Liquid chromatographic enantioseparation of three beta-adrenolytics using new derivatizing reagents synthesized from (S)-ketoprofen and confirmation of configuration of diastereomers.

Diastereomers of racemic β-adrenolytic drugs [namely (RS)-propranolol, (RS)-metoprolol and (RS)-atenolol] were synthesized under microwave irradiation with (S)-ketoprofen based chiral derivatization reagents (CDRs) newly synthesized for this purpose. (S)-Ketoprofen was chosen for its high molar absorptivity (εo  ~ 40,000) and its availability as a pure (S)-enantiomer. Its -COOH group was activated with N-hydroxysuccinimide and N-hydroxybenzotriazole; these were easily introduced and also acted as good leaving groups during nucleophilic substitution by the amino group of the racemic β-adrenolytics. The CDRs were characterized by UV, IR, 1 H-NMR, HRMS and CHNS. Separation of diastereomers was achieved by RP HPLC and open column chromatography. Absolute configuration of the diastereomers was established with the help of 1 HNMR supported by developing their optimized lowest energy structures using Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G* basis set (based on density functional theory), and elution order was established. RP HPLC conditions for separation were optimized and the separation method was validated. The limit of detection values were 0.308 and 0.302 ng mL-1 . Copyright © 2016 John Wiley & Sons, Ltd.

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.

Diastereomers of (RS)-propranolol were synthesized using (S)-levofloxacin-based new chiral derivatizing reagents (CDRs). Levofloxacin was chosen as the pure (S)-enantiomer for its high molar absorptivity (εo  ∼ 24000) and availability at a low price. Its -COOH group had N-hydroxysuccinimide and N-hydroxybenzotriazole, which acted as good leaving groups during nucleophilic substitution by the amino group of the racemic (RS)-propranolol; the CDRs were characterized by UV, IR, (1) H-NMR, high resolution mass spectrometry (HRMS) and carbon, hydrogen, nitrogen, and sulphur fundamental elemental components analyser (CHNS). Diastereomers were separated quantitatively using open column chromatography; absolute configuration of the diastereomers was established and the reagent moiety was detagged under microwave-assisted acidic conditions. (S)- and (R)-propranolol as pure enantiomers and (S)-levofloxacin were separated, isolated and characterized. Optimized lowest-energy structures of the diastereomers were developed using Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G* basis set (based on density functional theory) for explanation of elution order and configuration. In addition, RP HPLC conditions for separation of diastereomers were optimized with respect to pH, concentration of buffer, flow rate of mobile phase and nature of organic modifier. HPLC separation method was validated as per International Conference on Harmonization guidelines. With the systematic application of various analytical techniques, absolute configuration of the diastereomers (and the native enantiomers) of (RS)-propranolol was established. Copyright © 2016 John Wiley & Sons, Ltd.

Alwera S ，Bhushan R 《-》

RP-HPLC enantioseparation of β-adrenolytics using micellar mobile phase without organic solvents.

Enantioseparation of a few commonly administered racemic β-adrenolytics (namely, carvedilol, betaxolol, salbutamol and bisoprolol) has been achieved using a water micellar mobile phase containing surfactants (sodium dodecyl sulphate and Brij-35) without organic solvents as a new approach in RP-HPLC. Two chiral derivatizing reagents based on enantiomerically pure (S)-(-)-levofloxacin were synthesized using N-hydroxysuccinimide and N-hydroxybenzotriazole as the activation auxiliaries. Diastereomeric derivatives of the chosen β-adrenolytics were synthesized under microwave irradiation in a very short reaction time. The (S)-(-)-levofloxacin moiety enhanced molar absorbance of the diastereomeric derivatives resulting in very low limit of detection (1.618 and 4.902 ng/mL, respectively, for diastereomeric derivatives of (RS)-betaxolol and better resolution with lower retention times (for all the analytes), in comparison to literature reports. There was 15-20 times less consumption of mobile phase because of lower retention time.

Alwera S ，Bhushan R 《-》

Enantioresolution of five β-blockers by reversed-phase high-performance liquid chromatography using fifteen chiral derivatizing reagents having amino acids or their amides as chiral auxiliaries on a cyanuric chloride platform.

Enantioseparation of five β-blockers, namely, (R,S)-atenolol, (R,S)-propranolol, (R,S)-bisoprolol, (R,S)-metoprolol and (R,S)-carvedilol, was achieved as their diastereomers prepared with chiral derivatizing reagents (CDRs) synthesized on a cyanuric chloride platform. Fifteen CDRs were synthesized by nucleophilic substitution of the Cl atom in cyanuric chloride or its 6-methoxy derivative with amino acids (namely, L-Leu, L-Val, D-Phg, L-Met and L-Ala) or their amides as chiral auxiliaries. The diastereomers were synthesized under microwave irradiation for 70 or 100 s at 85% power. Separation of diastereomers was carried out on a C(18) column and gradient eluting mixtures of methanol with aqueous trifluoroacetic acid with UV detection at 230 nm. Separation efficiencies of the reagents were compared on the basis of effect of chiral auxiliaries (i.e. amino acids or amino acid amides) and achiral substituents (i.e. chlorine or methoxy group) in the CDRs. The method was validated for detection limit, linearity, accuracy and precision.

Bhushan R ，Dixit S 《-》

Development of liquid chromatographic methods for enantioseparation and sensitive detection of β-adrenolytics/β2-agonists in human plasma using a single enantiomer reagent.

Enantioseparation of four commonly used β-adrenolytics (bisoprolol, salbutamol, and carvedilol, marketed as racemic mixtures) has been achieved by both TLC and RPHPLC via an indirect approach. A new chiral reagent, (S)-naproxen benzotriazole ester, was synthesized and it was characterized by UV, IR, 1HNMR, elemental analysis and polarimetry. It was used to synthesize diastereomeric derivatives of the three β-adrenolytics under microwave irradiation. TLC separation of diastereomeric derivatives was achieved which were then isolated by preparative approach; these were characterized and were used as standard reference for determining absolute configuration of diastereomeric derivatives and for establishing validated HPLC method for enantioseparation and sensitive detection of the three β-adrenolytics in human plasma. Mobile phase in gradient mode containing methanol and aqueous triethylaminephosphate (TEAP) was successful for HPLC separation; conditions with respect to pH, flow rate, and buffer concentration were optimized. The method is capable to accurately quantitate β-adrenolytics in human plasma with minimal sample clean-up and rapid separation by TLC and RPHPLC. The limit of detection values were 0.97 and 0.87ng/mL for diastereomeric derivatives of (S)- and (R)-bisoprolol, respectively, which are very low in comparison to literature reports.

Malik P ，Bhushan R 《-》