Characterization and Application of Membrane-emulsified HLB Microspheres for Quantification of 25-Hydroxy Vitamin D2 and 25-Hydroxy Vitamin D3 in Serum by UPLC-MS/MS

Membrane emulsification can obtain microspheres with relatively uniform particle size and pore size, which has unique advantages in the synthesis of polymer microsphere adsorbents and the enrichment and separation of organic compounds. In this study, the Hydrophilic Lipophilic Balance (HLB) solid phase extraction microspheres were prepared by membrane emulsification technique and was used in the pretreatment of samples to detect 25-hydroxy vitamin D₂ (25OH-VD₂) and 25-hydroxy vitamin D₃ (25OH-VD₃) in serum by UPLC-MS/MS for the first time. To confirm the efficacy of membrane-emulsified (ME) HLB microspheres, three types of microspheres, including ME HLB microspheres, non-membrane-emulsified (non-ME) HLB microspheres and commercial HLB microspheres, were compared for the pretreatment of 25OH-VD₂ and 25OH-VD₃. The performance of HLB microspheres was characterized based on the sample recovery values acquired by UPLC-MS/MS analysis. Results showed that among the three types of microspheres, ME HLB microspheres showed the best performance and demonstrated good adsorption properties for 25OH-VD₂ and 25OH-VD₃. The recoveries range of 25OH-VD₂ and 25OH-VD₃ were 96.7–101.4 and 98.7–104.9%, respectively, and the coefficients of variation were 0.67–1.24 and 1.39–2.28%, respectively, which were superior to those of commercial HLB microspheres and non-ME HLB microspheres. Additionally, due to their excellent homogeneity, the ME HLB microspheres exhibited good precision in the determination of low values of 25OH-VD₂ and 25OH-VD₃, indicating accurate and simple detection of 25OH-VD₂ and 25OH-VD₃ with a broad potential for further development.