MSACL 2016 EU Abstract

Development of a New Methodology for the Simultaneous Determination of Cholecalciferol and 24,25(OH)2D3 by LC-MS/MS

Neus Fabregat-Cabello (Presenter)
University of Liège, CHU de Liège.

Bio: I graduated from University Jaume I (Castellón, Spain) in 2010 with a degree in Chemistry. After that, I completed a Master in Applied Chromatographic Techniques at the same university. Part of this study was undertaken in the research group on Enriched Stable Isotopes at the University of Oviedo, Spain. In March 2012 I was back at the University Jaume I and I began at my PhD. Later, in 2013, I performed a four-month research stay at RIKILT, Institute of Food Safety (Wageningen, The Netherlands). I have finished my PhD recently, in June 2015 and at my actual position is as a Post-doc at the Department of Clinical Chemistry at the University Hospital of Liège, in Belgium.

Authorship: Neus Fabregat-Cabello, Loreen Huyghebaert, Ana González-Antuña, Caroline Le Goff, Etienne Cavalier.
Departament of Clinical Chemistry, CHU de Liège, University of Liège, Belgium

Short Abstract

In the present work an ultra-high-pressure liquid chromatography–tandem mass spectrometry (UHPLC–MS/MS) method for the simultaneous determination of cholecalciferol and 24,25(OH)2D3 in serum has been developed and validated. The validation was carried out by analyzing serum fortified at five concentration levels. In all cases, recoveries ranging from 89-109% for cholecalciferol and 85-111% for 24,25(OH)2D3 and CV below 7% were attained with this approach. As a consequence, this procedure is ready to assess the rates of synthesis of cholecalciferol and 24,25(OH)2D3 in the skin either by sun exposition or by oral absorption.

Long Abstract


The major source of vitamin D3 in the human body is its synthesis from its precursor 7-dehydroxycholesterol in skin, with exposure to solar UVB (290-315 nm) acting as catalyst. This synthesis accounts for around 90 percent of vitamin D3 present in the organism. The remaining 10 percent is acquired by food intake, mostly from oily fishes such as salmon, sardine, cod liver, etc. In humans, vitamin D can be present in two main forms, vitamin D3 (cholecalciferol) and vitamin D2 (ergocalciferol), which is obtained from plants or from supplements[1]. Once in the bloodstream, both vitamin D types are transported by different ligands according to their lipophilicity, VDBP (Vitamin D Binding Protein) being the most common. At this level, both forms of vitamin D are firstly hydroxylated in the liver to 25-hydroxyvitamin D (25(OH)D)and secondly in the kidneys to produce 1,25-dihydroxyvitamin D (1,25(OH)2D) the active form of vitamin D. Alternatively, 25(OH)D can also enter into a degradation pathway to its hydroxylation by the 24-hydroxylase to give 24,25-dihydroxyvitamin D (24,25(OH)2D).

There are several factors than can be barriers to successful vitamin D synthesis, including age, pigmentation of the skin, wearing sunscreen and sun avoidance habits[2]. As a consequence it is important to carry out studies where the impact of sunlight exposure on skin production of vitamin D as well of 24,25(OH)2D3 can be evaluated. On the other hand, the degradation pathway of 25(OH)D can also influent 24,25(OH)D levels and may need further investigation in some cases where a supplementation does not reach its target.

In order to be able to determine cholecalciferol levels different analytical strategies have been adopted. Cholecalciferol analysis has been commonly performed by either commercially available ELISA essays or by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), but only a few methods have been proposed for the later approach. The main drawback of these LC-MS methods are either the use of uncommon ionization sources, like atmospheric pressure photo ionization[3], or the validation at very high levels (15 ng/mL)[4]. However, to the best of our knowledge the simultaneous determination of 24,25(OH)2D3 and cholecalciferol by LC-MS has only been described recently by Mena-Bravo et al.[4] but is applicability is difficult due to the use of automatic solid phase extraction(SPE)

In the present study, we developed and validated a new methodology for the quantitative measurement of cholecalciferol and 24,25(OH)2D3 in serum samples by ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS). For optimal ionization efficiency, a derivatization with the Amplifex diene reagent has been applied, for the first time to our knowledge, to cholecalciferol determination.

Materials and Methods

Materials. Standard of cholecalciferol was purchased from Sigma-Aldrich (Setinheim, Germany). Stable isotopes d6-cholecalciferol and d6-24,25(OH)2 D3, as well as the natural form of 24,25(OH)2 D3 were was supplied by Medical Isotopes Inc. (Pelham, NH, USA). Vitamin D Free Human serum purchased from Golden West Biologicals (Temecula, CA, USA). SPE selected was Oasis HLB 96-well µElution Plate, with 2 mg of sorbent (Waters, Milford, MA, USA). Amplifex reagent was obtained from Sciex (Concord, ON, Canada). Methanol, acetonitrile and water solvents (LC-MS grade) were provided by Biosolve (Dieuze, France) and formic acid by Fisher Chemicals (Madrid, Spain).

Sample preparation. to 100 µL of homogenized sample, 20 µL of the internal standard solution containing 24,25(OH)2D3-d6 and were added, followed by 100 µL of 4% H3PO4. Samples were equilibrated between additions. Analytes were then extracted using Oasis® HLB microelution SPE 96-well plates. Sample extracts were washed with a 5% methanol solution in water before elution with acetonitrile. Prior to the derivatisation with the Amplifex® reagent, extracts were evaporated to dryness. Finally the reaction was performed using 50 µL of reagent solution during 30 minutes, after which 50 µL of water were added in order to stop the reaction.

Liquid chromatography-mass spectrometry. Identification and quantification of analytes were carried out using an Sciex 6500 triple quadrupole MS/MS (Framingham, MA, USA) equipped with LC-30A Nexera ultra-performance liquid chromatography (UHPLC) system (Shimadzu Co., Kyoto, Japan).The mass spectrometer was operated in electrospray ionization with positive polarity and selected reaction monitoring (SRM) mode. Identification and quantitation of cholecalciferol was performed by recording the transitions (m/z) 716 → 657 (quantifier ion, Q) and 716 → 217 (qualifier ion, q) with d6-cholecalciferol as an internal standard by monitoring 722 → 663. For 24,25(OH)2D3 the transitions recorded were 748→689 (Q), 748 → 217(q) and 754 →695 for d6-24,25(OH)2D3.

Chromatographic separation was performed with an Acquity UPLC BEH column (1.7 μm, 2.1 mm x 100 mm) with C18-bonded silica stationary phase. Gradient conditions were used, being the mobile phases water and acetonitrile, both modified with formic acid.


Evaluation of the extraction methods. In a preliminary optimization different liquid-liquid extraction conditions, including extraction with ethyl acetate, methyl-terc-butyl-ether and hexane, were tested. In this step of the optimization concentrations of 100 ng/mL for both analites were employed in order to avoid the derivatization step. Recoveries circa 100% were obtained in all conditions for spiked water samples but no peaks were observed in serum extracts. After rejecting this approach, different SPE cartridges were tested. This study included 2, 10 and 30 mg OASIS HLB and from Waters. Despite of the better retention for the 10 and 30 mg cartridges, microelution SPE containing 2 mg was selected in order to match a 100 uL sample volume as well as to minimize the elution solvent to evaporate before the derivatization step.

Method validation. Prior to its application, the overall analytical procedure was satisfactorily validated for considering the following parameters: linearity, precision and accuracy, Limits of Quantification/Detection (LOQ and LOD) and Q/q ratios between transitions (Q: quantifier, q: qualifier) used for confirmation. These Analytical characteristics of the developed method were evaluated in free human serum. Recovery experiments were performed in triplicate at five concentration levels, ranging from 2-80 ng/mL for vitamin D and from 1-40 ng/mL for 24,25(OH)2D3, during three different days.

The mean recoveries obtained were within 89-109% for cholecalciferol and 85-111% for 24,25(OH)2D3. The LOQ (signal to noise ratio=10) and LOD(signal to noise ratio=10) were 1 and 0.3 ng/mL for vitamin D and 0.8 and 0.2 ng/mL for 24,25(OH)2D3. These values were obtained by quintuplicate experiments at lowest validated level. In order to confirm the specificity of the assay the Q/q ratios from neat standards prepared in solvent were compared with those obtained in the studied samples. All tested samples showed deviations always below 30%. In this line, the chromatographic separation of the isobaric interferences 23,25(OH)2D3, 1,25 (OH)2D3 and 7-dehydrocholesterol has been also tested. The precission was evaluated by the relative Coefficent of Variation (CV%), obtaining values lower than 7% for the intraday variability and a 6% for the interday variability for both compounds.

The degree of ion suppression/enhancement attributed to the sample matrix was estimated in a separate set of experiments. Five serum as well as five water samples matching the validated levels were extracted as described. Ion suppression was calculated by comparing analyte peak areas obtained from spiked water samples solutions with those obtained from spiked serum samples, in both cases added before extraction. While a 40 percent of ion suppression occurred for cholecalciferol, a 60 percent of signal enhancement was observed for 24,25(OH)2D3.


This work proposes a new methodology for the quantification of vitamin D and 24,25(OH)2D3 by LC–MS/MS in serum samples, taking profit of the already known advantages of this analytical tool, including high selectivity and sensitivity, substantial reduction of sample treatment, and reliable quantification and confirmation at the low concentrations required in the clinical laboratory.

The method is prepared for being used in studies where the rate of formation of vitamin D in the skin (or obtained by oral absorption) as well as rates of formation and degradation of 24,25(OH)2D3.

References & Acknowledgements:

[1] C. Le Goff, E. Cavalier, J.-C. Souberbielle, A. González-Antuña, and E. Delvin, “Measurement of circulating 25-hydroxyvitamin D: A historical review,” Pract. Lab. Med., vol. 2, no. MAY, pp. 1–14, 2015.

[2] L. Y. Matsuoka, L. Ide, J. Wortsman, J. A. MacLaughlin, and M. F. Holick, “Sunscreens suppress cutaneous vitamin D3 synthesis,” J. Clin. Endocrinol. Metab., vol. 64, no. 6, pp. 1165–1168, 1987.

[3] J. Adamec, A. Jannasch, J. Huang, E. Hohman, J. C. Fleet, M. Peacock, M. G. Ferruzzi, B. Martin, and C. M. Weaver, “Development and optimization of an LC-MS/MS-based method for simultaneous quantification of vitamin D2, vitamin D3, 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3,” J. Sep. Sci., vol. 34, no. 1, pp. 11–20, 2011.

[4] A. Mena-Bravo, C. Ferreiro-Vera, F. Priego-Capote, M. A. Maestro, A. Mouriño, J. M. Quesada-Gómez, and M. D. Luque de Castro, “Quantitative analytical method to evaluate the metabolism of vitamin D,” Clin. Chim. Acta, vol. 442, pp. 6–12, 2015.

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