Analysis of 25-Hydroxyvitamin D in serum by automated sample cleanup and LC-MS/MS

Vitamin D stands for a group of fat-soluble vitamins, the so-called calciferols. These include the most important forms vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D plays an essential role in regulating the calcium level in the blood and in bone formation. It is also involved in other metabolic processes and in the protein biosynthesis.
For several years, it has been suspected that there are links between vitamin D levels in the body and chronic diseases. To date, however, evidence is lacking.
The majority of vitamin D (approx. 80-90%) is formed in the skin with the help of sunlight (UV-B radiation); approx. 10-20% is contributed by diet, e.g. via fatty sea fish, eggs, edible mushrooms. Therefore, especially in the winter months with little sunlight, there is a risk of undersupply and resulting low vitamin D levels. Therefore, the vitamin D value is also a common parameter in clinical diagnostics. Vitamin D level is determined by measuring 25-hydroxyvitamin D, or 25(OH)D, in blood serum. 25(OH)D is a precursor of active vitamin D. It is often also called "storage vitamin D". There are various reference values for the purpose of classifying the measured values. The Robert Koch Institute, for example, refers to the classification of the Institute of Medicine (IOM) in the USA.

For analysis, the combination of solid phase extraction (SPE) with subsequent HPLC-MS/MS analysis is expedient. Due to the large number of samples, automation of sample preparation is an obvious solution. For this purpose, MACHEREY-NAGEL has developed an automated sample preparation in 96-well format on an Eppendorf epMotion® 5075vt robot. CHROMABOND® HLB serves as a sorbent for analyte preconcentration. High recoveries and good reproducibilities were achieved. 

More details about the application can be found in the following note:
Analysis of 25-Hydroxyvitamin D in serum by automated sample cleanup and LC-MS/MS

Robert-Koch-Institute website, available at: (retrieval date: 06/29/2023)