min, for KM-1 and KM-2 were 5.014 and 6.651 min, and for IM-1 and IM-2 were 8.431 and 10.87 min. Table-1 showing the chromatographic separation data of all diastereomeric derivatives of (RS)-Mex, it reflects micellar based mobile phase is sufficient to achieve good separation. Optimization of WMP system for RP-HPLC: Literature5-7 shows that separation capacity increases when increasing concentration of SDS in constant-concentration solution of Brij-35 as well as when increasing concentration of Brij-35 in constant-concentration solution of SDS. Taking into account the literature reports,5-7, 29 a reference aqueous mobile phase was prepared containing 0.005 M SDS and 0.0015 M Brij-35 in aqueous phase. In the present studies, the retention time and resolution behaviour of the diastereomeric derivatives of (RS)-Mex were observed. It was observed, when the concentration of SDS was increased to a particular value (0.0005 to 0.005 M) the resolution of analyte was also increased and after that resolution was decreased. Similarly, when the concentration Brij-35 was increased to a particular value (0.00015-0.0015 M) resolution was increased and after that resolution was decreased (Figure-4). Also, when the concentration of the SDS and Brij-35 increasing in the mobile phase the retention time of the analyte were decreased (Figure-5). Increasing the concentration of SDS (0.0005 M to 0.005 M) in the mobile phase containing a fixed concentration of Brij-35 at 0.0015 M the retention time of analyte was decreased but peak height was increased. Similarly, it was also observed that the retention time decreased on increasing the concentration of Brij-35 (0.00015 M to 0.0015 M) in the mobile phase having a fixed concentration of SDS at 0.005 M. Thus, the mixed system provided good resolution with lower retention times in comparison to the mobile phase having single surfactant. “Successive additions of the non-ionic surfactant gradually reduced the retention time, though to a smaller extent, in comparison to the addition of SDS to a mobile phase containing a fixed amount of Brij-35”.5,6


Page 12 of 22

Page 13 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Chromatogram showing separation of diastereomeric derivatives of (RS)-Mex prepared with (S)Lfx.

RP-HPLC of diastereomeric derivatives of (RS)-Mex WMP-TEAP (80:20) was used as mobile phase for RP-HPLC under isocratic mode for 60 min, at a flow rate of 0.70 mL/min. The pH of TEAP buffer was maintain in between 3.5-4.0. WMP-TEAP as mobile phase was found successful for separation of all pair of diastereomeric derivatives of (RS)-Mex and in terms of selectivity and reproducibility; values for retention factor (k), separation factor (α) and resolution (Rs) under the optimized HPLC conditions are given in Table-1. Figure-6 showing separation chromatograms of LM-1 and LM-2 (as a representative) and Figure-S3 shows separation peaks of the rest of the diastereomeric derivatives. Preparative separation and hydrolysis of diastereomeric derivatives The mobile phase containing “TEAP buffer-MeOH in a ratio of 30:70 in the isocratic mode at a flow rate of 5 mL min-1 was found to be optimal” for the isolation of the diastereomeric derivatives of (RS)-Mex. The isolated derivatives by preparative HPLC were characterized by “the 1H-NMR, UV, and IR spectra, specific rotation, and melting point, and CHN analysis results” and the characterization data are given in the supporting information. The microwave irradiated acid hydrolysis of diastereomeric derivatives was performed to isolate native enantiomers of (RS)-Mex. “The acid hydrolysis did not involve any reaction at the stereogenic centre. Therefore, racemization was neither theoretically expected nor experimentally observed”. The recoveries of the native enantiomers of (RS)-Mex obtained in good yields. The



native enantiomers that were isolated using preparative HPLC and purified enantiomers were characterized by “specific rotation and spectroscopic techniques such as 1H-NMR, HRMS, and IR spectroscopies”. The characterization data of the obtained enantiomers are presented in the supporting information. Optimized lowest energy structures and elution order Geometry optimized ‘lowest energy’ structures of all diastereomeric derivatives of (RS)-Mex were developed using the “program Gaussian 09 Rev. A.02 and hybrid density functional B3LYP with 6-31G* basis set”. DFT optimized lowest energy structures of LM-1 and LM-2 are given in Figure-7 and rest DFT optimized structures are given in Figure-S4 and Figure-S5 as ‘Supporting Information’.

Figure 7. Optimized lowest energy structures of diastereomeric derivatives of (RS)-Mex prepared with (S)Lfx, using the “program Gaussian 09 Rev. A.02 and hybrid density function B3LYP with 6-31G*”.


Page 14 of 22

Page 15 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


These lowest energy optimized structures of diastereomeric derivatives (Figure-7) showing different spatial nearness in between phenyl of (RS)-Mex and piperazinyl of (S)-Lfx, and this leads to have different size, different spatial arrangements and different hydrophobic interactions with C18-column of both diastereomeric derivatives. Figure-7 clearly showing size difference in between both diastereomeric derivatives (Size of LM-1 and LM-2, respectively is, 17.21 Å and 14.34 Å); and these structures were used to determine elution order of first and second eluting diastereomeric derivatives as pervious literature6,7 and described in supporting information. Method validation As followed by ICH guideline44 validation studies were carried out for diastereomeric derivatives of (RS)-Mex prepared with (S)-Lfx, as a representative, in a concentration range between 40-4000 ng mL−1. The LOD and LOQ were found, respectively, 0.126 ng mL−1 and 0.385 ng mL−1 for diastereomers of (RS)-Mex. The validation data is given in Table-S1. The detailed description of validation is given as Supporting Information.

Table-1: Chromatographic separation data of diastereomeric derivatives of (RS)-Mex. Diastereomeric derivatives of (RS)-Mex prepared with

Chromatographic separation data k1

k2

α

RS

1.

(S)-Lfx

1.791

3.170

1.770

8.28

2.

(S)-Kpf

4.571

6.389

1.397

10.91

3.

(S)-Ibf

6.664

8.881

1.332

8.86

The green assessment of the developed method The Analytical Eco-Scale method25 and Green Analytical Procedure Index method (GAPI)26 were used to measure the green aspects of the developed method. The Figure-8 showing GAPI assessment of the developed method and the Figure-S6 showing the Analytical Eco-Scale optimization of the steps of the developed methods using “Green alternatives for different steps”, both of the figures clearly showing that the developed method is a green method. Also, the penalty points for developed method were calculated (Table-S2) and the obtained green assessment score 15 ACS Paragon Plus Environment


value is 76 points, and according to the Analytical Eco-Scale the score value >75 represent the excellent green analysis.25

Figure 8. GAPI assessment of the green profile of the developed method

Comparison of present work with literature reports The current method could be considered green analytical method when compared with all available literature reported on enantioseparation. Previous literature on enantioseparation revels the use of huge amount of hazardous organic solvents during chromatographic separation, while in current studies only aqueous surfactant base non-hazardous mobile phase (WMP) was used. Also, the single step in-situ covalent derivatization of (RS)-Mex reduced the consumption of organic solvent and minimize the waste, and leads to fast derivatization as compared to reported methods15-24 [reported methods

require, the synthesis of the CDRs and then synthesis of diastereomeric

derivative; multi-step synthesis]. Also, the developed method is examined by Analytical Eco-Scale method25 and Green Analytical Procedure Index method (GAPI)26 and it is observed that the developed method is an excellent green analytical method (green score value is 76). Present work shows very good enantioseparation of the diastereomeric derivatives of (RS)-Mex in terms of resolution (8.28-10.91), separation factor (1.39-1.77), and retention times (2.512-10.87 min). This is observed calculated values of resolution (Rs), separation factor (α) and retention time were better than diastereomeric derivatives of (RS)-Mex prepared with Marfey’s reagent,15,16 cyanuric chloride,17,18 chloroformates,19 isothiocyanate,19,20 naproxen,16,23 (S,S)-O,O’-di-p-toluoyl 16 ACS Paragon Plus Environment

Page 16 of 22

Page 17 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


tartaric acid anhydride,21 divinyl dicarboxylates,22 (S)-(−)-(N)-trifluoroacetyl-prolyl chloride,23 (1S)-(−)-camphanic chloride,23 2-anthroyl chloride24 based chiral reagents; and also better than direct enantioseparation methods in which different chiral stationary phased used.45-47 The current method providing very low values of LOD and LOQ when comparing with literature.15-24,45-47 Also, the method provided decreasing elution time for diastereomeric derivatives of (RS)-Mex as compared to literature,

15-24,45-47

resulting into lesser consumption of mobile phase. During this

whole work only aqueous WMP mobile phase was used for HPLC analysis so this method is not showing any negative impact on environment as like pervious reported methods.

CONCLUSION This is the first report on the synthesis of three pairs of diastereomeric derivatives of (RS)-Mex, under single step in-situ derivatization condition, and their enantioseparation using micellar liquid chromatography (indirect approach of enantioseparation). Covalently bonded moiety [(S)-Lfx/(S)Kpf/(S)-Ibf] in the diastereomers play a good role in elution and enhances their molar absorbance resulting into very low LOD (0.126 ng mL−1). An aqueous solution of SDS (0.005 M) and Brij-35 (0.0015 M) was used with TEAP buffer as mobile phase and found sufficient to separate prepared diastereomeric derivatives completely, and considered as a green mobile phase. Besides being ‘green mobile phase’, it is less expensive and less hazardous in the absence of organic solvents. The retention time and resolution behaviour of diastereomeric derivatives of (RS)-Mex were strongly affected by varying the concentration of SDS and Brij-35. Increased the concentration of surfactant [SDS (0.0005 to 0.005 M) or Brij-35 (0.00015 to 0.0015 M)] in mobile phase resulted in decreasing retention time with increasing resolution. The method provided very good resolution (8.28-10.91), separation factor (1.39-1.77) and very low retention time (2.512-10.87 min). The synthesized diastereomeric derivatives were hydrolysed in order to obtain native enantiomers of (RS)-Mex. The isolated enantiomers were characterised by spectroscopic techniques. The developed method was validated according to ICH guidelines for linearity, accuracy and limit of detection (LOD). The developed method was measured with green assessment methods and scored 76 green assessment points, and represents an excellent green analytical method for the determination of the enantiopurity of Mexiletine. Also, developed method can be used for determination of enantiomeric purity of amino group-containing compounds racemic mixture obtained from organic syntheses or 17 ACS Paragon Plus Environment


the pharmaceutical industries. The method leads to a new opportunity and scope to modify liquid chromatographic chiral separations in greenways.

Acknowledgment Author is grateful to the University Grants Commission of India (UGC), New Delhi, for the award of a senior research fellowship (to Shiv Alwera).

References 1. Yuan, X.; Kim, E. G.; Sanders, C. A.; Richter, B. E.; Cunningham, M. F.; Jessop, P. G.; Oleschuk, R. D. CO2-modified solvents for chromatographic separation. Green Chem. 2017, 19, 1757–1765. 2. Yuan, X.; Richter, B. E.; Jiang, K.; Boniface, K. J.; Cormier, A.; Sanders, C. A.; Palmer, C.; Jessop, P. G.; Cunningham, M. F.; Oleschuk, R. D. Carbonated water for the separation of carboxylic compounds: a chromatography approach. Green Chem. 2018, 20, 440-448 3. Shen, Y.; Chen, B.; Beek, T. A. V. Alternative solvents can make preparative liquid chromatography greener. Green Chem. 2015, 17, 4073–4081. 4. Funari, C. S.; Carneiro, R. L.; Khandagale, M. M.; Cavalheiro, A. J.; Hilder, E. F. Acetone as a greener alternative to acetonitrile in liquid chromatographic fingerprinting. J. Sep. Sci. 2015, 38, 1458–1465. 5. Ruiz-Angel, M. J.; Peris-García, E.; García-Alvarez-Coque, M. C. Reversed-phase liquid chromatography with mixed micellar mobile phases of Brij-35 and sodium dodecyl sulphate: a method for the analysis of basic compounds. Green Chem. 2015, 17, 3561– 3570. 6. Alwera, S.; Bhushan, R. Micellar liquid chromatography for enantioseparation of βadrenolytics using (S)-ketoprofen-based reagents. J. Liq. Chromatogr. Relat. Technol. 2017, 40(14), 707–714. 7. Alwera, S.; Bhushan, R. RP‐HPLC enantioseparation of β‐adrenolytics using micellar mobile phase without organic solvents. Biomed. Chromatogr. 2017, 31(11), e3983. https://onlinelibrary.wiley.com/doi/abs/10.1002/bmc.3983 8. Rentsch, K. M. The importance of stereoselective determination of drugs in the clinical laboratory. J. Biochem. Biophys. Methods 2002, 54(1-3), 1-9. 9. Katzung, B. G. The nature of drugs. In: Basic and clinical pharmacology. 9th ed. New York: Lange Medical Books/McGraw Hill. 2004; 3-5. 10. Nguyen, L. A.; He, H.; Pham-Huy, C. Chiral drugs: an overview. IJBS, 2006, 2(2), 85-100.


Page 18 of 22

Page 19 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


11. Dunn, P. J. The importance of green chemistry in process research and development, Chemical Society Reviews. Chem. Soc. Rev. 2012, 41, 1452–1461. 12. Welch, C. J.; Wu, N.; Biba, M.; Hartman, R.; Brkovic, T.; Gong, X.; Helmy, R.; Schafer, W.; Cuff, J.; Pirzada, Z.; Zhou, L. Greening analytical chromatography. Trends. Analyt. Chem., 2010, 29(7), 667-680. 13. Esteve-Romero, J.; Carda-Broch, S.; Gil-Agustí, M.; Capella-Peiró, M. E.; Bose, D. Micellar liquid chromatography for the determination of drug materials in pharmaceutical preparations and biological samples. Trends Anal. Chem., 2005, 24, 75–91. 14. Stępnik, K. E. A concise review of applications of micellar liquid chromatography to study biologically active compounds. Biomed. Chromatogr. 2017, 31, e3741. 15. Bhushan, R.; Vashistha, V. K. Synthesis of variants of Marfey’s reagent having d-amino acids as chiral auxiliaries and liquid-chromatographic enantioseparation of (RS)-Mexiletine in spiked plasma: Assessment and comparison with l-amino acid analogs. J. Chromatogr. A 2015, 1379, 43–50. 16. Bhushan, R.; Tanwar, S. Synthesis of dinitrophenyl-L-Pro-N-hydroxysuccinimide ester and four new variants of Sanger’s reagent having chiral amines and their application for enantioresolution of mexiletine using reversed-phase high-performance liquid chromatography. J. Chromatogr. A 2009, 1216, 5769–5773. 17. Bhushan, R.; Dixit, S.; Reversed-phase high-performance liquid chromatographic separation of diastereomers of (R,S)-mexiletine prepared by microwave irradiation with four new chiral derivatizing reagents based on trichloro-s-triazine having amino acid as chiral auxiliaries and 10 others having amino acid amides. J. Chromatogr. A 2010, 1217, 7669– 7676. 18. Batra, S.; Bhushan, R. Amino acids as chiral auxiliaries in cyanuric chloride-based chiral derivatizing agents for enantioseparation by liquid chromatography. Biomed. Chromatogr. 2014, 28, 1532–1546. 19. Büschges, R.; Linde, H.; Mutschler, E.; Lagguth, H. S. Chloroformates and isothiocyanates derived from 2-arylpropionic acids as chiral reagents: synthetic routes and chromatographic behaviour of the derivatives. J. Chromatogr. A 1996, 725, 323-334. 20. Kleidernigg, O. P.; Lindner, W. Synthesis of new stable aliphatic isothiocyanate based chiral derivatizing reagent and application in indirect separation of chiral amino and thiol compounds. Chromatographia 1997, 44, 465–472. 21. Bhushan, R.; Tanwar, S.; Dixit, S. Indirect enantioresolution of (R,S)- mexiletine by reversed-phase high-performance liquid chromatography via diastereomerization with ((S,S)-O,O′-di-p-toluoyl tartaric acid anhydride), (S)-naproxen and nine chiral reagents synthesized as variants of Marfey’s reagent. Biomed. Chromatogr. 2011, 25, 398–404.



22. Zheng, C.; Zhang, D.; Wu, Q.; Lin, X. High performance liquid chromatography enantioseparation of the novel designed mexiletine derivatives and its analogs. Chirality 2011, 23, 99–104. 23. Dixit, S.; Dubey, R.; Bhushan, R. High-performance liquid chromatography for analytical and small-scale preparative separation of (RS)-mexiletine using (S)-(−)-(N)- trifluoroacetylprolyl chloride and (1S)-(−)-camphanic chloride and recovery of native enantiomer by detagging. Acta Chromatogr. 2014, 26(4), 625–636. 24. Kowk, D. K.; Igwemezie, L.; Kerr, C. R.; McErlane, K. M. High-performance liquid chromatographic analysis using a highly sensitive fluorogenic reagent, 2-anthroyl chloride, and stereoselective determination of the enantiomers of mexiletine in human serum. J. Chromatogr. B 1994, 661(2), 271-80. 25. Gałuszka, A.; Konieczka, P.; Migaszewski, Z. M.; Namieśnik, J. Analytical Eco-Scale for assessing the greenness of analytical procedures. Trends Analyt. Chem. 2012, 37, 61-72. 26. Płotka-Wasylka, J. A new tool for the evaluation of the analytical procedure: Green Analytical Procedure Index. Talanta 2018, 181, 204–209. 27. Rezgui, F.; Gaied, M. M. E. DMAP-catalyzed hydroxymethylation of 2-cyclohexenones in aqueous medium through Baylis-Hiilman Reaction. Tetrahedron Lett.1998, 39, 5965-5966. 28. Alwera, S.; Bhushan, R. (RS)-Propranolol: enantioseparation by HPLC using newly synthesized (S)-levofloxacin-based reagent, absolute configuration of diastereomers and recovery of native enantiomers by detagging. Biomed. Chromatogr. 2016, 30, 1223–1233. 29. Zhu, L.; Ding, L.; Zhang, Q.; Wang, L.; Tang, F.; Liu Q.; Yao, S. Direct analysis of cryptotanshinone and tanshinone IIA in biological samples and herbal medicinal preparations by a green technique of micellar liquid chromatography. Green Chem. 2009, 11, 132–137. 30. Ogino, K.; Uchiyama, H.; Abe, M. Mixed surfactant systems, Marcel Dekker, New York, 1993. 31. Berthod, A.; GarcÍa-Alvarez-Coque, C. Micellar liquid chromatography, Marcel Dekker, New York, 2000, Vol. 83. 32. Scamehorn, J. F. Phenomena in mixed surfactant systems. 1985, Phenomena in mixed surfactant systems; scamehorn, J.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986. 33. Scamehorn, J. F.; Schechter, R. S.; Wade, W. H. Adsorption of surfactants on mineral oxide surfaces from aqueous solutions. II: binary mixtures of anionic surfactants. J. Colloid Interface Sci. 1982, 85, 479-493. 34. Williams, R. J.; Phillips, J. N.; Mysels, K. J. The critical micelle concentration of sodium lauryl sulphate at 25° C. Trans. Faraday Soc. 1955, 51, 728-737. 20 ACS Paragon Plus Environment

Page 20 of 22

Page 21 of 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


35. Zana, R.; Benrraou, M.; Rueff, R. Alkanediyl-α,Ω.-bis(dimethylalkylammonium bromide) surfactants. 1. Effect of the spacer chain length on the critical micelle concentration and micelle ionization degree. Langmuir 1991, 7(6), 1072–1075. 36. Xuan, J.; Hamblin, M. N.; Stout, J. M.; Tolley, H. D.; Maynes, R. D.; Woolley, A. T.; Hawkins, A. R.; Lee, M. L. Surfactant addition and alternating size current electrophoretic oscillation during fractionation of nanoparticles segments in channels with two or three different height. J. Chromatogr. A 2011, 1218(50), 9102-9010. 37. Qazi, M. J.; Liefferink, R. W.; Schlegel, S. J.; Backus, E. H. G.; Bonn, D.; Shahidzadeh, N. Influence of surfactants on sodium chloride crystallization in confinement. Langmuir 2017, 33, 4260−4268. 38. Determination of critical micelle concentration values using capillary electrophoresis instrumentation. Anal. Chem. 1997, 69, 4271-4274. 39. Thermo Scientific™, Brij™-35 surfact-amps™ detergent solution, https://www.thermofisher.com/order/catalog/product/28316 (accessed June 20, 2018). 40. Hait, S. K.; Moulik, S. P. Determination of critical micelle concentration (cmc) of nonionic surfactants by donor–acceptor interaction with iodine and correlation of cmc with hydrophile–lipophile balance and other parameters of the surfactants. J. Surfactants Deterg. 2001, 4(3), 303-309. 41. Kahlweit, M. Kinetics of formation of association colloids. J. Colloid Interface Sci. 1982, 90, 92-99. 42. Griffiths, I. M.; Bain, C. D.; Breward, C. J. W.; Colegate, D. M.; Howell, P. D.; Waters, S. L. On the predictions and limitations of the Becker–Döring model for reaction kinetics in micellar surfactant solutions. J. Colloid Interface Sci. 2011, 360, 662–671. 43. Aniansson, E. A. G.; Wali, S. N. On the kinetics of step-wise micelle association. J. Phys. Chem. 1974, 78, 1024-1030. 44. ICH. Q2B Document: Validation of analytical procedures. International conference of harmonization: Geneva, 1996. 45. Jin, K. B.; Kim, H. E.; Hyun, M. H. Liquid chromatographic resolution of mexiletine and its analogs on crown ether-based chiral stationary phases. Chirality 2014, 26, 272–278. 46. Perera, R. W.; Lough, W. J. Assessment of chiral stationary phases for suitability for combined enantiomeric impurity/related substances assays. J. Chromatogr. A 2011, 1218, 8655–8663. 47. McErlane, K. M.; Igwemezie, L.; Kerr, C. R. Stereoselective analysis of the enantiomers of mexiletine by high-performance liquid chromatography using fluorescence detection and study of their stereoselective disposition in man. J. Chromatogr. B 1987, 415(2), 335-346.



Graphical Abstract 26x13mm (300 x 300 DPI)

ACS Paragon Plus Environment

Page 22 of 22

In Situ Derivatization of (RS)-Mexiletine and Enantioseparation Using

Recommend Documents