Analysis of Vitamin D Metabolites by Mass Spectrom

Authors: Dietrich A Volmer Caroline S Stokes

Publish Date: 2016

Volume: , Issue: , Pages: 1-20

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Abstract

This chapter provides an overview of the stateoftheart of mass spectral analysis of vitamin D and its metabolites Most activities in the vitamin D analytical field today center around clinical measurements and emphasis in the following sections is therefore placed on vitamin D in human samples mostly from serum or plasma Virtually all modern mass spectrometry analyses of vitamin D are performed using hyphenated liquid chromatographytandem mass spectrometry LCMS/MS techniques Measurements using gas chromatography GCMS are now rarely performed mainly because of problems with compound degradations during analysis For those readers interested in GCMS of vitamin D compounds the present authors refer to an excellent review by Vouros and coworkers Gathungu et al 2013 Food analysis is also not addressed in this chapter as requirements are quite different usually focusing only on fortified or unfortified levels of vitamin D2 and D3 in food components Thomson and Cressey 2014 whereas clinical analyses are mostly concerned with concentrations of vitamin D metabolitesThe chapter begins with a short introduction to vitamin D photosynthesis and metabolism in humans and abundances of vitamin D metabolites in various compartments of the body followed by a discussion of sample matrices for clinical measurements and appropriate sample preparation procedures Next liquid chromatography of vitamin D is briefly summarized leading to mass spectrometry ionization and analyzer techniques The mass spectrometry section concludes with a discussion on selectivity and accuracy issues and certified reference materials Finally selected clinical applications of vitamin D metabolite analysis are highlighted at the end of the chapterThe 25OHD3 metabolite is present in abundant concentrations and can be easily measured in blood samples Moreover the 25OHD3 metabolite has the longest halflife of all readily measureable metabolites thus representing the status marker of vitamin D Holick 2009 25OHD3 is however not the most active of vitamin D metabolites which is produced under renal conversion so as to form 125dihydroxyvitamin D 125OH2D3 when 25OHD3 reaches the kidneys Fig 1 This active metabolite exerts its actions via the vitamin D receptor VDR however it is present in much smaller concentrations and has a comparably shorter halflife than 25OHD3 several hours as compared to 3 weeks Jones 2008 Nagpal et al 2005 The smaller concentrations of 125OH2D3 in blood also pose a challenge in its analytical determination Volmer et al 2015Both 25OHD3 and 125OH2D3 conversion occurs via the vitamin D catabolites 2425dihydroxyvitamin D3 2425OH2D3 and 12425trihydroxyvitamin D3 12425OH3D3 respectively Both catabolites are formed through what is known as the C24hydroxylation process and are excreted in bile Nagpal et al 2005 The 2425OH2D3 compound can be analytically determined and is present in higher concentrations than 125OH2D3 Currently these vitamin D metabolites/catabolites are quantified in blood samples There are however indications that other body tissues for example adipose tissue which also contains VDR ClementePostigo et al 2015 may prove interesting targets for vitamin D quantification Adipose tissue is known to sequester and thus store vitamin D Wortsman et al 2000 yet recently the active 125OH2D3 metabolite has been implicated as having significant effects on adipogenesis and inflammation on a cellular level in adipose tissue Mutt et al 2014 Therefore the extension of vitamin D metabolite quantification beyond that in serum and plasma samples may prove to be biologically significant in the near futureVirtually all published assays for 25OHD3 and other vitamin D metabolites measure from serum or plasma as metabolites circulate in blood The various mass spectrometric analyses of vitamin D metabolites which are discussed in the following sections of this chapter are based on serum or plasma samplesVariations of sample matrix have included dried blood spots DBS in particular for samples of infants Eyles et al 2009 2010 Heath et al 2014 Higashi et al 2011 Hoeller et al 2015 Newman et al 2009 As sampling for DBS on filter paper is minimally invasive the technique has also been successfully used for vitamin D status screening of adult cohorts in particular outside hospital environments “unsupervised sampling” Hoeller et al 2015 In addition DBS can usually be transported and stored at room temperature over extended periods of time thus offering significant advantages over venous blood taking and low temperature transport and storageAn interesting alternative as sample matrix is saliva which was shown to contain sufficient levels of 25OHD3 for successful quantification by LCMS/MS after chemical derivatization Higashi et al 2008 Other liquid matrices that were used were urine Ogawa et al 2014 – where 2425OH2D3 dominated over 25OHD3 a reverse situation to serum – and cerebrospinal fluid CSF Holmøy et al 2009TOFSIMS images of visceral adipose tissue biopsies size 258 × 258 μm2 from one obese individual lateral resolution ≈1 μm a–d Distribution of secondary ions of sodium Na+ a and potassium K+ b phosphatidylcholine headgroup C5H15PNO4+ m/z 1842 c and diacylglycerol DAG at m/z 5512 d e Vitamin D3 MOH+ at m/z 3671 f 25OHD3 MOH+ at m/z 3832 g 125OH2D3 MOH+ at m/z 3998 h overlay images of vitamin D3 MOH+ red 25OHD3 green and 125OH2D3 blue Adipocytes are indicated by arrows in panel d The color bar ranges from black representing 0 intensity to white indicating highest signal intensity Reprinted with permission from Malmberg et al 2014LCMS/MS assays for vitamin D from serum or plasma predominantly apply liquid/liquid extraction LLE solidphase extraction SPE or combinations of the two techniques to remove the hydrophobic vitamin D compounds from the sample matrix van den Ouweland et al 2013 Volmer et al 2015 Often protein precipitation is performed solely or as part of further extraction steps Ding et al 2010 Herrmann et al 2010 Kushnir et al 2010 Furthermore techniques such as ballistic turbulent flow chromatography TFC Bunch et al 2009 Singh et al 2006 supported liquid extraction SLE Geib et al 2015 Jenkinson et al 2016 or immunoenrichment Laha et al 2012 have been applied to vitamin D sample preparation

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