Quantitative Analysis of Total Plasma Homocysteine by LC‐MS/MS

Libin Yuan1, J. Daniel Sharer2

1 Department of Human Genetics, Emory University School of Medicine, Decatur, Georgia, 2 Department of Genetics, University of Alabama-Birmingham School of Medicine, Birmingham, Alabama
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 17.21
DOI:  10.1002/0471142905.hg1721s89
Online Posting Date:  April, 2016
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Homocysteine is a nonessential, sulfur‐containing amino acid involved in one‐carbon (folate) metabolism. A number of inherited and acquired conditions cause increased accumulation of this metabolite in blood (homocysteinemia) and other biofluids. Homocysteinemia is a risk factor for cardiovascular disease, including recurrent thrombosis. Accurate measurement of total plasma homocysteine is an important element in the diagnostic evaluation of these disorders. While a number of different methods have been developed for this purpose, the focus of this unit will be on a specific technique utilizing liquid chromatography‐tandem mass spectrometry, which provides several advantages in terms of speed, sensitivity, and specificity. © 2016 by John Wiley & Sons, Inc.

Keywords: homocysteine; homocystine; homocysteinemia/homocystinuria; methionine synthase; cardiovascular disease; cystathionine β‐synthase; folate; cobalamin; HPLC‐MS/MS

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Table of Contents

  • Basic Protocol 1:  
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1:  

  • Acetonitrile (Fisher Scientific)
  • DL‐Homocystine (3,3,3′,3′,4,4,4′,4′‐D8, 98%; Cambridge Isotopes Laboratories, cat. no. DLM‐3619‐1)
  • Internal standard working solution (see recipe)
  • Human serum protein calibrator matrix (Monobind)
  • 10 mM homocysteine (Hcys) stock solution (see recipe)
  • 0.5 M DL‐dithiothreitol (DTT; dissolve 38.56 mg DTT [Sigma‐Aldrich] in 0.5 ml Ultrapure water by vortexing 1 min; make fresh for every run)
  • Formic acid (Sigma‐Aldrich)
  • Costar 96‐well polypropylene microtiter plates (Fisher Scientific)
  • 1.5‐ml centrifuge tube (Fisher Scientific)
  • Aluminum foil (Fisher Scientific)
  • Supelcosil LC‐CN HPLC (4.6 × 33 mm column; Sigma‐Aldrich)
NOTE: Quality controls (QC), calibration standards, patient specimens, and working solutions of standards and internal standards should be removed (as needed) from the −20°C freezer at least 30 min prior to preparation and thawed at room temperature. Both low‐level and high‐level QCs should be included with each assay.
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Literature Cited

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