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A Novel LC-MS/MS Method for the Quantification of Levothyroxine (T4) in Human Serum

02 Feb 2018

Structure: Method:

Levothyroxine or L-thyroxine is a synthetic thyroid hormone that is chemically identical to thyroxine (T4), which is naturally secreted by the follicular cells of the thyroid gland. It is used to treat thyroid hormone deficiency, and occasionally to prevent the recurrence of thyroid cancer.

Levothyroxine is an endogenous molecule, hence requires specially treated matrix for the preparation of Calibration Curve and Quality Control samples. The novel LC-MS/MS method developed at Lambda involves the usage of charcoal stripped human serum as surrogated matrix for the preparation of CC and QC samples. Briefly, the method involves solid-phase extraction technique using Levothyroxine (T4)-13C9, 15N as an internal standard (ISTD).  Chromatographic separation was achieved on Poroshell120 EC-C18 2.7µm 4.6*100mm column with a mobile phase consisting of 0.01% formic acid solution (v/v) and methanol with gradient time program, and with a total run-time of 8 min. Detection was carried out in positive electro-spray ionization mode using MRM transitions of 777.60→731.65 for Levothyroxine (T4) and 787.60→740.75 for Levothyroxine (T4)-13C9, 15N (ISTD).

The method was fully validated in terms of linearity, stabilities, limit of detection (LOD), carry-over effect, reinjection reproducibility, precision and accuracy. To mimic the study samples, which are normal human serum, QC samples were prepared from normal human serum for evaluating the precision and accuracy, and also to evaluate the stability at different conditions like freeze and thaw, bench top and others. The method is also selective from endogenous Levothyronine (T3). The validated method has a linearity range from 5 to 300ng/mL. Levothyroxine is found to be stable in serum under the assay condition. The method exhibited good precision and accuracy in both the matrices with precision better than 6.7% and accuracy greater than 88.0%. Also, there was no significant matrix effect in both charcoal as well as normal human lots.

The validated method is suitable for the analysis of sample from a single oral dose of 600 mcg administration to humans and has already been applied to analysis of study samples.

To conclude, a simple, precise, accurate and reproducible LC-MS/MS method is now available for quantification of Levothyroxine in human serum.

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