Thyroid Hormones in Serum and Plasma Using SPE Extraction and UHPLC–MS-MS Analysis

This application note describes the development of an analytical method for the sensitive and accurate determination of free and total thyroid hormones in serum and plasma using a mixed-mode SPE extraction procedure and UHPLC–MS-MS analysis. Chromatographic separation, including baseline resolution of triiodothyronine and reverse triiodothyronine, is conducted in 3 min using a Selectra® DA column. Ultra-low hormones and steroids serum (Golden West Biologicals, Inc.®) were used to generate accuracy and precision data. The method was evaluated at two concentrations (0.2 and 1 ng/mL) using procedures for free and total hormones. Recoveries of the three hormones ranged from 87% to 100% and reproducibility was ≤3.5%.

Extraction/Analytical Materials

Procedure

1. Sample Preparation for free hormones:

(a) To 1 mL of plasma add 1 mL of 0.1 M HCl.

(b) Vortex for 30 s.

Sample Preparation for total hormones

(a) To 1 mL of plasma add 2 mL of acetonitrile.

(b) Vortex briefly and let stand for 5 min.

(c) Centrifuge at ≥1500 rcf for 5 min.

(d) Transfer supernatant to a clean glass tube.

(e) Add 2 mL of 0.1 M HCl and vortex for 30 s

2. Condition Cartridge

(a) 1 × 3 mL methanol.

(b) 1 × 3 mL 0.1 M HCl.

(c) Apply the sample to the SPE cartridge.

3. Wash Cartridge

(a) 1 × 3 mL 0.1 M HCl.

(b) 1 × 3 mL methanol.

(c) Dry cartridges for ~30 s to remove excess methanol.

4. Elution

(a) Elute with 1 × 3 mL methanol containing 2% ammonium hydroxide.

(b) Evaporate to dryness/Reconstitute in 100 µL of methanol.

Instrumental

LC–MS-MS: Thermo Scientific Dionex Ultimate 3000® LC System/ Thermo Scientific TSQ Vantage tandem MS

Column: UCT Selectra® DA LC column, 100 × 2.1 mm, 1.8 µm

Guard Column: UCT Selectra® DA guard column, 10 × 2.1 mm, 1.8 µm

Injection Volume: 10 µL

Mobile Phase A: 0.1% formic acid in D.I. H₂O

Mobile Phase B: 0.1% formic acid in methanol

Column Flow rate: 0.40 mL/min

Results

Table I: Accuracy and precision data in plasma using free hormone procedure (n=6 each).

Table II: Accuracy and precision data in plasma using total hormone procedure (n=6 each).

Conclusion

Excellent recoveries ranging from 87.1% to 99.8% were obtained using UCT's mixed-mode DAU extraction column (CSDAU203) and procedure in conjunction with the Selectra® DA UHPLC column (SLDA100ID21-18UM) fothe analysis of free and total thyroid hormones in human serum and plasma.

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