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Basic Amino Acids
In Synthetic or Human Urine
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printable Application Sheet
Notes: The “cleanup” procedure used proved additionally advantageous by eliminating the use of C-18 solid phase extraction
columns required by techniques described in the literature. The level of amino acids in biological fluids can be correlated
with several neurological (Alzheimer’s disease, ischemic stroke and others) and metabolic disorders (argininemia,
phenyloketonuria, maple syrup urine disease and others).
Sample preparation: 400 µL of acetonitrile was added to 100 µL of synthetic or human urine and the sample was
centrifuged (3000 g). Next 20 µL of the supernatant was mixed with 10 µL of the 50% acetonitrile/50%DI water +
0.1% formic acid.
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L-Arginine, L–Lysine and L–Histidine retained and separated in ANP
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: 90% acetonitrile + 0.1% formic acid + 0.005% TFA |
| Mobile Phase |
Gradient:
| Time |
%B |
Time |
%B |
| 0.0 |
100 |
9.0 |
85 |
| 5.0 |
100 |
10.0 |
85 |
| 6.0 |
95 |
12.0 |
70 |
| 7.0 |
95 |
12.1 |
100 |
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| Post Time |
5 min. |
| Flow Rate |
0.4 mL/min. |
| Sample |
Synthetic human urine |
| Peaks |
1. L – Arginine 175 m/z RT = 12.83 min
2. L – Lysine 147 m/z RT = 13.49 min
3. L - Histidine 156 m/z 14.42 min |
| Detection |
ESI – neg - Agilent 6210 MSD TOF mass spectrometer |
Discussion
A “cleanup” procedure for the isolation of the basic amino acids was used. No derivatization procedure was used. Three basic amino
acids were separated using gradient Aqueous Normal Phase (ANP) chromatography.
The advantages of this method are: (1) isolation and stable recovery (>95%) of the desired basic amino acids, (2) sensitivity of
detection (low pmol range), (3) complete resolution of non-derivatized amino acids via ANP LCMS and (4) limited amount of sample
required for analysis.
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