{"id":16608,"date":"2022-03-23T09:10:12","date_gmt":"2022-03-23T09:10:12","guid":{"rendered":"https:\/\/clinlabint.com\/?p=16608"},"modified":"2022-03-23T09:18:08","modified_gmt":"2022-03-23T09:18:08","slug":"rapid-supercritical-fluid-chromatography-tandem-mass-spectrometry-sfc-ms-ms-for-the-routine-quantification-of-retinol-and-%ce%b1-tocopherol-in-human-serum-and-plasma","status":"publish","type":"post","link":"https:\/\/clinlabint.com\/rapid-supercritical-fluid-chromatography-tandem-mass-spectrometry-sfc-ms-ms-for-the-routine-quantification-of-retinol-and-%ce%b1-tocopherol-in-human-serum-and-plasma\/","title":{"rendered":"Rapid supercritical fluid chromatography\u2013tandem mass spectrometry (SFC-MS\/MS) for the routine quantification of retinol and \u03b1-tocopherol in human serum and plasma"},"content":{"rendered":"
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Rapid supercritical fluid chromatography\u2013tandem mass spectrometry (SFC-MS\/MS) for the routine quantification of retinol and \u03b1-tocopherol in human serum and plasma<\/h1>\/ in Featured Articles<\/a> <\/span><\/span><\/header>\n<\/div><\/section>
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As interest in vitamins A and E has increased in recent years, so too has the need for rapid and reliable methods by which these compounds can be assayed. The development of a supercritical fluid chromatography\u2013tandem mass spectrometry (SFC-MS\/MS)-based method with an automated extraction procedure provides faster analysis than other chromatographic-based methods and improved specificity.<\/strong><\/h4>\n

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Introduction<\/strong><\/h4>\n

In the clinical laboratory, retinol and \u03b1-tocopherol are most commonly measured in serum (or plasma) to estimate the nutritional status of vitamins A and E, respectively. Chromatographic-based methods, such as high-performance liquid chromatography (HPLC) and liquid chromatography\u2013tandem mass spectrometry (LC-MS\/MS), have been used since the 1980s to perform the analysis but involve long and tedious extraction procedures, and the use of large volumes of solvents and serum. Our objective was to develop a selective and sensitive rapid method based on supercritical fluid chromatography\u2013tandem mass spectrometry (SFC-MS\/MS) with an automated extraction procedure that required the minimal use of solvent and patient material.<\/p>\n

Biological functions of vitamins A and E Vitamin A<\/strong><\/h4>\n

Vitamin A occurs in a variety of forms that can be divided into two groups: retinoids and provitamin A carotenoids. Naturally occurring retinoids include retinol (Fig. 1), retinol esters, retinaldehyde and retinoic acid, whereas the major provitamin A carotenoid is \u03b2-carotene [1]. Retinol is found in the circulation and transported by retinol binding proteins (RBP) [2]. It is a precursor for two essential molecules: 11-cis-retinol and all-transretinoic acid. The most understood action of vitamin A is its role in vision. In brief, retinol is transported in the blood by RBP to the retina of the eye. Retinol is then esterified to form retinyl esters, which can be stored in retinal pigment epithelial cells. When needed, retinyl esters are hydrolysed and isomerized to 11-cis-retinol and then oxidized to 11-cis-retinal. The 11-cis form of retinal is a component of the visual pigment rhodopsin, which is found in the rods of the retina. On absorption of light, rhodopsin isomerizes to transretinal and subsequently releases opsin that results in a series of conformational and other changes, leading to the generation of an electrical signal to the optic nerve to relay visual imagery to the brain [3]. All-transretinoic acid modulates gene expression by activation of nuclear receptors, of which there are two groups: retinoic acid receptors (RAR) that bind all-trans-retinoic acid (and some other retinoids); and retinoid X receptors (RXR) that bind 9-cis-retinoic acid. Other metabolic roles of vitamin A include maintenance of epithelial barriers, immune competence, reproduction and embryonic growth and development [3].<\/p>\n

Vitamin E <\/strong><\/p>\n

Compounds with vitamin E activity contain a chromanol ring attached to a long phytyl side chain (Fig. 2). There are eight naturally occurring forms: \u03b1-, \u03b2-, \u03b3-, and \u03b4-tocopherols and -tocotrienols [1]. \u03b1-Tocopherol is the most abundant form of vitamin E in the circulation, and accounts for about 90% of the vitamin E in human tissue [4]. It is a potent antioxidant and aids the prevention of lipid peroxidation, such as phospholipids within cell membranes. The mechanism by which it exerts its protective effect is through the absorption of peroxyl radicals, which results in the termination of the free radical chain reaction. Vitamin E is transported to and from the liver via various lipoproteins and incorporated into cell membranes where it is anchored by its hydrophobic tail. It is suggested that vitamin E antioxidant properties help to prevent or delay disease and disorders linked to oxygen free radicals. These include cardiovascular disease in certain patient subgroups such as those on hemodialysis or in diabetic patients [1] and patients with cognitive impairment and neurological diseases such as Alzheimer\u2019s in which amyloid-\u03b2 protein causes cytotoxicity through oxidative stress [4].<\/p>\n

Vitamin E also modulates the transcription of certain genes, inhibits platelet aggregation and vascular smooth muscle proliferation and has an effect on cell signalling in the immune system. Of note, \u03b1-tocopherol has a significant role in the prevention of miscarriage in humans [5].<\/p>\n

Assessing vitamin A and E status<\/strong><\/h4>\n

Requests for the determination of vitamin A and vitamin E status are most commonly made so that patients with a deficient state can be identified. Deficiency is almost exclusively seen in patients with an impaired ability to absorb fat-soluble vitamins, for example in patients with cystic fibrosis and short bowel syndrome, or those that have impairment of fat absorption or metabolism, such as pancreatic insufficient cystic fibrosis patients. Other requests for vitamin A and E status evaluation include patients that are on supplementation, such as enteral and parenteral nutrition patients [6].<\/p>\n

As interest in vitamins A and E has increased in recent years, so too has the need for rapid and reliable methods by which these compounds can be assayed. Determination of fat-soluble vitamins in serum typically involves lengthy sample preparation (such as saponification and extraction), followed by HPLC coupled to ultraviolet (UV) or fluorescence detection. Vitamins A and E are often determined co-currently in the same analytical procedure because of their similar sample preparation requirements [6]. The authors\u2019 laboratory previously used reversed phase chromatography with UV detection which featured long chromatographic run times requiring considerable time and solvent usage during sample analyses. In order to meet the increasing demand for analysis, we validated and implemented a SFC-MS\/MS-based method for the assessment of retinol and \u03b1-tocopherol.<\/p>\n

HPLC-UV method limitations<\/strong><\/p>\n