B-360 Development and Evaluation of LC-MS/MS Method for Fat-Soluble Vitamins with Magnetic Bead-Based Pretreatment

Abstract

Fat-soluble vitamins (FSVs)-vitamin A (VA), 25-hydroxyvitamin D [25(OH)D], and vitamin E (VE)-are primarily soluble in lipids and are closely associated with cardiovascular and endocrine systems, as well as bone health. Both deficiency and excess of FSVs pose significant clinical risks, ranging from metabolic dysregulation to fatal toxicity. Consequently, the accurate assessment of FSV levels is crucial for clinicians to formulate appropriate nutritional plans and mitigate these risks. However, complex sample matrices and cumbersome pretreatment operations limit the efficiency of current liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods in clinical vitamin detection. As an emerging pretreatment technology, the magnetic bead method utilizes the specific binding capacity of functionalized magnetic particles to achieve efficient enrichment and purification of target analytes. This approach offers operational simplicity, high recovery rates, and ease of automation, which can significantly enhance the efficiency and quality of FSV detection. This study aims to establish and systematically evaluate a novel method for detecting FSVs, integrating magnetic bead pretreatment with LC-MS/MS. Through a scientifically rigorous experimental design, we will comprehensively assess key parameters, including accuracy, limit of quantification (LOQ), and linearity. The evaluation seeks to provide an experimental foundation for the standardized application of this enhanced technology in clinical laboratory practice. Firstly, an equal volume of isotopically labeled internal standard (IS) was added to the serum sample. Via a plate-based format, the mixed sample was adsorbed onto the magnetic beads and subsequently processed through sequential steps-loading, wash I, wash II, and elution. The eluate was quantitatively analyzed by LC-MS/MS. A standard curve was established using the known calibrator concentrations and their corresponding IS peak area ratios to quantify the concentration of target analytes. Following the method described above, the methodological performance was further evaluated, including, but not limited to, the limit of quantification (LOQ), linearity, precision, and trueness. And, the evaluation criteria were derived from the industry standard YY/T 1789. The results demonstrated that the correlation coefficient (r) of the linear standard curve regression equation was greater than 0.9900, indicating excellent linearity for the method. Regarding the LOQ, the results was shown in Table 1. For the precision and trueness, all measured precision values were within ± 15.0%. The method’s trueness, determined by recovery rates, was 96.86%-104.97% for VA, 92.59%-104.76% for 25(OH)D2, 98.60%-104.29% for 25(OH)D3, and 99.30%-102.74% for VE, all of which conform to the acceptable criteria of 85.0%-115.0%. This validated that the established assay was fit for the purpose of quantifying fat-soluble vitamins. This study established a novel, magnetic bead-based pretreatment method for FSV testing, demonstrating superior efficiency and consistency compared to conventional techniques. Notably, our semi-automated platform achieved high throughput, processing over 30 samples in a single batch within just 20 minutes. Furthermore, the method could be designed for seamless integration with fully automated pretreatment systems, meeting the demands of high-throughput clinical settings for automation, standardization, and highly reproducible results.