Testing-Validation of bioactive VITAMIN D (25-OH D2/D3/Calcitriol) AND VITAMIN B12 (MMA) BY LCMSMS

RATL is the only institutional lab to be assessed, audited and approved for assay/ testing of Vitamin B12 & Bioactive Vitamin D (25-OH D2/D3/Calcitriol) (LC-MS-MS) by the ISO 17025 / NABL accreditation authority.

VITAMIN D

Testing of Bioactive Vitamin D levels has been validated with a high degree of accuracy, precision, repeatability and sensitivity with lowest detection levels (1.0 ng/ml) by the gold-standard method - LCMSMS using a high-end Waters XEVO-TQD-UPLC system, one of the most advanced in the world today.

The US Food and Drug Administration stipulates that bioactive Vitamin D metabolites are the ultimate indicator of Vitamin D deficiency.

Laboratories which measure a single component (D2 or D3) render patients prone to dosage errors because the bioactive component present is ignored. Hence it is very important to select an analytical method that will accurately estimate calcitriol and not just independent levels of Vitamin D2 or D3 or 'Total' Vitamin D

Analyzing bioactive vitamin D by LC/MS/MS is an ideal method for high-throughput, accurate, low-level detection and fast analysis times. This allows assessment of the source of the deficiency and also facilitates treatment & monitoring in a manner that is far more accurate and complete.

VITAMIN B12

Vitamin B12 status is typically assessed via serum or plasma vitamin B12 levels. Values below approximately 170–250 picogram/mL (120–180 picomoles/L) for adults and below 200 picogram/mL in children indicate a vitamin B12 deficiency. Evidence, however,  suggests that serum vitamin B12 concentrations might not accurately reflect intracellular concentrations. Homocysteine (HC) and methyl malonic acid (MMA) levels are better indicators of vitamin B12 deficiency; MMA being a more specific and sensitive indicator than homocysteine. Homocysteine has poor specificity as its measurement is confounded by other factors such as low B6 or folate levels.

Increased MMA or HC levels may indicate vitamin B12 deficiency. Normal MMA levels are in the range of 0.073 - 0.271 μmoles/mL, while HC levels are in the range of 2.2 - 13.2 μmoles/mL. MMA levels are generally reported as μmoles/moles creatinine, with values below 3.6 μmoles/moles creatinine being within range. MMA values greater than 3.6 μmoles/moles creatinine would be indicative of vitamin B12 deficiency.

COMPARISON OF LC-MS-MS vs. LIGAND-BINDING IMMUNOASSAY

SAMPLE INFORMATION

LC-MS-MS

  • LC-MS-MS provides 25-OH-VitD2 and 25-OH-VitD3 levels in addition to the total vitamin D level. This allows assessment of the source of the deficiency and also facilitates treatment monitoring
  • Measures low concentrations with high accuracy and precision using LC–MS-MS
  • LC-MS-MS approach is more suitable for simultaneous determination of multiple analytes/metabolites
  • Matrix interference can be minimized by adopting selective sample preparation techniques
  • LC-MS-MS methods are more robust while performing lab-to-lab and assay-to-assay comparisons with other MS-MS
  • The provided laboratory development test method overcomes the issues related to lot-to-lot variation, reagent availability issues, or licensing fees associated with proprietary kits.
  •  It has earned a reputation as the analytical “gold standard,” delivering high quality and specificity, particularly for small molecule analytes including vitamin D metabolites.

 

LIGAND-BINDING IMMUNOASSAY

  • Ligand-binding assays cannot distinguish between 25-OH-VitD2 and 25-OH-VitD3. Therefore, it is difficult to judge whether patients are compliant with therapies or suffer from vitamin D malabsorption.
  • All immunoassays show high cross reactivity with 24,25dihydroxyvitamin D (24, 25(OH)2D) that increases in concentration in blood with increasing sun exposure and as 25OHD increases.
  • Automated assays show good precision at high concentrations but havedifficulties to meet analytical goals at low concentrations.
  • Some assays significantly overestimate vitamin D levels; others significantly underestimate them
  • Ligand-binding immunoassays are fundamentally difficult because some assays work best in an aqueous environment; 25-OH-VitD2 and 25-OH-VitD3, however being poorly soluble in water (Manual extraction assays can overcome solubility problems but suffer from increased imprecision).
  • Lack of result comparability occurs between different assays and often between the same assays performed in different laboratories.

SAMPLE INFORMATION

VITAMIN D2

  • Linearity: 1 - 40 ng/mL
  • LoQ: 1 ng/mL
  • Method of Assay: LC-MS-MS
Linearity between 1- 40 ng/mL and extracted chromatogram of VITAMIN D2
Linearity between 1- 40 ng/mL and extracted chromatogram of VITAMIN D2
  • Sampling time: Morning of sampling day; fasting optional
  • Specimen: 2ml serum
  • Container: Red top serum Vacutainer® tube, alternatively serum separator tube (SST®)
  • Collection: Routine venipuncture
  • Special processing: Red top: allow blood to clot at room temperature, centrifuge and separate serum from cells immediately and transfer serum to a plastic screw-cap vial; SST tube: allow to clot at room temperature, centrifuge and remove from the gel within 48 hours and transfer to a plastic screw-cap vial.

 

  • Storage/Transport temperature: Refrigerated
  • Unacceptable conditions: Grossly hemolyzed, lipemic, or icteric specimens, serum not separated from Serum Separator Tube (SST®) gel or clot within 24 hours.
  • Stability: After separation from cells, ambient: 24 hours; refrigerated: 1 week; frozen: 3 months
  • Reference value: <10 ng/ml (severe deficiency), 10-19 ng/ml (mild to moderate deficiency), 20-50 ng/ml (optimum levels), 51-80 ng/ml (increased risk of hypercalciuria), >80 ng/ml (toxicity possible)

VITAMIN D3

  • Linearity: 1 - 40 ng/mL
  • LoQ: 1 ng/mL
  • Method of Assay: LC-MS-MS
Linearity between 1 - 40 ng/mL and extracted chromatogram of VITAMIN D3
Linearity between 1 - 40 ng/mL and extracted chromatogram of VITAMIN D3
  • Sampling time: Morning of sampling day; fasting optional
  • Specimen: 2ml serum
  • Container: Red top serum Vacutainer® tube, alternatively serum separator tube (SST®)
  • Collection: Routine venipuncture
  • Special processing: Red top: allow blood to clot at room temperature, centrifuge and separate serum from cells immediately and transfer serum to a plastic screw-cap vial; SST tube: allow to clot at room temperature. Centrifuge and remove from the gel within 48 hours to a plastic screw-cap vial.

 

  • Storage/Transport temperature: Refrigerated
  • Unacceptable conditions: Grossly hemolyzed, lipemic, or icteric specimens, serum not separated from Serum Separator Tube (SST®) gel or clot within 24 hours.
  • Stability: After separation from cells, ambient: 24 hours; refrigerated: 1 week; frozen: 3 months
  • Reference values: <10 ng/ml (severe deficiency), 10-19 ng/ml (mild to moderate deficiency), 20-50 ng/ml (optimum levels), 51-80 ng/ml (increased risk of hypercalciuria), >80 ng/ml (toxicity possible)

VITAMIN B12 (MMA)