Unstable Norepinephrine and Epinephrine Peaks - Large Spikes
Posted: Tue Jan 03, 2012 8:59 pm
First time on here. I am a grad student working in a lab and was tasked with trying to get a new HPLC going, including the extraction procedure, to analyze human plasma samples of rest and exercise conditions. I am looking at norepinephrine and epinephrine in the analysis and it appears that I am getting intermittent large spikes for both norepinehprhrine and epinephrine. The retention times are right on for both catecholamines, but the voltage reading of the peaks are sometimes very high, such as a resting sample is 100x larger than an exercise sample, which isn't possible. I have two of these machines and both read the injected sample identically, so I know that it probably doesn't have to do with the HPLCs, but probably more with the extraction procedure. Also, I can run extracted standards all day long with no issues (all peak heights are stable and reproducible). It is only when I inject extracted plasma samples that I get these higher-than-normal readings. This led me to believe that the interaction of plasma and some component in the extraction process is having some type of influence on the pH or voltage reading. My procedure is as follows:
1) 30 mg alumina, 200 ul plasma, 1 ml Tris (add all components to a 1.5 ml eppendorf tube).
2) Inverting rocker for 30 mins. at room temp. to bind catecholamines to alumina.
3) 3 washes of the alumina with DI water (1 ml DI water per each wash).
4) Add 300 ul DI water to the tube and transfer the alumina and 300 ul water to filter cup.
5) Spin for 3 mins.
6) Transfer filter cup with dried alumina to another eppendorf tube, then add 200 ul of 4% acetic acid.
7) Inverting rocker for 10 mins. at room temp.
Spin for 3 minutes.
9) Inject 15 ul of sample.
I have tried eluting with both acetic acid and perchloric acid and still obtain similar results. It also seems that epinephrine is much less stable and more prone to reading at a high voltage.
Any ideas or recommendations on extraction procedure or what component in the extraction process may be causing the electrochemical detector to read much higher than normal?
Thanks. I appreciate all the help I can get.
1) 30 mg alumina, 200 ul plasma, 1 ml Tris (add all components to a 1.5 ml eppendorf tube).
2) Inverting rocker for 30 mins. at room temp. to bind catecholamines to alumina.
3) 3 washes of the alumina with DI water (1 ml DI water per each wash).
4) Add 300 ul DI water to the tube and transfer the alumina and 300 ul water to filter cup.
5) Spin for 3 mins.
6) Transfer filter cup with dried alumina to another eppendorf tube, then add 200 ul of 4% acetic acid.
7) Inverting rocker for 10 mins. at room temp.
Spin for 3 minutes.9) Inject 15 ul of sample.
I have tried eluting with both acetic acid and perchloric acid and still obtain similar results. It also seems that epinephrine is much less stable and more prone to reading at a high voltage.
Any ideas or recommendations on extraction procedure or what component in the extraction process may be causing the electrochemical detector to read much higher than normal?
Thanks. I appreciate all the help I can get.