{"id":2905,"date":"2019-07-31T08:11:30","date_gmt":"2019-07-31T08:11:30","guid":{"rendered":"https:\/\/www.gyanvihar.org\/journals\/?p=2905"},"modified":"2019-07-31T10:07:25","modified_gmt":"2019-07-31T10:07:25","slug":"2905","status":"publish","type":"post","link":"https:\/\/www.gyanvihar.org\/journals\/2905\/","title":{"rendered":"ENANTIOSEPARATION OF ORCIPRENALINE, USING HPLC AND CHIRAL REAGENT BASED ON DFDNB"},"content":{"rendered":"

\u00a0PP. 1-18<\/strong><\/p>\n

1<\/sup><\/u>Hariom Nagar<\/u>* 1<\/sup>Renu Vyas, 2<\/sup>Shiv Alvera,<\/p>\n

1<\/sup>School of Applied Sciences, Suresh Gyan Vihar University, Jaipur-302017<\/p>\n

2<\/sup>Department of Chemical Science, Indian Institute of Science Education and Research Mohali, Punjab-140306<\/p>\n

e-mail: hariomnagariitr@gmail.com<\/a>; renuvyas6@gmail.com<\/a>; Kumalvera@gmail.com<\/a><\/p>\n

\u00a0<\/strong><\/p>\n

ABSTRACT<\/strong><\/p>\n

\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0\u00a0 <\/strong>Orciprenaline is a moderately selective \u03b2<\/em>-adrenergic receptor agonist and used in the treatment of asthma. Generally, the (S<\/em>)-( \u0336 )-enantiomer of \u03b2<\/em>-blockers is pharmacologically effective, show about 50\u2013500-fold higher activities than its (R<\/em>)-( \u0336 )-enantiomer. Diastereomers of orciprenaline were synthesized using a chiral derivatizing reagents that was prepared by substituting one fluorine atom in 1,5-difluoro-2,4-dinitrobenzene with chirally pure L-amino acid; L-Phenylalanine. Characterization of the chiral derivatizing reagent was made by using UV, IR, CHN, and 1<\/sup>H NMR. Diastereomers were synthesized under microwave irradiation 45 s at 80% (of 800W) and also by stirring for 50 min at 45 \u00b0C. Furthermore, the diastereomers were separated by high-performance liquid chromatography in reversed phase condition on a C18<\/sub> column with detection at 340 nm. Acetonitrile and aqueous trifluoroacetic acid (TFA) were used as the mobile phase components. The conditions of derivatization and chromatographic separation were optimized to achieve good results. The method validation was made for accuracy, precision, limit of detection and limit of quantification.<\/p>\n

KEYWORDS<\/strong> Orciprenaline, 1,5-Difluoro-2,4-dinitrobenzene, Enantioseparation, Liquid Chromatography,<\/p>\n

INTRODUCTION <\/strong><\/p>\n

Orciprenaline (Orc) is a moderately selective \u03b2<\/em>2<\/sub>-adrenergic receptor agonist and a bronchodilator used in the treatment of asthma. Generally, the (S<\/em>)-(\u2212)-enantiomer of \u03b2<\/em>-blockers is pharmacologically effective, than its (R<\/em>)- form and shows about 50\u2013500 fold higher activities (Lee and William 1990). But in most cases \u03b2<\/em>-blockers are administered as racemic mixtures.<\/p>\n

Many clinical pharmacologists and biologists tend to deal the drug with mixtures of isomers as if one compound were involved and also a physician,\u00a0 presented with such a drug under a brand name is unaware of isomers and generally he then makes mistakes (Ari\u00ebns 1984). It is mostly because of non availability of single enantiomer as in enantiomerically purified form. In most of cases the undesired enantiomer can have side effects or even toxic effects. So it is prime importance and challenging task to determine optical purity of the drugs and if it is not enantiomerically pure then to resolve racemic mixtures in to its different enantiomers.<\/p>\n

\u00a0In literature, for separation of such drugs (\u03b2<\/em>-blockers) there have been developed and used two approaches one is direct (no any type of chemical derivatization of enantiomers) and other is indirect approach (derivatization of enantiomers with chiral derivatizing reagent (CDR) to form their diastereomers) to achieve enantiomeric separation of \u03b2<\/em>-blockers using the high performance liquid chromatographic (HPLC) techniques. HPLC techniques have been also utilised previously via indirect method for enantiomeric separation of various active pharmaceuticals; amino acids (Bhushan and Nagar 2013) and selenomethionine (Bhushan and Nagar 2014a) using CDRs based on (S<\/em>)-naproxen moiety<\/strong>, isoxsuprine (Bhushan and Nagar 2015) and selenomethionine (Nagar and Bhushan 2014) using CDRs based on difluorodinitrobenzene moiety<\/strong>. \u00a0\u00a0\u00a0\u00a0\u00a0<\/strong><\/p>\n

In literature, certain chiral stationary phases (CSPs) have been used for direct separation of enantiomers of Orc are; sulfobutyl ether \u03b2<\/em>-cyclodextrin (SBECD) (Ngim et al. 2012), methyl-\u03b2<\/em>-cyclodextrin (met-\u03b2<\/em>-CD) (Amini et al. 2000) and sulfated-\u03b2<\/em>-cyclodextrin (S-\u03b2<\/em>-CD) (Yang et al. 2005) as chiral selector on C18<\/sub> column, by direct approach in HPLC. L-Glutamic acid was used as chiral inducing reagent (CIR) for resolution of enantiomers of Orc (Bhushan and Nagar 2015) by direct approach in thin layer chromatography (TLC).<\/p>\n

Besides above literature, Bhushan and Nagar (2014b) have been carried out enantioseparation of Orc by using 1,5-difluoro-2,4-dinitrobenzene (DFDNB) based chiral derivatizing reagents (CDRs) having amino acid and amines as chiral moiety.<\/p>\n

DFDNB namely as Sanger\u2019s reagent a bifunctional variant was first time used for cross linking of proteins such as wool, silk and insulin (Zahn and Meienhofer 1958). Marfey (1984) carried out the reaction of DFDNB with L-alanine amide and formed its chiral variant namely 1-fluoro-2,4-dinitrophenyl-L-alaninamide (FDNP-L-Ala-NH2<\/sub>, Marfey\u2019s reagent), in this reaction one fluorine atom in DFDNB was substituted with L-alanine amide. This chiral derivatizing reagent (CDR) was used for HPLC enantioseparation of five DL-amino acids. DFDNB based CDRs have also been used in Bhushan laboratory for the separation of \u03b2<\/em>-blockers (Bhushan and Tanwar 2008; 2009; Bhushan and Dixit 2012) other than the Orc, \u03b1<\/em>-amino acids (Bhushan and Kumar 2008) and \u03b1<\/em>-amino alcohols (Bhushan and Kumar 2009a). There have appeared certain review articles on use of Marfey\u2019s reagent (B’Hymer et al. 2003; Bhushan and Br\u00fcckner 2004; 2011) as CDR.<\/p>\n

In view of the characteristics of DFDNB, the literature cited above and the references cited therein on the synthesis of DFDNB based CDRs, L-Phe was chosen as chiral amino acid for synthesis of DFDNB based CDR because the non bonding electron pair of amino group of L-Phe would show resonance by direct conjugation, with the para positioned \u2013NO2<\/sub> group and would be expected to further increase molar absorptivity of dinitrobenzene moiety. The chiral auxiliary in this CDR contain \u2013CH2<\/sub>C6<\/sub>H5<\/sub> as the bulky hydrophobic group which enhance the hydrophobicity of the CDR and are thus expected to influence the separation.<\/p>\n

The CDR was used for liquid chromatographic enantioseparation of Orc compound since there are no reports on its enantioseparation using this DFDNB based CDR. The method was validated for linearity, accuracy, limit of detection (LOD) and limit of quantification (LOQ). To the best of the authors’ knowledge, this is the \ufb01rst report on use of this CDR for enantioseparation of Orc compound (Fig. 1).<\/p>\n

EXPERIMENTAL <\/strong><\/p>\n

Chemicals and Reagents<\/strong><\/p>\n

1,5-Difluoro-2,4-dinitrobenzene, L-Phenylalanine (L-Phe), Orciprenaline sulphate as Alupent tablets (Zydus Healthcare, East Sikkim, India) was obtained from the local market. Trifluoroacetic acid (TFA), phosphoric acid (H3<\/sub>PO4<\/sub>), triethylamine (TEA), sodium hydrogen carbonate (NaHCO3<\/sub>) and hydrochloric acid (HCl) of analytical reagent grade, acetonitrile (MeCN) and methanol (MeOH) of HPLC grade were obtained from E. Merck (Mumbai, India). Aqueous\u2013TFA (0.1%) and TEAP (50 mM) buffers were prepared in Millipore water.<\/p>\n

Apparatus<\/strong><\/p>\n

The C18<\/sub> column (Promosil, 250 \u00d7 4.6 mm I.D., 5 \u03bcm) was from younglin. HPLC consisting of a 10 mL pump head, vaccum degasser, Acme UV\/Vis detector, younglin manual injection valve and Autochro-3000 operating software<\/strong> was from younglin (Gyeonggi-do, korea). UV-1601 spectrophotometer (Shimadzu), FT-IR spectrometer 1600 (Perkin-Elmer, USA), NMR spectrometer 500 MHz (Bruker, Germany), pH meter Cyberscan 510 (Singapore) and Microwave-Multiwave 3000 (800W, Perkin-Elmer, USA) equipments were also used for experimental studies.<\/p>\n

Extraction of Orc from Commercial Tablets<\/strong><\/p>\n

Orc was extracted from commercially available \u201cAlupent tablets\u201d in local market. The extraction process was carried out as per the method described in literature (Bhushan and Nagar 2014b). The purity of the product was confirmed by determination of its melting point and UV absorption (\u03bbmax<\/sub>) spectra which were matching with literature reports. The recovery of compound was found in order of 93\u201395% of the quantities reported on the commercial labels. The purified compound was used as racemic standard.<\/p>\n

Preparation of Stock Solutions<\/strong><\/p>\n

Solutions of NaHCO3 <\/sub>(1 M) and HCl (2 M) were prepared in purified water. Stock solution of racemic Orc was prepared by dissolving appropriate amount in 1 M NaHCO3<\/sub> and used for<\/p>\n

derivatization reactions. Solutions and samples were filtered by using 0.45 \u03bcm filters. Acetone was used as solvent to prepare solutions of CDR (25 mM) and then CDR stored in refrigerator at 0\u20134 \u00b0C for further uses.<\/p>\n

Synthesis of CDR <\/strong><\/p>\n

Due to sensitivity of the reagent synthetic procedures were protected from light. The CDR was synthesized by introducing optically pure L-Phe via substitution of one fluorine atom in DFDNB as per method described in literature (Bhushan and Kumar 2009b). These were characterized and stored at 4 \u00b0C. A representative procedure for synthesis of CDR is given below. Following solutions were prepared.<\/p>\n