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Chromatographic Separation of Methionine Pathway Metabolites and Methylmalonic Acid in Plasma on Raptor Polar X

LC_CF0787
PeakstR (min)Precursor IonProduct Ion 1Product Ion 2Polarity
1.L-Methionine0.75150.0104.1+
2.L-Homocysteine0.81136.290.1118.1+
3.L-Cysteine1.00122.176.0+
4.Succinic acid1.33117.072.955.1-
5.dl-Cystathionine1.45221.3119.8113.9-
6.Methylmalonic acid2.92117.055.172.9-
ColumnRaptor Polar X (cat.# 9311A12)
Dimensions:100 mm x 2.1 mm ID
Particle Size:2.7 µm
Pore Size:90 Å
Guard Column:Raptor Polar X EXP Guard Column Cartridge 5 mm, 2.1 mm ID, 2.7 µm (cat.# 9311A0252)
Temp.:40 °C
Standard/Sample
Conc.:100 ng/mL
Inj. Vol.:5 µL
Mobile Phase
A:Water, 0.5% formic acid
B:Acetonitrile
Time (min)Flow (mL/min)%A%B
0.000.61585
1.000.64555
3.000.69010
3.010.61585
4.000.61585
DetectorSCIEX 4500
Ion Source:Electrospray
Ion Mode:ESI+/ESI-
InstrumentShimadzu Nexera X2
Sample PreparationA 100 ng/mL standard mix of all of the analytes on this method was prepared in plasma. A 100 µL aliquot was taken from the standard and mixed with 20 µL of 0.5 M DTT. The Sample was vortexed for 10 seconds and then left to incubate at room temperature in darkness for 30 minutes. After 30 minutes, 300 µL of methanol was added, the sample was vortexed for 10 seconds, and then centrifuged for 10 minutes at 4000 rpm. 100 µL of the supernatant was added to a 2 mL vial (cat.# 21142) containing a vial insert (cat. # 21776) capped with a short screw cap (cat.# 24498), and injected for LC-MS/MS analysis.