Validation of Automated Liquid-Liquid Extraction of 25-hydroxy vitamin D

Validation of Automated Liquid-Liquid Extraction of 25-hydroxy vitamin D

The following study validates the capacity for automated vitamin D extraction (25-hydroxy) from whole blood/spiked BioCell material (artificial blood) samples. With the VERSA Liquid-Liquid Extraction (LLE) system , LC/MS was able to make a determination.


Vitamin D is important to bone health as you can associate its deficiency with several health conditions. From osteoporosis and rickets, cardiovascular disease, diabetes, and autoimmune disorders to depression, stroke, chronic pain, osteoarthritis, and many forms of cancer. Measurement of circulating levels of 25-hydroxy vitamin D is also important in the diagnosis of intestinal mal-absorption and vitamin D deficiency or intoxication in humans. Research has indicated that vitamin D deficiency is widespread in the general population. This has led to exponential growth in clinical diagnosis tests for 25-hydroxy vitamin D also known as [25(OH)D] in blood serum. However, this form of vitamin D in the blood has a half-life of four to six hours, and is tightly bound to a transport protein in circulation. Therefore, testing methods must process samples rapidly, and must be capable of dislodging 25(OH)D from its carrier proteins.

Several methods are available to assess vitamin D sufficiency through measurement of serum 25-hydroxy vitamin D. Since two forms of vitamin D contribute to the overall status of an individual (one derived primarily from vitamin D produced in the skin and the other from diet or supplementation), you must measure both forms equally. Antibody (ELISA) or other protein-binding assays are available, but proficiency testing and comparison studies have raised questions about the ability of most existing assays to accurately measure the different forms of 25(OH)D, and by extension, the total vitamin D value that these assays report. Some larger laboratories have developed assays using LC-MS/MS tandem mass spectrometry or HPLC. However, manual sample preparation methods for such assays are very cumbersome. Research has shown that liquid-liquid extraction of 25(OH)D is a cost-effective option to familiar immunological Vitamin D extraction methods (Højskov et al, 2010).

Aurora’s VERSA automated LLE Workstation has received validation for the organic solvent extraction of 25-hydroxy vitamin D from blood serum using liquid-liquid extraction (partition chromatography). Validation data for the VERSA LLE’s ability to conduct automated vitamin D extraction appears in this technical-note.


Reliable reproducibility of peak height data from the unit suggests that biomolecules like 25(OH)D can be effectively extracted using this automation within acceptable values. Moreover, the automated vitamin D extraction takes 60 minutes for a set of 96 samples with no detectable cross-contamination. The vitamin D extraction process took approximately 17 minutes less than the manual procedure.


The process of automated vitamin D extraction, assisted by the VERSA Liquid-Liquid Extraction system, was a successful alternative to the cumbersome separatory funnel for the isolation of such compounds. 25-hydroxy vitamin D extraction using the VERSA LLE Workstation is just one of many solutions offered by Aurora. Numerous liquid handling applications have become automated, and each VERSA workstation remains customizable to accommodate unique needs.

Aurora provides complete lab automation solutions supporting life science, chemical analysis, and drug discovery research by supplying application support, reagents and instruments which offer higher throughput while improving quality, accuracy, and precision.

Højskov, CS., Heickendorff, L. and Holger J. 2010. High-throughput liquid-liquid extraction and LCMSMS assay for determination of circulating 25(OH) vitamin D3 and D2 in the routine clinical laboratory. Clinica Chimica Acta. 411(1-2): 114-116.