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Determination of Important Isobaric Metabolites in Urine
A Simple & Reproducible MS Friendly Method
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printable Application Sheet
Notes: The determination of metabolites and amino acids in biological fluids is an important problem in clinical biochemistry
and analytical chemistry. Changes in the concentrations of these compounds in urine, serum and other physiological fluids
has proved to be correlated with several neurological disorders such as Alzheimer’s disease, ischemic stroke as well as with a
number of metabolic disorders such as phenylketonuria, argininemia, maple syrup urine disease and others.
Chromatogram presented is adapted from: J.J.Pesek, M.T. Matyska, S. M. Fisher, T. R. Sana, Journal of Chromatography A, 1204
(2008) p55.
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Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-15P-2 |
| Dimensions |
2.1 x 150 mm |
| Solvents |
A: DI water + 0.1% formic acid
B: acetonitrile + 0.1% formic acid |
| Gradient |
0-0.2 min 95.0% B
0.2-30 min to 50% B
30-35 min 50% B
35.1 min to 95% B |
| Post time |
5 min |
| Flow rate |
0.4 mL/min. |
| Sample Prep |
Synthetic urine: 400 microL of acetonitrile was added to 100
microL of synthetic urine and sample was centrifuged (3000 g).
Next 20 microL of the supernatant was mixed with 10 microL of
50% acetonitrile/50%DI water + 0.1% formic acid.
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| Peaks |
1. methylsuccinic acid, 133 m/z (M + H)+, RT = 2.14 min
2. glutaric acid, 133 m/z (M + H)+, RT = 4.25 min
3. oxaloacetic acid, 133 m/z (M + H)+, RT = 10.40 min
4. creatine, 133 m/z (M + H)+, RT = 12.52 min
5. 4-hydroxyproline, 133 m/z (M + H)+, RT = 13.15 min
6. D,L - asparagine, 133 m/z (M + H)+, RT = 13.62 min
7. D,L - ornithine, 133 m/z (M + H)+, RT = 21.31 min |
| Detection |
ESI – pos - Agilent 6210 MSD TOF mass spectrometer. |
Discussion
This MS friendly method demonstrates the ability of Cogent Diamond Hydride™ column to separate a wide variety of polar
metabolites using mass spectrometry for detection. When monitoring the EIC at 133 m/z, seven peaks were positively detected in a urine
sample by comparison with standards or by using accurate mass.The method described requires only a small sample volume and needs
minimal manual sample preparation. Although primarily focused on the analysis of metabolites in urine, the method could also be applied
to the determination of these compounds in other physiological or biological fluids.
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