ABSTRACT
Drug analysis has evolved to cater to the requirements of drug discovery and pharmaceutical quality control. High-performance liquid chromatography (HPLC) is the most widely used separation technique for qualitative and quantitative determination of various chemicals in pharmaceutical industry. Prior to the 1970s, few reliable HPLC methods were available to the laboratory scientist. Till then, hydrophilic stationary phase and hydrophobic mobile phase were used as normal phase (NP) chromatography. Discovery of reversed-phase (RP) liquid chromatography in the late 1970s allowed improved separation for a wide variety of compounds. Since a substantial number of drugs in pharmaceutical research and discovery are ionic and polar hydrophilic, RP-HPLC methods may be dif“cult for highly polar compounds because of inadequate retention due to their highly polar nature. Achieving retention
16.1 Introduction .................................................................................................. 373 16.2 Analysis of Pharmaceutical Ingredients ....................................................... 374
16.2.1 Basic Amines in Cough-Cold Formulations .................................... 375 16.2.2 MFH and Related Compounds in Tablets ........................................ 375 16.2.3 BT in Ophthalmic Solution ............................................................... 375 16.2.4 SCG in Ophthalmic Solution ............................................................ 375
16.3 Retention Mechanism ................................................................................... 377 16.3.1 Effect of Organic Solvent ................................................................. 378
16.3.1.1 Choice of Organic Solvent ................................................. 378 16.3.1.2 Use of Alternate Solvent .................................................... 379
16.3.2 Mobile-Phase pH and Ion Exchange.................................................380 16.3.3 Ionic Strength ................................................................................... 386 16.3.4 Stationary Phase ............................................................................... 387 16.3.5 Diluent and Injection Volume ........................................................... 391 16.3.6 Validation of Methods ...................................................................... 392
16.4 Advantages .................................................................................................... 393 16.5 Conclusion .................................................................................................... 397 References .............................................................................................................. 397
and separation of polar compounds is an ongoing challenge for chromatographers.1-3 Such compounds need highly or 100% aqueous mobile phases but this can lead to wetting of stationary phases (e.g., C18).4,5 Attempts to reduce the risk of dewetting of C18 phases by means of polar embedding or polar end capping is not successful since these stationary phases provide lower retention than standard C18 phases.2 Lack of retention for hydrophilic compounds in RP-HPLC is largely due to solovophilic factors, where polar functional groups have an ability to form favorable dipolar bonds with the solvent to become solvated. Since nonpolar stationary phase cannot offer similar bonding, the solutes stay in solution and elute in void volume.6 In these cases, ion-exchange chromatography is the obvious choice where retention is accomplished by coulombic interaction mechanism. Another approach is the use of ion-pairing chromatography (IPC) where the ion-pair reagents in mobile phase mediate the retention of analytes on RP columns. Ion-pair reagents are soap-like ionic molecules having a charge opposite to that of molecule of interest, as well as a hydrophobic region to interact with stationary phase. The counter ion combines with the ions of the eluents, becoming ion pairs in the stationary phase, which results in retention. Several IPC methods have been reported for polar compounds but its use is limited due to expensive ion-pair reagents and its incompatibility with electrospray ionization mass spectrometry (ESI-MS).7 To retain compounds that are highly hydrophilic and uncharged, derivatization techniques are used where one or more of the functional groups are converted into hydrophobic groups by a chemical reaction.8 Derivatization may be limited to single-component analysis and not suitable for multicomponents where one or a few of the solutes may not be derivatized to give detectable molecules under similar conditions. Also, derivatization may not be suitable for closely eluting related compounds and the procedure is time consuming.
