Misleading measures in Vitamin D analysis: A novel LC-MS/MS assay to account for epimers and isobars
© Shah et al; licensee BioMed Central Ltd. 2011
Received: 29 January 2011
Accepted: 14 May 2011
Published: 14 May 2011
Recently, the accuracies of many commercially available immunoassays for Vitamin D have been questioned. Liquid chromatography tandem mass spectrometry (LC- MS/MS) has been shown to facilitate accurate separation and quantification of the major circulating metabolite 25-hydroxyvitamin-D3 (25OHD3) and 25-hydroxyvitamin-D2 (25OHD2) collectively termed as 25OHD. However, among other interferents, this method may be compromised by overlapping peaks and identical masses of epimers and isobars, resulting in inaccuracies in circulating 25OHD measurements. The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 through chromatographic separation of 25OHD from its epimers and isobars.
A positive ion electrospray ionisation (ESI) LC-MS/MS method was used in the Multiple Reaction Monitoring (MRM) mode for quantification. It involved i) liquid-liquid extraction, ii) tandem columns (a high resolution ZORBAX C18 coupled to an ULTRON chiral, with guard column and inlet filter), iii) Stanozolol-D3 as internal standard, and iv) identification via ESI and monitoring of three fragmentation transitions. To demonstrate the practical usefulness of our method, blood samples were collected from 5 healthy male Caucasian volunteers; age range 22 to 37 years and 25OHD2, 25OHD3 along with co-eluting epimers and analogues were quantified.
The new method allowed chromatographic separation and quantification of 25OHD2, 25OHD3, along with 25OHD3 epimer 3-epi-25OHD3 and isobars 1-α-hydroxyvitamin-D3 (1αOHD3), and 7-α-hydroxy-4-cholesten-3-one (7αC4). The new assay was capable of detecting 0.25 ng/mL of all analytes in serum.
To our knowledge, this is the first specific, reliable, reproducible and robust LC-MS/MS method developed for the accurate detection of 25OHD (Vitamin D). The method is capable of detecting low levels of 25OHD3 and 25OHD2 together with chromatographic separation from the co-eluting epimers and isobars and circumvents other instrumental/analytical interferences. This analytical method does not require time-consuming derivatisation and complex extraction techniques and could prove very useful in clinical studies.
Vitamin D3 is formed from its precursor 7-dehydrocholesterol in the skin by ultraviolet B light (medium wavelength, 290-315 nm) and Vitamin D2 originates from dietary sources together with some fraction of D3. In the liver, Vitamins D3 and D2 undergo hydroxylation reactions catalyzed by 25-hydroxylase, which leads to the formation of pharmacologically active metabolites 25OHD3 and 25OHD2 respectively (collectively termed as 25OHD). Further metabolism (in the presence of 1α,hydroxylase) in the kidney produces the pharmacologically active metabolites 1-alpha,25-dihydroxyvitamin-D3 (1α,25(OH)2D2) and 1-alpha,25-dihydroxyvitamin-D2 (1α,25(OH)2D3) along with the minor metabolite 24,25(OH)2D3 [8–10].
Since 25OHD has significant effects on health and wellbeing, there has been a substantial interest in improving the relevant analytical techniques [11–30]. Owing to a long serum half-life, measurement of total 25OHD (25OHD2 and 25OHD3) is the routinely used approach for assessing the total circulating Vitamin D status [10–14]. In immunoassay techniques, a measure of total metabolite concentration and equivalent detection of both 25OHD2 and 25OHD3 is challenging, as binding proteins show a higher affinity for 25OHD3 than 25OHD2 [15–18]. Reports have shown inter-laboratory and inter-method variations in results for Vitamin D determinations [19–21].
LC-MS/MS is currently the best technique available for the correct quantification of 25OHD3 and 25OHD2 [22, 23] and it also has the capability to overcome most of the problems associated with protein binding assays. LC-MS/MS is a more favourable technique because sample derivatisation is not required, run time is very short and an internal standard is used which usually compensates for any matrix related and instrumental effects [24–32].
The epimerization of 25OHD3 and 1α,25-(OH)2D3 results in the formation of 3-epi-25OHD3 and 3-epi-1α,25(OH)2D3 epimers respectively as shown in Figure 2. The epimers of 25OHD differ in configuration at third carbon atom (C-3) (shown by dashed highlights in Figure 2) that is attached to a hydroxyl group. Hydroxylation of 3-epi-25OHD3 forms 3-epi-1α,25(OH)2D3 .
The aim of this study was to develop a novel LC-MS/MS method that can accurately identify and quantitate 25OHD3 and 25OHD2 and chromatographically separate epimers and isobars.
Materials and methods
Blood samples (100 mL) were obtained from 5 healthy, male, Caucasian volunteers of age ranging from 22 to 37 years, weight ranging from 72.1-98.1 kg (mean weight 84.86 ± 11.3 kg) and height ranging from 165-190 cm (mean height 179.5 ± 8.6 cm). Blood samples were centrifuged at 1500 g and serum was extracted. The serum samples were frozen individually in labelled, amber, plastic vials . To minimise assay variations, a multilevel serum calibrator set (Chromsystems, Germany) was used for preparation of calibration curves and quality controls. The lyophilised calibrators (based on human serum) consisted of 3 high and 1 low-level calibrators and were handled in the same manner as volunteer specimens. According to the assay procedures, the calibrators were analysed along with routine samples to meet the standards outlined by National Institute of Standards and Technology (NIST) [44, 45].
Standards and reagents
GV-65C (3 mL syringe) mixed bed cation exchange columns were obtained from Biochemical Diagnostics, (New York, USA), Bond Elut-SI, Bond Elut Plexa, Bond Elut LMS, Bond Elut PPL, SampliQ OPT and SampliQ DVB solid phase extraction cartridges were purchased from Agilent Technologies (Cheshire, UK). 25OHD3, 25OHD2, 1-α-hydroxyvitamin-D3 (1αOHD3), 3-epi-25OHD3, hexane, isopropanol, methanol, dichloromethane, deionised water, formic acid, acetonitrile, ammonium hydroxide, pentane and ether were obtained from Sigma Aldrich (Poole, UK). 7-α-hydroxy-4-cholesten-3-one (7αC4) and stanozolol-D3 (internal standard) were obtained from LGC standards (Teddington, UK). All chemicals and reagents were of HPLC grade.
Preparation of standards and samples
Stock solutions of all analytes were prepared in methanol to obtain a concentration of 1 mg/mL and stored in amber vials at -20°C in the dark. Under these conditions, the stock solutions were found to be stable for 3 months. The solutions were kept in the dark to minimise light induced degradation of Vitamin D [46, 47]. Working solutions were made in methanol by serial dilution of stock solutions. Working internal standard solution was also prepared by diluting the stock solution of internal standard with methanol to a final concentration of 1 μg/mL. Calibrators and internal standard solutions made in-house were stable for 2 weeks when stored at -20°C. The lyophilised serum calibrators were reconstituted in HPLC grade water and allowed to stand for 10 to 15 minutes at room temperature. The vials were then swirled to dissolve the contents until homogeneity. The lyophilized reference serum calibrants were stable for 3 months when stored at -20°C [46, 47]. The calibration curves and quality controls were prepared from the multilevel reference calibrator set in the range 0.5 to 84.4 ng/mL concentrations, respectively. Different methods and conditions for sample pretreatment and extraction were undertaken to optimise recovery, specificity and signal to noise ratio. The sample pretreatment and extraction methods [12–32] were adopted as follows.
Serum samples were thawed; vortex mixed and equilibrated at room temperature for 15 minutes and 25 μL of working solution of stanozolol-D3 (internal standard) was added to all samples. Formic acid (2 M, 50 μL) was added and the resultant solution vortexed. Then 3 mL of methanol/isopropanol (1:1, v/v) mixture was added and vortexed to release the protein bound analyte and to promote protein precipitation during a 15 minutes incubation at 4°C. The suspended matter was removed after centrifugation at 3500 g for 5 minutes at 4°C. The supernatants were transferred to clean amber glass tubes. The remaining solution was subjected to further sample purification, as follows.
Liquid-Liquid extraction (LLE)
Different solvent mixtures were tested for liquid-liquid extraction: namely; heptane, methanol, propanol, dichloromethane, acetonitrile and hexane. Hexane/dichloromethane (1:1, v/v) mixture was found to give optimum extraction recovery. For liquid-liquid extraction a 3 mL hexane/dichloromethane mixture (1:1, v/v) was added to the supernatant after protein precipitation and the solution was vigorously vortexed for 1 minute and then centrifuged at 3500 g for 5 minutes at 4°C. The supernatant layer was transferred to clean, amber, glass tubes. The residual lower layer of the serum sample was further extracted twice with the hexane/dichloromethane (1:1, v/v) mixture. The organic phase obtained was pooled and dried under a gentle stream of nitrogen at room temperature. It was then reconstituted in 200 μL of HPLC grade methanol/water (1:1, v/v) mixture. Solid phase extraction (SPE) was also tested as an alternative to the liquid-liquid extraction technique.
Solid phase extraction (SPE)
The mass spectrometer was operated in positive electrospray ionisation (ESI) mode at a spray voltage of 5000 V and capillary temperature of 450°C. The generated protonated molecules of 25OHD3, 25OHD2, 3-epi-25OHD3, 1αOHD3, 7αC4 and stanozolol-D3 (internal standard) were used as precursor ions for collision activated dissociation (CAD) into product ions in MS-MS analysis.
Summary of assay validation results.
Linear range (ng/mL)
Recovery (%) at 50 ng/mL
Under- or over-estimation of actual 25OHD3 concentrations may occur owing to co-eluting epimers (e.g.3-epi-25OHD3) and isobars (e.g.7αC4) [39–42]. The use of high resolution microbore ZORBAX column in tandem with chiral ULTRON column not only facilitated accurate determination of the analyte ions but also chromatographically separated all isobars and epimers from 25OHD co-eluting peaks.
Retention times, MRM transitions and ESI conditions of analytes.
Retention time (min)
Transition (m/z) Precursor→ Product
Collision energy (eV)
401.3 → 383.1
401.3 → 365.1
401.3 → 159.2
413.3 → 395.5
413.3 → 377.2
413.3 → 355.5
401.3 → 383.1
401.3 → 365.1
401.3 → 159.2
401.3 → 383.1
401.3 → 365.1
401.3 → 159.2
401.3 → 383.1
401.3 → 365.1
401.3 → 159.2
332.2 → 81.2
Vitamin D serum results.
The percentage of interfering epimer (E) and isobars (I) in serum.
These results confirm the complexities involved in measuring Vitamin D status and reinforce the need for the uniform adoption of improved accurate assays. Measurements of 25OHD in human serum using competitive immunoassays are difficult owing to lipophilicity/tight binding to the Vitamin D-binding protein (DBP), even at very low serum concentrations . In addition, immunoassays for 25OHD have been reported to cross-react with 24,25(OH)2D3, an intermediate product during the formation of 1,25(OH)2D3 in the kidney [16, 17] (Figure 1). Many commercial immunoassays can only measure 25OHD3 and are not suitable to monitor supplementation with Vitamin D2, which is derived from plant sources and widely used in many countries for fortification of foods . Hence, LC-MS/MS analysis is preferred but it has been shown that it is also subjected to interference issues [19–28]. In our new method, the use of an electrospray ionisation technique has overcome the problems associated with in-source transformation of 25OHD3-sulfate and other metabolites to 25OHD3 which may occur when using atmospheric pressure chemical ionisation mode .
The use of tandem column technology, an optimised mobile phase composition and a modified extraction method not only separate the epimers and isobars but also eliminates the interferences caused by early eluting salts (e.g. sodium) and the late eluting phospholipids, which may interfere with analyte ionisation [29–34]. Moreover, the installation of a low dispersion inlet filter together with a chiral guard column also minimises the interferences resulting from early eluting amino acids and late eluting xenobiotics. The column also separates interfering compounds present at higher concentration which have been shown to overlap or share exact mass transitions with 25OHD e.g. detergents and phthalates etc [29–31].
The separation of epimers and isobars from the target analyte is critical because they can overlap 25OHD peaks and form the same masses upon ionisation, thus compromising the true status of 25OHD in circulation [38–43]. The epimer of 25OHD3 is known to have the same effects on suppressing parathyroid hormone (PTH) secretion, but it has negligible calcium-producing effects [40, 41]. Recently, it was concluded that the absence of external standardisation for the 25OHD assay might lead to greater variations and false results . To minimise the inter-laboratory and inter-method variations in our LC-MS/MS analysis, we have used reference materials for preparation of our calibrants and quality controls, along with the introduction of a new internal standard. The latter was required as, when using the internal standard hexadeuterated-25OHD3, certain fragment transitions of the parent ions could lead to greater isobaric interferences such as the transition 407.7 > 389.7 . Stanozolol-D3 as a new internal standard prevents isobaric interferences by using the transition 332.2 > 81.2.
To date, only one transition of the parent ion to product ion has been investigated, instead of our three-precursor ions to fragment ions transitions (according to the qualifier-qualifier principle) . To our knowledge, this is first time that three transitions of precursor ions to product ions have been used for quantification of 25OHD3, 25OHD2, 3-epi-25OHD3, 1αOHD3 and 7αC4 as shown in Table 2. The sum of the three transitions not only increases the sensitivity of the assay but also minimises isobaric interferences.
To summarise, this is the first LC-MS/MS method for the determination of 25OHD which includes separation and quantification of epimers and isobars. It is a fast liquid-liquid extraction method, which does not require complicated derivatisation procedures, hence reducing assay time and variability. This method can quantify the different 25OHD forms accurately, which is difficult to achieve using immunoassay methods. This methodology can be used for accurate blood testing to prevent falsely elevated Vitamin D levels being reported.
The LC-MS/MS method was free from all types of interferences arising due to epimeric, isobaric, instrumental and matrix components, which may interfere with analyte ionisation. Thus the LC-MS/MS method provides a robust, specific, reliable and reproducible technique as a solution to the problems identified in relation to current assays for Vitamin D. Removal of uncertainties in Vitamin D measurement, is required in order to progress the current understanding of the roles of Vitamin D in health and disease via more rigorous clinical trials .
The authors thank the volunteers who participated in this study. Authors also thank Dr Julian Swinden for his technical help.
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