Arginine Lysine and Histidine Analyzed with LCMS - AppNote
May 15, 2012
/
/
/
Date: 15-MAY-2012 Last Updated: 31-AUGUST-2026
Basic Amino Acids In Synthetic or Human Urine Can be Analyzed
A “Cleanup” procedure for the isolation of the Basic Amino Acids in Urine was developed in this Method and no derivatization procedure was used. Three Basic Amino Acids were Separated using an inverse gradient such as HILIC Chromatography.The advantages of this Method are: (1) Isolation and stable recovery (>95%) of the desired Basic Amino Acids, (2) Sensitivity of detection (low pico mole range), (3) Complete resolution of non-derivatized Amino Acids and (4) Low amount of Sample required for Analysis.
The “cleanup” procedure used proved additionally advantageous by eliminating the use of C-18 Solid Phase Extraction Columns normally required by techniques described in the Literature.
1. L – Arginine 175 m/z RT = 12.83 minutes
2. L – Lysine 147 m/z RT = 13.49 minutes
3. L - Histidine 156 m/z 14.42 minutes
Column: Cogent Diamond Hydride™, 4 μm, 100 Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150 mm
Mobile Phase:
-- A: DI Water / 0.1% Formic Acid
-- B: 95% Acetonitrile / 0.1% Formic Acid / 0.005% Trifluoroacetic Acid (TFA)
Gradient:
| Time (minutes) | %B |
| 0 | 100 |
| 5 | 100 |
| 6 | 95 |
| 7 | 95 |
| 9 | 85 |
| 10 | 85 |
| 12 | 70 |
| 12.1 | 100 |
Flow rate: 0.4 mL / minute
Detection: ESI – pos - Agilent 6210 MSD TOF Mass Spectrometer
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 50:50 Acetonitrile / DI Water / 0.1% Formic Acid.
Notes: 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).
