Quantitative analysis of s-adenosylmethionine and s-adenosylhomocysteine in neurulation-stage mouse embryos by liquid chromatography tandem mass spectrometry

Abstract

The potential importance of the methylation cycle during embryonic development necessitates the establishment of methodology to detect alterations in the relative abundance of s-adenosylmethionine (SAM) and s-adenosylhomocysteine (SAH) in an embryonic experimental system. We have developed a precise and sensitive method for measurement of SAM and SAH based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in single neurulation-stage mouse embryos. Use of a penta-fluorinated high-performance liquid chromatography (HPLC) stationary phase gave enhanced sensitivity due to optimal ionisation in organic mobile phase and increased retention time compared to standard reversed-phase separation. Calibration curves suitable for the analysis of neurulation-stage mouse embryos (SAM 0.02-25.0microM, SAH 0.01-10.0microM) were linear (r(2)>0.997) with limits of detection for SAM and SAH of 10 and 2.5nmol/L, respectively.

Fig. 1

Product ion spectra of protonated molecules used for quantification. Precursor ions were at m/z 401.2 for [²H₃]-SAM (a), 399.2 for SAM (b) and 385.2 for SAH (c). In each case the MS/MS conditions were optimised to favour the transition to a major product ion at m/z 136.1, thought to correspond to adenine [13].

Fig. 2

LC-MS/MS chromatograms obtained in SRM mode. Representative chromatograms are shown for, (a) 1 µmol/L aqueous standards and, (b) endogenous SAM and SAH in an E10.5 mouse embryo, containing measured concentrations of 0.766 µmol/L SAM and 0.121 µmol/L SAH. Retention times are indicated above peaks.

Fig. 3

Calibration curves of peak-area ratios plotted against SAM (a) and SAH (b) concentration. Calibrators were made up in a pooled sample of homogenised embryos with 1.0 µmol/L of [²H₃]-SAM (internal standard) added to each sample. SAM data points correspond to 0.5, 1, 2.5, 5, 10, 25 µM of SAM and the SAH data points correspond to 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 µM. Each sample was run in triplicate (n=3).