quantification of organic acids

[chandra]

Hello to everyone,
I had prepared calibration curve of 14 organic acids of my interest. For preparing the calibration curve i had taken the four concentrations of the organic acids i.e 25, 50, 75 and 100 umol/l. When i run the urine sample , i found that when the peak of organic acid is more sharp and peak height is large than the quantification result says that the particular organic acid is not detected in the sample.I don't know how to quantitate the quantities that are above or below the calibration curves range.Please help.


Thanks
Chandra

[unmgvar]

what is the software that you use?

[Don_Hilton]

I'll add another question to that posted above:

Did the software integrate the peak and exclude the result or did it fail to even integrate the peak? If the peak is not integrated, there is an issue with integration parameters - and if you are analyzing underivatized organic acids by GC, this is not surprising because of the issues of peak shape with column loading.

If the peak was integrated, the next question is: Is the retention time within the window of expected retention time for the compound?

If the compund is outside of the range of the calibration, caution!! Most instrument software includes the capability (and it may be the default option) to not report results outside of the range of the calibration curve or a range based on the calibration curve. This is not an accidental fault in the software. Because of various issues related to detectors, peak shape, and such, you do not know the quality of results obtained for a peak outside the calibration curve -- and the result you obtain may be significanly incorrect.

If you find that you have analytes with response above the range of your calibration curve and wish to report values, let me strongly reccomend that you extend the range of the curve.

[chandra]

Yes the software integrates the peak and Retention time is within the window of expected retention time.
When i tried to extend the range of curve the peaks of the compound became saturated and they give <25% match with the compound.


Thanks
Chandra

[Don_Hilton]

Ah! you must be using GC/MS? This makes a difference.

(I take the clue from the lack of match.)

When you have a saturated detector, the detector is unable to count ions as fast as they arrive, so recorded area is low relative to the quantity of compound present - and as concentrations continue to increase, the response factor continues to drop.

You need to dilute your samples or use a split injection to avoid saturation of the detector. Using a mass spec, it is possible to pick an alterative ion that does not saturate the detector. But, becareful of saturation of the ion source.