A new method has been developed for the combined analysis of Vitamin A, Vitamin D, Vitamin E and Vitamin K from serum using LC-MS/MS. The method uses supported liquid extraction (SLE) with a novel sample load which could also potentially be employed for many other highly non-polar analytes that have limited water solubility and are highly protein bound within the body.
Vitamin monitoring (especially Vitamin D) is becoming more popular in the healthcare industry but analysis of more lipid soluble vitamins (A, E and K) can be analytically challenging. The vitamin types are not structurally related, exist as many forms in the body and at significantly different concentrations making combination analysis difficult. Because of their non polarity fat soluble vitamins are either highly protein bound or when unbound have solubility limitations that make them difficult to stay in solution or chromatograph.
A method has been developed using ISOLUTE® SLE+ supported liquid extraction products to measure circulating levels of the most commonly available varieties of fat soluble vitamins. These include the Vitamin A components Retinol and Beta Carotene, Vitamin D components 25 OH D2 and 25 OH D3, Vitamin E component Alpha Tocopherol and Vitamin K components K1 and K2d4.
The procedure includes a number of novel steps not normally carried out in other SLE applications but performed here to maximise the recovery of these highly non-polar molecules.
Procedure step by step:
1. The serum sample is precipitated with isopropylalcohol (IPA) in a heptane rich mixture. The heptane creates an environment where the now unbound hydrophobic vitamins stay in solution.
2. The sample is thoroughly mixed immediately before loading. Since IPA is soluble in aqueous and water immiscible environments a well-mixed sample can be loaded on to the SLE bed as a single suspension.
3. The volume loaded is greater than the quoted capacity of the SLE. SLE columns and plates are generally referred to by the typical maximum aqueous sample that can be loaded. As the sample being loaded in this method is a combination of aqueous and water immiscible solvent the SLE capacity for this sample is increased.
4. Samples are reconstituted in pure IPA. As the pure extracted analytes have little water solubility and only a small injection volume is employed, water can be excluded from this step to improve extract stability.
5. The organic mobile phase contains a combination of methanol and IPA. The presence of IPA gives improved chromatography for beta carotene, a compound so hydrophobic it has poor solubility in both water and methanol. Another benefit of using an IPA rich mobile phase is that the method run time can be completed relatively quickly.
6. Alpha Tocopherol is present in the body at levels significantly higher than the other vitamins. This is measured by intentionally detuning the LC-MS away from the optimum electrospray and transition conditions and by making sure that no other vitamins elute close to it.
Biotage has plenty of interesting material about sample preparation and blogs like the following ones:
The application note Extraction of Fat-Soluble Vitamins from Human Serum Using ISOLUTE® SLE+ Prior to UHPLC/MS-MS Analysis describes the process I presented in this blog. Click on the button below to download it: