title: Measurement of total homocysteine in plasma and blood spots using liquid chromatography-tandem mass spectrometry: comparison with the plasma Abbott IMx method.
abstract: BACKGROUND
  Current sampling for total homocysteine (tHcy) is problematic, requiring plasma separation within 15 min. The aim of this study was to develop a liquid chromatographic-tandem mass spectrometric (LC-MS/MS) method for the measurement of tHcy in plasma and dried blood spots and to determine whether the dried blood spot concentration could be used to predict plasma concentrations of tHcy.
  
  
  METHODS
  LC-MS/MS methodology was optimized to measure tHcy in plasma and dried blood spots. Fifty blood samples collected from heart transplant patients were used to form dried blood spots and for plasma analysis. Plasma tHcy was also measured using the Abbott IMx method and values were compared to the tHcy concentrations determined in plasma and dried blood spots using LC-MS/MS methodology.
  
  
  RESULTS
  The plasma tHcy LC-MS/MS results compared well with the IMx values: LC-MS/MS=1.18(IMx)-0.44 (r(2)=0.915). The within-batch precision (n =10) of the plasma LC-MS/MS method was < 2.0% at 14.6 and 37.7 micromol/L, respectively; the between-batch precision (n=10) was 5.0 and 8.0%, respectively, at these concentrations. The method was found to be sensitive down to 1 micromol/L and linear up to at least 100 micromol/L. Dried blood spot LC-MS/MS results were considerably lower than the plasma IMx values (P < 0.0001): dried blood spot LC-MS/MS=0.33IMx+1.77 (r(2)=0.682). The within-batch precision (n=20) of the dried blood spot LC-MS/MS method was 7.3% and 4.7% at concentrations of 4.0 and 7.9 micromol/L, respectively; the between-batch precision was 12.6% and 7.9% at concentrations of 5.1 and 8.0 micromol/L, respectively. To assess whether dried blood spots are suitable as a screening test to predict plasma tHcy concentrations, arbitary cut-off levels were compared. If it is assumed that a plasma tHcy concentration of >15 micromol/L is raised, a dried blood spot result of >6.8 micro mol/L has a sensitivity and specificity in detecting a raised plasma tHcy of 83.3% and 96.2%, respectively, and a positive and negative predictive value of 95% and 86%, respectively, with an efficiency of 90%. Use of a dried blood spot cut-off concentration of 6.2 micromol/L for predicting high plasma tHcy concentrations (above 15 micromol/L) has a sensitivity and specificity of 95.8% and 73.1%, respectively, positive and negative predictive values of 76% and 95%, respectively, and an efficiency of 84%.
  
  
  CONCLUSIONS
  We have developed a precise and accurate LC-MS/MS method for measuring plasma tHcy concentrations, which uses a small volume of plasma and is suitable for routine use. A satisfactory LC-MS/MS method for the measurement of tHcy in dried blood spots was also developed; this method might be useful in routine screening for raised plasma concentrations of tHcy.
authors.affiliation: Department of Clinical Biochemistry, Wythenshawe Hospital, Wythenshawe, Manchester M23 9LT, UK. steve.mccann@smuht.nwest.nhs.uk
authors.forename: Steven J
authors.initials: SJ
authors.lastname: McCann
authors.forename: Scott
authors.initials: S
authors.lastname: Gillingwater
authors.forename: Brian G
authors.initials: BG
authors.lastname: Keevil
authors.forename: Donald P
authors.initials: DP
authors.lastname: Cooper
authors.forename: Michel R
authors.initials: MR
authors.lastname: Morris
mesh_terms: D002624:Chemistry, Clinical / Q000379:methods; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid / Q000379:methods*; D006710:Homocysteine / Q000097:blood*; D006801:Humans; D007118:Immunoassay / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods*; D010949:Plasma; D012044:Regression Analysis; D013997:Time Factors
chemical_list: D006710:Homocysteine