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Determination of S-Adenosylmethionine and S-Adenosylhomocysteine by LC-MS/MS and evaluation of their stability in mice tissues.

Krijt J, Dutá A, Kozich V - J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2009)

Bottom Line: No significant matrix effects were observed by analysing the tissue extracts on LC-MS/MS.Storage of liver tissues at -80 degrees C for 2 months resulted in decrease of SAM/SAH ratio by 40%.These results demonstrate that preanalytical steps are critical for obtaining valid data of SAM and SAH in tissues.

View Article: PubMed Central - PubMed

Affiliation: Institute of Inherited Metabolic Disorders, Charles University in Prague - 1st Faculty of Medicine, Prague, Czech Republic. jkrijt@LF1.cuni.cz

ABSTRACT
S-Adenosylmethionine (SAM) serves as a methyl donor in biological transmethylation reactions. S-Adenosylhomocysteine (SAH) is the product as well as the inhibitor of transmethylations and the ratio SAM/SAH is regarded as the measure of methylating capacity ("methylation index"). We present a rapid and sensitive LC-MS/MS method for SAM and SAH determination in mice tissues. The method is based on chromatographic separation on a Hypercarb column (30 mm x 2.1 mm, 3 microm particle size) filled with porous graphitic carbon stationary phase. Sufficient retention of SAM and SAH on the chromatographic packing allows simple sample preparation protocol avoiding solid phase extraction step. No significant matrix effects were observed by analysing the tissue extracts on LC-MS/MS. The intra-assay precision was less than 9%, the inter-assay precision was less than 13% and the accuracy was in the range 98-105% for both compounds. Stability of both metabolites during sample preparation and storage of tissue samples was studied: the SAM/SAH ratio in liver samples dropped by 34% and 48% after incubation of the tissues at 4 degrees C for 5 min and at 25 degrees C for 2 min, respectively. Storage of liver tissues at -80 degrees C for 2 months resulted in decrease of SAM/SAH ratio by 40%. These results demonstrate that preanalytical steps are critical for obtaining valid data of SAM and SAH in tissues.

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Chromatograms of SAM and SAH in mouse liver. The panels show selected reaction monitoring of the transitions m/z 399.3 → 250.3 for SAM (a); 402.3 → 250.3 for [2H3]-SAM (b); 385.3 → 136.3 for SAH (c) and 390.3 → 136.3 for [13C5]-SAH (d).
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fig1: Chromatograms of SAM and SAH in mouse liver. The panels show selected reaction monitoring of the transitions m/z 399.3 → 250.3 for SAM (a); 402.3 → 250.3 for [2H3]-SAM (b); 385.3 → 136.3 for SAH (c) and 390.3 → 136.3 for [13C5]-SAH (d).

Mentions: S-Adenosylmethionine is a polar compound and as such it is poorly retained on standard reversed-phase columns. High water content in the mobile phase and the presence of interfering substances close to the hold-up time (t0) result in poor ionization efficiency in the mass spectrometric detection. Some authors use ion-pairing additives in the mobile phase to improve the retention and also to increase the content of organic solvent in the mobile phase, however, these additives may cause loss of sensitivity on mass spectrometer [31]. We aimed at developing a simple LC method for separation of SAM and SAH without the use of ion-pairing reagents in the mobile phase. From the several tested columns we selected a column packed with porous graphitic carbon, which is a strong adsorbent offering retention mechanisms different from C18 stationary phases [32]. SAM was retained on the PGC column washed with 0.1% formic acid and was eluted by increasing the concentration of acetonitrile in the mobile phase. At 20% acetonitrile the retention of SAM dropped below 1 min, which we regarded as insufficient retention considering possible ionization suppression by compounds eluting at the t0. Because the retention factor of SAH was too high (more than 15 min) at acetonitrile concentrations lower than 20% in the mobile phase, we finally adopted a gradient elution to keep the retention of both metabolites in optimal range. After optimization of chromatographic conditions, the retention times of SAM and SAH were 5.9 and 7.8 min, respectively. The total chromatographic run time including column regeneration was 15 min. Typical chromatogram of mouse liver extract is shown in Fig. 1.


Determination of S-Adenosylmethionine and S-Adenosylhomocysteine by LC-MS/MS and evaluation of their stability in mice tissues.

Krijt J, Dutá A, Kozich V - J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. (2009)

Chromatograms of SAM and SAH in mouse liver. The panels show selected reaction monitoring of the transitions m/z 399.3 → 250.3 for SAM (a); 402.3 → 250.3 for [2H3]-SAM (b); 385.3 → 136.3 for SAH (c) and 390.3 → 136.3 for [13C5]-SAH (d).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2724122&req=5

fig1: Chromatograms of SAM and SAH in mouse liver. The panels show selected reaction monitoring of the transitions m/z 399.3 → 250.3 for SAM (a); 402.3 → 250.3 for [2H3]-SAM (b); 385.3 → 136.3 for SAH (c) and 390.3 → 136.3 for [13C5]-SAH (d).
Mentions: S-Adenosylmethionine is a polar compound and as such it is poorly retained on standard reversed-phase columns. High water content in the mobile phase and the presence of interfering substances close to the hold-up time (t0) result in poor ionization efficiency in the mass spectrometric detection. Some authors use ion-pairing additives in the mobile phase to improve the retention and also to increase the content of organic solvent in the mobile phase, however, these additives may cause loss of sensitivity on mass spectrometer [31]. We aimed at developing a simple LC method for separation of SAM and SAH without the use of ion-pairing reagents in the mobile phase. From the several tested columns we selected a column packed with porous graphitic carbon, which is a strong adsorbent offering retention mechanisms different from C18 stationary phases [32]. SAM was retained on the PGC column washed with 0.1% formic acid and was eluted by increasing the concentration of acetonitrile in the mobile phase. At 20% acetonitrile the retention of SAM dropped below 1 min, which we regarded as insufficient retention considering possible ionization suppression by compounds eluting at the t0. Because the retention factor of SAH was too high (more than 15 min) at acetonitrile concentrations lower than 20% in the mobile phase, we finally adopted a gradient elution to keep the retention of both metabolites in optimal range. After optimization of chromatographic conditions, the retention times of SAM and SAH were 5.9 and 7.8 min, respectively. The total chromatographic run time including column regeneration was 15 min. Typical chromatogram of mouse liver extract is shown in Fig. 1.

Bottom Line: No significant matrix effects were observed by analysing the tissue extracts on LC-MS/MS.Storage of liver tissues at -80 degrees C for 2 months resulted in decrease of SAM/SAH ratio by 40%.These results demonstrate that preanalytical steps are critical for obtaining valid data of SAM and SAH in tissues.

View Article: PubMed Central - PubMed

Affiliation: Institute of Inherited Metabolic Disorders, Charles University in Prague - 1st Faculty of Medicine, Prague, Czech Republic. jkrijt@LF1.cuni.cz

ABSTRACT
S-Adenosylmethionine (SAM) serves as a methyl donor in biological transmethylation reactions. S-Adenosylhomocysteine (SAH) is the product as well as the inhibitor of transmethylations and the ratio SAM/SAH is regarded as the measure of methylating capacity ("methylation index"). We present a rapid and sensitive LC-MS/MS method for SAM and SAH determination in mice tissues. The method is based on chromatographic separation on a Hypercarb column (30 mm x 2.1 mm, 3 microm particle size) filled with porous graphitic carbon stationary phase. Sufficient retention of SAM and SAH on the chromatographic packing allows simple sample preparation protocol avoiding solid phase extraction step. No significant matrix effects were observed by analysing the tissue extracts on LC-MS/MS. The intra-assay precision was less than 9%, the inter-assay precision was less than 13% and the accuracy was in the range 98-105% for both compounds. Stability of both metabolites during sample preparation and storage of tissue samples was studied: the SAM/SAH ratio in liver samples dropped by 34% and 48% after incubation of the tissues at 4 degrees C for 5 min and at 25 degrees C for 2 min, respectively. Storage of liver tissues at -80 degrees C for 2 months resulted in decrease of SAM/SAH ratio by 40%. These results demonstrate that preanalytical steps are critical for obtaining valid data of SAM and SAH in tissues.

Show MeSH
Related in: MedlinePlus