Sensitive UHPLC-MS/MS Technique for Monitoring Levothyroxine (T4) in Human Serum Against Endogenous Thyroxin Level

Background: Levothyroxine is a synthetic thyroid hormone that is chemically identical to Thyroxine (T4), which is secreted by the follicular cells of the thyroid gland. Levothyroxine is used to treat deficiency of thyroid hormone and prevent the recurrence of thyroid cancer. Levothyroxine is present endogenously in the human body. Methods: It requires a treated matrix for the preparation of calibration curve standard and quality control samples. The method was developed using LC-MS/MS and validated in human charcoal stripped serum. Charcoal stripped matrix was used for the preparation of Calibration curve standards and Quality control samples. The method involves Solid-Phase Extraction technique. Levothyroxine D3 is used as an internal standard (ISTD). Results: Chromatographic separation was achieved using reversed phase analytical column Gemini NX-C18 110Å, 3μm (50x3.6) mm. Mobile phase consisted of acetonitrile and water in a ratio of 70:30 with 150μL of formic acid in 1000 mL of the mobile phase. Mobile phase achieved shorter run-time of 0.9 minute due to the use of Ultra-high performance liquid chromatography (UHPLC). Positive electro-spray ionization technique detected MRM ion pair transitions 777.60→731.65 for Levothyroxine and 780.70→734.6 for Levothyroxine- D3 (ISTD) were used. AB SCIEX Triple Quad™ API-4000 LC-MS/MS system and the bioanalytical method with 10ng/mL as the limit of quantification have been applied successfully to pharmacokinetics studies. Conclusion: Chromatographic separation was achieved using reversed phase analytical column Gemini NX-C18 110Å, 3μm (50x3.6) mm. Mobile phase consisted of acetonitrile and water in a ratio of 70:30 with 150μL of formic acid in 1000 mL of the mobile phase. Mobile phase achieved shorter run-time of 0.9 minutes due to the use of Ultra-high performance liquid chromatography (UHPLC). Positive electro-spray ionization technique detected MRM ion pair transitions 777.60→731.65 for Levothyroxine and 780.70→734.6 for Levothyroxine- D3 (ISTD). AB SCIEX Triple Quad™ API-4000 LC-MS/MS system and the bioanalytical method with 10ng/mL as the limit of quantification have been applied successfully to pharmacokinetics studies.