Impurity C of Calcifediol, also known as 25-Hydroxyvitamin D3 23-olefin, is a specified impurity of Calcifediol (25-Hydroxyvitamin D3). It is a structural isomer of Calcifediol, differing in the position of the double bond in the side chain. This compound is a key process-related impurity formed during the synthesis of Calcifediol, an important active pharmaceutical ingredient (API). As a well-characterized impurity, it is primarily used as a reference standard in pharmaceutical quality control. Its main application is in the analytical testing of Calcifediol drug substances and finished products to monitor and control impurity levels, ensuring product safety, efficacy, and compliance with regulatory guidelines (e.g., ICH Q3A/B). It is essential for method development, validation, and routine analysis using techniques like HPLC and LC-MS. The availability of this impurity standard is critical for the pharmaceutical development and manufacturing of vitamin D analogs, which are used to treat and prevent conditions related to calcium and phosphate metabolism, such as osteoporosis and renal osteodystrophy. Handling requires standard laboratory precautions for potentially light- and air-sensitive compounds.