4.6.1. (Ultra) High-Performance Liquid Chromatography ((U)HPLC)

During different stages of pharmaceutical development, including synthesis and isolation of API, dosage form development, and pharmacological testing, gas or liquid chromatographic methods of analysis are used for quantitative and/or qualitative purposes [133].

High-Performance Liquid Chromatography (HPLC) is based on the principles and theories of gas chromatography and is a rapid and efficient analytical procedure [134,135]. In the broadest sense, chromatographic separations can be classified as normal (NPC) and reversed-phase (RPC), where the stationary phase is more polar than the mobile phase for NPC, whereas the converse is true for RPC [136].

RP-HPLC is the method of choice for in vitro analysis of API due to the stability and reproducibility of stationary phases used for analyses [137]. A large number of aqueous components in the mobile phase, in addition to the ease of reproducing analytical methods in different laboratory settings, make RP-HPLC an ideal analytical method for product development and quality control purposes in the pharmaceutical industry [137].

The use of stationary phases that have a silica-based backbone chemically bonded to a variety of different organic functional groups, form the basis for separations in RP-HPLC [138]. Separation is achieved when the sample to be analysed partitions between an organic modifier used in the mobile phase and the stationary phase or column [139,140]. In general, non-polar compounds are attracted to hydrophobic stationary phases and polar compounds preferentially partition into polar mobile phase components [139]. The specificity and selectivity of an analytical method is the consequence of interaction(s) between analyte(s) and binding sites of the stationary phase and partitioning in the mobile phase [141]. Silica-based columns with C3, C4, C8, or C<sup>18</sup> alkyl chains bonded to the backbone are the commonly used non-polar stationary phases in RP-HPLC. Silica-based phases are compatible, do not swell in water, and some organic solvents are, therefore, suitable for use with mobile phases comprised of these solvents [139,141,142].

The mobile phase in RP-HPLC is usually water or a buffered solution with an organic modifier such as methanol or acetonitrile added to the composition in order to reduce the polarity of the mobile phase, thereby facilitating the achievement of appropriate retention characteristics for the compounds of interest [138]. Other factors that affect the retention time of analytes include temperature, pH of the buffer and/or mobile phase, stationary phase properties, polarity of the mobile phase, flow rate, and mobile phase composition [139,140].
