Chromatography Forum

Hi, I'm new to this forum.. and I've got 2 questions hoping for answers.

Q1: I am running an assay for cefuroxime in plasma and facing two problems: I can't get Empower to correctly quantitate my sample peaks (although I have used it to generate a calibration curve from 9-point standards). everytime i try to qunatitate an unknown, it gives me fixed amounts for the IS, usually 1.00, and calculates an erroneous amount for cefuroxime based on quantitation of QC's. I can't get it to correctly reflect the actual amount of the IS (and not the amount pre-entered by me) based on the area its founding on each run. and I'm thinking thats the reason it won't give correct numbers for my unknown either. notice that I have:
- entered default amounts (for IS and analyte) in the processing method parameters.
- specefied that I want single internal (vs. external) calibration in the processing method wizard.
-

Q2: after running ~ 300 samples (QCs, stds, and unknown). I decided to rest my original column (for cleaning an regeneration) and use a new same column (both symmetry C8, 5um, 3.9x150). Now my new column is showing ~ 2.3 minute different RT (earlier) than my old one for cefuroxime, and just ~ 0.3 min difference for IS.. what would be a cause? proteins clogging the column? I should also note that I'm not using a guard column (didn't have any available).. but both columns were out-of-the-box. any answers will be greatly appreciated..

1) (not sure on this one) Single Internal doesn't sound like what you would want if you're calibrating against a 9 point curve. If you're getting 1.00, are you reporting the IS vs. itself?

2) You'll have to provide more details about your mobile phase composition, sample prep routine and gradient /run conditions if you want help diagnosing a column problem. If you look at the very first run on the original column, are those RTs the same as the last runs on that column, or are they pretty close to the times you see on the fresh column?

1) Not sure, but could this be an "amount" versus "concentration" issue? We have to be careful as to how we enter our stds and specify amount or concentration.

We don't use internal std methods much here, but wouldn't you expect the method to report the IS as the predefined amount, because, after all, isn't that what an IS is for? That is, the area would change from sample to sample due to varying recovery, but that recovery always signifies the amount of IS you put in. Wouldn't you have to quantitate the IS as an unknown in order for it to be reported with avarying amount?

Wish I could be of more help, but maybe this will spur some thoughts from someone else who can help.

Best of luck,
Jon

DR,
1) single internal standard is the only option regarding internal calibration I get in the processing method wizard of Empower, the other being external standard. Not sure what u mean by reporting the IS against itself, but I specify in the wizard that I'm using an IS, and it asks which component in the chromatogram represents the IS and I specify that. now when i get to quantification, it reports the IS amount as 1.00, no matter what the actual area it got in that particular run.

2) about the conditions: I'm using 12:88 ACN: 25mM phosphate buffer @ pH~3. the very first run on the column had RT's between 9-10 min. by now, (i'm in ~450th sample) its eluting at 13-14 min. still, the IS peak is sharp and defined, the drug peak has gained a very faint tail but not too obvious. I can still do the curve and stuff, but its bothering me if it keeps shiftling like that indefinetily.

JZeszotarski, can you please clarify more, isn't the IS technique supposed to calculate the area of the drug over that of the IS and report the response?
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It appears that the new column is behaving in the same way as the old column: earlier retention when not yet used much...

1) Then all is well with Empower. It doesn't care what the amoount of IS is for a given vial. It assumes that all IS concentrations are constant, then it reports the ratio of peak area/IS area for everything. In the case of IS peaks, this is always 1.0. To check your calculations, you can collect these ratios for your samples & standards and treat them exactly as you would the area counts for an external standard method.

Medkemist,

I can't really understand the description of your problem, but here's what I do when I use Internal Std quantifiation in Empower.

1. Standard - Go to the channels tab and select your standard. Then select Alter Sample. Go to the toolbar and select Amount. Then the Component Editor appears. All your ES and IS peak names should appear. If they don't, key them in, or copy them from the processing method. Find your IS peak, and key in its quantity here (say, "4.6mM") . Click OK/Save. Repeat for all your standards.

2. Unknown - Do the same, but in the Component Editor, only the IS peak name should be there. Key in its quantity ("4.6mM") . Click OK/Save. Repeat for all your unknowns.

Calibrate and quantitate as usual.

The internal standard quantity that appears in your results should not be 1.0, but should be whatever you keyed in.

The bottom line is, regardless of the default IS quantity entered in the processing method, you can still change the IS quantity for each standard and unknown individually.

Whew! Hope this helps.