Title:Sensitive UHPLC-MS/MS Technique for Monitoring Levothyroxine (T4)
in Human Serum Against Endogenous Thyroxin Level
Volume: 9
Author(s): Sanjay Kumar Yadav, Diwakar Pareek, Sanjeev Mishra, Sanjay Jagannath Gurule, Arshad Khuroo and Dipanjan Goswami*
Affiliation:
- Clinical Pharmacology and Pharmacokinetics, Sun Pharmaceuticals Industries Limited Vill. Sarhaul, Sector-18, Gurugram-
122 015, Haryana, India
Keywords:
Levothyroxine, serum, LC-MS/MS, validation, bioavailability studies, thyroid hormone.
Abstract:
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.
