This chapter aims to explain the key parameters of analytical method development using the chromatography techniques which are used for the identification, separation, purification, and quantitative estimation of complex mixtures of organic compounds. Mainly, the versatile techniques of ultra−/high-performance liquid chromatography (UPLC/HPLC) are in use for the analysis of assay and organic impurities/related substances/degradation products of a drug substance or drug product or intermediate or raw material of pharmaceuticals. A suitable analytical method is developed only after evaluating the major and critical separation parameters of chromatography (examples for UPLC/HPLC are selection of diluent, wavelength, detector, stationary phase, column temperature, flow rate, solvent system, elution mode, and injection volume, etc.). The analytical method development is a process of proving the developed analytical method is suitable for its intended use for the quantitative estimation of the targeted analyte present in pharmaceutical drugs. And it mostly plays a vital role in the development and manufacture of pharmaceuticals drugs.

Midostaurin (MDS) is used for the treatment of acute myeloid leukemia, myelodysplastic syndrome, and advanced systemic mastocytosis. MDS softgel capsule samples were subjected to stress testing per International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use guidelines for impurity profiling study. MDS underwent extensive degradation under stress testing (acid, alkaline, oxidative, photolytic, thermolytic, and hydrolysis conditions) and formed four degradation products (DPs). MDS and its DPs were separated well from one another with good resolution using reserved-phase HPLC using an Inertsil ODS-3V column (250 Â 4.6 mm, 5 μm) and a mobile phase of ammonium formate (40 mM) and acetonitrile. The stability-indicating characteristic of the newly developed method was proven for the estimation of MDS assay, and its organic impurities were free from interference. The validated method exhibited excellent linearity, accuracy, precision, specificity, detection limit, and quantitation limit within 25 min run time. Stress testing, robustness, and solution stability were performed to ensure the continuous performance of the developed method. The peak fractions of DPs formed under stress testing were isolated and characterized using LC-MS, 1 H and 13 C NMR, IR, and UV-Vis. The structure of the major DPs was predicted as DP1 based on the spectral data. The proposed method is effectively used for MDS in bulk drug and finished formulations in the pharmaceutical industry.

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A simple, short and stability-indicating reverse phase-ultra-performance liquid chromatography method was developed and validated for the quantitative determination of related impurities of halobetasol propionate in halobetasol propionate 0.05% cream formulation. The proposed method was developed on an ACQUITY UPLC™ BEH Phenyl (2.1 × 100 mm, 1.7 µm) column at 40°C with a mobile phase containing a gradient mixture of potassium hydrogen phosphate buffer and acetonitrile and methanol as modifiers with a runtime of 13.0 min at a monitored wavelength of 242 nm. A simple preparative method and liquid chromatography-mass spectrometry-compatible UPLC method also were developed for the isolation and identification of impurities and degradation products. The drug was subjected to forced-degradation conditions and found to degrade significantly. The stability-indicating capability of the developed method is established by analyzing forced-degradation samples in which the spectral purity of halobetasol propionate is ascertained along with the separation of degradation products from the analyte peak. The developed method was validated as per International Conference on Harmonization guidelines. The developed method is precise (%relative standard deviation <2.0) and is capable of detecting and quantifying all the six impurities at a level of 0.01 and 0.03%, respectively, with respect to test concentration. The wide linearity range, sensitivity, accuracy, short retention time and simple mobile phase imply that the method is suitable for routine quantification of halobetasol propionate and its related substances.

Apalutamide, an androgen receptor inhibitor, is used to treat prostate cancer. A stability-indicating high-performance liquid chromatography method was developed for the estimation of assay and organic impurities of apalutamide in drug substance and in tablet dosages using Design of Experiments. The chromatographic separation was achieved within 30 min using Atlantis dC 18 , 100 Â 4.6 mm, 3.0 μm and the binary gradient program (10 mM KH 2 PO 4 , pH 3.5; acetonitrile). The detection wavelength, flow rate, column temperature and injection volume used were 270 nm, 1.0 ml/min, 45 C and 10 μl, respectively. The interaction of independent variables (pH, column temperature and flow rate) and their influences on HPLC parameters were studied using a central composite design, and then the peak separation and elution behaviors between apalutamide and its seven impurities were determined. The method validation was performed for linearity, detection limit, quantitation limit, accuracy, precision and robustness as per the International Conference on Harmonization. A high-quality recovery with good precision (91.7-106.0%) and correlations (r 2 > 0.997) within a linear range of 0.12-2.24 μg/ml (0.05-0.3%, w/w) were achieved consistently for assay and organic impurities of apalutamide. The stabilityindicating characteristics of the proposed method were assessed through forced degradation and mass balance studies. An effort was made to figure out the chemical structures of newly formed degradation products (DP1-DP5) using LC-MS/MS.