Quantitative analysis of vitamin D using m/MALDI-TOF mass spectrometry based on a parylene matrix chip

Vitamin D deficiency is associated with various disorders and is diagnosed based on the concentration of 25-hydroxy vitamin D3 (25(OH)D₃) in serum. The parylene matrix chip was fabricated to reduce the matrix background noise, and the homogenous distribution of the matrix was retained for the quantitative analysis of 25(OH)D₃. The Amplex Red assay was performed to confirm that the sample-matrix mixing zone of the parylene matrix chip was formed below the surface of the parylene-N film. The homogeneous distribution of the matrix was verified from the fluorescence image. For effective analysis using a parylene matrix chip, 25(OH)D₃ was modified through the nucleophilic addition of betaine aldehyde (BA) to form a hemiacetal salt. Such modified 25(OH)D₃ with a positive charge from BA could be effectively analyzed using MALDI-TOF mass spectrometry. Serum 25(OH)D₃ was extracted by liquid–liquid extraction (LLE) and quantified using MALDI-TOF mass spectrometry based on the parylene matrix chip. The intensity of the mass peak of 25(OH)D₃ was linearly correlated (r² = 0.992) with the concentration of 25(OH)D₃ spiked in serum, and the LOD was 0.0056 pmol/μL. Energy drinks and vitamin D₃ tablets were also employed for the real sample analysis. Finally, the results of the chemiluminescence binding assay and MALDI-TOF mass spectrometry were statistically analyzed to determine the applicability of the method using the Bland–Altman test and Passing–Bablok regression.