The common reagents like ammonium dihydrogen orthophosphate, hydrochloric acid, sodium hydroxide, and hydrogen peroxide were all of AR grade, procured from Merck (India). The gradient grade acetonitrile was procured from Merck, Mumbai, India. HPLC grade water obtained from Millipore system (Millipore Inc., USA) was used throughout the analysis. The potential process related impurities viz., Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7 and Imp-8 and test sample of Apremilast were received from synthetic laboratory of Megafine Pharma (P) Ltd., Nashik, India.

HPLC (Agilent 1200, Agilent Technologies, Germany) equipped with photodiode array detector was used for method development, forced degradation studies and method validation. The photodiode array detector was used to determine the peak purity of stressed samples. Synergi Max-RP 80 A (150 × 4.6 mm ID), 4 µ HPLC column thermostated at 40˚C was used for the separation. Mobile phase A was prepared by dissolving 5.75 ± 0.10 g ammonium dihydrogen orthophosphate and 1 ml triethylamine in 1000 mL of Water, filtered through 0.45 µm membrane filter (0.45 µ, Millipore) and degassed in ultrasonic bath prior to use. Acetonitrile was used as mobile phase B. The flow rate and injection volumes were 1.0 mL∙min⁻¹ and 10 µl respectively and auto sampler temperature was kept at 5˚C. The analysis was carried out under gradient conditions such as: time (min)/A (v/v): B (v/v); T_0.01/70:30, T_3.0/70:30, T_18.0/45:55, T_24.0/45:55, T_26.0/70:30 and T_30.0/70:30. The data was acquired at 230 nm for 30 min and processed by using EZ Chrom Elite software Ver. 3.2.1.

The mixture of 50% acetonitrile and 50% water was used as diluent for preparation of analytical solutions. The test sample solution having concentration of 500 µg∙mL⁻¹ was prepared for the determination of process and degradation related impurities of Apremilast. Individual stock solutions of each impurity at concentration about 75 µg∙mL⁻¹ (Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7, and Imp-8) was prepared in diluent which was further diluted adequately to study the validation attributes. The stock solution of Apremilast reference standard of a concentration 50 µg∙mL⁻¹ was prepared and further diluted with impurity stock solution to get the 0.75 µg∙mL⁻¹ and 0.50 µg∙mL⁻¹ concentrations of impurities and Apremilast respectively as a standard solution preparation as shown in Figure 2(b). The specification limits used for validation studies for known impurities was 0.15% w/w for the related substances viz., Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7 and Imp-8. Apremilast reference standard solution (500 µg∙mL⁻¹) spiked with all impurities at a specification level (0.15% w/w) was used as system suitability solution (SST) as shown in Figure 2(c).

The blank (diluent), SST, six replicate injections of standard solution and test sample solution were separately injected and chromatographed. The relative standard deviation (RSD) of NMT 5.0% for Apremilast, Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7, and Imp-8 peak areas obtained from six replicate injections of standard solution was used to verify the system precision of method. The resolution NLT 1.5 between Apremilast and Imp-5 was set as system suitability criteria in SST. The all known related substances in test sample solution were determined against the mean area of respective impurities obtained from replicate injections of standard solution.

Forced degradation studies were also performed on Apremilast drug substance to provide an indication of the stability-indicating property and specificity of the proposed method. The stress conditions employed for the degradation study included light (1.2 million lux hours), heat (105˚C), acid hydrolysis (0.1 M HCl), base hydrolysis (0.1 M NaOH) and oxidation (15% v/v H₂O₂). For heat sample was exposed for 5 days, for acid, base and oxidation samples were treated for 15 min at RT. The degradation was observed in acid and alkali solution as shown in Figure 3. The mass balance was calculated for all the stressed samples. The mass balance is a process of adding together the assay value and the levels of degradation products to see how closely these add up to 100% of initial value with due consideration of the margin of analytical error [17] [18] [19] . A photodiode array detector was employed to check and ensure the homogeneity and purity of Apremilast peak in all the stressed sample solutions.

Few crude samples of Apremilast were analyzed for identification of process related unknown impurities by LCMS. Based on the observed m/z, starting material and reactants used in the synthetic scheme (Figure 1) the possible structures of process related impurities were assigned. The impurities (Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7and Imp-8) were synthesized and co-in- jected with the Apremilast to confirm the retention times.

The quality of product can be determined by good analytical method therefore

s, singlet; m, multiplet; t, triplet; d, doublet.

objective of method development was to separate Apremilast and its process and degradation related impurities (Imp-1 to Imp-8) in a short run time with good resolution and good peak shape. The resolution between Imp-5 and Apremilast was critical during the method development. Hence selection of proper stationary phase was a major role during method development to achieve the good resolution and peak shape. Initial method development trials were conducted on different stationary phases like C₈, C₁₈, Phenyl, Bi-Phenyl and Phenyl hexyl along with the optimization of other chromatographic conditions like detection of wavelength, the type and quantity of organic/inorganic buffer, pH of mobile phase, thermostat and column oven temperature. The analytical method development flow diagram has been depicted below.

Few trials were carried on gradient mode by using reversed phase C18 column and a mixture of acetonitrile-buffer (0.05 M NH₄H₂PO₄) in the different ratio. The system was found not suitable to separate the Apremilast and its Imp-3 and Imp-5, where the peak of Apremilast was co eluted with Imp-3 and closely separated by Imp-5. To increase the resolution between Apremilast and its Imp-3 and Imp-5, the percentage of acetonitrile in the mobile phase was decreased along with the screening of different columns. To overcome the lower resolution of Apremilast and its impurities (Imp-5) stationary phase was changed from C18 to Synergi Max-RP 80A with gradient mode.

Every time system suitability criteria was evaluated during the different trial runs of method development to ensure the strength of developed method. Gradient mode was preferred than the isocratic mode to achieve the good resolution and elution of late-eluting (non-polar) impurities. Finally satisfactory resolution and good peak shape of analyte was observed on Synergi Max-RP 80A_, (150 x 4.6 mm ID), 4 µ column at flow rate 1.0 mL∙min⁻¹ , λ 230 nm, column oven temperature 40˚C and injection thermostat 5˚C and mobile phase was consisting phosphate buffer, and acetonitrile. It was observed that Apremilast, Imp-1, Imp-2, Imp-3, Imp-4, Imp-5, Imp-6, Imp-7 and Imp-8 were well separated under the optimized conditions with a resolution greater than 1.5 and indicated the specificity of the developed HPLC [20] . The representative chromatograms of spiked and unspiked test preparations are shown in Figure 4.

The optimized method was validated as per ICH guidelines. The developed method was complying system suitability criteria during the analytical method validation and batch analysis which itself indicate the good strength of developed analytical method. The results of system suitability criterion are depicted in Table 2.

The analysis of bulk drug sample indicated that the method is specific, selective, and rapid for determination of related substances of Apremilast (Table 7).

ND, Not detected.