Data for the measurement of serum vitamin D metabolites in childhood acute lymphoblastic leukemia survivors

This article describes data related to a companion research paper entitled “Vitamin D nutritional status and bone turnover biomarkers in childhood acute lymphoblastic leukemia (cALL) survivors.” (Delvin et al., submitted for publication) [1]. Various methods for the measurement of serum 25OHD3, the accepted biomarker for assessing vitamin D nutritional status, have been described (Le Goff et al., 2015; Jensen et al., 2016) [2], [3]. This article describes a novel mass spectrometry-QTOF method for the quantification of circulating 25OHD3, 3-epi-25OHD3 and 24,25(OH)2D3. It provides the description of the extraction, chromatography and mass spectrometry protocols, a sample of mass spectra obtained from standards and extracted serum, and a comparison with another HPLC-MS/MS (Jensen et al., 2016) [3] method for the measurement of serum concentrations of 25OHD3.


Subject area
Biology, More specific subject area Clinical Chemistry Type of data Tables, figures How data was acquired Liquid Chromatography coupled to a quadrupole time-of-flight mass spectrometer (Waters UPLC-MS system (Xevo G2 quadrupole time-offlight)) Data format Mass spectral analysis, analyzed Experimental factors Extracted serum samples, blank and standards were analyzed by Liquid Chromatography coupled to a quadrupole time-of-flight mass spectrometry.

Value of the data
The data describes a novel LC/MS-QTOF method for the measurement of serum vitamin D metabolites, providing the possibility of profiling.
The details given enable other researchers to reproduce this method. This technology will be useful for vitamin D profiling in future clinical studies involving vitamin D supplementation.

Data
The data shared in this article include the description of the extraction, chromatography and mass spectrometry protocols as well sample mass spectra obtained from standards and extracted serum. The validation procedure of the method and results are also described.

Experimental design, materials and methods
The sample preparation method was adapted from Jones et al. [4]. In brief, 300 μL of a blank, consisting of charcoal-stripped plasma (cat. # 1131-00) purchased from Biocell (Rancho Dominguez, CA, USA), sample, calibrator or control in glass tubes were spiked with 75 μL of a mixture of deuterated internal standards (IS) consisting of [25OHD 3   A Waters UPLC system with a Waters BEH phenyl, 2.1 × 50 mm column with 1.7 µm particle preceded by a guard column was used for the chromatographic separations. Mobile phase A and B consisted of 2 mM ammonium acetate/0.1% formic acid in water and methanol, respectively. Initial conditions were 35% phase A and 65% phase B with a 5 min-gradient to reach 90% phase B, followed by a one-minute equilibration time. The flow rate was 300 µL/min, the column temperature was set at 40°C, and the auto-sampler at 4°C. A Waters Xevo G2 QTOF was used to detect and quantify the vitamin D metabolites. The instrument was operated in positive mode using the sensitivity mode. Capillary voltage was set at 1.0 kV, cone voltage at 35 V, with a source temperature of 150°C, a desolvation gas temperature of 650°C, with a flow rate of 900 L/h. Mass spectra were acquired in the target-enhanced mode with an acquisition time of 1 s. The chromatographic retention times and ionic transition masses are listed in Table 1. Fig. 1 illustrates superimposed representative chromatographic profiles of a blank charcoalstripped plasma, a charcoal-stripped plasma spiked with the deuterated internal standards [ 6 d 2 ]-25OHD 3 , a charcoal-stripped plasma spiked with a standard and a patient sample. The inset in Fig. 1 shows the profile for [ 3 d 2 ]-3-epi-25OHD 3 in a patient serum extract analyzed in the conditions described above. Note that the derivatization of the different vitamin D metabolites yielded, for each,    The analysis was performed in the scan mode from ions 100-1000 m/z. The data acquisition time was 1 s in the continuum mode.
2 DMEQ-TAD enantiomers (R & S) due to an asymmetric carbon (Fig. 2). The major peak was used for quantification. The method validation was performed with modified CLSI Guidelines [5,6]. Briefly, the Lower Limit of quantification (LLoQ) was estimated by the serial dilution of the standard solution (n ¼ 5 per dilution) and was defined as the concentration at which precision was r 20%. Linearity was evaluated by serially diluting a pool of high 25OHD 3 concentration samples with charcoal-stripped serum to generate 8 samples of intermediate concentrations that were measured in duplicate. Within assay imprecision was characterized by 5 measurements of a plasma sample pool spiked with 3-epi-25OHD 3 , 25OHD 2 , and 24,25(OH) 2 D 3 . Between-assay imprecision was assessed by analyzing 1 reference sample at 2 different concentrations in each batch over 14 months. Bias was determined by using UTAK vitamin D controls and a DEQAS test sample set. Table 2A summarizes the performance characteristics of the method. Linear responses were observed up to 462 nM for 25OHD 3 , 158 nM for 25OHD 2 , 148 nM for 3-epi-25OHD 3 and 149 nM for    24,25(OH) 2 D 3 . The LLOD spanned from 0.1 to 0.3 nM and the LLOQ from 2.0 to 2.5 nM for the 4 vitamin D metabolites. The intra-assay imprecision ranged from respectively 4.2% to 7.0% and the inter-assay imprecision from 8.9% to 10.2% depending on the metabolite measured. The recovery of spiked samples ranged from 92% for 24,25(OH) 2 D 3 to 118% for 25OHD 3 . As shown in Table 2B, using DEQAS samples, the mean bias for 25OHD 3 ranged between 6.0 and − 3.1% at 85.7 and 80.0 nM respectively, within the limits set by the Vitamin D Standardization Program [7]. Fig. 3 is the Deming regression plot comparing serum 25OHD 3 concentration observed in the 248 serum samples of cALL survivors (1) by the present QTOF method and by HPLC-MS/MS (3). Table 3 lists the 25OHD 3 , 3-epi-25OHD 3 and 24,25(OH) 2 D 3 serum concentrations for the same samples.