title: Mechanism of error caused by isotope-labeled internal standard: accurate method for simultaneous measurement of vitamin D and pre-vitamin D by liquid chromatography/tandem mass spectrometry.
abstract: BACKGROUND
  Bias of up to 25% has been observed for vitamin D3 and D2 samples exposed to heating during sample preparation, even when isotope-labeled internal standards are used. The goals of this study were to identify the mechanism of the positive bias observed in measuring vitamin D3 and D2 by liquid chromatography/tandem mass spectrometry (LC/MS/MS) and determine a way to eliminate the error source.
  
  
  METHODS
  Several internal standards with varying locations of labeling were used for comparison in this study. Additionally, different temperatures (25, 37, 55, and 75 °C) and different treatment times were investigated for sample preparation and a LC/MS/MS method capable of simultaneously measuring vitamin D and pre-vitamin D was developed.
  
  
  RESULTS
  It was demonstrated that the different conversion behaviors of the analyte and the internal standard were the cause of the positive bias. This bias was eliminated when internal standards with labeling remote from the double-bond area of the molecules were used. Additionally, sample preparation was shortened from overnight saponification at room temperature to 0.5 h at 75 °C.
  
  
  CONCLUSIONS
  The use of an internal standard with labeling remote from the conjugated area eliminated the error source and gave accurate correction at all of the temperatures investigated. Heating may be used for rapid sample preparation as an alternative to overnight saponification at room temperature. This work not only describes the mechanism of an inaccurate internal standard correction, but also establishes a rapid LC/MS/MS method for simultaneous measurement of vitamin D and pre-vitamin D.
authors.affiliation: Aegis Sciences Corporation, 365 Great Circle Road, Nashville, TN, 37228, USA.
authors.forename: Min
authors.initials: M
authors.lastname: Huang
authors.forename: Amy B
authors.initials: AB
authors.lastname: Cadwallader
authors.forename: Rebecca
authors.initials: R
authors.lastname: Heltsley
mesh_terms: D002762:Cholecalciferol / Q000032:analysis* / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D003903:Deuterium / Q000032:analysis / Q000737:chemistry; D004872:Ergocalciferols / Q000032:analysis* / Q000737:chemistry; D006801:Humans; D007223:Infant; D041943:Infant Formula / Q000737:chemistry; D016014:Linear Models; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013696:Temperature
chemical_list: D004872:Ergocalciferols; D002762:Cholecalciferol; D003903:Deuterium