Unique Method Development Strategy for Polar Compounds – AppNote

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ARTICLES, APPNOTES, FAQ (9187)
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May 17, 2012
Bill Ciccone

AppNotes ANP/HILIC Methods / AppNotes Cogent Diamond Hydride / AppNotes Food and Beverages / AppNotes Library / ARTICLES, APPNOTES, FAQ

Anatoxin-a Analyzed with LCMS

Figures below illustrate an example of the work flow in developing a Method for the Analysis of a Polar Compound using Cogent Diamond Hydride™ Columns and the impact of Aqueous Normal Phase HPLC.

The simplified steps of Method Development are as follows:
—A. Injection of the Sample at 50:50 Solvent A / Solvent B Mobile Phase Composition results in Figure A. In this case, Anatoxin-a has considerable retention.
—B. Injection of the Sample at 40:60 Solvent A / Solvent B Mobile Phase Composition results in Figure B. As expected, the Retention of Anatoxin-a is longer and the Peak Shape is broader with higher organic content.
—C. Based on the above results, a simple Linear Gradient is designed to achieve the desired Retention of the Compound and excellent Peak Shape (Figure C). If shorter Retention time is desired it can be accomplished by changing the starting concentration of Solvent B to 60%, designing a steeper gradient, or using a shorter Column such as 2.1 x 50mm.

Peak:
Anatoxin-a, 166.1226 m/z (M+H)+

Method Conditions
Column: Cogent Diamond Hydride™, 4μm, 100Å
Catalog No.: 70000-15P-2
Dimensions: 2.1 x 150mm
Mobile Phase:
—A: 50% Methanol / 50% DI Water / 0.1% Formic Acid
—B: Acetonitrile / 0.1% Formic Acid
Gradient:

Time (minutes)	%B
0	70
5	30
6	30
7	70

Temperature: 25˚C
Post time: 5 minutes
Injection vol.: 1μL
Flow rate: 0.4mL / minute
Detection: ESI – POS – Agilent 6210 MSD TOF Mass Spectrometer
t0: 0.9 minutes

Note: Anatoxin-a (ANTX-A) is a cyanobacterial neurotoxin, implicated in many animal and human poisoning incidents. ANTX-A blocks neurotransmission causing death by respiratory arrest. The presence of this toxin in freshwater has to be monitored in order to prevent fatalities.

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