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

	Peaks	t_R (min)	Precursor Ion	Product Ion 1	Product Ion 2	Polarity
1.	L-Methionine	0.75	150.0	104.1	-	+
2.	L-Homocysteine	0.81	136.2	90.1	118.1	+
3.	L-Cysteine	1.00	122.1	76.0	-	+
4.	Succinic acid	1.33	117.0	72.9	55.1	-
5.	DL-Cystathionine	1.45	221.3	119.8	113.9	-
6.	Methylmalonic acid	2.92	117.0	55.1	72.9	-

Column

Raptor Polar X (cat.# 9311A52)

Dimensions:

50 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

Water, 0.5% formic acid

Acetonitrile

Time (min)	Flow (mL/min)	%A	%B
0.00	0.6	15	85
1.00	0.6	45	55
3.00	0.6	90	10
3.01	0.6	15	85
4.00	0.6	15	85

Detector	SCIEX 4500
Ion Source:	Electrospray
Ion Mode:	ESI+/ESI-
Instrument	Shimadzu Nexera X2
Sample Preparation	A 100 ng/mL standard mix of all of the analytes in this method was prepared in plasma. A 100 µL aliquot was taken from the standard and mixed with 20 µL of 0.5 M dithiothreitol (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, and 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) and capped with a short screw cap (cat.# 24498).

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