Chiral recognition of polysaccharide based CSP for separation of enantiomers and regio isomers of Prasugrel and its related impurities

Article history: Received on: 24/04/2017 Accepted on: 09/06/2017 Available online: 30/07/2017 The current study focuses on the efficiency of chiral chromatographic conditions for separation of critical enantiomers and regio-isomeric pairs of Prasugrel, an antiplatelet drug and its related impurities. Totally, 10 chiral compounds were screened and well resolved using Phenominex-LUX-2-Amylose based chiral HPLC column (polysaccharide CSP). Chiral separations were achieved using Isopropanol, ethanol as organic modifiers and trimethylamine (TEA), trifluoro acetic acid (TFA) as polar modifier. Impact assessment for selection of superior chromatographic conditions to achieve best resolution was evaluated by varying column temperatures and concentrations of organic and polar modifiers. Significant enantiomeric separations were achieved with low alcohol and high TFA concentrations as modifiers in mobile phase. With optimized conditions, not only Prasugrel’s enantiomers, two process related impurities (3-floro and 4-floro prasugrel) and major degradant i.e., Des acetyl impurity which exhibits keto-enol tautomerism in solution state were also separated into respective individual enantiomeric forms. Hence, the current work can be implemented for evaluation of chiral impurities of Prasugrel during synthesis as well as in Active Pharmaceutical Ingredient.


INTRODUCTION
Recently, optically active compounds gained attention towards various fields including pharmaceutics, agrochemicals, ferroelectric, liquid crystals, etc., and therefore their preparation, identification and analysis became increasingly important (Lein et al., 2007, Ulrich et al., 2012).Recent Advances in optical resolution and chiral separations by high-performance liquid chromatography (HPLC) has progressed rapidly, and has become a powerful methodology whose applications were not only limited for determining optical purity but also for purification of optical isomers (Stevenson et al., 1991).It is well known that biological systems are specific in chiral recognitions and impose significant impact on drug's pharmacological and toxicological responses (Waldeck et al., 1993, Lein Ai et al., 2006).
Prasugrel is a racemic mixture and chemically described as (RS)-5-[2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4, 5,6,7tetrahydrothieno [3,2-c]pyridin-2-yl acetate.It is a new oral thienopyridine anti-platelet drug from the same class as clopidogrel and has a more rapid and potent effect on platelet inhibition than does clopidogrel (Bhatt et al., 2003, Z.-M. Wang et al., 2010).It has an empirical formula of C 20 H 20 FNO 3 S, a molecular weight of 373.44 g/mole.Both the prasugrel's isomers have same biological activity and active metabolites inhibits platelet activation and aggregation by irreversibly blocking P2Y12 adenosine diphosphate receptors.Prasugrel is also approved for treating acute coronary syndrome (moderate to high risk unstable angina, ST-segmentelevation myocardial infarction (STEMI) or non-ST-segmentelevation myocardial infarction (NSTEMI) in combination with aspirin in people who are to undergo percutaneous coronary intervention (PCI).As per thorough literature survey, few research articles were published till date which discusses about quantification of prasugrel and its enantiomeric metabolites in human plasma.
One method discusses about Stereo selective Metabolism of Prasugrel in Humans Using a Novel Chiral Liquid Chromatography-Tandem Mass Spectrometry Method (Enaksha et al., 2007).Another work reveals about Electrospray ionization LC-MS/MS validated method for the determination of the active metabolite (R-138727) of prasugrel in human plasma and its application to a bioequivalence study (Ojikumar et al., 2011).Few more research articles discusses about impurity profiling and comparison of forced degradation behavior of prasugrel by HPLC and UPLC (Singh et al., 2015, Malati et al., 2012, Kapendra et al., 2011).But, till date no work represents enantiomeric separation of prasugrel in active pharmaceutical ingredient's perspective, which basically add core value to manufacturing sector.No Official monograph is available at present, for drug substance and drug product which supports chiral separation of prasugrel.
Hence, the aim of our study is to develop a chromatographic method to separate racemic mixture of prasugrel and its positional isomers as well as its process related impurities and major degradant into individual enantiomers.The effect of the steric structure of selected CSP and concentration of the mobile phase modifier on the retention (k') and stereo selectivity (α) of a series of prasugrel related enantiomers along with impact of column temperature on retention and resolution were studied and represented in this work.

Instrumentation and Chromatographic conditions
Method development and verification studies were carried on Waters alliance 2489 HPLC system equipped with a photo diode array detector (PDA).The output signal was monitored at UV wavelength of 215nm and data processed using Empower-2 software.The chromatographic separation was achieved on a Lux amylose-2, 250 mm x 4.6 mm, 5 m column supplied by Phenomenex, USA, using a mobile phase containing n-hexane -ethanol -TEA -TFA (90 : 10 : 0.1 : 0.2,v/v/v/v).The flow rate of the mobile phase was kept at 1.0 mL/min in iso-cratic mode.The column oven temperature was maintained at 35C and the injection volume was set at 10L.

Test Sample preparation
Absolute Ethanol was used as diluent during the test samples preparation.Prior to sample preparation, glassware used for study were thoroughly washed and rinsed with diluent to restrict contamination.Weighed and transferred appropriate quantities of racemic mixtures of Prasugrel (PRA-E1 and PRA-E2), 3-fluoro impurity (PRA-3F-E1 and PRA-3F-E2) and 4-fluoro impurity (PRA-4F-E1 and PRA-4F-E2) to a 10mL amber volumetric flask.Vortexed to dissolve completely and further dilutions were made with ethanol to meet final concentration of 0.8mg/mL.This initial concentration was chosen to evaluate the elution pattern of selected compounds.Refer to Figure-1 for structural details of compounds.

Optimization of chromatographic conditions
Several authors demonstrated that the acidic and basic mobile phase additives can have a significant impact in addition to the classical factors such as temperature, pH and type of organic modifier, which can influence the enantio separation in LC (Matthijs et al., 2006, Perrin et al., 2002, Ye Y et al., 2001, Levin et al., 1993, Khatuna et al., 2015, Gübitz et al., 2006).These studies mostly carried by normal-phase chromatography, as these additives were supposed to minimize the non-specific interactions between the analytes and the free silanol groups of the CSP.Hence study design was done by selecting normal-phase mode containing n-Hexane as major solvent, ethanol, isopropanol as organic modifiers and triethyl amine (TEA) and trifluoroacetic acid (TFA) as polar modifiers as main constituents of the mobile phase.Initial trials were performed to select suitable chiral recognition using available polysaccharide-based CSPs.Chiralcel OD-H (Cellulose tris (3,5-dimethylphenylcarbamate) and other amylose based columns like Chiralpak-IA (Immobilised Amylose tris-(3,5dimethylphenylcarbamate) and Chiralcel AD-H (Amylose tris-(3,5-dimethylphenylcarbamate) were screened, which resulted in improper separation between certain pair of the enantiomers.Lux amylose-2 column which possess amylose tris(5-chloro-2methylphenyl carbamte) as function group has shown good selectivity over the other amylose based column, where promising results were noticed in respect to resolution and peak shape.
Prior to testing, HPLC system was well equilibrated with chromatographic conditions at least for 3 hours, and baseline was monitored.Desacetyl impurity being major degradant exists in racemic mix like prasugrel, but exhibits keto-enol tautomerism in solution state (PRA-DA-1,2,3,4).

Effect of the column temperature
The influence of the temperature on the enantioresolution (Rs) of the studied chiral drugs was investigated.Based on observations, the change in enantioresolution with column temperature seems to be depend on chiral center of compound.For those which exhibited an increased enantioresolution with temperature, a significant efficiency enhancement was observed at 35°C.However, low enantioresolution is observed at 25°C and 45°C.Hence, was decided to select 35°C and the separation found unaffected at ±3°C of finalized temperature.The results are illustrated in Table-1, figure-5.

Effect of proportion of the acidic additive (TFA)
Acidic additives has significant impact on enantioresolution and selectivity.Addition of Triflouroacetic acid (TFA) to the mobile phase yields separation between the isomers, this may be due to the ion-dipole interactions.It can be assumed that the introduction of chlorine on the phenyl moiety of Lux-2 CSP is supposed to reduce non-specific interactions with CSP in presence of acidic additive in the mobile phase.
During development, enantio resolution and selectivity were found to increase with increase in acidic additive concentration.As % TFA in mobile phase found directly proportional to increased interactions, longer retention times for isomer peaks were observed.Therefore, This might be explained by a The results are illustrated in Table-2 and figure-6 which evaluates the influence of the proportion of TFA on the enantio resolution and selectivity within a range of 1.0ml/L to 2.0ml/L.Influence of temperature on resolution Influence of TFAA Conc. on resolution

Effect of basic additive and concentration of organic modifier
The influence of the basic modifier such as triethyl amine was studied.Addition of 0.1% triethyl amine (TEA) improved the peak shape by reducing the secondary interactions with the stationary phase but addition of TEA doesn't yielded any significant resolution among the stereo isomers.Effect of ethanol and isopropanol on the resolution and selectivity was also studied.During study, it was observed that the resolutions were dropped and shown broader peaks when isopropanol was used as a polar modifier.At the same time, retentions of the isomer peaks were increased with increase in polar nature of the alcohol, hence ethanol was selected along with both TFA and TEA as additives.Greater than 15% of ethanol concentration in mobile phase affected the resolution between PRA-4F-E1 (Compound-5) and PRA-3F-E1(Compound-3) and PRA-E1(Compound-1) and PRA-4F-E2(Compound-6).The relationship of enantiomeric resolution (Rs) values with either hydrophobicity or bulkiness of the alcohols, as well as the effect of IPA and ethanol on interaction of the analytes were studied.The best resolution was achieved with the use of 10%ethanol in the mobile phase.The results are illustrated in .

CONCLUSIONS
Baseline separation with resolution greater than 2.0 was achieved between the enantiomers of any regioisomer within 30 min.Refer to Figure-3 and 4, for chromatograms generated with finalized chromatographic conditions.The effect of organic modifiers and temperature on resolution and retention of enantiomers have been evaluated and the mobile phase composition has been optimized.The results of the study indicates the enantiomeric separation was found to be an enthalpy driven process.Organic modifiers like Isopropanol and polar modifiers like triethylamine and trifluoroacetic acid competes with the solutes for achiral and chiral binding sites.As the steric bulk around the hydroxyl moiety of the modifier plays a role in the mobile phase competition, increased steric bulk resulted in increased k' .However, the results also suggests that the effect of the alcoholic mobile phase modifiers on stereoselectivity may also be due to binding to achiral sites near or at the chiral cavities of the CSP which alters the steric environment of these cavities.This method was designed to determine the relative contribution of each stereoisomer, based on statistical analysis of each stereoisomer's chromatographic peak areas.Although, this work has been done with research purpose and not fully validated, but can be implemented for verification and quality check of chiral impurities of Prasugrel during synthesis, reaction monitoring as well as in finalized Active Pharmaceutical Ingredient.

Fig. 5 :
Fig. 5: Graphical representation of effect of column temperature on chiral selectivity.

Fig. 6 :
Fig. 6: Graphical representation of Influence of TFA Concentration on resolution.

Table - 1
: Effect of temperature on chiral selectivity.

Table - 2
: Influence of TFA Concentration on resolution.

Table 3 :
Effect of Ethanol (Polar organic modifier) concentration on retention times.