Analysis of Vitamin D metabolites in Human PlasmaAfter the last post on the use of gas chromatography-mass spectrometry in bioanalysis research studies (link to post), I am pleased to feature a recently released solid-phase extraction (SPE) and LC-MS/MS method for the analysis of the sunshine vitamin, Vitamin D, in human plasma.

Did you know that the rickets disease–resulting from the deficiency of Vitamin D, calcium, and phosphorus–was reported in infants by the Greek physician Soranus of Ephesus as early as the first and second centuries CE? (Source & link Wikipedia). In addition, Vitamin D deficiency causes osteoporosis in adults and, hence, the presence and quantity of Vitamin D is widely monitored in human plasma. As stated in the application note, Vitamin D occurs in two forms: Vitamin D2 (ergocalciferol) is found naturally in plants and is commonly used as a dietary supplement; Vitamin D3 (cholecalciferol) occurs naturally in mammals and is formed in the skin by exposure to sunlight.

Another interesting bit of information I learned from the application note is that regulatory bodies differ on the optimum concentration of Vitamin D in the human body. As per the Endocrine Society, 30–100 ng/mL is sufficient and 0–20 ng/mL is deficient and as per the U.S. Food and Nutrition board, >20 ng/mL is sufficient and 0−11 ng/mL is deficient.

Application Note 20936, SPE and LC-MS/MS Method for the Determination of 25-Hydroxyvitamin D2 and 25-Hydroxyvitamin D3 from Human Plasma, (downloadable PDF) describes the development of the 2-min method for the analysis of 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 from human plasma. The method features a fast and efficient sample preparation using one of our hydrophobic reversed phase well plates (Thermo Scientific SOLA SPE Well Plate), one of our HPLC systems (Thermo Scientific Dionex UltiMate 3000 HPLC system), and a silica HPLC column (Thermo Scientific Syncronis C18 HPLC column). The mass spectrometry detection was provided by one of our MS systems (Thermo Scientific TSQ Vantage triple stage quadrupole mass spectrometer).

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By the way, the advantage in using the column in this method is provided by the high surface area silica, which results in extra retention of analytes in comparison to chromatography columns with a lower surface area. This was significant within the Vitamin D application, as additional retention was required to shift the peaks of interest away from isobaric interferences to increase sensitivity. The method shows good precision with %RSD (n=6) <4.2% for both compounds.

Additional Resources

  • Our recently released Chromatography Solution Online Center features many useful and complimentary chromatography tools which can help speed up your analysis. The site is updated on a monthly basis; therefore, do check out the Archives section to see what was previously featured.

Let us know if you have any questions on the method, instruments, and consumables in the Comments box below. We look forward to hearing from you.