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.