Abstract
Background
The current millennium has seen an explosion in vitamin D testing with the overarching aim of requests to clinically stratify patients as replete or deficient in vitamin D. At a population level, dried blood spot (DBS) sampling offers a less invasive and more practical application for assessment of vitamin D status. We have therefore aimed to develop a sensitive and robust DBS vitamin D method that is traceable to serum for use in population-based studies.
Methods
Blood spots, calibrators and controls were prepared by punching a 3.2 mm DBS from filter paper and placed into a 96-well micro-plate. The DBS disk was eluted with a combination of water-methanol and internal standard (ISTD) solution followed by supported-liquid extraction and derivatisation. The extract was analysed by liquid-chromatography tandem-mass spectrometry in positive electrospray-ionisation mode with 732.5 > 673.4 and 738.4 > 679.4 m/z ion-transitions for derivatised vitamin D and the ISTD, respectively. Vitamin D results were made traceable to the National Institute of Standards and Technology reference material through the inclusion of Chromsystems vitamin D calibrators.
Results
25-Hydroxy-vitamin D3 and its related ISTD were detected at a retention time of 7 min. The seven-point calibration-curve consistently demonstrated a coefficient of determination of 0.99 with an experimentally determined reportable range of 0.5–376 nmol/L. Method validation studies using DBS samples demonstrated 12.9% between-assay imprecision at 45 nmol/L, 84% average recovery and high correlation with plasma vitamin D (correlation coefficient = 0.86).
Conclusions
We have successfully developed an analytical method for vitamin D quantitation from DBSs which will be applied to our population-based vitamin D research study. This approach improves traceability of DBS results and potentially could be used broadly for other DBS measurands that require comparison to serum/plasma for their interpretation.
Funding source: National Health and Medical Research Council – Centre of Excellence in Paediatric Food Allergy and Food-related Immune Disorders
Award Identifier / Grant number: 1041420
Funding statement: This work was supported by the National Health and Medical Research Council – Centre of Excellence in Paediatric Food Allergy and Food-related Immune Disorders [Grant ID 1041420].
The diagram demonstrating internal standard preparation procedure for liquid and dried blood samples analysis.
Acknowledgments
We wish to thank Dr. Chris Fouracre (Agilent Technologies) for his assistance in configuring the LC-MS/MS system for micro-sampling. We are grateful to Mr. Nick Crinis (Austin Pathology) and Mrs. Lidia De Rosa (Royal Children’s Hospital Melbourne), for their assistance in retrieving the discarded patient samples used for the method validation.
Author contributions: RZ performed the studies as part of her PhD project and wrote the first draft of the manuscript. KA, JK, RG and PR supervised this project as part of RZ’s PhD candidature. RG and RZ together developed and validated the method. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Guarantor: Ronda F. Greaves.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
References
1. Wolf G. The discovery of vitamin D: the contribution of Adolf Windaus. J Nutr 2004;134:1299–302.10.1093/jn/134.6.1299Search in Google Scholar PubMed
2. Zakariaeeabkoo R, Allen KJ, Koplin JJ, Roche P, Greaves RF. Are vitamins A and D important in the development of food allergy and how are they best measured? Clin Biochem 2014;47:804–11.10.1016/j.clinbiochem.2014.01.033Search in Google Scholar PubMed
3. Vitamin D nutrition with a focus on the prevention of rickets and vitamin D deficiency in pregnant women [Internet]. World Health Organization 2015 [cited 27/06/2018]. http://www.who.int/nutrition/events/2015_vit_d_workshop_pregnantwomen_21to24Apr15/en/.Search in Google Scholar
4. Zakaria R, Allen KJ, Koplin JJ, Roche P, Greaves RF. Advantages and challenges of dried blood spot analysis by mass spectrometry across the total testing process. EJIFCC 2016;27:288–317.Search in Google Scholar
5. Eyles DW, Morley R, Anderson C, Ko P, Burne T, Permezel M, et al. The utility of neonatal dried blood spots for the assessment of neonatal vitamin D status. Paediatr Perinat Epidemiol 2010;24:303–8.10.1111/j.1365-3016.2010.01105.xSearch in Google Scholar PubMed
6. Heath AK, Williamson EJ, Ebeling PR, Kvaskoff D, EylesDW, English DR. Measurements of 25-hydroxyvitamin D concentrations in archived dried blood spots are reliable and accurately reflect those in plasma. J Clin Endocrinol Metab 2014;99:3319–24.10.1210/jc.2014-1269Search in Google Scholar PubMed
7. Jensen BP, Saraf R, Ma J, Berry S, Grant CC, Camargo Jr CA, et al. Quantitation of 25-hydroxyvitamin D in dried blood spots by 2D LC-MS/MS without derivatization and correlation with serum in adult and pediatric studies. Clin Chim Acta 2018;481:61–8.10.1016/j.cca.2018.02.024Search in Google Scholar PubMed
8. Eyles DW, Anderson C, Ko P, Jones A, Thomas A, Burne T, et al. A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots. Clin Chim Acta 2009;403:145–51.10.1016/j.cca.2009.02.005Search in Google Scholar PubMed
9. Higashi T, Suzuki M, Hanai J, Inagaki S, Min JZ, Shimada K, et al. A specific LC/ESI-MS/MS method for determination of 25-hydroxyvitamin D3 in neonatal dried blood spots containing a potential interfering metabolite, 3-epi-25-hydroxyvitamin D3. J Sep Sci 2011;34:725–32.10.1002/jssc.201000911Search in Google Scholar PubMed
10. Newman MS, Brandon TR, Groves MN, Gregory WL, Kapur S, Zava DT. A liquid chromatography/tandem mass spectrometry method for determination of 25-hydroxy vitamin D2 and 25-hydroxy vitamin D3 in dried blood spots: a potential adjunct to diabetes and cardiometabolic risk screening. J Diabetes Sci Technol 2009;3:156–62.10.1177/193229680900300118Search in Google Scholar PubMed PubMed Central
11. Larkin EK, Gebretsadik T, Koestner N, Newman MS, Liu Z, Carroll KN, et al. Agreement of blood spot card measurements of vitamin D levels with serum, whole blood specimen types and a dietary recall instrument. PLoS One 2011;6:e16602.10.1371/journal.pone.0016602Search in Google Scholar PubMed PubMed Central
12. Hoeller U, Baur M, Roos FF, Brennan L, Daniel H, Fallaize R, et al. Application of dried blood spots to determine vitamin D status in a large nutritional study with unsupervised sampling: the Food4Me project. Br J Nutr 2016;115:202–11.10.1017/S0007114515004298Search in Google Scholar PubMed
13. Makowski AJ, Rathmacher JA, Horst RL, Sempos CT. Simplified 25-hydroxyvitamin D standardization and optimization in dried blood spots by LC-MS/MS. J AOAC Int 2017;100:1328–36.10.5740/jaoacint.17-0086Search in Google Scholar PubMed
14. Rola R, Kowalski K, Bienkowski T, Kolodynska-Goworek A, Studzinska S. Development of a method for multiple vitamin D metabolite measurements by liquid chromatography coupled with tandem mass spectrometry in dried blood spots. Analyst 2018;144:299–309.10.1039/C8AN01422ASearch in Google Scholar
15. Durazo-Arvizu RA, Tian L, Brooks SP, Sarafin K, Cashman KD, Kiely M, et al. The vitamin D standardization program (VDSP) manual for retrospective laboratory standardization of serum 25-hydroxyvitamin D data. J AOAC Int 2017;100:1234–43.10.5740/jaoacint.17-0196Search in Google Scholar PubMed
16. Munns CF, Shaw N, Kiely M, Specker BL, Thacher TD, Ozono K, et al. Global Consensus Recommendations on Prevention and Management of Nutritional Rickets. J Clin Endocrinol Metab 2016;101:394–415.10.1210/jc.2015-2175Search in Google Scholar PubMed PubMed Central
17. Braegger C, Campoy C, Colomb V, Decsi T, Domellof M, Fewtrell M, et al. Vitamin D in the healthy European paediatric population. J Pediatr Gastroenterol Nutr 2013;56:692–701.10.1097/MPG.0b013e31828f3c05Search in Google Scholar PubMed
18. Paxton GA, Teale GR, Nowson CA, Mason RS, McGrath JJ, Thompson MJ, et al. Vitamin D and health in pregnancy, infants, children and adolescents in Australia and New Zealand: a position statement. Med J Aust 2013;198:142–3.10.5694/mja11.11592Search in Google Scholar PubMed
19. Hannon WH. NBS01-A6: Blood collection on filter paper for newborn screening programs; approved standard–Sixth Edition [Approved Guideline]. Wayne, PA, USA: Clinical and Laboratory Standards Institute, 2013 [updated 2013. 52].Search in Google Scholar
20. Mei JV, Zobel SD, Hall EM, De Jesus VR, Adam BW, Hannon WH. Performance properties of filter paper devices for whole blood collection. Bioanalysis 2010;2:1397–403.10.4155/bio.10.73Search in Google Scholar PubMed
21. Hall EM, Flores SR, De Jesus VR. Influence of hematocrit and total-spot volume on performance characteristics of dried blood spots for newborn screening. Int J Neonatal Screen 2015;1:69–78.10.3390/ijns1020069Search in Google Scholar PubMed PubMed Central
22. Kadjo AF, Stamos BN, Shelor CP, Berg JM, Blount BC, Dasgupta PK. Evaluation of amount of blood in dry blood spots: ring-disk electrode conductometry. Anal Chem 2016;88:6531–7.10.1021/acs.analchem.6b01280Search in Google Scholar PubMed
23. Albahrani AA, Rotarou V, Roche PJ, Greaves RF. A simultaneous quantitative method for vitamins A, D and E in human serum using liquid chromatography-tandem mass spectrometry. J Steroid Biochem Mol Biol 2016;159:41–53.10.1016/j.jsbmb.2016.02.019Search in Google Scholar PubMed
24. Biotage. Extraction of vitamin D metabolites from human serum using ISOLUTE®SLE+ in 96-fixed well plate format prior to LC-MS/MS analysis. In: Biotage, editor. Application note AN757. Sweden: Biotage AB, 2012.Search in Google Scholar
25. Müller MJ, Stokes CS, Lammert F, Volmer DA. Chemotyping the distribution of vitamin D metabolites in human serum. Sci Rep 2016;6:21080.10.1038/srep21080Search in Google Scholar PubMed PubMed Central
26. Clinical Laboratory Improvement Amendments (CLIA) [Internet]. Federal Government-Centers for Medicare & Medicaid Services 2017 [cited 27/06/2018]. https://www.cms.gov/Regulations-and-Guidance/Legislation/CLIA/index.html?redirect=/Clia.Search in Google Scholar
27. Dietrich AV, Jessome LL. Ion suppression: a major concern in mass spectrometry. Chromatography Online 2006;24:498–510.Search in Google Scholar
28. Furey A, Moriarty M, Bane V, Kinsella B, Lehane M. Ion suppression; a critical review on causes, evaluation, prevention and applications. Talanta 2013;115:104–22.10.1016/j.talanta.2013.03.048Search in Google Scholar PubMed
29. Ricos C, Alvarez V, Cava F, Garcia-Lario JV, Hernandez A, Jimenez CV, et al. Current databases on biological variation: pros, cons and progress. Scand J Clin Lab Invest 1999;59:491–500.10.1080/00365519950185229Search in Google Scholar PubMed
30. Stöckl D, Sluss PM, Thienpont LM. Specifications for trueness and precision of a reference measurement system for serum/plasma 25-hydroxyvitamin D analysis. Clin Chim Acta 2009;408:8–13.10.1016/j.cca.2009.06.027Search in Google Scholar PubMed
31. Fraser CG. Biological variation: from principles to practice, Illustrated ed. Washington, DC, USA: American Association for Clinical Chemistry, 2001.Search in Google Scholar
32. Little J. Simple method (IS-MRM) to monitor lysophospholipids and phospholipids during LC-MS method development via in-source CID. Kingsport, TN, USA: Eastman Chemical Company, 2005.Search in Google Scholar
33. Ogawa S, Ooki S, Morohashi M, Yamagata K, Higashi T. A novel Cookson-type reagent for enhancing sensitivity and specificity in assessment of infant vitamin D status using liquid chromatography/tandem mass spectrometry. Rapid Commun Mass Spectrom 2013;27:2453–60.10.1002/rcm.6708Search in Google Scholar PubMed
34. Kvaskoff D, Heath AK, Simila HA, Ko P, English DR, Eyles DW. Minimizing matrix effects for the accurate quantification of 25-hydroxyvitamin d metabolites in dried blood spots by LC-MS/MS. Clin Chem 2016;62:639–46.10.1373/clinchem.2015.251538Search in Google Scholar PubMed
35. O’Mara M, Hudson-Curtis B, Olson K, Yueh Y, Dunn J, Spooner N. The effect of hematocrit and punch location on assay bias during quantitative bioanalysis of dried blood spot samples. Bioanalysis 2011;3:2335–47.10.4155/bio.11.220Search in Google Scholar PubMed
36. Kvaskoff D, Ko P, Simila HA, Eyles DW. Distribution of 25-hydroxyvitamin D3 in dried blood spots and implications for its quantitation by tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2012;901:47–52.10.1016/j.jchromb.2012.05.040Search in Google Scholar PubMed
37. Zakaria R, Allen KJ, Koplin JJ, Crinis N, De Rosa L, Roche P, et al. Determination of haemoglobin derivatives in aged dried blood spot to estimate haematocrit. Clin Chem Lab Med 2019;57:1026–34.10.1515/cclm-2018-0753Search in Google Scholar PubMed
38. Aronov PA, Hall LM, Dettmer K, Stephensen CB, Hammock BD. Metabolic profiling of major vitamin D metabolites using Diels-Alder derivatization and ultra-performance liquid chromatography-tandem mass spectrometry. Anal Bioanal Chem 2008;391:1917–30.10.1007/s00216-008-2095-8Search in Google Scholar PubMed PubMed Central
39. Eyles D, Anderson C, Ko P, Jones A, Thomas A, Burne T, et al. A sensitive LC/MS/MS assay of 25OH vitamin D3 and 25OH vitamin D2 in dried blood spots. Clin Chim Acta 2009;403:145–51.10.1016/j.cca.2009.02.005Search in Google Scholar PubMed
40. Hedman CJ, Wiebe DA, Dey S, Plath J, Kemnitz JW, Ziegler TE. Development of a sensitive LC/MS/MS method for vitamin D metabolites: 1,25 dihydroxyvitamin D2&3 measurement using a novel derivatization agent. J Chromatogr B Analyt Technol Biomed Life Sci 2014;953954:62–7.10.1016/j.jchromb.2014.01.045Search in Google Scholar PubMed PubMed Central
41. Jones G, Kaufmann M. Vitamin D metabolite profiling using liquid chromatography-tandem mass spectrometry (LC-MS/MS). J Steroid Biochem Mol Biol 2016;164:110–4.10.1016/j.jsbmb.2015.09.026Search in Google Scholar PubMed
42. Method Comparison and Bias Estimation Using Patient Samples; Approved Guideline [Internet]. Wayne, PA, USA: Clinical and Laboratory Standards Institute 2013. https://clsi.org/media/1435/ep09a3_sample.pdf.Search in Google Scholar
43. The Correlation Coefficient: Definition [Internet]. DM STAT-1 Consulting [cited July 25, 2018]. http://www.dmstat1.com/res/thecorrelationcoefficientdefined.html.Search in Google Scholar
44. Bioanalytical Method Validation Guidance for Industry [Internet]. Food and Drug Administration 2018 [cited 25/07/2018]. https://www.fda.gov/downloads/drugs/guidances/ucm070107.Pdf.Search in Google Scholar
45. Vesper HW, Thienpont LM. Traceability in laboratory medicine. Clin Chem 2009;55:1067–75.10.1373/clinchem.2008.107052Search in Google Scholar PubMed
46. ISO 15193:2009.In vitro diagnostic medical devices – Measurement of quantities in samples of biological origin – Requirements for content and presentation of reference measurement procedures. Validation of a reference measurement procedure. Switzerland: SAI Global, 2009.Search in Google Scholar
©2020 Walter de Gruyter GmbH, Berlin/Boston
