|
|
Domoic Acid, Amino Acids, & Ornithine in Cell Extracts
LC-MS analysis without analyte derivatization
Click here to view
printable Application Sheet
|
Note: Domoic acid, a compound known for some 30 years, was found to have neurological effects when ingested by humans. Upon
ingestion, it causes diarrhea, dizziness, seizures, permanent loss of short-term memory and sometimes death.
|
|
Method Conditions
| Column |
Cogent Diamond Hydride™, 4µm, 100A |
| Catalog No. |
70000-15P-2 |
| Dimensions |
2.1 x 150 mm |
| Solvents |
| A: | 50% DI H2O/ 50% MeOH/ 0.1% formic acid |
| B: | Acetonitrile/ 0.1% formic acid |
|
| Gradient |
| time (min.) |
%B |
time (min.) |
%B |
| 0 |
95 |
10 |
20 |
| 7 |
20 |
11 |
95 |
|
| Post Time |
5 min |
| Injection Vol. |
1 microL |
| Flow rate |
0.4 mL/min. |
| Detection |
ESI – POS - Agilent 6210 MSD TOF mass spectrometer |
| Sample |
Methanolic extracts of proprietary cell cultures A and B, isolated by filtration |
| Peak |
1. Domoic acid 312.1442 m/z (M +H)+
2. L-aspartic acid 134.0453 m/z (M +H)+
3. L-asparagine 133.0608 m/z (M +H)+
4. Ornithine 133.0972 m/z (M +H)+ |
| t0 |
0.9 min |
Discussion
The potent toxin domoic acid (DA) is a highly polar compound which is poorly retained on most reversed phase columns. When a Diamond
Hydride™ column was used, not only DA was retained but also two isobaric compounds (L-asparagine and ornithine) were retained and separated.
In addition, the peak obtained for aspartic acid was very symmetrical. From these chromatograms, it is clear that the two cell extracts show
differences in the ratios between the analyzed compounds (Figure A vs. B). The presented method can be used in studies of cell extracts. In
addition, it is robust and reproducible. Cogent columns require a very short equilibration time between gradient runs.The developed method based
on ANPHPLC and MS detection does not require derivatization of DA, amino acids or ornithine.
|
