SCIEX Vitamin D 200M Assay for the Topaz System

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1 The First FDA-Cleared LC-MS/MS Assay for Vitamin D SCIEX Vitamin D 200M Assay for the Topaz System The first FDA-cleared LC-MS/MS assay for Vitamin D Vitamin D is an important building block for human health, and has been linked in the literature to several clinically important diseases. Thus, it is necessary to provide physicians with accurate results to allow for confident assessment of Vitamin D sufficiency. With the SCIEX Vitamin D 200M Assay for the Topaz System, your lab can now harness the power of mass spectrometry to deliver gold standard accuracy without the complexity. Benefits CDC certified (VDSCP): high correlation with <% bias FDA-cleared: proven performance in multiple patient sample studies Excellent sensitivity: LLMI = 4. ng/ml enables low level detection High specificity: confirmed by extensive interference testing Consistent and reproducible: traceable to NIST international standards Wide linear dynamic range: minimizes retests and sample dilution Simple adoption: locked assay method for rapid implementation The Pinnacle of Diagnostic Accuracy Unlike standard Vitamin D testing methods that are subject to inaccuracy and bias due to cross-reactivity and interferences, LC-MS/MS provides direct measurement of the analytes of interest to deliver extremely accurate quantitation. The Vitamin D 200M Assay for the Topaz system individually quantitates both the D2 and D3 forms to generate a precise quantitation of total Vitamin D; in addition, the assay separates out potential interferences, such as the D3 epimer, to minimize the risk of assay bias and false results. 25-OH-Vitamin D3 25-OH-Vitamin D3 25-OH-Vitamin D2 3-epi-25-OH- Vitamin D3 Fig. The Topaz Vitamin D Assay directly measures both 25-OH-Vitamin D2 and D3 forms individually, to ensure accurate quantitation of total Vitamin D. Fig 2. The Topaz Vitamin D Assay removes interference from the 3-epi-25-OH- Vitamin D3 epimer, preventing overestimation of total Vitamin D levels.

2 Certified Gold Standard The Vitamin D 200M assay underwent a rigorous method comparison study against the CDC standard method. The assay exhibited excellent correlation, showing <% bias versus this method. It has been certified within the CDC Vitamin D Standardization Program. A Complete Solution With the Topaz Vitamin D 200M Assay Kit, you get everything you need to run the assay from the start. The kit eliminates any concerns regarding component sourcing and compatibility. All components required, from calibrators and controls, columns, mobile phases and even test tubes for sample preparation are included within the kit. All individual components were validated together, ensuring seamless and highly accurate quantitation of total Vitamin D levels. Fig 3. Bland-Altman plot showing method comparison data between the Vitamin D 200M Assay and the CDC reference method Eliminate method development to start running your own Vitamin D test in weeks, not months. Included in the Vitamin D 200M Assay Kit Calibrator Set (7 Points) Controls (3 Levels) Internal Standard System Suitability Mix Analytical Column Trap Column Precipitation Reagent Mobile Phase A and B Rinsing Solution PBS Solution Sample Prep Reaction Vials Key Specifications Sample Material/Volume Human Serum; 00 µl LC-MS/MS Testing Time Calibration Sample Preparation Method Lower Limit of Measuring Interval (LLMI) Measuring Range Precision 6.2 minutes (injection to injection) 6 points (D2); 7 points (D3) Protein Precipitation 25-OH-Vitamin D2:.9 ng/ml 25-OH-Vitamin D3: 2.2 ng/ml 25-OH-Vitamin D: 4. ng/ml 25-OH-Vitamin D2:.9 to 65 ng/ml 25-OH-Vitamin D3: 2.2 to 60 ng/ml 25-OH-Vitamin D: 4. to 325 ng/ml For 25-OH-Vitamin D over the entire range: Repeatability: 3.8% - 6.3% Reproducibility: 5.8% - 0.4% Ordering Information Vitamin D 200M Assay for the Topaz System; 000 tests AB Sciex is doing business as SCIEX. 207 AB Sciex. For in vitro Diagnostic Use. Not available in all countries. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. IVD-MKT A

3 Development and Validation of a Fully Automated Analytical Solution for the Analysis of 25-Hydroxyvitamin D in Human Serum Utilizing the Vitamin D 200M Assay and Topaz LC-MS/MS System Dan Blake, Scott Daniels2, and Subhasish Purkayastha 2 SCIEX, UK; 2SCIEX, Framingham, MA Introduction Vitamin D consists of a group of secosteroid compounds that are involved in the regulation of calcium and phosphorus levels in the human body. The two major, physiologically relevant forms, Vitamin D2 and Vitamin D3, differ in their side-chain structures. Vitamin D3, or cholecalciferol, is synthesized in the skin upon exposure to sunlight, or may be obtained from nutritional sources, especially fatty fish. Vitamin D2, or ergocalciferol, is not synthesized by the human body and must be obtained from plant and other nutritional sources. Vitamin D itself has no biological activity, however the hydroxy metabolites 25-hydroxyvitamin D2 and 25-hydroxyvitamin D3 are converted to the biologically active,25-dihydroxyvitamin D2 and D3. It is this metabolite that is associated with calcium homeostasis and other functions. The levels of the monohydroxy metabolites are generally considered to be the best indication of an individual s Vitamin D levels. We introduce here a fully automated, end-to-end in vitro diagnostic solution to be used in clinical laboratories for the quantification of these main metabolites of Vitamin D3 and D2, 25hydroxycholecalciferol and 25-hydroxyergocalciferol, in patient serum samples by liquid chromatography-tandem mass spectrometry. This approach utilizes the Vitamin D 200M Assay Kit and Topaz LC-MS/MS System Liquid chromatography Chromatographic separation of the metabolites analyzed was achieved using a 2D LC configuration on the Topaz system; a step gradient with combined flow at 0.8 ml/min was utilized. Mass spectrometry Acquisition was performed on the Topaz system fitted with a Turbo V source. Data processing Data processing was carried out using ClearCore MD software version.., and an automated Vitamin D 200M Assay software plug-in. ClearCore MD has been specifically developed for the clinical lab, allowing enhanced ease of use and minimal operator training requirements to simplify the process from sample to result.. Materials and methods All reagents utilized for this method are included as part of the Vitamin D 200M Assay Kit for the Topaz system. Sample preparation The sample preparation involves the addition of a protein precipitation reagent and internal standard to a tube containing 00 µl of the sample (calibrator, control, blank, or unknown), followed by incubation for 0 min at 4 C, centrifugation for 5 min at 5,000 x g, and transfer of the supernatant to autosampler vials. Sample preparation can be automated. Figure Home screen of ClearCore MD.. user interface. Results and Discussion Full multi-site validation protocols have been carried out on the proposed workflow on patient samples provided by multiple laboratories. A summary of the validation data acquired is presented here. p

4 Accuracy and precision A 5 day precision study was performed at 3 sites and data was analyzed in accordance with CLSI guideline EP05-A3. The same set of samples was used at all the sites. Seven serum samples were used in this study with 25-OH-vitamin D3 and 25-OH-vitamin D2 concentrations approximating low (4 ng/ml) and high (90 ng/ml) assay levels as well as medical decision points (deficient/insufficient- 0 ng/ml, insufficient/optimal- 5 ng/ml, optimal/increased risk of toxicity- 30 ng/ml, and toxic- 50 ng/ml). ANOVA analysis was used to calculate the variance components used to determine the site-specific repeatability, within-laboratory precision, and reproducibility estimates for 25-OH-vitamin D3, 25- OH-vitamin D2, and total 25-OH-vitamin D. The assay meets the repeatability (<0% CV at all concentrations except for the lowest where is should be <5%) and reproducibility (<5% CV at all concentrations except for the lowest where is should be <20%) goals. The maximum repeatability %CV estimate of all sample concentrations is 7.3% for 25-OH-vitamin D3, 6.6% for 25-OH-vitamin D2, and 6.3% for total 25-OH-vitamin D. The maximum reproducibility %CV estimate of all sample concentrations is 9.8% for 25-OH-vitamin D3, 3.4% for 25-OHvitamin D2, and 0.4% for total 25-OH-vitamin D. Summary data for all acquired instances is given in Table 3 Linearity Studies were performed to determine whether the relationship between quantitative output of the assay and the analyte concentration was linear across the reporting range. The design of the linearity study was based on CLSI guideline EP06-A. A serum pool with the analyte concentrations at the lowest level and a serum pool with analyte concentrations at the highest level were prepared. These two pools were then used to prepare 9 linearity samples with equally spaced concentrations from 2.2 ng/ml to 60 ng/ml for 25-OH-vitamin D3 and from.9 ng/ml to 90 ng/ml for 25-OH-vitamin D2. The studies were performed at a single internal site using 3 instruments with 3 kit lots and 3 operators. The data was analyzed by regression analysis of the linear and polynomial fits. The repeatability of each analysis was also calculated with a goal of <0% for the pooled repeatability estimate. The assay was linear over the range from 2.2 ng/ml to 60 ng/ml for 25-OH-vitamin D3 and.9 ng/ml to 65 ng/ml for 25-OHvitamin D2 which encompasses the desired assay measuring range of 4. ng/ml to 325 ng/ml for total 25-OH-vitamin D. The pooled repeatability estimate was <6% for all data sets. Visual data summary is shown in Figure 2 Sample Analyte Mean (ng/ml) Repeatability Within- Laboratory Reproducibility SD %CV SD %CV SD %CV % % % % % % % %.0 7.3% % % % % % % % % % %.0 7.%.0 7.% % %.0 7.% % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % % 2 BMR N/A N/A N/A N/A N/A N/A % % % Table - Summary data of precision and reproducibility data acquired Figure 2 - Linearity data for 25(OH) Vitamin D3 (top) and 25(OH) Vitamin D2 (bottom) Limit of quantitation Studies were performed to establish the limit of quantitation (LoQ) for each analyte measured in the assay as well as for total 25-OHvitamin D (by addition of 25-OH-vitamin D3 and 25-OH-vitamin D2 values). The design of the study was based on a precision profile approach described in CLSI guideline EP7-A2 and C62-A using samples with known reference concentrations. p 2

5 The LoQ was determined to be 2. ng/ml for 25-OH-vitamin D3 and.2 ng/ml for 25-OH-vitamin D2. The reported values for the method are the maximum of the 2 kit lots analyzed The signal-to-noise ratio at the LoQ for 25-OH-vitamin D3 and 25- OH-vitamin D2 using 90 determinations from 2 kit lots and 3 instruments exceeds the desired minimum value of 0. Summary data of the LoQ investigations, including an example chromatogram at the proposed LoQ, is given in figure 3 Analyte LoQ (ng/ml) 20 Mean Signal- Passing-Bablok fit Kit Lot Kit Lot Method Noise Ratio 0 A B 25-OH-Vitamin D Reference Method (ng/ml) 25-OH-Vitamin D OH-Vitamin D n/a Parameter Estimate 95% CI Intercept to Test Method (ng/ml) Slope to.056 Figure 4 - Passing-Bablock fit and data for method comparison exercise LLMI of 25-OH- VitD3 LLMI of 25-OH- VitD2 30% 20% 0% Figure 3 - Limit of Quantitation summary data and chromatograms Comparisons with currently accepted reference method A method comparison study was performed following CLSI guideline EP09-A3. A total of 20 unique natural patient samples (2% modified to achieve difficult to obtain concentrations), with total 25-OH-vitamin D concentrations ranging from 5.6 ng/ml to 54 ng/ml, were tested on the Vitamin D 200M Assay for the Topaz System and the CDC and University of Ghent vitamin D2 and D3 Reference Method. Analysis of the comparison data gave a Pearson correlation coefficient of 0.99 and Passing-Bablok analysis gave a line with a slope of.0 indicating a high correlation between the candidate device method and reference method across the measuring range of the candidate device. Passing-Bablock and Bland-Altman summary data of this exercise are given in figures 4 and 5 respectively. Note data provided on total 25(OH) Vitamin D to ensure direct comparison with the current reference method. Difference (%) 0% -0% -20% -30% -40% (Reference Method + Test Method)/2 (ng/ml) Parameter Estimate 95% CI SE Mean difference -0.6% -.64% to.32% 0.747% 95% Lower LoA -6.2% -8.7% to -3.7%.28% 95% Upper LoA 5.9% 3.3% to 8.4%.28% SD 8.9% Figure 5 - Bland-Altman plot and data for method comparison exercise Interference Identity Mean 95% LoA Extensive interference testing was conducted to ensure that the assay performed with high specificity for the analytes of interest. Studies were performed to determine if certain metabolites, drugs, supplements, and isobaric compounds (or compounds with isobaric fragments), when added to the sample matrix, interfere with the quantitation of the analytes of interest. 79 compounds were evaluated in total. The design of the study was based on CLSI guideline EP07-A2. p 3

6 None of the potential endogenous and exogenous interfering substances tested or the sample collection tube were found to cause interference with the analytes of interest greater than a 0% difference between the test and control samples, or suppression of the signal greater than a 30% difference between the test and control samples. References Holick, M.F. and Chen, T.C. Vitamin D deficiency: a worldwide problem with health consequences. Am J Clin Nutr. 2008; 87(suppl):080S-086S.. The % interference due to crosstalk between the analytes and internal standard was within the guidelines stated in CLSI C62-A. Conclusions Based on the data presented herein, the following conclusions can be drawn. The SCIEX Vitamin D 200M assay for the Topaz system delivers the precision required to accurately quantitate 25- OH-vitamin D over the entire measuring range. The assay is linear over a wide range of concentrations resulting in good accuracy at very low and very high 25-OHvitamin D concentrations. The assay delivers the sensitivity required to accurately quantitate low levels of 25-OH-vitamin D. The assay shows a high degree of correlation with the standard reference method (same method used for the CDC Vitamin D Standardization Program). The overall bias between the reference and candidate methods is less than %. The method has also been certified by the CDC Vitamin D Standardization Program. The assay exhibits excellent specificity for the analytes of interest. None of the 79 potential interfering substances (including isobaric compounds) or collection tubes tested in this study were found to cause interference with the analyte of interest greater than a 0% difference between the test and control samples. In final conclusion, we present here a robust and highly accurate in vitro diagnostic workflow solution for the analysis of 25- hydroxyvitamin D by LC-MS/MS. This solution is fully validated for use on the SCIEX Topaz System within clinical diagnostic laboratories. AB Sciex is doing business as SCIEX. 207 AB Sciex. For IN VITRO Diagnostic Use. Not available in all countries. The trademarks mentioned herein are the property of AB Sciex Pte. Ltd. or their respective owners. AB SCIEX is being used under license. Document number: IVD-MKT B p 4

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