STM Article Repository

Accurate measurement of ergocalciferol in biological samples has become easier using high end mass spectra. Currently, various LC-MS/MS methods have been developed to quantify ergocalciferol. Use of LC-MS/MS has accomplished to develop accurate method that could quantify ergocalciferol in large number of samples where it should be analysed in the blood immediately after dosage. The objective of this study is to optimize chromatographic conditions and evaluate the LC-MS/MS method for accurate quantification of dosage form of ergocalciferol in human plasma. Extraction and separation of ergocalciferol in human plasma was achieved using methanol liquid–liquid extraction and by a reverse phase C8 column, respectively. The residue after liquid-liquid extraction was dissolved using PTAD reagent along with the reconstituted solution to detect and quantify ergocalciferol by LC-MS/MS. Detection was performed on a quadrupole tandem mass spectrometer (LC-MS/MS) equipped with atmospheric pressure chemical ionization (APCI) source. The method was validated according to FDA guidelines and certified reference materia l was used. The LOQ for analyte tested was 5.44 ng/mL, in medium-MQC it was 65.123 ng/mL and in high-HQC it was 120.598 ng/mL and the quality control samples did not exceed 20 and 15% CV, respectively. Accuracy of the method for determination of ergocalciferol was validated and found to be in the range of 95.40–103.09%. This method can be used for quantification of ergocalciferol present in the systemic circulation via external consumption in the form of tablets. LC-MS/MS assay can be used for quantifying ergocalciferol native form from external consumption and study the metabolism of each individuals enrolled in the clinical trials.