Candidate Reference Measurement Procedure for the Determination

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Candidate Reference Measurement Procedure for the Determination of (24R),25-Dihydroxyvitamin D3 in Human Serum Using IsotopeDilution Liquid Chromatography−Tandem Mass Spectrometry Susan S.-C. Tai* and Michael A. Nelson Chemical Sciences Division, National Institute of Standards and Technology, Gaithersburg, Maryland 20899, United States ABSTRACT: The two major forms of vitamin D, vitamin D3 and vitamin D2, are metabolized in the liver through hydroxylation to 25-hydroxyvitamin D species, and then further hydroxylated in the kidney to various dihydroxyvitamin D species. (24R),25-Dihydroxyvitamin D3 ((24R),25(OH)2D3) is a major catabolite of 25hydroxyvitamin D metabolism and is an important vitamin D metabolite used as a catabolism marker and indicator of kidney disease. The National Institute of Standards and Technology has recently developed a reference measurement procedure for the determination of (24R),25(OH)2D3 in human serum using isotope-dilution LC−MS/MS. The (24R),25(OH)2D3 and added deuterated labeled internal standard (24R),25(OH)2D3-d6 were extracted from serum matrix using liquid−liquid extraction prior to LC−MS/MS analysis. Chromatographic separation was performed using a fused-core C18 column. Atmospheric pressure chemical ionization in the positive ion mode and multiple reaction monitoring were used for LC−MS/MS. The accuracy of the measurement of (24R),25(OH)2D3 was evaluated by recovery studies of measuring (24R),25(OH)2D3 in gravimetrically prepared spiked samples of human serum with known (24R),25(OH)2D3 levels. The recoveries of the added (24R),25(OH)2D3 averaged 99.0% (0.8% SD), and the extraction efficiencies averaged 95% (2% SD). Excellent repeatability was demonstrated with CVs of ∼1%. The limit of quantitation at a signal-to-noise ratio of ∼10 was 0.2 ng/g. Potential isomeric interferences from other endogenous species and from impurity components of the reference standard were investigated. LC baseline resolution of (24R),25(OH)2D3 from these isomers was achieved within 35 min. This method was used for value assignment of (24R),25(OH)2D3 in Standard Reference Materials of Vitamin D Metabolites in Human Serum, which can serve as an accuracy base for routine methods used in clinical laboratories.

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treatment of diseases. Measurements of (24R),25(OH)2D3 in human serum are complicated by low concentration levels as well as the presence of several endogenous dihydroxyvitamin D species that have identical molecular masses.5,14−20 Mass spectrometric-based methods may yield biased results for (24R),25(OH)2D3 if chromatographic separation of this species from its isomers is not achieved. In particular, the presence of structurally related 3-epi-(24R),25(OH)2D3,5,14−17 a diastereomer of (24R),25(OH)2D3 that differs by the position of the hydroxyl group on C3, can pose problems for accurate measurement of (24R),25(OH)2D3 using liquid chromatography−tandem mass spectrometry (LC−MS/MS) methods. Many recent studies suggest that the presence of the 3-epimer of 25(OH)D3 can bias results of 25(OH)D3 measurements for LC−MS-based methods if 3-epi-25(OH)D3 cannot be chromatographically separated from 25(OH)D3.21−24 Similarly, chromatographic separation of (24R),25(OH)2D3 from its 3-epimer is crucial for accurate measurement of (24R),25(OH)2D3. Furthermore, (24S),25(OH)2D, a synthetic diastereomer of (24R),25(OH)2D3 differing by the

he two major forms of vitamin D, vitamin D3 and vitamin D2, are metabolized in the liver through hydroxylation to 25-hydroxyvitamin D (25(OH)D) species. Levels of these vitamin D metabolites in human serum are considered the best biomarkers of vitamin D status.1,2 25(OH)D is further metabolized through a second hydroxylation in the kidney to various dihydroxyvitamin D species.1,3 (24R),25-Dihydroxyvitamin D3 ((24R),25(OH)2D3) is a major catabolite of 25(OH)D metabolism and is primarily bound to vitamin D binding protein in circulation.1 The half-life of (24R),25(OH)2D3 is ∼7 days,4 and in healthy individuals, its concentration is generally in the range of 1 ng/mL to 5 ng/ mL.5−7 Levels of this vitamin D metabolite are significantly lower in individuals who have chronic kidney diseases.8,9 As such, (24R),25(OH)2D3 is an important biomarker for vitamin D metabolite catabolism and an indicator of kidney disease.5,8−10 Recent studies also suggest that there is a strong correlation between 25(OH)D and (24R),25(OH)2D. Serum (24R),25(OH)2D, particularly when expressed as a molar ratio to 25(OH)D, provides an additional index of vitamin D status and may mark the presence of hypercalcemic diseases caused by genetic mutations in 25-hydroxyvitamin D-24-hydroxylase.11−13 Accurate and precise quantitative evaluations of (24R),25(OH)2D3 are important for more reliable diagnosis and better © XXXX American Chemical Society

Received: May 18, 2015 Accepted: July 14, 2015

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DOI: 10.1021/acs.analchem.5b01861 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry position of the hydroxyl group on C24,25 generally exists as an impurity in the (24R),25(OH)2D3 primary standard. This isomer is an artifact of the (24R),25(OH)2D3 synthesis process and can introduce a positive bias if chromatographic separation of the R and S forms is not achieved. A limited number of LC-based methods (LC−MS/MS) for serum (24R),25(OH) 2 D3 have been reported in the literature.11,24,26−30 These are high-throughput methods capable of simultaneously measuring multiple vitamin D metabolite species. Their precision and trueness meet the performance specification suggested for routine testing of vitamin D metabolites, but not for reference measurement procedures (RMPs).31 Most critically, chromatographic separation of (24R),25(OH)2D3 from multiple endogenous dihydroxyvitamin D3 species and from a synthetic structural analogue, has not been investigated and demonstrated in these published methods; thus, there is potential that these results are positively biased. There is need for a thoroughly evaluated RMP to assess the methods used in clinical laboratories. At the request of the Vitamin D Standardization Program (VDSP)32,33 coordinated by the National Institutes of Health, Office of Dietary Supplements, National Institute of Standards and Technology (NIST) recently developed and critically evaluated a RMP with high accuracy and high precision using an isotopedilution (ID) LC−MS/MS method for the determination of (24R),25(OH)2D3 in human serum. The potential interferences from structural analogues of (24R),25(OH)2D3 were investigated. Chromatographic separation of (24R),25(OH)2D3 from its isomers was demonstrated. The requirements of RMPs for clinical diagnostic markers are outlined in the International Organization for Standardization (ISO) 15193.31 The Joint Committee for Traceability in Laboratory Medicine (JCTLM)34 reviews candidate RMPs and recognizes the methods that meet the requirements of ISO 15193. Currently, no RMPs for serum (24R),25(OH)2D3 have been approved by the JCTLM. This ID LC−MS/MS method is the first reported candidate RMP for the measurement of serum (24R),25(OH)2D3. All of the criteria established by JCTLM as a RMP have been met. This method was used to value assign (24R),25(OH)2D3 in SRMs of Vitamin D Metabolites in Human Serum, which can serve as an accuracy base for routine methods used in clinical laboratories.

SRMs of Vitamin D Metabolites in Human Serum. An Ascentis Express C18 column (4.6 mm (i.d.) × 15 cm, 2.7-μm particle diameter) was obtained from Sigma (Milwaukee, WI). Solvents used for LC−MS/MS measurements were HPLC grade, and all other chemicals were reagent grade. Purity Assessment of Reference Standard. The purity value of the (24R),25(OH)2D3 reference standard used for measurement calibrations was assessed using quantitative nuclear magnetic resonance spectroscopy (qNMR) with a traceable internal standard technique, as well as a limited mass balance approach that implemented LC with UV detection (LC/UV) and thermogravimetric (TGA) analytical techniques. qNMR served as a primary ratio direct measurement method to determine the traceable mass fraction of (24R),25(OH)2D3. The supplementary results via the mass balance approach were determined as the sums of relative observed impurities, quantified using relative LC/UV chromatographic peak area ratios and the mass fraction of volatiles via TGA, subtracted from 100%. Adjustments were made to qNMR results for 1H spectral peak bias associated with (24S),25(OH)2D3 (0.6%), as detected and quantified with LC/UV. Given the higher metrological order and direct measurement of the (24R),25(OH)2D3 species by the qNMR method, the corresponding purity results were held with greater confidence than those assessed with the indirect mass balance approach. As such, an asymmetric consensus purity probability density was evaluated as a two-piece normal distribution35 with a mode (μ) equal to the qNMR result. The left half of this distribution is Gaussian with μ and σ parameters equal to the qNMR mean result and standard uncertainty, respectively, and the right half is Gaussian with the same μ, but the standard deviation increased such that the 97.5th percentile is equal to the mass balance result. The purity value of the (24R),25(OH)2D3 reference standard was evaluated to be 94.1% (u = 0.8%). The ID LC−MS/MS measurement results were adjusted to reflect the assessed mass fraction purity of the (24R),25(OH)2D3 primary standard. Preparation of Calibration Solutions. Three standard stock solutions of (24R),25(OH)2D3 at concentrations ranging from 11 to 16 μg/g were gravimetrically prepared by dissolving ∼1−2 mg of (24R),25(OH)2D3 in 100 mL of anhydrous ethanol. A working standard solution was gravimetrically prepared from each stock solution by ∼150-fold dilution with anhydrous ethanol, yielding concentrations of (24R),25(OH)2D3 ranging from 88 to 105 ng/g. A solution of an isotopically labeled internal standard, (24R),25(OH)2D3-d6, at a concentration of 49.32 ng/g was prepared using the same procedure as was used for the unlabeled (24R),25(OH)2D3. Six calibrants were gravimetrically prepared for calibration. Two aliquots ranging from 164 to 366 μL from each of the three working standard solutions were spiked with 500 μL of the internal standard solution, yielding calibrants with mass ratios of unlabeled to labeled (24R),25(OH)2D3 ranging from 0.7 to 1.3. The mixtures were dried under nitrogen at ∼45 °C and reconstituted with 180 μL of methanol for LC−MS/MS analysis. Sample Preparation. The liquid−liquid extraction procedure described previously for 25(OH)D22 was used to isolate (24R),25(OH)2D3 from serum matrix due to the similarity of these metabolites’ chemical structures. For each serum sample, ∼2 g of serum was accurately weighed into a glass centrifuge tube and gravimetrically spiked with appropriate amounts of (24R),25(OH)2D3-d6 to get an approximately 1:1 ratio of

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EXPERIMENTAL SECTION (24R),25(OH)2D3 and its isotopically labeled internal standard are light-sensitive. All experiments were performed under minimal exposure of light (incandescent light at reduced intensity was used). Materials. A reference standard used for calibration of (24R),25(OH)2D3 measurements and an isotopically labeled compound (24R),25(OH)2D3-d6 (isotopic purity of >99%) were custom-synthesized by IsoSciences (King of Prussia, PA). The following compounds were analyzed to evaluate the likelihood that they were sources of bias for the measurement methods implemented during this study: 23,25-(OH)2D3 and (24S),25(OH)2D3 obtained from Sigma (Milwaukee, WI); 1,25-(OH)2D3 obtained from USP (Rockville, MD); 25,26(OH)2D3 and 3-epi-(24R),25(OH)2D3 custom-synthesized by IsoSciences. Frozen human serum materials from individual donors were obtained from Interstate Blood Bank, Inc. (Memphis, TN). A vitamin D metabolites-stripped serum was a gift from DiaSorin (Stillwater, MN). Three pooled human serum materials used for the repeatability study were candidate B

DOI: 10.1021/acs.analchem.5b01861 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

time required for (24R),25(OH)2D3 to completely equilibrate with the spiked internal standard (24R),25(OH)2D3-d6, and recovery of added (24R),25(OH)2D3 to serum (used to evaluate accuracy of the method). A vitamin D metabolites-stripped serum was used to assess absolute recovery of (24R),25(OH)2D3 from the serum matrix. Two groups of serum samples were prepared to evaluate the extraction efficiency, with each group consisting of six samples. One sample group was spiked prior to extraction to achieve ∼5 ng/g concentrations of both unlabeled and labeled compounds, and the other sample group was spiked with unlabeled compound before extraction and labeled compound after extraction. The results from the two groups of samples were compared to determine the absolute recovery of (24R),25(OH)2D3 from serum. A serum material from a single donor containing endogenous (24R),25(OH)2D3 at a concentration of ∼2.5 ng/g was used for an equilibration study. Four groups of samples for the four equilibration time periods of 0.5, 1, 2, and 3 h were prepared, each consisting of three replicates. The results of the four groups were compared. A serum material from a single donor containing endogenous (24R),25(OH)2D3 at a concentration of ∼0.8 ng/g was used to evaluate the accuracy of the method. Four groups of samples were prepared, each consisting of three replicates. No unlabeled (24R),25(OH)2D3 was spiked to the first group of samples to determine the endogenous concentration of (24R),25(OH)2D3. The other three groups of samples were spiked with unlabeled (24R),25(OH)2D3 at three concentrations of (24R),25(OH)2D3; ∼2.5, 3.7, and 8.3 ng/g. For each concentration level, the amount recovered and the amount expected (equals the sum of the amount of endogenous (24R),25(OH)2D3 and the amount spiked) were compared. The percent recovery was calculated from the amount detected divided by the amount expected, then multiplied by 100. Three pooled human sera containing endogenous (24R),25(OH)2D3 at concentration levels of 1.6, 2.6, and 3.1 ng/g were used to evaluate the repeatability of the method. For each level, samples were prepared in three different sets (each set on a different day), each set consisting of four subsamples of each level. Method repeatability (within-set precision) and intermediate precision (between-set precision) were evaluated. Structural analogues of (24R),25(OH)2D3 were tested as potential interferences. Endogenous isomers 23,25-(OH)2D3, 1,25-(OH) 2 D3, 25,26-(OH) 2 D3, and 3-epi-(24R),25(OH)2D3,5,14−20 and a synthetic structural isomer (24S),25(OH)2D3 were tested. All these compounds were evaluated by comparing their retention times with that of (24R),25(OH)2D3 under the LC−MS/MS conditions described above.

analyte to internal standard. After equilibration with the spiked (24R),25(OH)2D3-d6 at room temperature for 1 h, the sample pH was adjusted to pH 9.8 ± 0.2 with a 0.1 g/mL pH 9.8 carbonate buffer solution. The (24R),25(OH)2D3 was extracted twice with hexane−ethyl acetate (50:50, volume fraction) by vigorous mechanical shaking. The combined organic layer extract was dried under nitrogen at 45 °C and subsequently reconstituted with 100 μL of methanol for LC− MS/MS analysis. LC−MS/MS Analysis. Analyses were performed on an AB Sciex API 4000 LC−MS/MS system (Framingham, MA) equipped with an Agilent 1100 series LC system (Wilmington, DE). Chromatographic separation was achieved using an isocratic method with an Ascentis Express C18 column at 35 °C and with a 30:70 (volume fraction) water−methanol mobile phase flowing at a rate of 0.75 mL/min. At the completion of each run (35 min), the column was rinsed with 100% methanol for 15 min and then equilibrated at the initial mobile phase condition for 12 min. The samples were held at 5 °C in the auto sampler, and the injection volume was 10 μL. Selective ion detection was achieved using atmospheric pressure chemical ionization (APCI) in the positive ion mode and multiple reaction monitoring (MRM) mode. The transitions at m/z 417 → m/z 381, and m/z 423 → m/z 387 were monitored for (24R),25(OH)2D3 and (24R),25(OH)2D3-d6, respectively. The dwell times were 0.25 s for MRM. Both the curtain gas and the collision gas were nitrogen and had settings of 207 kPa (30 psi) and 34 kPa (5 psi), respectively. The ion source gas 1 was air at a setting of 448 kPa (65 psi). The needle current was set at 6 μA, and the temperature was maintained at 350 °C. The declustering potential and entrance potential were set at 70 and 10 V, respectively. The collision energy and collision exit potential were set at 16 and 26 V, respectively. For each group of four to six samples, the following measurement protocol was used for LC−MS/MS analysis: single measurements of each of the six calibrants were performed prior to the single measurement of each of the serum extract samples. The samples and calibrants were reanalyzed in reverse order. For each group, a linear regression was calculated using a slope−intercept model (y = mx + b), which was used to calculate analyte concentrations. Uncertainty Evaluation. The Guide to the Expression of Uncertainty in Measurement (GUM),36,37 along with Practical Statistics for the Analytical Scientist: A Bench Guide,38 were implemented for estimation of uncertainties of the measurement results. Potential sources of uncertainty were evaluated, and significantly contributing factors were assessed to calculate the standard uncertainty. The standard uncertainty of the measurement was attributable to two types of uncertainties: type A and type B. For the type A component of measurement uncertainty, single-factor analysis of variance (ANOVA) was performed on the measurement data to obtain the standard deviation of the mean. Other uncertainty components (type B) include the uncertainties of the purity of the reference standard, the weighing of the reference standard, and an estimate of unidentified systematic errors. Type A and type B uncertainty components were combined quadratically to determine the standard uncertainty, uc, which was multiplied by a coverage factor, k, to calculate the expanded uncertainty, U. Method Validation. The approaches of method validation of RMPs, as described previously in published papers for 25(OH)D22 and steroid hormones,39−41 were used to determine absolute recovery from serum (extraction efficiency),

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RESULTS AND DISCUSSION Complete equilibration of (24R),25(OH)2D3 with the spiked internal standard is critical to obtain accurate measurement results. During the equilibration study, determined levels of (24R),25(OH)2D3 agreed within 1% for the four time periods (0.5, 1, 2, and 3 h), indicating that equilibration is complete within 0.5 h. Similar to 25(OH)D22 and steroid hormones,38−40 (24R),25(OH)2D3 is primarily bound to serum protein. Vigorous mechanical shaking with a solvent containing hexane/ethyl acetate (50:50 by volume) at high pH (pH 9.8) was performed to unbind (24R),25(OH)2D3 from protein and to extract (24R),25(OH)2D3 from the serum. The absolute recovery of C

DOI: 10.1021/acs.analchem.5b01861 Anal. Chem. XXXX, XXX, XXX−XXX

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Analytical Chemistry

gravimetrically determined “expected” concentrations and the respective RMP-determined “detected” concentrations were in very good agreement with mean recovery of 99.0% (0.8% SD). Efforts have been made to critically evaluate structural isomers that could potentially cause significant measurement bias. Compounds that were not readily available were customsynthesized through commercial sources. Endogenous structural isomers of (24R),25(OH)2D3 (23,25-(OH)2D3, 1,25(OH)2D3, 25,26-(OH)2D3, and 3-epi-(24R),25(OH)2D3), having the same molecular masses and fragmentation patterns as (24R),25(OH)2D3, were analyzed to determine whether they could interfere with measurements using the LC−MS/MS method described above. (24S),25(OH)2D3, a synthetic structural analogue of (24R),25(OH)2D3 that is not naturally present in serum25 but often exists as an impurity from the (24R),25(OH)2D3 chemical synthesis process, was also tested. Complete chromatographic separation of the R and S forms is essential for accurate measurement of (24R),25(OH)2D3. Various columns, including CN, Chiral γ-CD BR, C18-PFP, fused core F5 (PFP), and fused core C18 columns, were investigated in the preliminary studies. Chromatographic separation of 23,25-(OH)2D3, 1,25-(OH)2D3, and 25,26(OH)2D3 from (24R),25(OH)2D3 can be achieved using most of the columns investigated; however, separation of (24S),25(OH) 2 D3 and 3-epi-(24R),25(OH) 2 D3 from (24R),25(OH)2D3 is a major measurement challenge. An F5 column can separate (24R),25(OH)2D3 from 3-epi-(24R),25(OH)2D3, but (24R),25(OH)2D3 and (24S),25(OH)2D3 completely coelute. A CN column only partially separates

(24R),25(OH)2D3 from the serum with this extraction method averaged 95% (2% SD, n = 6). Absolute recovery