Reversed-phase-HPLC enantioseparation and control of enantiomeric purity of duloxetine using a new chiral reagent and recovery of enantiomers.

This study reports a rapid and low-cost LC method for control of enantiomeric purity of duloxetine. Though duloxetine, as marketed and administered, is expected to be a single (S)-enantiomer, the analysis of a few commercial branded samples by the method developed and presented here showed that they contain a relatively high percentage of (R)-enantiomer (e.g., 2.71-5.42%, which is undesirable in drug formulations). A new chiral derivatizing reagent [isatinyl-(S)-naproxen amide] was synthesized on (S)-naproxen platform. Diastereomeric derivatives were synthesized under microwave irradiation and were separated using reversed-phase-HPLC on a C18 column. A combination of acetonitrile and triethylammonium phosphate buffer (9 mM, pH 4) as the mobile phase and detection at 273 nm were found successful. The diastereomeric derivatives at preparative scale were separated using open column chromatography, and the native enantiomers were obtained and characterized. The HPLC separation method was validated for detection limit, linearity, accuracy, and precision. The limits of detection of (S,R)-diastereomer and (S,S)-diastereomer were found to be 12 and 16 pg/mL, respectively, for the 20-µL injected volume. The method so developed has a practical significance and greater societal impact in establishing the control of enantiomeric purity and in ensuring the enantiomeric purity of the drug meant for human consumption.

Resolution and isolation of enantiomers of (±)-isoxsuprine using thin silica gel layers impregnated with L-glutamic acid, comparison of separation of its diastereomers prepared with chiral derivatizing reagents having L-amino acids as chiral auxiliaries.

Thin silica gel layers impregnated with optically pure l-glutamic acid were used for direct resolution of enantiomers of (±)-isoxsuprine in their native form. Three chiral derivatizing reagents, based on DFDNB moiety, were synthesized having l-alanine, l-valine and S-benzyl-l-cysteine as chiral auxiliaries. These were used to prepare diastereomers under microwave irradiation and conventional heating. The diastereomers were separated by reversed-phase high-performance liquid chromatography on a C18 column with detection at 340 nm using gradient elution with mobile phase containing aqueous trifluoroacetic acid and acetonitrile in different compositions and by thin-layer chromatography (TLC) on reversed phase (RP) C18 plates. Diastereomers prepared with enantiomerically pure (+)-isoxsuprine were used as standards for the determination of the elution order of diastereomers of (±)-isoxsuprine. The elution order in the experimental study of RP-TLC and RP-HPLC supported the developed optimized structures of diastereomers based on density functional theory. The limit of detection was 0.1-0.09 µg/mL in TLC while it was in the range of 22-23 pg/mL in HPLC and 11-13 ng/mL in RP-TLC for each enantiomer. The conditions of derivatization and chromatographic separation were optimized. The method was validated for accuracy, precision, limit of detection and limit of quantification.

Indirect enantioseparation of selenomethionine by reversed-phase high-performance liquid chromatography using a newly synthesized chiral derivatizing reagent based on (S)-naproxen moiety.

(S)-Naproxen was reacted with N-hydroxyphthalimide in the presence of coupling reagent dicyclohexylcarbodiimide, and a new chiral derivatizing reagent, phthalimidyl-(S)-naproxen ester, was synthesized. It was characterized and was used for synthesis of diastereomers of selenomethionine via microwave irradiation or vortexing. The reaction conditions were optimized. Diastereomeric pairs synthesized by two approaches were successfully separated by reversed-phase high-performance liquid chromatography using binary mixtures of aqueous triethylammonium phosphate and acetonitrile. Detection was carried out at 231 nm. The limit of detection was found to be 0.11 and 0.10 pmol/mL for diastereomers of d- and l-SeMet, respectively. The method was validated for accuracy, precision and limit of detection. The new chiral derivatizing reagent was capable of enantioseparation of dl-SeMet in the form of diastereomers having higher stability, enhanced resolution and lower limits of detection in comparison to the diastereomers prepared with other chiral derivatizing reagents reported in the literature. Optimized structures of the two diastereomers were drawn using the Gaussian 09 Rev. A.02 program and hybrid density functional B3LYP with 6-31G basis set to explain the separation mechanism.

Indirect enantioseparation of proteinogenic amino acids using naproxen-based chiral derivatizing reagent and HPLC.

Diastereomers of 18 proteinogenic amino acids were synthesized under microwave irradiation and by vortexing using (S)-naproxen-benzotriazole as chiral derivatizing reagent. The diastereomers synthesized by two approaches were found to be identical in terms of their characterization and chromatographic data. A linear gradient of triethylammonium phosphate (pH 3.5)-acetonitrile (30-65%, within 35 min) was found to be successful using reversed-phase high-performance liquid chromatography for their separation. Detection was carried out at 231 nm and sharp peaks were obtained. The method was validated for accuracy, precision and limit of detection.