Methylmalonic Acid in SeraFlx BIOMATRIX on Force C18 by LC-MS/MS

	Peaks	t_R (min)	Precursor Ion	Product Ion
1.	Succinic acid	1.69	117.3	73.1
2.	Methyl-D3-malonic acid	2.02	120.2	76.2
3.	Methylmalonic acid	2.03	117.3	73.1

Column

Force C18 (cat.# 963431E)

Dimensions:

100 mm x 3.0 mm ID

Particle Size:

3 µm

Pore Size:

100 Å

Guard Column:

Force C18 EXP guard column cartridge 3 mm ID, (cat.# 963450253)

Temp.:

35 °C

Standard/Sample

Conc.:

50 ng/mL methylmalonic acid in SeraFlx BIOMATRIX

Inj. Vol.:

3 µL

Mobile Phase

0.5% Formic acid in water

0.5% Formic acid in methanol

Time (min)	Flow (mL/min)	%A	%B
0.00	0.7	95	5
0.50	0.7	95	5
3.00	0.7	5	95
3.01	0.7	95	5
5.00	0.7	95	5

Detector	MS/MS
Ion Mode:	ESI-
Mode:	MRM
Instrument	HPLC
Sample Preparation	100 μL of sample (SeraFlx BIOMATRIX) was aliquoted for extraction. The sample was fortified at 50 ng/mL with methylmalonic acid. It was also fortified with 5 μL of succinic acid (20 μg/mL in water, for validation purposes only). 5 μL of the internal standard (2500 ng/mL MMA-D3 in water) was added to the sample. The sample was precipitated using 300 μL of 0.5% formic acid in methanol followed by a 10 second vortex at 3000 rpm. The sample was then centrifuged at 4000 rpm for 10 minutes at 10 °C. 250 μL of the supernatant was filtered using a Thomson SINGLE StEP standard filter vial (PVDF, 0.2 μm, Restek cat.# 25895) prior to analysis.

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