فهرست نشریات علمی دسترسی آزاد ایران

The development and validation of analytical methods are fundamental processes in pharmaceutical discovery, formulation, quality control, and regulatory compliance. High-performance liquid chromatography (HPLC) and liquid chromatography–mass spectrometry (LC-MS) are among the most widely employed techniques for drug quantification, metabolite profiling, and bioanalytical studies due to their sensitivity and versatility. However, several challenges arise during method development and validation, including poor solubility and instability of active pharmaceutical ingredients (APIs), pKa-dependent ionization, mobile phase incompatibility, column degradation, pump or injector malfunctions, and peak shape distortions. In bioanalytical studies, additional issues such as matrix effects, ion suppression, and analyte instability complicate accurate quantification. Addressing these challenges often requires optimization of chromatographic parameters, use of suitable mobile phase modifiers, application of advanced sample preparation techniques, and adoption of stability-indicating assays. Tandem mass spectrometry (LC-MS/MS) further enhances selectivity and sensitivity, particularly for trace-level detection. Robustness and ruggedness testing ensure that methods remain reliable under varying laboratory conditions and across multiple operators. Emerging innovations, including 3D-printed chromatographic columns, high-resolution mass spectrometry (HRMS), and machine learning approaches for robustness prediction, represent promising future directions. This review discusses current challenges, highlights effective remedies, and emphasizes advanced strategies to improve reproducibility, sensitivity, and regulatory compliance in HPLC and LC-MS workflows.