Merck
  • Home
  • Search Results
  • Accurate and reliable quantification of 25-hydroxy-vitamin D species by liquid chromatography high-resolution tandem mass spectrometry.

Accurate and reliable quantification of 25-hydroxy-vitamin D species by liquid chromatography high-resolution tandem mass spectrometry.

Journal of lipid research (2015-04-03)
Gerhard Liebisch, Silke Matysik
ABSTRACT

In general, mass spectrometric quantification of small molecules in routine laboratory testing utilizes liquid chromatography coupled to low mass resolution triple-quadrupole mass spectrometers (QQQs). Here we introduce high-resolution tandem mass spectrometry (quadrupole-Orbitrap) for the quantification of 25-hydroxy-vitamin D [25(OH)D], a marker of the vitamin D status, because the specificity of 25(OH)D immunoassays is still questionable and mass spectrometric quantification is becoming increasingly important. Liquid chromatography coupled to high-resolution tandem mass spectrometry (LC-MS/HR-MS) was used to quantify 25-hydroxy-cholecalciferol [25(OH)D3], 25-hydroxy-ergocalciferol [25(OH)D2], and their C3-epimers 3-epi-25(OH)D3 and 3-epi-25(OH)D2. The method has a run time of 5 min and was validated according to the US Food and Drug Administration and the European Medicines Agency guidelines. High mass resolution was advantageously applied to separate a quasi-isobaric interference of the internal standard D6-25(OH)D2 with 3-epi-25(OH)D3. All analytes showed an imprecision of below 10% coefficient of variation (CV), trueness between 90% and 110%, and limits of quantification below 10 nM. Concentrations measured by LC-MS/HR-MS are in good agreement with those of the National Institute of Standards and Technology reference methods using LC-MS/MS (QQQ). In conclusion, quantification of 25(OH)D by LC-MS/HR-MS is applicable for routine testing and also holds promise for highly specific quantification of other small molecules.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
2-Propanol, electronic grade, 99.999% trace metals basis
Sigma-Aldrich
2-Propanol, BioReagent, for molecular biology, ≥99.5%
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, ACS spectrophotometric grade, 95.0%
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5%
Sigma-Aldrich
2-Propanol, ACS reagent, ≥99.5%
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, meets USP testing specifications
Sigma-Aldrich
Formic acid, ≥95%, FCC, FG
Sigma-Aldrich
2-Methylheptane, 98%
Sigma-Aldrich
2-Propanol, anhydrous, 99.5%
Sigma-Aldrich
Methanol-12C, 99.95 atom % 12C
Sigma-Aldrich
2-Propanol, BioUltra, for molecular biology, ≥99.5% (GC)
Sigma-Aldrich
2-Propanol, HPLC Plus, for HPLC, GC, and residue analysis, 99.9%, poly coated bottles
Supelco
Methanol solution, contains 0.10 % (v/v) formic acid, UHPLC, suitable for mass spectrometry (MS), ≥99.5%
Supelco
Ethanol standards 10% (v/v), 10 % (v/v) in H2O, analytical standard
Sigma-Aldrich
Ammonium acetate solution, for molecular biology, 7.5 M
Sigma-Aldrich
Ammonium acetate, reagent grade, ≥98%
Sigma-Aldrich
Methanol, anhydrous, 99.8%
Sigma-Aldrich
Chloroform, ACS reagent, ≥99.8%, contains amylenes as stabilizer
Supelco
Chloroform, suitable for HPLC, ≥99.8%, contains 0.5-1.0% ethanol as stabilizer
Sigma-Aldrich
Chloroform, anhydrous, ≥99%, contains 0.5-1.0% ethanol as stabilizer
Sigma-Aldrich
Isopropyl alcohol, ≥99.7%, FCC, FG
Sigma-Aldrich
Ammonium acetate, 99.999% trace metals basis
Sigma-Aldrich
Chloroform, ACS reagent, ≥99.8%, contains 0.5-1.0% ethanol as stabilizer
Sigma-Aldrich
o-Cresol, ≥99%
Sigma-Aldrich
Ammonium acetate, for molecular biology, ≥98%
Sigma-Aldrich
Ammonium acetate, BioXtra, ≥98%
Sigma-Aldrich
Chloroform, anhydrous, contains amylenes as stabilizer, ≥99%
Sigma-Aldrich
Methanol, HPLC Plus, ≥99.9%, poly-coated bottles
Sigma-Aldrich
Chloroform, ≥99%, PCR Reagent, contains amylenes as stabilizer