Stable-isotope dilution liquid chromatography-electrospray injection tandem mass spectrometry method for fast, selective measurement of S-adenosylmethionine and S-adenosylhomocysteine in plasma.
BACKGROUND
It has been postulated that changes in S-adenosylhomocysteine (AdoHcy), a potent inhibitor of transmethylation, provide a mechanism by which increased homocysteine causes its detrimental effects. We aimed to develop a rapid and sensitive method to measure AdoHcy and its precursor S-adenosylmethionine (AdoMet).


METHODS
We used stable-isotope dilution liquid chromatography-electrospray injection tandem mass spectrometry (LC-ESI-MS/MS) to measure AdoMet and AdoHcy in plasma. Acetic acid was added to prevent AdoMet degradation. Solid-phase extraction (SPE) columns containing phenylboronic acid were used to bind AdoMet, AdoHcy, and their internal standards and for sample cleanup. An HPLC C(18) column directly coupled to the LC-MS/MS was used for separation and detection.


RESULTS
In plasma samples, the interassay CVs for AdoMet and AdoHcy were 3.9% and 8.3%, and the intraassay CVs were 4.2% and 6.7%, respectively. Mean recoveries were 94.5% for AdoMet and 96.8% for AdoHcy. The quantification limits were 2.0 and 1.0 nmol/L for AdoMet and AdoHcy, respectively. Immediate acidification of the plasma samples with acetic acid prevented the observed AdoMet degradation. In a group of controls (mean plasma total Hcy, 11.2 mumol/L), plasma AdoMet and AdoHcy were 94.5 and 12.3 nmol/L, respectively.


CONCLUSIONS
Stable-isotope dilution LC-ESI-MS/MS allows sensitive and rapid measurement of AdoMet and AdoHcy. The SPE columns enable simple cleanup, and no metabolite derivatization is needed. The instability of AdoMet is a serious problem and can be prevented easily by immediate acidification of samples.
Gellekink
Henkjan
H
Laboratory of Pediatrics and Neurology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
van Oppenraaij-Emmerzaal
Dinny
D
van Rooij
Arno
A
Struys
Eduard A
EA
den Heijer
Martin
M
Blom
Henk J
HJ
D002853:Chromatography, Liquid; D003903:Deuterium; D006801:Humans; D011865:Radioisotope Dilution Technique; D012016:Reference Values; D012435:S-Adenosylhomocysteine / Q000097:blood*; D012436:S-Adenosylmethionine / Q000097:blood*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization
D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D003903:Deuterium