Measurement of Serum Vitamins B2 and B6 in Patients with Hashimoto’s Thyroiditis by LC–MS/MS Method

Vitamin B2 and vitamin B6 are cofactors and coenzymes in many biochemical reactions, and their sufficient serum concentrations may be crucial for the proper functioning of the thyroid gland. The aim of the study was to develop and validate a novel high-performance liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the determination of vitamin B2 and vitamin B6 in the serum of patients with Hashimoto’s thyroiditis (HT). Detection of the analytes was performed on a triple-quadrupole MS with multiple reaction monitoring using electrospray ionization. The mobile phase consisted of water and methanol, both containing 0.1% (v/v) formic acid. The analytes were isolated from serum by the one-step sample preparation of the trichloroacetic acid precipitation. Validation parameters, including linearity, accuracy, precision, limits of detection and quantification, and stability, were determined and fulfilled the criteria for analyzing compounds in biological fluids. The method was applied for the determination of the above vitamin concentrations in serum samples of 74 patients with HT and 33 healthy controls. The concentration of vitamin B2 was significantly lower in patients with HT compared to the control group (7.20 ± 7.86 ng/mL vs. 12.03 ± 9.62 ng/mL) and the concentration of vitamin B6 was also lower, but without a statistically significant difference (30.09 ± 19.16 ng/mL vs. 37.29 ± 23.02 ng/mL). We concluded that the vitamins concentration measurement may be helpful in the diagnosis of vitamin deficiencies and the supplementation strategy.