Chromatography Forum

Good afternoon.

I've been trying to apply NMKL (NORDIC COMMITTEE ON FOOD ANALYSIS) 196 - 2013 for the detection of histamine in standart solutions.

I am reproducing exactly as mentioned. However, by increasing and decreasing the concentration of the samples, there's not a peak that reproduces the expected behavior for histamine.

What could I ve been doing wrong?

Mobile phases are Ammonium acetate 0.1M (A) and Acetonitrile (B). Flow rate 0.9 mL/min; UV detector 254 nm; gradient 50% B to 90% B in 19 minutes. My column is a C18 - 250mm*4,6mm*10um.

Derivatization is prepared with sodium hydroxide solution, saturated sodium bicarbonate, dansyl chloride solution, ammonia and ammonium acetate:acetonitrile mixture ( 4.8 ). Time and temperature for reaction are respected. Final filtration too.

Any ideas or tips? Doesnt anyone know a better method?

You need to inject your compound with and without the column and compare peak areas. If you see several peaks with the column use the sum of them. This will give you indication if your injector is working and if you are eluting everything from the column. This would be your first step.