TY - JOUR
AU - Bhushan,, Ravi
AB - Abstract Direct resolution of (RS)-ketorolac and (RS)-etodolac has been achieved by ligand exchange thin layer chromatography. Cu(II) has been used as a complexing ion with three enantiomerically pure amino acids (namely, l-tryptophan, l-histidine and l-phenylalanine) as chiral dopants. Chromatograms were developed using different combinations of solvent systems in different ratio having no chiral additive. Iodine vapors were used for location of spots. Different experimental factors, i.e., effect of temperature, mole ratio of Cu(II) to l-amino acids and solvent ratio were optimized in order to improve the separation efficiency. Results have been compared (for RF and Rs). Limit of detection for each enantiomer of both the racemic analytes (ketorolac and etodolac) was found to be 0.6 and 0.8μg. Introduction The significance of enantiomeric separation cannot be overemphasized in pharmaceutical and pharmacological work because it is extremely important in order to obtain higher drug efficiency and to alleviate undesirable side effects since enantiomers of drug compounds may possess quite different pharmacokinetics and pharmacodynamics. To control the enantiomeric purity and to increase the number of enantiopure drugs into the market (as per requirements of regulatory agencies) the need for fast and efficient enantioseparation methods with minimal costs is becoming more compelling. Enantioseparation remains challenging and fascinating; it is expected that the development of methods for chiral analysis, particularly for enantioseparation, will continue in life sciences, the pharmaceutical industry and asymmetric synthesis. According to “Transparency Market Research” (1) the global market of chiral analysis in 2010 was estimated at US $7.34 million while it was assumed to grow to $10.6 billion in 2017. Among the various methods and approaches, chiral ligand-exchange chromatography (CLEC) is one of the earliest and the most effective method of direct enantioseparation. It was developed (during 1968–1971) when Davankov and Rogozhin modified commercial HPLC columns by coating alkyl derivatives of α-amino acids such as n-decyl-l-histidine or n-hexadecyl-l-proline, onto C18 silica for enantiomeric resolution (2–4). Various aspects of ligand-exchange chromatography have been reviewed (5–7). Based on the work of Davankov, ligand exchange TLC was developed during the period 1984–1986 at Degussa AG by Günther, Martens et al. (8–11), and resolution of enantiomeric amino acids was achieved. The RP C18 silica gel plates were impregnated by sequential immersion in the solutions of Cu(II) acetate and the new chiral selector namely (2S,4 R,2′RS)-N-(2′-hydroxy dodecyl)-4-hydroxy proline (12). During the same year (1984) Weinstein (13) reported TLC enantiomeric separation of all the dansyl protein amino acids, except proline, based on ligand exchange using RP plates pre-treated with a copper(II) complex of N,N-di-n-propyl-l-alanine. Although the procedure differed from that reported by Günther, Martens et al. (8–11), in the choice of chiral selectors and range of applicability, both had a very similar methodology. Application of ligand exchange and use of different chiral selectors as impregnating reagents has been described for TLC resolution of enantiomers of a variety of compounds (14). Bhushan et al. (15) resolved a few racemic amino acids, certain β-blockers (16, 17), and bupropion and baclofen (18–20) via ligand exchange on silica plates using a complex of copper (II)-l-proline or other l-amino acids. Thin layer chromatography (TLC) is an economically viable yet a consistent technique in various situations for analysis of drugs, pharmaceuticals, products of enantioselective synthesis and detection of counterfeit drugs for regulatory purposes. It is a simple, convenient, inexpensive and rapid separation method requiring little instrumentation. The compounds separated by TLC can be identified and quantified in many ways, using either off-line or online detection. Separation, detection, isolation, identification, purification and quantification, of each of the enantiomers of racemic analytes comprise an example of the comprehensive use of analytical TLC. The method holds considerable significance especially in resource limited settings. The overall methodology of CLEC used both in TLC or HPLC remains advantageous and efficient and a technique of choice for serial determinations of enantiomeric compositions as it neither requires expensive chiral columns nor requires any pre-column derivatization with chiral reagent. The non-steroidal anti-inflammatory drugs (NSAIDs) are the most often used ones for human health care since they are available over the counter. It is a group of drugs that reduces pain, decrease fever, and, in higher doses, decreases inflammation; side effects include an increased risk of stomach ulcers. Every day more than 30 million Americans use them to soothe headaches, sprains, arthritis symptoms, and other daily discomforts. Both ketorolac (Ket) and etodolac (Etd) belong to category NSAIDs. Ket, [(RS)-5-benzoyl-1, 2-dihydro-3H-pyrrolo [1,2a] pyrrole-1-carboxylic acid] (Figure 1), produces analgesia and decreases inflammation by inhibiting the enzyme cyclooxygenase, resulting in the decrease in formation of prostaglandins and sensitization to pain at sites of inflammation. It is more effective than other NSAIDs in reducing pain from both inflammatory and non-inflammatory causes. Its rapid onset of action, effectiveness, lack of opiate action and safety makes it an attractive agent for general-purpose analgesia. It also has antipyretic properties 20 times more potent than those of aspirin. The physiological activity of Ket resides almost exclusively with the (S)-(−)-enantiomer while the drug is marketed and administered as a racemic mixture. Figure 1. View largeDownload slide Structures of (RS)-Ket and (RS)-Etd. Figure 1. View largeDownload slide Structures of (RS)-Ket and (RS)-Etd. Etd, [(RS)-1,8-diethyl-1,3,4,9-tetrahydropyrano[3,4-b]-indole-1-acetic acid] (Figure 1) is also marketed and administered as racemate, as analgesic and for the treatment of rheumatoid arthritis and osteoarthritis. It also decreases synthesis of peripheral prostaglandins involved in mediating inflammation by inhibition of cyclooxygenase (COX) enzyme. As compared to other NSAIDs, it produces less gastrointestinal toxicity. The biochemical and pharmacological effects of (RS)-Etd are due to (S)-enantiomer, while the (R)-enantiomer lacks COX-inhibitory activity. The (R)- and (S)-enantiomers of Etd are not metabolically interconvertible (21). Starek et al. (22) reviewed TLC methods and new TLC techniques, along with patent developments, which were developed and used for determination of NSAIDs (including Ket and Etd) in bulk drugs, formulations and biological fluids for the period from 1990 to 2008. Direct enantiomeric resolution of Ket was achieved on anamylose-based chiral stationary phase under the normal phase mode and using ornidazole as internal standard (23). Radio-chemical purification of (the synthesized) tritium-labeled rac-ketorolac by RPHPLC followed by direct resolution using a chiral α1-acid glycoprotein HPLC column afforded labeled enantiomers of high specific activity (24). Analytical and semi-preparative HPLC enantioresolution of (RS)-Ket from pharmaceutical formulation and in human plasma using chiral columns (25), and HPLC resolution using polysaccharide based CSP (26) has been reported. In the recent past, direct enantiomeric resolution of (RS)-Etd by achiral phase TLC (27), LC–MS studies of diastereomeric derivatives of (RS)-Etd prepared from chirally pure amines (28), and TLC separation of diastereomeric amides of Etd (29) have been reported from this laboratory. These reports and the literature cited therein (on enantiomeric resolution of Etd and Ket) clearly suggest that (i) there is no report on TLC resolution of (RS)-Etd or (RS)-Ket by using CLEC, and (ii) CLEC (as described above) is an important and successful method for enantioresolution. Taking into account (i) and (ii), experiments were designed and performed to achieve enantioresolution of the two title analytes by TLC using chiral ligand exchange reagent (LER) as “chiral additive in achiral stationary phase” and the chromatograms were developed using mobile phase having no chiral additive. LER was mixed with the slurry of silica gel while preparing the TLC plates. Three enantiomerically pure amino acids [namely, l-ryptophan (Trp), l-histidine (His) and l-phenylalanine (Phe)] were used as chiral ligands with Cu(II) as a bivalent complexing ion. The enantiomers of each of the two analytes, so separated, were located with iodine vapors. The present work is different from existing reports on enantiomeric resolution of (RS)-Etd or (RS)-Ket in terms of the following aspects (i) it is a direct approach using CLEC, (ii) the ligand exchanged diastereomeric complexes corresponding to each of the enantiomers (of the two racemates) were isolated via preparative TLC and their specific rotations were determined, and (iii) the native enantiomers were obtained from the isolated ligand exchanged diastereomeric complexes. Experimental Chemicals and instrumentation The pharmaceutical tablets of (RS)-ketorolac marketed as Ketorol DT (Dr Reddy Laboratories Ltd, Hyderabad, India) containing 10 mg active pharmaceutical ingredient and (RS)-etodolac marketed as Etova (Ipca Laboratories Ltd, Mumbai, India) containing 400 mg active pharmaceutical ingredient were purchased from pharmaceutical shop in the local market. Solvents employed, i.e., ethanol (EtOH), acetonitrile (MeCN), dichloromethane (CH2Cl2), methanol (MeOH) and ethyl acetate (EtOAc) of analytical reagent grade were obtained from SISCO Research Laboratory (Mumbai, India), E. Merck (Mumbai, India) and BDH (Mumbai, India). Silica gel G (pH 7.0) having 13% calcium sulfate as binder and 0.02% iron, chloride and lead impurities in a 10% aqueous suspension, was purchased from Merck (Mumbai, India). The equipment/instrument used for the present experiments are, Milli-Q system from Millipore (Bedford, MA, USA) to obtain purified water (18.2 MΩ cm3) for preparing stock solutions, Cyberscan 510 pH meter (Singapore), Shimadzu UV-1601PC UV–Visible Scanning Spectrophotometer, Krüss model P3001RS polarimeter (Germany) and FT-IR spectrometer 1600 (Boardman, OH, USA). Isolation and purification of racemic analytes from commercial formulations The coating of 10 Ketorol DT tablets (each containing 10 mg of (RS)-ketorolac tromethamine, for example) was scratched out and were finely powdered. It was sonicated for ~10 min in 20 mL methanol at room temperature. It was filtered and the residue obtained was further extracted with methanol. Both combined filtrates were concentrated and ketorolac tromethamine salt was obtained. The sample was protected from light throughout the experiments, and was preserved in tight, light-resistant containers at room temperature (30). Etodolac was also extracted, purified and characterized by following the same procedure. Separation of tromethamine from ketorolac (RS)-Ket was extracted by sonicating ketorolac tromethamine salt (as obtained above) for ~5 min in dichloromethane (DCM). It was filtered. Though Ket is miscible in DCM while tromethamine is not; the filtrate was concentrated and kept in refrigerator untilcrystals appeared. The crystals were washed with cold DCM and dried in vacuum desiccator. The isolated and purified Ket was characterized by UV (λmax ∼ 316 nm) and IR. Preparation of standard solutions Stock solutions of (RS)-Ket and (RS)-Etd (each 10 mM) were prepared in MeOH and further diluted with MeOH for required working solutions. All the solutions were filtered through a 0.45 μm filter. The solutions were scanned for determination of λmax. Five solutions in the range 1 × 10−4 to 5 × 10−4 M were prepared by dilution. Their absorbance was recorded and a calibration plot was constructed. Copper (II) acetate (2 mM) was prepared in purified water–methanol (95:5). Preparation of Cu (II)–l-amino acid complex (the LER) and TLC plates l-Trp, l-His and l-Phe were chosen as chiral selectors. Following combinations of varying molar concentrations of l-amino acid(s) and Cu(II) were used to prepare TLC plates by preparing the slurry of silica gel in these solutions. The plates were prepared by, (i) using 1, 2, 4 and 6 mM concentrations of each of the amino acids with 2 mM of Cu (II) acetate; this provided the l-amino acid-Cu (II) ratio of 1:2, 1:1, 2:1 and 3:1, (ii) using a fixed concentration of amino acid (4 mM) and varying the concentrations of Cu(II) acetate (2, 4 and 6 mM); in this case, the ratio of l-amino acid-Cu(II) was 2:1, 1:1 and 2:3, respectively, (iii) keeping the ratio of l-amino acid-Cu(II) as 2:1, the following combinations were also tried, 2 mM l-amino acid-1 mM Cu(II), 4 mM l-amino acid–2 mM Cu(II), 6 mM l-amino acid-3 mM Cu(II) and 8 mM l-amino acid–4 mM Cu(II). In this manner experiments were performed using all three AAs, i.e., l-Trp, l-His and l-Phe (18). The UV spectra and pH for each of the solutions mentioned at (i), (ii) and (iii), above, were recorded. TLC plates (10 × 20 cm2 × 0.5 mm thick) were prepared in the laboratory by spreading the slurry of silica gel G (25 g), with a Stahl-type applicator. The plates were allowed to set at room temperature and then were activated for 8–10 h at 60 ± 2°C. These were considered to be impregnated plates. Development of chromatograms and isolation of native enantiomers The 10 μL solution of each of the racemic analytes [(RS)-Ket or (RS)-Etd] were spotted on TLC plates with a 25 μL Hamilton syringe. The chromatograms were developed in a completely dried, paper-lined, pre-equilibrated rectangular glass chamber at different temperatures. The chamber was pre-equilibrated for 15 min with mobile phase at different temperatures, 16, 20, 24, 28 and 32°C (when the room temperature (RT) was <28°C). Each temperature was maintained/controlled using an incubator. Experiments were performed with binary, ternary and quaternary mixtures of solvents such as ethyl acetate, ethanol, methanol, water, dichloromethane and acetonitrile to achieve enantiomer separation. Chromatograms were dried at 40°C in an oven for 10 min and cooled to room temperature. The spots were located in an iodine chamber and RF values were measured. The effect of copper ion concentration to the concentration of the l-amino acids and temperature on chiral separation was investigated and evaluated. Preparative TLC For preparative work, TLC plates (10 × 20 cm2 × 2.0 mm thick) were prepared using the molar concentrations of l-amino acid(s) and Cu(II) that was found successful (Table I) for enantiomeric separation. On each plate, 10 spots in parallel were applied (50 μL of each solution of each racemate per spot). The silica gel corresponding to two separated spots of the ligand exchanged complex (LEC) was scrapped from the plates. It was extracted with 90% ethanol by sonication for a few minutes and the insoluble silica was removed by filtration. The filtrates were dried under vacuum. The residues were dissolved individually in a little amount of DCM. Table I. The hRF (RF× 100), Resolution (Rs) Values and Solvent Systems for Successful Resolution of (RS)-Ket and Etd Under LEC Approach Mobile phase Solvent ratio (v/v) hRF values Rs Specific rotation values [α]D25 =(c=1, MeOH) R S Upper spot Lower spot Ketorolac  Tryptophan MeOH-MeCN-DCM-H2O 3:6:1:0.5 52 75 2.2 +63 −101  Histidine MeOH-MeCN-EtOAc-H2O 2:4:3:1 53 75 2.0 +60 −104  Phenylalanine MeOH-MeCN-DCM 2:3:3 43 58 1.4 +62 −102 Etodolac  Tryptophan EtOH-MeCN-DCM 3:4:2 47 74 2.3 −7 −31  Histidine EtOH-MeCN-EtOAc-DCM 4:1:4:3 56 79 2.2 −10 −34  Phenylalanine MeOH-MeCN-DCM 1:5:2 42 57 1.5 −8 −32 Mobile phase Solvent ratio (v/v) hRF values Rs Specific rotation values [α]D25 =(c=1, MeOH) R S Upper spot Lower spot Ketorolac  Tryptophan MeOH-MeCN-DCM-H2O 3:6:1:0.5 52 75 2.2 +63 −101  Histidine MeOH-MeCN-EtOAc-H2O 2:4:3:1 53 75 2.0 +60 −104  Phenylalanine MeOH-MeCN-DCM 2:3:3 43 58 1.4 +62 −102 Etodolac  Tryptophan EtOH-MeCN-DCM 3:4:2 47 74 2.3 −7 −31  Histidine EtOH-MeCN-EtOAc-DCM 4:1:4:3 56 79 2.2 −10 −34  Phenylalanine MeOH-MeCN-DCM 1:5:2 42 57 1.5 −8 −32 Rs = resolution; hRF = RF × 100; detection, by iodine vapors, solvent front, 8.0 cm; development time, 15–20 min, temperature, 28°C (RT). The spots correspond to the diastereomeric complex species. Table I. The hRF (RF× 100), Resolution (Rs) Values and Solvent Systems for Successful Resolution of (RS)-Ket and Etd Under LEC Approach Mobile phase Solvent ratio (v/v) hRF values Rs Specific rotation values [α]D25 =(c=1, MeOH) R S Upper spot Lower spot Ketorolac  Tryptophan MeOH-MeCN-DCM-H2O 3:6:1:0.5 52 75 2.2 +63 −101  Histidine MeOH-MeCN-EtOAc-H2O 2:4:3:1 53 75 2.0 +60 −104  Phenylalanine MeOH-MeCN-DCM 2:3:3 43 58 1.4 +62 −102 Etodolac  Tryptophan EtOH-MeCN-DCM 3:4:2 47 74 2.3 −7 −31  Histidine EtOH-MeCN-EtOAc-DCM 4:1:4:3 56 79 2.2 −10 −34  Phenylalanine MeOH-MeCN-DCM 1:5:2 42 57 1.5 −8 −32 Mobile phase Solvent ratio (v/v) hRF values Rs Specific rotation values [α]D25 =(c=1, MeOH) R S Upper spot Lower spot Ketorolac  Tryptophan MeOH-MeCN-DCM-H2O 3:6:1:0.5 52 75 2.2 +63 −101  Histidine MeOH-MeCN-EtOAc-H2O 2:4:3:1 53 75 2.0 +60 −104  Phenylalanine MeOH-MeCN-DCM 2:3:3 43 58 1.4 +62 −102 Etodolac  Tryptophan EtOH-MeCN-DCM 3:4:2 47 74 2.3 −7 −31  Histidine EtOH-MeCN-EtOAc-DCM 4:1:4:3 56 79 2.2 −10 −34  Phenylalanine MeOH-MeCN-DCM 1:5:2 42 57 1.5 −8 −32 Rs = resolution; hRF = RF × 100; detection, by iodine vapors, solvent front, 8.0 cm; development time, 15–20 min, temperature, 28°C (RT). The spots correspond to the diastereomeric complex species. Following experiments were performed further. Each of the solutions was examined by UV spectrophotometer and polarimeter to ascertain concentration (using the standard plot as described above) and to calculate specific rotation (of both eluted complexes, e.g., [l-AA-(R)-Ket-Cu] and [l-AA-(S)-Ket-Cu]). The same approach was adopted for the separated spots of complexes of (RS)-Etd. Thus, there were six samples for each of the analytes corresponding to the complex species obtained from the chromatograms. One drop of conc. HCl was added to each of the solutions. The solution(s) were irradiated under microwave (80% at 800 W) for 1 min. The resulting solutions were lyophilized. Each residue was extracted with DCM. The solutions corresponding to the both enantiomers were concentrated. UV spectrum and optical rotations were measured for each of the solutions. There were six samples corresponding to native enantiomer dissociated from the complex, for each of the racemates. Method validation Different solutions of known concentration (200, 600 and 900 μg mL−1) of both racemic analytes were applied three times on impregnated TLC plates to determine repeatability of the method. The accuracy was determined by replicate TLC analysis (n = 3) of the racemic mixture. Results The purity of the racemic analytes isolated from commercial formulations was confirmed by determining the melting point and UV absorption (λmax), and IR spectra which were in agreement with literature reports (31, 32). In both the cases the recovery was of the order of 98% of the quantity reported on the commercial label. Both the purified compounds were used as racemic standards. The λmax for Cu(II) solution was 670 nm, i.e., in the visible region (33). The λmax for l-Trp, l-His and l-Phe were found to be 280, 213 and 260 nm, respectively. These values are in agreement with the literature values (34) for their λmax. The UV spectrum for each of the solution by Cu(II) mixed with the l-AA(s) was also recorded and the λmax was found to be different than that of the Cu(II) solution and any of the AAs individually. This confirmed /indicated the formation of a Cu(II) complex with the respective amino acid, i.e., LER. Mobile phase and hRF (RF × 100) values Various solvent systems, i.e., the combinations of different solvents, enabling successful resolution along with the hRF (RF × 100) and the resolution (Rs) values are reported in Table I. Though both the racemates resolved well into their enantiomers, a different combination was found successful for resolution of (RS)-Ket and (RS)-Etd when there was a different amino acid in the LER as the chiral ligand. The pH of the solvent system (the mobile phase) in each case was measured and found to be between 5.8 to7.5. Application of l-Trp as the chiral ligand in the LER provided the best resolution (Rs) for (RS)-Etd among the various situations mentioned in Table I. hRF values are averages of at least three runs on different plates under identical conditions on the same day and on different days (to evaluate reproducibility). The resolution was calculated by dividing the distance between the center of two spots by the sum of the radii of both the spots. The resolution (Rs) varied from the lowest 1.4 for (RS)-Ket with l-Phe (as the chiral ligand in the LER) to 2.3 for (RS)-Etd with l-Trp (as the chiral ligand in the LER). Representative photographs of actual chromatograms showing resolution of the two racemates are shown in Figure 2. Figure 2. View largeDownload slide Actual photographs of chromatograms showing resolution of (RS)-Ketorolac and (RS)-Etodolac by use of Cu(II)–l-amino acids complex by impregnation method. The plates at (i), (ii) and (iii) show resolution of (RS)-Ket using (i) L-Trp (ii) L-His and (iii) L-Phe, respectively, as the chiral agents for ligand exchange. The plates at (iv), (v) and (vi) show resolution of (RS)-Etd using the same set of L-amino acids. Figure 2. View largeDownload slide Actual photographs of chromatograms showing resolution of (RS)-Ketorolac and (RS)-Etodolac by use of Cu(II)–l-amino acids complex by impregnation method. The plates at (i), (ii) and (iii) show resolution of (RS)-Ket using (i) L-Trp (ii) L-His and (iii) L-Phe, respectively, as the chiral agents for ligand exchange. The plates at (iv), (v) and (vi) show resolution of (RS)-Etd using the same set of L-amino acids. The racemates under study resolved well into their enantiomers at 28°C using LERs having any of the three L-amino acids as chiral ligand. Experiments were performed in a range of temperature systematically until its effect was noted in terms of either tailing or figure-of-eight shaped spots or clear resolution. Experiments were carried out at 16, 20, 24, 28 and 32°C using the successful solvent systems. The decrease in temperature to 16°C showed eight shaped structures or no resolution (with a change of solvent system) and the increase in temperature to 32°C resulted in tailing of spots. Effect of mole ratio of Cu(II) to amino acid The TLC experiments were carried out using varying molar concentration of Cu(II) and l-AAs (as described under experimental section) which showed that the best resolution of the each of the two racemates was achieved when Cu(II) (2 mM) and l-amino acid (4 mM) were in 1:2 molar ratio. Polarimetry Specific rotation values for all the corresponding complexes of upper spots of Ket and Etd (obtained by preparative TLC and as described under (A), above) were calculated which are summarized in Table I. Specific rotation values for enantiomers recovered from the complex species (via acid hydrolysis, as described under (B), above) were found to be [α]D25 = +163° (c = 1, MeOH) for pure enantiomer of Ket (recovered from the upper spots) and [α]D25 = +24° (c = 1, MeOH) for Etd (recovered from the upper spot of the corresponding complex). The specific rotation values for the enantiomer of Ket and Etd recovered from the complex corresponding to lower spot were identical in magnitude with an opposite rotation. By observing specific rotation values, it is concluded that the (R)-isomer was found to have a higher RF value than the (S)-isomer. The polarimetry measurements also showed that the two isomers were in the ratio of 1:1. These results also confirmed the elution order. Method validation The average hRf (Rf × 100) values for the first and second eluting LEC are 7 and 59 for Ket and 6 and 61 for Etd, respectively (Tables IIa and IIb). The results were averages of at least three identical runs on different days and at different time. Table IIa hRF Values Obtained Corresponding to Different Concentrations for Both of the Ligand Exchanged Complexes (LEC) of (RS)-Ket Concentration of diastereomeric mixture (μg mL−1) hRF1 for first eluting diastereomeric ligand exchanged complex hRF2 for second eluting diastereomeric ligand exchanged complex 200 7.3 59.1 600 7.0 57.8 900 7.1 60.3 Concentration of diastereomeric mixture (μg mL−1) hRF1 for first eluting diastereomeric ligand exchanged complex hRF2 for second eluting diastereomeric ligand exchanged complex 200 7.3 59.1 600 7.0 57.8 900 7.1 60.3 Table IIa hRF Values Obtained Corresponding to Different Concentrations for Both of the Ligand Exchanged Complexes (LEC) of (RS)-Ket Concentration of diastereomeric mixture (μg mL−1) hRF1 for first eluting diastereomeric ligand exchanged complex hRF2 for second eluting diastereomeric ligand exchanged complex 200 7.3 59.1 600 7.0 57.8 900 7.1 60.3 Concentration of diastereomeric mixture (μg mL−1) hRF1 for first eluting diastereomeric ligand exchanged complex hRF2 for second eluting diastereomeric ligand exchanged complex 200 7.3 59.1 600 7.0 57.8 900 7.1 60.3 Table IIb hRf Values Obtained Corresponding to Different Concentrations for Both of the Ligand Exchanged Complexes (LEC) of (RS)-Etd Concentration of diastereomeric mixture (μg mL−1) hRFI for first eluting diastereomeric ligand exchanged complex hRF2 for second eluting diastereomeric ligand exchanged complex 200 6.2 61.0 600 5.7 60.4 900 6.0 61.8 Concentration of diastereomeric mixture (μg mL−1) hRFI for first eluting diastereomeric ligand exchanged complex hRF2 for second eluting diastereomeric ligand exchanged complex 200 6.2 61.0 600 5.7 60.4 900 6.0 61.8 Table IIb hRf Values Obtained Corresponding to Different Concentrations for Both of the Ligand Exchanged Complexes (LEC) of (RS)-Etd Concentration of diastereomeric mixture (μg mL−1) hRFI for first eluting diastereomeric ligand exchanged complex hRF2 for second eluting diastereomeric ligand exchanged complex 200 6.2 61.0 600 5.7 60.4 900 6.0 61.8 Concentration of diastereomeric mixture (μg mL−1) hRFI for first eluting diastereomeric ligand exchanged complex hRF2 for second eluting diastereomeric ligand exchanged complex 200 6.2 61.0 600 5.7 60.4 900 6.0 61.8 The relative standard deviation (RSD) were calculated using the formula, R.S.D.=100∗S.D.μ and found 1.40 and 1.81% and 0.71 and 0.49% for first and second eluting LEC of Ket and Etd, respectively. Here “S.D.” stands for standard deviation and “μ” is the average value calculated using different hRf values obtained at different times for both the spots. Standard deviation was found 0.10 and 1.07 and 0.04 and 0.3 for the first and second eluting LEC of Ket and Etd, respectively, and calculated by the formula, S.D.=∑|μ−θ|2N where “N” is the number of hRf values obtained and “θ” is the experimentally observed hRf value corresponding to each concentration of individual LEC. The change in the Rf values were found to be 0.2 ± 0.6–0.8 which showed that the method was robust. The sensitivity of the method was established in terms of limit of detection (LOD). Limits of detection were determined by spotting the different known concentrations and known volume of each analyte to develop the chromatogram under the established conditions. Dilution of both samples of analytes was done until the spot was detected on TLC plate or visible in UV light. The detection was successful up to 0.6 and 0.8 μg mL−1 for Ket and Etd, respectively. Recovery of the enantiomers was in the range 97–99%. Discussion Ligand exchange TLC resolution of (RS)-Ket and (RS)-Etd Separations by means of CLEC are based on the formation of labile ternary metallic complexes in the mobile phase and/or in the stationary phase. Separation models developed for ligand exchange HPLC (35, 36) are also valid for TLC. The mechanism of enantioselectivity in the CLEC depends on whether the chiral ligand is linked on the stationary phase or it is added in the mobile phase since there is involved a series of complexation equilibria in the mobile phase and in the stationary phase. Thermodynamic enantioselectivity of the system, i.e., the difference in the stability of the above two diastereomeric ternary complexes is considered to be responsible for the observed enantioresolution. The stability of the diastereomeric complexes formed in LEC is higher than the stability of the diastereomeric adducts formed by other chiral selectors (37). Schmid et al. (38) proposed formation of ternary mixed metal complexes in CLEC between the chiral selector, the complexing metal ion, and the enantiomer to be recognized. In general, two chiral selector amino acid molecules act as chelating ligands for the Cu(II) ion. In the course of the enantioseparation, one chelating amino acid molecule is replaced by the competing enantiomer molecule of the separated enantiomeric mixture (as depicted in Figure 3). As a result, there occurs formation of diastereomeric complexes of the two enantiomers, so separated, having different stability and different retention through the interaction with the normal silica gel in TLC (or column packing in HPLC). Figure 3. View largeDownload slide Scheme showing formation and structure of ternary complex and the ligand exchanged complex on resolution of racemic analytes. Cu(II) aq. represents complexed state of Cu(II) in aq. medium. Figure 3. View largeDownload slide Scheme showing formation and structure of ternary complex and the ligand exchanged complex on resolution of racemic analytes. Cu(II) aq. represents complexed state of Cu(II) in aq. medium. The denting strength (arising from the functional group causing chelation) of the ligand strongly influences retention and hence separation. The amino acids (i.e., l-Trp, l-His and l-Phe) used in the present studies are the chiral selectors for developing chiral LERs have bidentate chelating properties due to their carboxylic and amino functional groups which are sufficiently strong for complexation with the Cu(II) metal ion and are thus successful for direct enantioseparation of the two racemic analytes. The explanation described in the literature is well satisfactory for enantioseparation of (RS)-Ket and (RS)-Etd under study. It was expected that the complex broke down into constituent units and only the (R)- and (S)-Ket would go into solution since copper ion and L-amino acid are insoluble in DCM. The diastereomeric complexes separated by TLC were isolated and were hydrolyzed. It was expected that the complex broke down into constituent units and only the (R)- and (S)-Ket (and also (R)- and (S)-Etd) would go into solution since copper ion and l-amino acid are insoluble in DCM. Specific rotation values for enantiomers (of each of the two analyte racemates) obtained from the complex species confirmed formation of diastereomeric LEC and recovery of the native enantiomers. Thus the isolation of native enantiomers characterized by their specific rotation values confirms direct resolution of both the racemic analytes, i.e., (RS)-Ket and (RS)-Etd. Conclusion The TLC method using the ligand-exchange approach is very simple, direct, fast, and sensitive and can be applied for resolution, detection and control of enantiomeric purity of (RS)-Ket and (RS)-Etd with low LODs in planar mode. The use of home-made plates is quite easy and simple for successful and economical resolution of the pharmaceutical analytes. Though, obtaining native enantiomers from the LEC may not always be required. But, the experiments so described are clearly an evidence for the formation of such complexes and could be an approach for obtaining native enantiomers. It is the first report to isolate pure enantiomers after doing ligand exchange chromatography. Isolation of native enantiomers is otherwise not feasible in other approaches or becomes very expansive using preparative chiral HPLC. The approach presented herein has additional advantage that the commonly available amino acids, as a pool of chiral selectors, containing opposite D-configuration can be easily used as chiral ligands; this extends the scope of the investigations to understand the chiral mechanism by using the same chiral ligand in different configurations; only a small amount of ligand is required. Overall, it is less expensive and does not require any spray reagent as spots are visible with iodine vapors. Chromatographic separation and detection take place separately in TLC which enables us to carry out the analysis at different times and to make full use of detection techniques for the analysis of constituents. The method is self-sustained and can be realized even in a small laboratory. It opens up an area to control enantiomeric purity of chiral drugs in industry and analytical laboratories (especially associated with regulatory agencies). 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TI - Ligand Exchange Thin Layer Chromatographic Enantioresolution of (RS)-Ketorolac and (RS)-Etodolac and Recovery of Native Enantiomers
JO - Journal of Chromatographic Science
DO - 10.1093/chromsci/bmz023
DA - 2019-07-01
UR - https://www.deepdyve.com/lp/oxford-university-press/ligand-exchange-thin-layer-chromatographic-enantioresolution-of-rs-rK5ByBi8ch
SP - 511
VL - 57
IS - 6
DP - DeepDyve
ER -