Chromatography Forum

I am having problem with the reproducibility of LC/MS signals. I use API 5500. I analyze the stability of small drug molecules (MW 200-1000 dalton) in blood or plasma after incubation at 37C for a period of time. The sample was prepared by quenching 20ul blood or plasma using 600ul mixture of Acetonitrile and methanol (50:50). After centrifugation at 4000 rpm for 25 minutes, 70ul supernatant was removed and mixed with 210ul water. Then the sample is ready for injection on LC/MS/MS.

For most of the compounds, I got good results. However, some compounds have bad reproduciabilities. When I repeat 15 injections from the same blood or plasma samples, I see decreasing trend in the MS responses. Can anyone tell me what can be the possible reasons? I know it can be due to matrix effect. Can it be due to dirty MS source? Thanks in advance.

Have you tried something like extracting a blood sample, (using your 50/50 ACN/MeOH ppt) leaving it on the benchtop for about an hour or two, then extracting another sample from the same blood sample, and injecting the 2 hour old ample, then the fresh extract?
In other words, does your analyte/sample have stability over the period you are assessing it?

Alp

Sorry for the delay in my response. Our area was hit by thunderstorm. We lost power for four days. Stability was not an issue for the compounds having decreasing trend. I know those compounds are stable. Sometimes I also see increasing trend. For example, carbutamide (my internal standard) has good reproducibility in positive mode. But sometimes when I switch to negative mode, it shows increasing trend.

I am wondering what are the possibilities that cause this kind of problem. I know matrix effect can, what else? Thanks.