Samples of Iloperidone and its eight impurities viz., Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7, and Imp-8 (Figure 1) were received from synthetic laboratory, Megafine Pharma (P) Ltd., Nashik, India. HPLC grade ammonium dihydrogen orthophosphate and triethylamine AR grade were purchased from Merck, Mumbai, India. Acetonitrile, gradient grade was purchased from J.T. Baker, Mumbai, India. High purity deionised water was obtained from Millipore, Milli-Q (Bedford, MA, USA) purification system.

Nexera UFLC system (Shimadzu Corporation Kyoto, Japan) equipped with quaternary gradient pump, auto sampler, column oven, and photodiode array detector was used for method development and method validation. The output signal was monitored and processed using LabSolutions version 5.53. The UPLC columns used during method development were Acquity UPLC^® HSS C18 (2.1 mm × 100 mm, 1.8 micron), Acquity CSH Phenyl-hexyl (2.1 mm × 100 mm, 1.7 micron), and Acquity^® HSS Cyano (2.1 mm × 100 mm, 1.8 micron). Acquity UPLC^® HSS C18 (2.1 mm × 100 mm, 1.8 micron) column thermostated at 35˚C was used for the separation. Mobile phase-A comprising phosphate buffer (aqueous solution of 5mM ammonium dihydrogen orthophosphate and 0.1% triethylamine) and mobile phase-B was comprising mixture of acetonitrile and methanol in the ratio of 80:20 v/v were used. The flow rate and injection volume was 0.5 mL·min⁻¹ and 2 µL respectively. The analysis was carried out under the gradient conditions as time (min)/A (v/v): B(v/v);T_0.01/65:35, T_1.5/65:35,

T_5.0/35:65, T_7.0/35:65, T_8.0/65:35, and T_10.0/65:35.The data was acquired at 225 nm for 10 min and processed by using LabSolutions version 5.53. For the analysis of forced degradation study, the photo diode array detector was set to scan from 200 nm to 400 nm. The peak homogeneity was calculated in terms of peak purity.

The diluent used for the analysis was prepared using homogenous mixture of 700 mL of water and 300 mL acetonitrile. A solution of Iloperidone was prepared at concentration of 1000 µg·mL⁻¹ in diluent. Stock solutions of each impurity (Imp-1 to Imp-8) were prepared at concentration of about 150 µg·mL⁻¹ in diluent. These solutions were further diluted to prepare standard solution for quantification of impurities according to the specification limits (0.15%) of each impurity.

Sample of formulation drug was prepared by powdering twenty tablets of Iloperidone, dissolving an equivalent of 25 mg of active ingredient in diluent in a 25 mL volumetric flask and ultra sonicating for about 15 - 20 min. The volume was made up to the mark with the diluent, and filtered through Merck Nylon syringe filter having pore size 0.45 µm. The clear liquid was collected and used for the determination of related substances in the pharmaceutical dosage forms. This solution was ten times diluted for the assay determination in pharmaceutical dosage forms.

Iloperidone with concentration of 1000 µg·mL⁻¹ was spiked with Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7 and Imp-8 at specification level i.e., 0.15% and the obtained solution was used as a system suitability mixture. 2 µL each of blank preparation, system suitability mixture (SSM), 6 replicates of standard solution and test solution are injected and separately chromatographed under the given chromatographic conditions. Resolution condition of not less than 1.5 between Imp-4 and Imp-5 was set as system suitability parameter for SSM. The relative standard deviation of not more than 5.0% for areas obtained from six replicate injections of all impurities was used to verify the system precision.

The forced degradation study was planned to investigate specificity and stability indicating property of the established method [22] -[24] . The API was deliberately stressed in solution state at acidic and alkaline pH, under oxidation conditions, in solid state at thermal and photolytic conditions. Solutions for degradation were prepared by dissolving the API in diluent at specification level i.e. 1000 µg·mL⁻¹ in diluent and then treating with conc. hydrochloric acid (24hr heating at 110˚C), with 5 N sodium hydroxide (24 hr heating at 110˚C), and with 30% aqueous hydrogen peroxide (15 min at room temperature). The solutions after treatment were diluted with diluents up to the mark to desired concentration of 1000 µg·mL⁻¹ and were injected in the established method. For thermal degradation, the solid API was heated at 105˚C for 7 days in calibrated oven. For photolytic degradation, the sample was exposed to UV source as per the ICH guidelines for 10 days. The treated samples were diluted with diluent to the desired concentration of 1000 µg·mL⁻¹ and injected in the established method. All the stressed samples are quantified for Iloperidone and impurities. Photodiode array detector was used to establish peak purity and thus the homogeneity of Iloperidone peak under every stress condition. Mass balance of the degraded samples was carried out to correlate the loss of a parent drug and the increase in the amount of degradation products.

The present study was aimed to develop a chromatographic system capable of eluting and resolving Iloperidone, its process related and degradation related impurities (Imp-1 to Imp-8) in short run time which complies with the general requirement of system suitability. During HPLC method development initial trials were conducted for optimizing various parameters on the system suitability of the method. The parameter assessed include the detection wavelength, the type and quantity of organic modifier, the column, salt concentration, pH of mobile phase and column temperature.

Preliminarily, the gradient HPLC method was developed for separation of Iloperidone and all its related substances, which was then transferred to UPLC. Generally, gradient elution mode is always preferred over the isocratic elution mode in order to achieve improved peak shape and resolution, and for elution of late-eluting (non- polar) impurities. In-addition, C18 bonded phases are the most popular phases for HPLC/UHPLC because they offer good retention and selectivity for a wide variety of sample types. Additionally, they are perceived to be more rugged and reliable than other bonded phases. C18 bonded phases depend mainly on hydrophobic interactions (and occasionally shape selectivity) to achieve satisfactory separations. Although versatile, C18 phases offer a limited number of separation mechanisms and this may lead to less than optimum separations in some cases and complete lack of resolution of important peak pairs in others. Hence, during method development it was decided to use a gradient HPLC mode with C18 phase. The chromatographic separation was achieved on a Hypersil BDS C18 (250 × 4.6 mm), 5 µm column at flow rate 1.0 mL/min. The column oven temperature was maintained at 35˚C.

Finally, satisfactory peak shape and the resolution of closely eluting potential impurity (Imp-4 and Imp-5) was achieved on a Hypersil BDS C18 column (250 × 4.6 mm and 5.0 µm particle size), by using mobile phase consisting the mixture of phosphate buffer and acetonitrile. There was a good separation of Iloperidone from its known impurities, while it was found to be eluting at a higher retention time (around 16.5 min) with a total run time of 45 min. To reduce the run time, the gradient HPLC mode was switched over to a gradient UPLC mode with Acquity UPLC^® HSS C18 (2.1 mm × 100 mm, 1.8 µ) column. The response of Iloperidone and its related impurities were found to be adequate at 225 nm. The basic chromatographic conditions, such as the stationary phase, solvents, and UV detection employed in HPLC, were taken into account while developing the new UPLC method. The detection wavelength, column temperature, and solvent used in HPLC were kept constant. The C18 stationary phase was chosen in order to have similar chemistry to that used in the HPLC. In UPLC, an Acquity UPLC^® HSS C18 (2.1 mm × 100 mm, 1.8 µ) column was employed for the separation. The injection volume was set to 2 µL from 20 µL, as used in HPLC. The flow rate was scaled to 0.5 mL/min. Using these condition, a satisfactory separation was achieved within 10 min by maintaining maximum back pressure at 9000 psi.

Comparative data on chromatographic performance of HPLC and UPLC has been obtained by injecting a solution of system suitability. The performance parameters of both the systems are shown in Table 1. It is observed as compared to HPLC method the elution time of Iloperidone in UPLC was reduced from 17 min to 3 min. The resolution and theoretical plates obtained for Iloperidone in UPLC show comparatively better separation efficiency than HPLC. Theoretical plates obtained for Iloperidone in gradient mode HPLC was obviously higher than UPLC. The typical chromatograms obtained from the final HPLC and the UPLC conditions are depicted in Figure 2 and Figure 3 respectively.