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Stable-Isotope Dilution Liquid Chromatography–Electrospray Injection Tandem Mass Spectrometry Method for Fast, Selective Measurement of S-Adenosylmethionine and S-Adenosylhomocysteine in Plasma

¹ Laboratory of Pediatrics and Neurology (424) and ² Departments of Endocrinology (531) and Epidemiology and Biostatistics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
³ Metabolic Unit, Department of Clinical Chemistry, VU University Medical Centre Amsterdam, Amsterdam, The Netherlands.

^aAddress correspondence to this author at: Laboratory of Pediatrics and Neurology (424), Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Fax 31-24-3688754; e-mail h.blom{at}cukz.umcn.nl.

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 C₁₈ 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 µmol/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.

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