Analytical Performance of the RZ-500 HPLC-MS/MS for Serum 25(OH)D Quantification and Its Clinical Value for Thyroid Malignancy

Abstract Objective To verify the analytical performance characteristics of the RZ-500 high-performance liquid chromatography-tandem mass spectrometry (LC-MS/MS) system (Reigncom company, Shanghai, China) for serum 25-hydroxyvitamin D [25(OH)D] quantification and to evaluate its clinical value for thyroid malignancy. Methods In accordance with CNAS-GL037:2019, the precision, accuracy, linear range, lower limit of quantification, and carryover of the RZ-500 system were verified. A total of 101 patients with thyroid malignancy and 101 age- and sex-matched healthy controls were enrolled in the clinical study from May to September 2026. The fasting serum samples were collected from all the participants and measured by LC-MS/MS. 25(OH)D 2 , 25(OH)D3, and total 25(OH)D levels were compared between groups, and receiver operating characteristic (ROC) curve analysis was used to assess discriminative performance. Results All analytical performance indicators met the predefined acceptance criteria. Serum 25(OH)D 3 and total 25(OH)D were significantly lower in the thyroid malignancy group than those in controls (12.00 vs. 17.70 ng/mL for 25(OH)D 3 , P < 0.001; 14.27 vs. 20.06 ng/mL for total 25(OH)D, P < 0.001, respectively). And vitamin D insufficiency was present in 87.1% of patients and 48.5% of healthy controls ( P < 0.001). ROC analysis used for thyroid malignancy assessment showed that the area under the curve (AUC) was 0.853 for 25(OH)D 3 at the optimal cutoff value of 13.91 ng/mL, and 0.864 for total 25(OH)D at the optimal cutoff value of 16.42 ng/mL, respectively. 25(OH)D 2 exhibited no significant discriminative value for thyroid malignancy (AUC = 0.522). Conclusion The RZ-500 LC-MS/MS system exhibits excellent analytical performance. Owing to isomer-specific quantification, serum 25(OH)D measured by LC-MS/MS showed favorable discriminative performance (high specificity), and may serve as a reliable auxiliary biochemical marker for thyroid malignancy diagnosis.