ORIGINAL PAPER Metabolic profiling of major vitamin D metabolites using Diels���Alder derivatization and ultra-performance liquid chromatography���tandem mass spectrometry Pavel A. Aronov & Laura M. Hall & Katja Dettmer & Charles B. Stephensen & Bruce D. Hammock Received: 7 February 2008 /Revised: 20 March 2008 /Accepted: 25 March 2008 /Published online: 24 April 2008 # Springer-Verlag 2008 Abstract Biologically active forms of vitamin D are impor- tant analytical targets in both research and clinical practice. The current technology is such that each of the vitamin D metabolites is usually analyzed by individual assay. However, current LC-MS technologies allow the simultaneous meta- bolic profiling of entire biochemical pathways. The impedi- ment to the metabolic profiling of vitamin D metabolites is the low level of 1��,25-dihydroxyvitamin D3 in human serum (15���60 pg/mL). Here, we demonstrate that liquid���liquid or solid-phase extraction of vitamin D metabolites in combina- tion with Diels���Alder derivatization with the commercially available reagent 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) followed by ultra-performance liquid chromatography (UPLC)��� electrospray/tandem mass spectrometry analysis provides rapid and simultaneous quantification of 1��,25-dihydroxyvitamin D3, 1��,25-dihydroxyvitamin D2, 24R,25-dihydroxyvitamin D3, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in 0.5 mL human serum at a lower limit of quantification of 25 pg/mL. Precision ranged from 1.6���4.8 % and 5���16 % for 25-hydroxyvitamin D3 and 1��,25-dihydroxyvitamin D3, respectively, using solid-phase extraction. Keywords 1��,25-Dihydroxyvitamin D3 . 25-Hydroxyvitamin D3 . 24R,25-Dihydroxyvitamin D3 . UPLC . LC-MS . Metabolic profiling . Derivatization Introduction Metabolic profiling, defined here as the quantification of metabolites involved in the same metabolic pathway, has become an important tool for determining steady-state concentrations of metabolites and studying the regulation of the corresponding metabolic pathways [1, 2]. Metabolic profiling allows metabolic regulation to be surveyed in a minimally invasive manner using biofluids such as plasma or urine that are subsequently analyzed by GC-MS or LC-MS. The field of vitamin D metabolite analysis has been historically dominated by immunoassays and receptor binding assays [3], although there are examples of the application of LC-UV or LC-MS to the analysis of 25-hydroxyvitamin D2 (25(OH)D2) and 25-hydroxyvitamin D3 (25(OH)D3) [4���6]. LC-MS can potentially detect and measure more than 40 reported vitamin D metabolites [7]. If we draw parallels with other families of steroid hormones, many of the vitamin D metabolites may have other biological roles beyond being mere catabolic products. However, very little is known about the biological roles of most of the downstream vitamin D metabolites, and the development of a comprehensive profiling method would facilitate research on vitamin D metabolism. Currently, most research and diagnostic assays Anal Bioanal Chem (2008) 391:1917���1930 DOI 10.1007/s00216-008-2095-8 Electronic supplementary material The online version of this article (doi:10.1007/s00216-008-2095-8) contains supplementary material, which is available to authorized users. P. A. Aronov Department of Entomology, University of California, Davis, CA 95616, USA L. M. Hall : C. B. Stephensen Department of Nutrition and USDA Western Human Nutrition Research Center, University of California, Davis, CA 95616, USA K. Dettmer Institute of Functional Genomics, University of Regensburg, Regensburg, Germany B. D. Hammock (*) Department of Entomology and U.C. Davis Cancer Research Center, University of California, Davis, CA 95616, USA e-mail: bdhammock@ucdavis.edu

focus on 25(OH)D and 1��,25-dihydroxyvitamin D (1��, 25(OH)2D) produced in a series of oxidations by the cyto- chromes P450 from their dietary precursor vitamin D (Fig. 1) [8]. Most current analytical methods based on immunoassays are not able to separate forms of vitamin D with different side-chains (mainly D2 and D3). There is a growing body of evidence that the biological activities of these forms may be different [9, 10]. This illustrates a need for analytical methods that are selective for D2 and D3 forms. Analytical methods for vitamin D also are needed for regulatory, quality control and nutritional studies. Biologically, the conversion of vitamin D2 and D3 into corresponding 25 (OH)D forms is rapid, as estimated from the 36���48-hour half-life of vitamin D3 in human circulation [11]. Thus, there is usually little need to analyze the blood levels of vitamin D2 and D3 except supplementation studies. Subsequently, 25 (OH)D3 is converted into biologically active 1��,25(OH)2D3, which binds to the vitamin D nuclear receptor (genomic response) as well as to a putative membrane receptor (rapid response) to initiate a cascade of biological events related to calcium and phosphorus homeostasis, cancer and inflamma- tion [12]. Alternatively, 25(OH)D3 is thought to be deacti- vated via conversion into 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3) by 25-hydroxyvitamin D 24-hydroxylase, although independent biological effects of 24R,25(OH)2D3 are also known [13���15]. Furthermore, the same enzyme deactivates 1��,25(OH)2D3 via conversion into 1��,24R,25- trihydroxyvitamin D3 (1,24R,25(OH)3D3). These metabo- lites can undergo further metabolism by several pathways including further oxidation and conjugation [8]. The development of an LC-MS profiling method for vitamin D metabolites is impeded by their low concentra- tion in human circulation, particularly for 1��,25(OH)2D3, with concentrations ranging from 15 to 60 pg/mL. Recently, an LC-tandem MS method was introduced to measure nonderivatized 1��,25(OH)2D3, but this requires 2 mL of human serum [16]. Vitamin D metabolites have low ion- ization efficiencies in electrospray (ESI) or atmospheric pressure chemical ionization (APCI) sources because they lack easily charged groups, which would enhance ioniza- tion efficiencies. However, the conjugated diene group of vitamin D metabolites makes them a specific target for Diels���Alder derivatization. In fact, several highly reactive 4-substituted 1,2,4-triazoline-3,5-diones (TADs or Cookson- type reagents) have been reported in the literature for the analysis of vitamin D metabolites, their analogs and other dienes, including derivatization reagents for ESI-MS [6, 17��� 22]. The derivatization reagents introduce polar groups and thus typically result in a 100���1000-fold increase in sensitivity over nonderivatized compounds. However, no method for profiling the major vitamin D metabolites 1��,25(OH)2D2, 1��,25(OH)2D3, 24R,25(OH)2D3, 25(OH) D2 and 25(OH)D3 has been reported that can detect and quantify endogenous levels of 1��,25(OH)2D3. Here, we demonstrate an ultra-performance liquid chromatography (UPLC)���tandem MS method for the quantification of an array of the most biologically important vitamin D metab- olites after Diels���Alder derivatization with 4-phenyl-1,2, 4-triazoline-3,5-dione (PTAD). The method was validated for solid-phase extraction (SPE) and liquid���liquid extraction (LLE) of 1��,25(OH)2D3 and 25(OH)D3 from serum and applied to studies of vitamin D metabolism in humans. Experimental Chemicals Hexane, methyl tert-butyl ether, dichloromethane, acetoni- trile, ethyl acetate, methanol, formic acid, and K2HPO4 were purchased from Fisher Scientific (Pittsburgh, PA, USA). Deionized water (resistivity of 18.1 M��/cm) and distilled water were prepared in-house and used for mobile phase preparation and SPE extraction, respectively. PTAD was purchased from Fluka (St. Louis, MO, USA). Standards of vitamin D metabolites were purchased from Fluka, Sigma��� Aldrich (St. Louis, MO, USA) and BIOMOL (Plymouth Meeting, PA, USA) as indicated below. Deuterated surrogates of vitamin D metabolites were purchased from Synthetica (Oslo, Norway) and Medical Isotopes (Pelham, NH, USA). Vitamin D 25(OH)D 1 24R,25(OH)2D 24-hydroxylase 1 25-hydroxylase Diet UV/skin 24-hydroxylase 1 D2 and D3 D3 2 d D 3 HO D3 D2 1 24 25 24 25 1 ,25(OH) 2 D 24-hydroxylase 1 -hydrox oxyl 24-hydroxylase 1 ,24R,25(OH) 3 D ��,25(OH)2D ��-hydroxylase ��,24R,25(OH)3D Fig. 1 Metabolism of vitamin D. The metabolites measured in this study are highlighted in a darker font on the left. The two major forms of vitamin D and sites of hydroxylation are shown on the right 1918 Anal Bioanal Chem (2008) 391:1917���1930