维生素D2 D3候选参考方-1

Candidate Reference Measurement Procedures for Serum 25-Hydroxyvitamin D3 and 25-Hydroxyvitamin D2 by Using Isotope-Dilution Liquid Chromatography–Tandem Mass Spectrometry Hedwig C.M. Stepman,1 An Vanderroost, Katleen Van Uytfanghe,1 and Linda M. Thienpont1* BACKGROUND: 25-hydroxyvitaminD [25(OH)D] assays are characterized by poor between-assay comparabil- ity. This result emphasizes the need for reference mea- surement procedures (RMPs) to establish calibration traceability and assist in method validation. We aimed at developing candidate RMPs on the basis of isotope- dilution liquid chromatography–tandem mass spec- trometry (ID-LC-MS/MS) for separate quantification of serum 25(OH)D2 and 25(OH)D3. METHODS: Hexa-deuterated 25(OH)D3/D2 was added to serum. This mixture was extracted with n-hexane and fractionated on Sephadex LH-20 before 2-dimensional LC-MS/MS. In the first dimension, both procedures used a C4 column; however, in the second dimen- sion, the 25(OH)D2 procedure used a C18 and the 25(OH)D3 procedure used a Zorbax SB-CN column. Calibration was traceable to the NIST Standard Refer- ence Material (SRM) 2972. Validation comprised as- sessment of interference and limit of quantification/ detection. Imprecision and trueness were validated by analysis of the SRM 972 against specifications (CV �5% and bias�1.7%). The expanded uncertainty for quadruplicate measurements was estimated. RESULTS: Testing of potentially interfering substances was negative. Interference by 3-epi-25(OH)D3 was re- solved by sufficient chromatographic resolution. The limits of quantification/detection were 1.1 nmol/L and 0.09 pmol/L for 25(OH)D3 and 1.2 nmol/L and 0.05 pmol/L for 25(OH)D2.Mean total CVs and differences from the SRM 972 target (� 1-sided 95% CI) were 2.1%and1.1%�1.5%[25(OH)D3] and3%and1.3%� 0.6% [25(OH)D2], respectively. The respective expanded uncertainties were 3.4% and 3.9%. CONCLUSIONS: From the validation data, we conclude that we achieved our objective of 2 state-of-the-art can- didate RMPs for serum 25(OH)D3 and 25(OH)D2. © 2010 American Association for Clinical Chemistry There is evidence for major public health issues sur- rounding the vitamin D status in humans (1 ). Vita- min D exists in 2 forms and is obtained from 2 sources. Vitamin D3 is formed by exposure of the skin to sunlight, whereas the D2 form is obtained from the diet. Fortified food and dietary supplements con- tain both forms. The circulating liver metabolites 25- hydroxyvitaminD3 andD2 [25(OH)D3 and 25(OH)D2] 2 are used as markers of the vitamin D status (2 ). Severe deficiency has recently been implicated in a wide range of clinical disorders other than skeletal deformities (3–5). As a result of this emerging consensus, physi- cians increasingly monitor the vitamin D status in in- dividuals. This testing is donewith a variety ofmethods based on different measurement principles, of which the typical performance characteristics and limitations recently have been reviewed (6, 7 ). Immunoassays mainly may be limited by cross-reactivity of the anti- bodies and nonequimolar recognition of the D2 and D3 forms of the 25(OH) metabolite, whereas in chro- matographic methods, it is the resolving power and detection that are determinants for sufficient specific- ity. In this regard, liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS) inherently has the better potential. Note that in chro- matographic methods, in contrast to immunoassays, equimolarity of measurement is not an issue because these methods separate the D2 and D3 25(OH) metab- 1 Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Ghent, Belgium. * Address correspondence to this author at: Laboratory for Analytical Chemistry, Faculty of Pharmaceutical Sciences, Ghent University, Harelbekestraat 72, B-9000 Ghent, Belgium. Fax �32-9-264-81-98; e-mail linda.thienpont@ ugent.be. Received June 30, 2010; accepted December 28, 2010. Previously published online at DOI: 10.1373/clinchem.2010.152553 2 Nonstandard abbreviations: 25(OH)D3, 25-hydroxyvitamin D3; 25(OH)D2, 25- hydroxyvitamin D2; LC, liquid chromatography; MS/MS, tandem mass spectrom- etry; SI, Syste`me International d’Unite´s; SRM, Standard Reference Material; ID, isotope dilution; RMP, reference measurement procedure; UPLC, ultra perfor- mance liquid chromatography; Mr, relative molecular mass; LoQ, limit of quantification; LoD, limit of detection; S/N, signal-to-noise. Clinical Chemistry 57:3 441–448 (2011) Automation and Analytical Techniques 441 olites and sum up the results to the total 25(OH)D concentration. However, Singh et al. (8 ) showed that commonly used LC-MS/MS procedures may overesti- mate the 25(OH)D concentration when the C-3 epimer accounts for a substantial proportion of the cir- culating 25(OH)D3 concentration. This occurrence re- sults from insufficient chromatographic resolution to separate the 3-epi metabolite. Another major limita- tion that applies for all current 25(OH)D methods is the poor comparability of results, as demonstrated in dedicated proficiency testing schemes (9, 10). This is confounding for diagnosis against expert recommen- dations to maintain circulating 25(OH)D concentra- tions above a certain concentration for optimal health (11–14). This concern increasingly calls for standard- ization (15–17). There is general consensus that this should be done by using a higher-order reference mea- surement system to establish traceability to the Syste`me International d’Unite´s (SI) (18). SI traceability or true- ness is indeed the most valid basis for generation of laboratory data that serve the establishment of guide- lines and clinical diagnosis, long-term epidemiologic studies, and programs to evaluate the effect and safety of dietary supplements (19–21). It was in this regard that the Office of Dietary Supplements from the Na- tional Institutes of Health worked together with the NIST to develop standard reference materials (SRMs) for 25(OH)D3/D2, i.e., the serum-based SRM 972 (4 levels) and the SRM 2972 calibration solutions (22– 24). The latter was helpful in decreasing interlabora- tory variation of chromatographic procedures (25). The SRM 972, certified with isotope dilution (ID)-LC- MS/MS reference measurement procedures (RMPs) (26), was intended for validation of the trueness of hi- erarchically lowermethods (18). However, some levels of the SRM were deemed noncommutable when mea- sured with immunoassays and therefore not fit for trueness assessment (27). This problem is not expected for routine MS/MS procedures because they are based on the samemeasurement principle as the certification procedure. Because of the issue of noncommutability of reference materials, the approach of directly using unadulterated sera, assigned with values by an RMP, is the preferred alternative (18, 28). Of course, when the laboratory community advocates that in vitro diagnos- tic companies and ID-LC-MS/MS laboratories should make use of this approach, there is a need for sufficient capacity of laboratories offering RMP services. Here we report the development of 2 candidate ID-LC-MS/MS RMPs for quantification of serum 25(OH)D3 and 25(OH)D2, the first with sufficient chromatographic resolution to separate 25(OH)D3 and the 3-epi metabolite. As part of the process of crit- ically evaluating the procedures, we validated trueness and imprecision against previously derived specifica- tions and assessed the applicability formeasurement of native patient sera (29). Materials andMethods MATERIALS 25(OH)D3, 25(OH)D2, and 7-dehydrocholesterol were purchased from Sigma-Aldrich, and hexa- deuterated analogs [d6-25(OH)D3/D2] were obtained from Medical Isotopes. SRM 972 and SRM 2972 were obtained from NIST, Sephadex LH-20 from GE Healthcare, and horse serum from Invitrogen. The components tested for interference were a gift from Immunotech andH.DeLuca (University ofWisconsin, Madison). Chemicals and solvents were analytical grade, with the exception of methanol and water of ultra performance liquid chromatography (UPLC) grade (Biosolve). For details on the measured human serum samples, see File 1 in the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol57/issue3. CALIBRATION Three working solutions of approximately 60/21 nmol/L 25(OH)D3/D2 in ethanol were prepared. They were assigned with exact values from a comparison protocol with the SRM 2972, further referred to as “calibration transfer protocol.” The solutions of d6- 25(OH)D3/D2 were prepared similarly. For quantifica- tion, a 1-point calibration procedure at the 1:1 isotope ratio (� 25%) was used (30) (see Supplemental Data File 1). SAMPLE PREPARATION Sample preparation is fully described in online Supple- mental Data File 1. Typically, 250 �L serum (maxi- mum 500 �L) was sampled and diluted to 1 mL with 0.9% (g/g) sodium chloride solution. Subsequently, equal absolute amounts of either d6-25(OH)D3 or d6- 25(OH)D2 were added. After equilibration, the serum was alkalinized, extracted with n-hexane, and fraction- ated by Sephadex LH-20 chromatography. LC-MS/MS ANALYSIS A 2-dimensional Acquity® UPLC system connected to an Acquity® tandem quadrupole mass detector (both from Waters), operating in the positive electro- spray ionization mode, was used. The first dimension used an Acquity® BEH300 C4 column (2.1 � 50 mm, 1.7 �m, 300 Å). The second-dimension columns were method specific: the 25(OH)D2 candidate RMP used an Acquity BEH C18 column (2.1 � 50 mm, 1.7 �m, 130 Å), while the 25(OH)D3 used a Zorbax SB-CN col- umn (2.1� 250mm, 5�m, 80 Å; held at 30 °C, Agilent Technologies). The selected reaction-monitoring tran- sitions werem/z 401.33 159.3 [25(OH)D3 and 3-epi- 25(OH)D3] and m/z 407.33 159.3 [d6-25(OH)D3)], 442 Clinical Chemistry 57:3 (2011) andm/z 413.43 159.4 [25(OH)D2] andm/z 419.43 159.4 [d6-25(OH)D2]. Further details on the UPLC and MS conditions are given in online Supplemental Data File 1. METHOD VALIDATION Calibration. We estimated the uncertainty of the cali- bration transfer protocol from the imprecision of the experimental design used for value assignment. Fur- thermore, we assessed whether it was justified to do the calibration from direct analysis of the calibrators, i.e., without submission to the sample pretreatment proce- dure. The experimental protocol is described in online Supplemental Data File 1. Recovery and matrix effect on ionization. The absolute recovery of the sample preparation procedure for 25(OH)D3/D2 was investigated by comparison of the isotope ratios obtained for 2 sets of 6 aliquots from the same serum pool. To the first set, the isotopically la- beled analog was added before sample preparation; to the second set, it was added only after extraction and fractionation (see online Supplemental Data File 1). Matrix effect on ionization was investigated by com- parison of the area under the peak obtained fromdirect injection of the labeled analog vs injection after addi- tion to a processed serum matrix (n� 6). Interference. Structural analogs of 25(OH)D3/D2 were tested for interference (for the amounts injected, see online Supplemental Data File 1). The relative molec- ular mass (Mr) of the [M�H] � and [M�Na]� ions was a first criterion for potential interference at them/z valuesmonitored for 25(OH)D3/D2 and the d6 analogs. For the substances with a higher Mr, a full scan spec- trum was registered (m/z range of 350–450) to verify the presence of an interfering MS signal. The sub- stances testing positive were evaluated for their reten- tion time relative to 25(OH)D3/D2 in the first dimen- sion of the UPLC procedure only. In addition, 87 native serum samples, not supplemented with d6- 25(OH)D3/D2, were analyzed to assess interference. Limit of quantification (LoQ) and limit of detection (LoD). The LoQ for 25(OH)D2 was evaluated from analysis of SRM 972 level 1 [certified concentration: 1.46 � 0.49 nmol/L (expanded uncertainty)] and from an analytical recovery experiment with a human serum pool supplemented to a total concentration of 2.98 nmol/L. Note that the endogenous concentration was 1.49 (1.46–1.52) nmol/L (2-sided 95% CI). For 25(OH)D3, the LoQ was estimated from analysis of horse serum supplemented with 1.02 nmol/L. All sam- ples were quantified in duplicate on 5 independent days (n� 10), which allowed the estimation of the total CV and themean signal-to-noise (S/N) ratio. Note that the same protocol was used to determine the endoge- nous concentration in the sera of the recovery experi- ment. Our specifications for LoQwere a total CV�7% and amaximum absolute deviation�0.13 nmol/L (see Discussion). The estimation of the LoD at an S/N ratio of 3 was based on the LoQ samples. Imprecision and trueness. These characteristics were validated from analysis of the 4 levels of the SRM 972 against specifications of a maximum total CV of 5% and a maximum systematic deviation of 1.7% (29). Because of the limited volumes of the NIST SRM materials, measurements were done according to a re- duced CLSI EP 5 protocol, i.e., duplicates on 5 inde- pendent days (n� 10) (31). For each level, the within- run, between-run, and total CVs were calculated by 1-way ANOVA. The mean total CV was calculated as the square root of the mean of the quadratic total CVs. For the different internal quality control samples (see online Supplemental Data File 1), the total CV was similarly calculated using 1-way ANOVA. The CV for measurement of native serum samples was derived from the difference between the duplicates; however, it wasmeasured as singlicate on 2 independent occasions. The trueness was expressed as percent recovery of the NIST-certified values. Performance of native serum samples. The candidate RMPs were challenged with analysis of 87 native serum samples. After screening of the samples for the pres- ence of 25(OH)D2 and 3-epi-25(OH)D3, all 3 metabo- lites were determined in duplicate with rigorous inter- nal quality control (see online Supplemental Data File 1). In the reconstructed ion chromatograms, we deter- mined the typical S/N ratio, verified the presence of nonidentified interfering peaks, and evaluated the re- solving power to separate 3-epi-25(OH)D3 [at 3% of the 25(OH)D3 peak height]. The 25(OH)D2 and 25(OH)D3 concentrations were quantified in absolute terms, whereas for the 3-epi form, the content relative to 25(OH)D3 (%) was estimated. Expanded uncertainty of measurement. First, the com- bined uncertainty was calculated from propagating the uncertainty due to the imprecision, the calibration trans- fer protocol, the SRM 2972 certification, and unspecific interferences. To obtain a representative imprecision, the totalCVestimated fromanalysis of thedifferent SRM972 levelswas combinedwith theCVs calculated for the inter- nal quality control and native serum samples. This step was done by taking the square root of the mean of each quadratic CV value. Finally, the expanded uncertainty was estimated with k� 2 (95%). Statistical data analysis. Microsoft Office Excel® (ver- sion 2007) was used to perform the Grubbs outlier test, 25-Hydroxyvitamin D Candidate Reference Measurement Procedures Clinical Chemistry 57:3 (2011) 443 the 2-sided F-test, 1- or 2-sided (depending on the ap- plication) Student’s t-tests with equal or unequal vari- ances (based on the F-test), and a 1-way ANOVA, all at 95% probability. Results CALIBRATION The uncertainty of the calibration transfer protocol amounted to 0.7%. The isotope ratios measured in the 2 sets of calibrators (directly analyzed and after submis- sion to sample preparation) were not significantly dif- ferent [25(OH)D3: difference 0.86%, P2-sided t-test � 0.186; 25(OH)D2: 0.54%, P2-sided t-test � 0.665]. This result together with the evidence for absence of matrix effects on ionization (see below) showed that calibra- tion with directly analyzed calibrators was justified. RECOVERY AND MATRIX EFFECT ON IONIZATION The absolute recovery � 2-sided 95% CI values of the sample preparation procedures were 71% � 4% [25(OH)D3] and 70% � 8% [25(OH)D2]. The matrix effect on ionization was found to be sta- tistically not significant [25(OH)D3, observed dif- ference 15%, P2-sided t-test� 0.0516; 25(OH)D2, 18%, P2-sided t-test� 0.0574]. INTERFERENCE The results for the interference study are summarized in Table 1 (for the retention time of the examined com- ponents, see online Supplemental Table S3). Although the registered full scan spectra identified potential interference by some compounds, the relative reten- tion time allowed us to exclude it for all, except 3-epi- 25(OH)D3. As shown in Fig. 1, the 3-epi form is sepa- rated at 3% of the 25(OH)D3 peak height on the Zorbax SB-CN column. Analysis of the 87 samples without added d6 analogs confirmed absence of inter- ference on the transition m/z values 407.33159.3 and 419.43159.4. LoQ AND LoD The LoQ for 25(OH)D2, estimated from analysis of SRM 972 level 1, was 1.22� 0.05 nmol/L (2-sided 95% CI). The absolute difference from the target was�0.24 nmol/L, the total CVwas 5.3%, and themean S/N ratio was 76. The LoQ, estimated from the analytical recov- ery experiment, was 1.43� 0.05 nmol/L, with a devia- tion of�0.054 nmol/L from the target. The measured total 25(OH)D3 concentration in the supplemented horse serum was 1.12 � 0.05 nmol/L. The deviation from the target (1.23 nmol/L, because of supplementa- tion of the endogenous concentration at 0.21 � 0.03 nmol/L with 1.02 nmol/L) was�0.11 nmol/L, the total CVwas 6.3%, and themean S/N ratio was 40. The LoD, estimated from the above data, was 0.025 pmol (10 pg) and 0.015 pmol (6 pg) on columns for 25(OH)D3 and 25(OH)D2, respectively. IMPRECISION AND TRUENESS Tables 2 and 3 list the different imprecision and trueness data. The mean within-run and total imprecision (ex- pressed as%CV) estimated from analysis of the different levels of the SRM972were 1.9% and 2.1% for 25(OH)D3 and 2.9% and 3.0% for 25(OH)D2. The mean trueness was 101.1%� 1.5% (1-sided 95%CI) for 25(OH)D3 and Table 1. Vitamin D analogs tested for potential interference. Vitamin D metabolite or structural analog Mr Interfering m/z valuea Relative retention timeb Interference[M�H-H2O] � [M�H]� [M�Na]� Cholecalciferol 367.7 385.7 407.7 � r � 1.34 No Ergocalciferol 379.7 397.7 419.7 � r � 1.31 No 7-Dehydrocholesterol 367.6 385.6 407.6 � No 3-epi-25-Hydroxyvitamin D3 383.6 401.6 423.6 � r � 0.99 Yes 1�-Hydroxyvitamin D3 383.6 401.6 423.6 � r � 1.15 No 1�,25-Dihydroxyvitamin D2 411.7 429.7 451.7 � No 1�,25-Dihydroxyvitamin D3 399.6 417.6 439.6 � No 24,25-Dihydroxyvitamin D3 399.6 417.6 439.6 � No 25,26-Dihydroxyvitamin D3 399.6 417.6 439.6 � No 1�,24,25-Trihydroxyvitamin D3 415.6 433.6 455.6 � No a Codes used to describe interfering m/z value:�, no signal, obtained in the scan mode, at the m/z values corresponding to the relative molecular mass of [M�H]�: 401.6 [25(OH)D3], 407.6 [d6-25 (OH)D3], 413.7 [25(OH)D2], and 419.7 [d6-25(OH)D2]; �, because the molecule had an interfering m/z value (note: m/z values are in italics), we determined the relative retention time. Only the molecules that eluted from the first dimension column between 5.4 and 6.4 min [corresponding relative retention times: 0.92–1.09 min for 25(OH)D3 and 0.89–1.05 min for 25(OH)D2] are transferred to the second dimension column). b Relative retention time in comparison with 25(OH)D3 or 25(OH)D2, respectively. 444 Clinical Chemistry 57:3 (2011) 101.3%�0.6%for25(OH)D2.Noneof theobtainedper- cent recoveries of the NIST target values exceeded the 1.7% deviation limit, as confirmed by a 1-sided t-test against the limits for maximum deviation (i.e., 98.3% or 101.7%) (see Table 3 for the P values). PERFORMANCE ON NATIVE SERUM SAMPLES The concentrations of 25(OH)D3 in 87 samples ranged from 2.4 to 590 nmol/L, with amean of 73 nmol/