This chapter provides an overview of versatile and efficient chemical syntheses of vitamin D derivatives by application of either linear or convergent synthesis approaches. Synthesis of the most relevant naturally occurring vitamin D metabolites and their deuterated counterparts to use as calibration and reference standards in LC-MS/MS assays is also shown. The chapter then summarizes the most important mass spectrometric approaches to quantify important vitamin D metabolites in human biofluids. In addition, new developments are described that are aimed at the pathobiological interpretation of the measured vitamin D metabolite distributions in various human diseases.
- vitamin D deficiency
- low abundant vitamin D metabolites
- assay development
- disease risk prediction
Vitamin D is mostly known for its role in the regulation of calcium and phosphorous homeostasis [1–3]. Consequently, vitamin D deficiency may cause various disorders related to bone mineralization . Drugs based on vitamin D analogs are commonly used to treat bone diseases (osteoporosis, osteomalacia, and rickets) or psoriasis. More recently, it has been suggested that vitamin D deficiency is also connected to a wide range of other diseases beyond bone mineralization, such as diabetes, autoimmune diseases, cardiovascular diseases, and cancer, as various clinical and epidemiological studies have shown [5–9]. However, the development of drugs for treatment of these diseases based on appropriate vitamin D analogs has mostly failed, either due to their rapid metabolic clearance or their calcemic effects. Vitamin D analogs are usually hormonally active compounds with pleiotropic functions and their levels in the body are strictly regulated by the hormonal system. In cases of oversupply, they are enzymatically degraded to avoid harmful effects such as calcemia, leading mainly to inactivation and conversion into water soluble degradation products suitable for renal clearance. Consequently, prevention of vitamin D deficiency rather than therapy of a vitamin D-related disease is a promising approach. Due to low concentration and short half-life of the active metabolite, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3, calcitriol) (
2. Metabolism of vitamin D
Vitamin D3 (
Finally, the corresponding metabolites of vitamin D2 (
3. Synthesis of low abundant vitamin D metabolites
The commercial unavailability of many relevant vitamin D3 and D2 species has limited the scope of LC-MS/MS assays in the past. For use as reference standards, vitamin D3 and D2 metabolites, as well as their corresponding stable isotopes (labeled with 2H (D) or 13C), have to be synthesized by application of a versatile and cost-effective methodology. A large variety of chemical syntheses of vitamin D derivatives has been developed in the last few decades by several academic and commercial groups [1, 25–27], mainly with the aim of synthesizing new analogs for drug discovery and development purposes. These efforts have resulted in a large number of more than 3000 synthesized compounds . Critical evaluation of these methods reveals, however, that only a few of them are suitable to reproducibly generate metabolites of interest in gram quantities at reasonable costs within short time frames. In this chapter, we will review some of the more suitable strategies that have been successfully applied and optimized in our laboratory.
The synthesis of deuterated vitamin D3 and D2 metabolites is mostly accomplished using the same procedures as those developed for nondeuterated metabolites.
The biosynthetic and technical synthesis (starting from an appropriate steroid precursor (analog to
3.1. Linear synthesis of vitamin D metabolites
Following a linear synthesis (Scheme 2), the
Vitamin D2 metabolites are synthesized by application of analogous strategies as applied for their corresponding D3 counterparts, although they are more challenging to synthesize, for two reasons: vitamin D2 metabolites contain an olefinic double bond in C-22/23-position, which has to be arranged in
3.2. Convergent synthesis of vitamin D metabolites
Alternatively, a convergent approach can be applied (Scheme 3), which is more versatile than a linear synthesis and allows for more harsh reaction conditions and wider scope of suitable substrates and reagents. In a classical and widely applied strategy, vitamin D2 (
Two other low abundant metabolites, 23(S),25(R)-dihydroxyvitamin D3 26,23-lactone
3.3. Convergent Palladium-catalyzed synthesis of vitamin D metabolites
The A-ring building block is preferably synthesized
4. LC-MS/MS assays for vitamin D metabolites
In the second part of this chapter, an overview of the mass spectrometric analysis of vitamin D metabolites in biological samples is presented. Because of the chemical nature of these secosteroidal molecules, liquid chromatography-tandem mass spectrometry (LC-MS/MS) currently provides the optimum analytical platform for analysis of vitamin D metabolites. While mass spectrometry assays are often not as rugged and more expensive than most non-mass spectrometric assays (in particular immunoassays), they provide the ability to capture multiple metabolites simultaneously at very low concentration levels. Immunoassays measure the 25(OH)D3 and 25(OH)D2 metabolites only and have limitations with respect to detection sensitivity as well as selectivity and specificity issues.
In most published assays, the metabolites are determined from serum or plasma matrices. Other biological matrices have rarely been used, although vitamin D metabolites have successfully been quantified in saliva  and various soft tissues . In the following sections, we discuss the requirements and characteristics of LC-MS/MS of vitamin D metabolites; that is, chromatographic separation, ionization,
4.1. Liquid chromatographic separation of vitamin D metabolites
Due to the hydrophobic structure, vitamin D metabolites are generally easily separated on reversed-phase liquid chromatography stationary phases (e.g., octadecyl C18) materials utilizing hydrophobic interactions. The vitamin D metabolites generally elute in the order trihydroxylated < dihydroxylated < monohydroxylated metabolites, that is, for vitamin D3-related molecules, the order of chromatographic retention is 1,25(OH)2D3 < 25(OH)D3 < D3. Between corresponding D2 and D3 analogs, the D2 metabolites elute marginally later than the D3 versions. For the two important isomers of dihydroxylated vitamin D metabolites, 24,25(OH)2D3 and 1,25(OH)2D3, the order of retention is 24,25(OH)2D3 < 1,25(OH)2D3.
Possibly more interesting—from a chromatography point of view—are the biochemically formed isomers after stereochemical reversal (β→α) at C-3; for example, the epimers 25(OH)D3 and 3-
4.2. Ionization of vitamin D metabolites for mass spectrometric analysis
Mass spectrometric determination of vitamin D metabolites using liquid chromatography-mass spectrometry (LC-MS) is not trivial because of the structural limitations that the analytes provide with respect to attaching a charge to the molecules. Gas chromatography-mass spectrometry techniques have been used in the past for qualitative analysis and structure determinations, but these methods have been almost completely replaced by modern LC-MS methods. While analysis of transformation products such as vitamin D sulfates is relatively easy by LC-MS as the metabolites can be simply analyzed as deprotonated molecules, ionizing the relatively nonpolar vitamin D metabolites is not straightforward. For LC-MS, electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) are the most common ionization techniques, which usually result in the formation of [M+H]+ ions for most biological molecules. APCI has been successfully applied to steroids, as gas-phase chemical ionization often efficiently transfers a proton to these types of molecules. ESI usually relies on a charging mechanism in the liquid phase and one would therefore expect ESI to be less efficient than APCI for vitamin D metabolites. In reality this is not the case, however, and both techniques have equally been applied to vitamin D analysis, with the analytical figures of merit being quite similar between the two ionization techniques. There appears to be a slight trend toward application of ESI rather than APCI in recent years.
An important, often neglected consideration in the choice of ionization source is the (usually detrimental) impact of coionized sample components that coelute with vitamin D metabolites from the LC column. Here, ionization suppression effects caused by these coeluting molecules may impact the outcome even stronger than the differences seen in ionization efficiencies between ESI and APCI [58, 59]. However, no systematic comparison of ionization efficiency/ionization suppression effects between ESI and APCI has been performed yet. Furthermore, coionization of coeluting components also leads to the formation of isobaric interferences when sample matrices such as plasma or serum are analyzed, which will be discussed in more detail below.
Another useful ionization technique is atmospheric pressure photoionization (APPI). This technique is very common in fields such as petroleum analysis, but surprisingly, it is virtually unknown in the clinical community. To our knowledge, only one study has systematically compared APPI with APCI and shown that APPI generated significantly higher ion currents for 25(OH)D3 than APCI . A second study applied APPI to quantification of 25(OH)D3 without comparison to ESI or APCI .
Finally, another option to overcome problems of ionization efficiency and resulting problems with detection sensitivity is derivatization of vitamin D metabolites, to convert them into better responding transformation products. Such procedures are now quite common in the vitamin D analytical field; they comprise introduction of a chemical group that is readily ionized or is permanently charged. Cookson-type triazoline-diones and triazoline-dione-related reagents (e.g., 4-phenyl-1,2,4-triazoline-3,5-dione [PTAD]) are most often applied, which utilize the reaction of the reagent’s dienophile with the
Several promising PTAD assays have been described in the literature [39, 49, 62–64]. The advantage of using the
4.3 Mass analyzers for vitamin D analysis
Most clinical applications for vitamin D utilize quadrupole-based mass analyzers with low resolving powers. Because these mass spectrometers measure
By far the most common low resolution tandem mass spectrometer for vitamin D analysis in clinical laboratories is the triple quadrupole (QqQ) instrument. In a QqQ mass spectrometer, the [M+H]+ ions of vitamin D metabolites are selected in the first quadrupole, fragmented in a quadrupole collision cell, and the products analyzed in the final quadrupole mass analyzer. The acquisition mode that is almost always applied is the so-called selected reaction monitoring (SRM) mode. In this mode, the [M+H]+ precursor ion is isolated in Q1 (e.g.,
A few studies also report the use of a low resolution quadrupole ion trap (IT) [69, 70] in MS/MS mode for 25(OH)D3 and 25(OH)D2, but these instruments are rarely implemented in clinical environments. IT instruments are often not fit for purpose for quantitation of low abundant biological molecules, because of the large detrimental contributions of acquisition time overhead to the duty cycle, thus limiting detection sensitivity. There is also a general decline of this instrument type in modern mass spectrometry labs.
The most anticipated future development for vitamin D analysis is the introduction of high resolution mass spectrometry (HRMS), which is now firmly established in many other fields of modern mass spectrometry, in particular, in pharmaceutical applications and the proteomics field . This trend is mostly due to the availability of robust quadrupole-quadrupole-time-of-flight (QqTOF) and orbitrap mass analyzers in recent years, which have transformed many analytical approaches to mass spectrometry. The use of mass defect as metabolite-specific property, for example, is now an integral part of many metabolite identification routines for drug metabolites . While the number of applications of HRMS in the vitamin D field is still limited, the existing work has clearly demonstrated the potential of HRMS for vitamin D analysis. For example, orbitrap mass spectrometers in full scan [73, 74] and MS/MS [75, 76] modes have been applied successfully to the analysis of 25(OH)D3 in human serum and analytical performance has been shown to be equivalent or better than triple quadrupole and immunoassays. The important role of HRMS in the separation of isobaric interferences will be shown below.
4.4. Interferences during LC-MS/MS
The LC-MS/MS analysis of vitamin D metabolites is affected by various sources of error, which can affect both precision and accuracy. As with any other LC-MS/MS analysis from biological samples, ion suppression by coeluting sample components or chemical modifiers from the sample preparation or chromatography can lead to reduced analyte signals. There are several options to assess whether or not ion suppression is present, which have been summarized in many review articles, e.g., by Matuszewski et al. . Stable isotope standards of vitamin D metabolites (mostly deuterated analogs) can usually correct for accuracy errors from ion suppression effects, as long as it is guaranteed that protein binding for the isotope standard is the same as for the endogenous analyte, which requires implementation of a careful incubation routine . Importantly, deuterated isotope standards are commercially available for most of the relevant vitamin D metabolites but unfortunately not for all.
A second important source of analytical error originates from isobaric noise; that is, endogenous or exogenous metabolites that coelute with the vitamin D analytes and erroneously contribute to the analytical signal, if unspecific ions are used for mass spectral analysis. This has recently been described in detail by Qi et al. , who clearly demonstrated the presence of multiple isobars of 25(OH)D3 in human serum. The isobars have to be carefully removed in low resolution mass spectrometry, by application of appropriate MS/MS or ion mobility spectrometry routines . A number of exogenous and endogenous molecules have been identified as relevant metabolites in serum samples [50, 67, 79].
Many of these interferences can be eliminated by application of high resolution mass spectrometry using sufficiently high resolving powers; for example, through implementation of orbitrap or Fourier-transform ion cyclotron resonance (FTICR) mass spectrometers. For example, Liebisch and Matysik demonstrated that the orbitrap MS instrument in their study was able to separate an isobaric interference of 25(OH)D3 in the MS/MS domain; this interference was caused by fragmentation of the d6-25(OH)D2 isotope standard .
Importantly, the issues relating to isobaric noise have only been studied for the 25(OH)D3 analyte; other metabolites will likely be affected by similar interferences, the impact of which during quantification, in particular, in multimetabolite assays (see below), remains unknown.
4.5. Method accuracy and certified reference materials
The vitamin D analytical community is supported through the Vitamin D External Quality Assessment Scheme (DEQAS), a nonprofit organization that evaluates the performance of analytical assays of member laboratories for 25(OH)D3 and 1,25(OH)2D3  through round-robin analyses. DEQAS has clearly demonstrated that analytical performance of vitamin D analysis has greatly improved over the years between 1994 and 2009 .
The United States National Institute of Standards and Technology (NIST) provides certified standard solutions for 25(OH)D3 and 25(OH)D2. Furthermore, the NIST and the National Institutes of Health (NIH) Office of Dietary Supplements (ODS) have established the Vitamin D Metabolites Quality Assurance Program (VitDQAP), for interlaboratory comparison of measurement of 25(OH)D2, 25(OH)D3, and 3-
A number of commercial reference, calibration, and quality control materials are available from several companies that allow rapid implementation and validation of vitamin D analytical methodologies.
4.6. Vitamin D multimetabolite assays
One of the most important advantages of LC-MS/MS assays over clinical immunoassays is the ability to determine multiple vitamin D species independently and simultaneously. As a result, there are now several very capable LC-MS/MS assays described in the literature that provide the capability for profiling the most relevant vitamin D metabolites at the same time, within a single analytical run, and with sufficient dynamic range to allow measuring the required physiological levels, down to the picomolar range. The topic has recently been reviewed in detail and the interested reader is referred to Ref. . Briefly, most of these multimetabolite assays utilize derivatization techniques that transform all vitamin D species into better responding analogs (vide supra). This procedure in turn permits analysis of both high and low abundant vitamin D species with similar analytical figures of merit [62, 83].
The simultaneous acquisition of all important metabolite levels then provides the possibility of using these vitamin D metabolite distributions (“chemotypes”) as complex diagnostic or prognostic biomarkers for correlation with disease phenotype or clinical outcome of treatment. A few examples for such correlations are summarized in the last section.
5. Vitamin D fingerprints (chemotypes) in clinical applications
A number of recent studies have gone beyond the usual determination of 25(OH)D3—as marker for vitamin D status —and 1,25(OH)2D3—for diagnosis of renal diseases, hypercalcemic syndromes, and disorders of 25(OH)D3 metabolism . These broader profiling techniques are aimed at discovering dynamic effects of metabolites and catabolites, which are located further downstream the 25(OH)D3 metabolic cascade. Current studies highlight 24,25(OH)2D3 as important diagnostic marker, which was previously only considered a clearance product of vitamin D without activity. In fact, it has been shown that 24,25(OH)2D3 has crucial roles in bone metabolism  and renal diseases .
Capturing multiple vitamin D species and their dynamic changes allows for a better understanding of interindividual variations after vitamin D supplementation. Müller et al. recently demonstrated an inverse linear correlation between baseline 25(OH)D3 and response to supplementation for patients with chronic liver disease . The study also showed that lower baseline 24,25(OH)2D3 levels were linked to larger changes of 25(OH)D3 levels, and that those patients who exhibited greater response to vitamin D supplementation had lower levels of 3-
Berg et al. implemented the ratio of 24,25(OH)2D3 and 25(OH)D3 as a novel status marker for vitamin D . Wagner et al. used the same ratio and demonstrated that it was predictive of 25(OH)D3 response to supplementation . Binkley et al. measured multiple vitamin D species and developed a model to describe interindividual variation of 25(OH)D3 levels after supplementation ; the authors highlighted the role of absorption (as measured by the nonmetabolized vitamin D3 species) and degradation (via the 24,25(OH)2D3 species) and presented a treat-to-target regime for tailored serum levels of 25(OH)D3 .
Other important studies include the work by Bosworth et al.  and Stubs et al. , who utilized multimetabolite LC-MS/MS methods to characterize chronic kidney disease (CKD). Similarly, Duan et al.  studied patients with multiple sclerosis and observed comparable levels of 25(OH)D3 in the healthy control subjects and the patients; however, serum levels for 1,25(OH)2D3 and 24,25(OH)2D3 were lower in patients than controls.
The availability of assays for simultaneous capturing multiple vitamin D metabolites combined with reliable techniques for synthesis of the required metabolite standard compounds makes accurate measurement of metabolite distributions and subsequent correlation with disease phenotype readily possible; the outcome of these strategies is expected to be useful for diagnosis and risk prediction for various diseases. It may also allow for specific supplementation strategies in the future, which consider patient-specific dosage requirements and use of selected vitamin D metabolites or analogs.
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