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Sample Preparation of Vitamin D Using VERSA Mini Liquid-Liquid Extraction Workstation

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[h=2]Introduction[/thanks]Vitamin D is essential for healthy bones and a deficiency in this vitamin may lead to various different health conditions including rickets, osteoporosis, diabetes, *****ovascular disease, and numerous forms of cancer. The circulating level of 25-hydroxy vitamin D or [25(OH)D] is an important diagnostic marker of intestinal mal-absorption and vitamin D deficiency or intoxication.
[h=2]Measurement of Vitamin D Deficiency[/thanks]According to reports, vitamin D deficiency is a common condition in the general population, which has led to an increase in the number of tests to check the blood level of 25(OH)D .
As a biologically inert molecule, vitamin D requires two hydroxylation reactions for its activation. The first reaction takes place in the liver, to give 25(OH)D, and the second takes place in the kidney to generate the physiologically active 1,25-dihydroxyvitamin D or 1,25(OH)2D.
However, the circulating level of 1,25(OH)2D is not a reliable indicator of vitamin D status because the level is often normal or elevated when a person is in fact vitamin D deficient. Therefore, 25(OH)D is used as an indicator of vitamin D status in clinical diagnosis, but 25(OH)D only a has a half-life of four to six hours and is circulated in the body tightly bound to a transport protein. Ideally, a diagnostic technique is required that is fast to process and also allows 25(OH)D to be dislodged from the transport protein.
Different techniques currently exist for determining vitamin D status using the serum 25(OH)D level. Two forms of 25(OH)D are found in the body (skin-derived vitamin D3 and exogenous vitamin D2) and both need to be quantified to give the overall vitamin D status. Although several protein-binding assays such as ELISA are available for determining vitamin D status, comparison studies and proficiency tests have raised concerns about their accuracy in measuring the different forms of 25(OH)D and therefore the total vitamin D value.
Some major laboratories have designed assays that use LC-MS/MS tandem mass spectrometry or HPLC. However, the sample preparation needed for these advanced techniques can be cumbersome when performed manually. The
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(Figure 1) has been successfully tested for preparing samples of vitamin D using partition chromatography.

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Figure 1. The VERSA Mini LLE Workstation
[h=2]Sample Preparation Using VERSA Mini LLE Workstation[/thanks]For preliminary automation of liquid-liquid extraction of 25(OH)D from whole blood or artificial blood (spiked Biocell material) and subsequent measurement by LC/MS, 200
 
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