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

LC_CF0717
PeakstR (min)Precursor IonProduct Ion
1.Succinic acid1.69117.373.1
2.Methyl-D3-malonic acid2.02120.276.2
3.Methylmalonic acid2.03117.373.1
ColumnForce 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
Sample
Conc.: 50 ng/mL methylmalonic acid in SeraFlx BIOMATRIX
Inj. Vol.:3 µL
Mobile Phase
A:0.5% Formic acid in water
B:0.5% Formic acid in methanol
Time (min)Flow (mL/min)%A%B
0.000.7955
0.500.7955
3.000.7595
3.010.7955
5.000.7955
DetectorMS/MS
Ion Mode:ESI-
Mode:MRM
InstrumentHPLC
Notes100 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 (2,500 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 3,000 rpm. The sample was then centrifuged at 4,000 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.