Our method.
What we measure, how we measure it, and the validation data that makes the result trustworthy enough for your GP to act on. TGA-registered kit (ARTG 526367), made in Australia.
A reference-laboratory method, delivered at home.
Vitamin D testing in Australia ranges from $10 pharmacy strips to $80 hospital pathology. The choice of method matters more than the price tag suggests — because immunoassay-based methods (used by most pharmacy tests and even some clinical labs) carry well-documented bias from the inactive C-3 epimer of vitamin D, and cannot distinguish between vitamin D₃ and D₂.
Our test kit is manufactured in Australia and registered as a Class IIa medical device with the Therapeutic Goods Administration (ARTG 526367). Vitamin D Test partners with Masdiag,an LC-MS/MS reference laboratory, to deliver hospital-grade vitamin D analysis with at-home convenience. Masdiag's R&D and methodology team has published 30 peer-reviewed scientific papers covering dried blood spot methodology, vitamin D metabolite profiling, and clinical applications — with a cumulative Impact Factor of 143.
This page documents the analytical method, the validation performance, and the peer-reviewed publications behind it.
What the numbers actually mean.
The method comparison study analysed paired venous serum and capillary dried blood spot samples from 96 patients in a clinical setting. Serum was analysed using a DEQAS-certified reference LC-MS/MS method; the same patients' DBS samples were analysed using the at-home capillary method.
Passing-Bablok regression yielded a slope of 0.98 (95% CI 0.94–1.02), an intercept of 0.37 (95% CI −0.29 to 0.95), and an R² of 0.974 — indicating the DBS method is statistically interchangeable with venous serum analysis. Bland-Altman analysis showed a constant bias of essentially zero (−0.01 ng/mL) with 95% limits of agreement of ±14.8% — well within accepted clinical interchangeability margins.
Diagnostic performance evaluated against the standard 50 nmol/L (20 ng/mL) deficiency cut-off:
The full technical detail.
For clinicians, researchers, and anyone who wants the complete picture. All parameters are taken from the validation document for the analytical method (VITD DBS v2.0, September 2023).
| Parameter | Specification |
|---|---|
| Kit registration | TGA-registered Class IIa Medical Device — ARTG 526367 (sponsor & manufacturer: Masdiag Pty Ltd, Oak Park VIC 3046) |
| Method | Isotope Dilution Liquid Chromatography Tandem Mass Spectrometry (ID LC-MS/MS) |
| Sample type | Dried blood spot (DBS) — finger-prick capillary blood on collection card |
| Analytes measured | 25(OH)D₃, 25(OH)D₂, 24,25(OH)₂D₃ — C-3 epimers chromatographically separated and excluded |
| Separation | Reversed-phase chromatography |
| Detection | Electrospray ionisation (ESI), multiple reaction monitoring (MRM) |
| Quantification | Hematocrit-adjusted recalculation to serum/plasma equivalent |
| LOD (all analytes) | 0.1 ng/mL (S/N ≥ 3) |
| LOQ | 25(OH)D₃: 1 ng/mL · 25(OH)D₂ and 24,25(OH)₂D₃: 0.5 ng/mL (S/N ≥ 10) |
| Dynamic range | 0.5–150 ng/mL (extensible 10× with dilution) · R² ≥ 0.990 |
| VMR reference range | 4.3–14.4% (2.5%–97.5% CI, n=2,109 reference population) |
| Sample stability | Room temperature 18–25°C: up to 4 weeks · Refrigerated/frozen: up to 6 months |
| Quality assurance | DEQAS UK (Vitamin D External Quality Assessment Scheme) · ISO 15189 compliant operation |
| Analytical partner | Masdiag Pty Ltd — specialist LC-MS/MS reference laboratory |
The three things most other tests get wrong.
1. The C-3 epimer problem. About 5–10% of circulating "25(OH)D" measured by standard immunoassays is actually the inactive C-3 epimer of 25(OH)D₃. In infants and young children, this fraction can exceed 30%. Including it artificially inflates the apparent vitamin D level and can hide deficiency. Our LC-MS/MS method physically separates and excludes the C-3 epimer.
2. The D₂ problem. Vegan supplements, prescription ergocalciferol, and some plant foods contain vitamin D₂ rather than D₃. Most immunoassays detect D₃ well but underestimate D₂ by 30–70%. People supplementing with D₂ can therefore appear deficient on a standard test even when their true total 25(OH)D is adequate. Our method measures D₂ and D₃ separately and reports your true total.
3. The activation problem. Two people with identical 25(OH)D levels can have very different rates of conversion to the active vitamin D hormone. The standard test is blind to this. By measuring 24,25(OH)₂D₃ alongside 25(OH)D₃, the method calculates the Vitamin D Metabolite Ratio (VMR) — a marker of how efficiently your body is metabolising vitamin D. Available on our Vitamin D Plus report.
The literature behind the method.
The first paper below is the foundational method publication for the exact assay used in our at-home product. The remainder cover method refinements, clinical applications, and the broader scientific case for LC-MS/MS measurement and VMR analysis. These are publications from our analytical partner's R&D team plus key external supporting literature.
- Olędzka I, et al. Development of a method for multiple vitamin D metabolite measurements by liquid chromatography coupled with tandem mass spectrometry in dried blood spots. Analyst, 2019; 144: 299–309. doi:10.1039/C8AN01422A
- Masdiag R&D team. Improved sample preparation method for fast LC-MS/MS analysis of vitamin D metabolites in serum. Journal of Pharmaceutical and Biomedical Analysis, 2020; 190. doi:10.1016/j.jpba.2020.113529
- Masdiag R&D team. Application of Dried Blood Spots and Serum Samples for the Determination of Vitamin D Metabolites in the Group of Healthy Women and with Hashimoto's Thyroiditis. Chromatographia, 2021; 84: 695–701. doi:10.1007/s10337-021-04047-6
- Masdiag R&D team. Evaluation of different biological matrices to assess the vitamin D status in newborns using LC-MS/MS. Microchemical Journal, 2021; 168. doi:10.1016/j.microc.2021.106368
- Masdiag R&D team. Vitamin D status including 3-epi-25(OH)D₃ among adult patients with thyroid disorders during summer months. Endokrynologia Polska, 2018; 69(6): 653–660. doi:10.5603/EP.a2018.0065
- Masdiag R&D team. Determination of vitamin D status in singleton and twin gestations using CLIA and LC-MS/MS. Endocrine Connections, 2023; 12(11): e230201. doi:10.1530/EC-23-0201
- Masdiag R&D team. The Effect of a Single High Dose of Vitamin D on Serum Levels of Its Metabolites in the Elderly. Frontiers in Bioscience (Landmark), 2023; 27(10): 289. doi:10.31083/j.fbl2710289
- Masdiag R&D team. Single High-Dose Vitamin D Supplementation as an Approach for Reducing Ultramarathon-Induced Inflammation: A Double-Blind Randomized Controlled Trial. Nutrients, 2021; 13: 1280. doi:10.3390/nu13041280
- Herrmann M, et al. Assessment of vitamin D status — a changing landscape. Clinical Chemistry and Laboratory Medicine, 2017; 55(1): 3–26. doi:10.1515/cclm-2016-0264
- Ginsberg C, et al. The Vitamin D Metabolite Ratio is Associated with Changes in Bone Density and Fracture Risk in Older Adults. Journal of Bone and Mineral Research, 2021; 36(12): 2343–2350. doi:10.1002/jbmr.4426
How we know the answer is right.
TGA registration. Our test kit is registered on the Australian Register of Therapeutic Goods as a Class IIa Medical Device (ARTG 526367). The kit is manufactured in Australia at Suite 2080, 112 Snell Grove, Oak Park VIC 3046.
DEQAS participation. The analytical method is enrolled in the Vitamin D External Quality Assessment Scheme — the global gold-standard quality assurance programme for vitamin D measurement. Quarterly blind samples are sent to participating laboratories worldwide and results are benchmarked against reference values established by the US National Institute of Standards and Technology (NIST) and the Centers for Disease Control (CDC).
ISO 15189 compliant operation. Laboratory processes follow ISO 15189 (Medical Laboratories — Requirements for Quality and Competence). This is the international standard specifically designed for medical testing laboratories and covers sample handling, method validation, internal QC, traceability, and result reporting.
Internal QC. Every analytical batch includes blank controls, calibrators across the full dynamic range, and at least two levels of quality-control material. Out-of-range patient results trigger automatic technical review by a qualified laboratory scientist before report release.
Stable-isotope internal standards. Deuterated analogues of every measured vitamin D metabolite are added to each sample at extraction. This corrects for any sample-to-sample variation in recovery and ionisation, producing results that are quantitatively accurate, not just relatively comparable.
For clinicians and researchers.
If you're a healthcare professional, researcher, or institution evaluating our test for clinical or research use, we're happy to share more detail — including the full method validation document, current DEQAS performance data, and PDFs of any relevant publications.
For healthcare professionals
Our vitamin D test is used by allied health practitioners, naturopaths, integrative GPs, and sports medicine clinicians across Australia. We offer practitioner pricing for verified accounts and bulk ordering arrangements for clinics.
The full method validation document, current DEQAS performance data, ARTG documentation, and copies of any of our partner's publications are available on request. Email info@vitamindtest.com.au with your AHPRA / professional registration number.
Ready to test?You've now seen the science.
Same accredited method, same gold-standard analysis — delivered to your door from $59.95.