quantification of organic acids by deuterated standards

[chandra]

Hello to everyone,

I want to quantify isovalerylglycine ,hexaonylglycine, butrylglycine, propionylglycine and methylcrotonylglycine in urine sample. I need pure compound for identification of their Rt and for calibration curve but my senior who is the decision maker ordered the deuterated form of all these compound and asked me to quantify the above marker compounds on the basis of these deuterated standards.I don't know how to do it please help.

Chandra

[James_Ball]

Hello to everyone,

I want to quantify isovalerylglycine ,hexaonylglycine, butrylglycine, propionylglycine and methylcrotonylglycine in urine sample. I need pure compound for identification of their Rt and for calibration curve but my senior who is the decision maker ordered the deuterated form of all these compound and asked me to quantify the above marker compounds on the basis of these deuterated standards.I don't know how to do it please help.

Chandra

I would think he is asking to do isotope dilution type quantification. You should though also have the non-deuterated analogs in the standard to do this best. You can make an estimation comparing the area of the peaks for the deuterated analog to the area of the peaks of the non-deuterated targets and assume that they will both give the same area counts at the same concentration. The problem will be that the deuterated and non-deuterated compounds may have slightly different retention times and slightly different response versus concentration.

The best way to do it is to use the deuterated compound as the internal standard holding it at constant concentration in all samples and calibration standards this will help normalize the results for problems you encounter with extraction or ionization of samples versus calibration standards to give you more accurate results independent of matrix.

[Camisotro]

Hello to everyone,

I want to quantify isovalerylglycine ,hexaonylglycine, butrylglycine, propionylglycine and methylcrotonylglycine in urine sample. I need pure compound for identification of their Rt and for calibration curve but my senior who is the decision maker ordered the deuterated form of all these compound and asked me to quantify the above marker compounds on the basis of these deuterated standards.I don't know how to do it please help.

Chandra

Indeed it'd be best to also have the non-deuterated compounds. Fortunately if your boss was willing to spend whatever the deuterated versions cost, I'm sure the non-deuterated ones would be significantly cheaper to order as well.

Keep in mind that sometimes a particular ion mass comes from more than one possible fragment of the molecule. Once it's been isotopically substituted, these accidental overlaps will change and possibly others will be created. Hence you can't be confident that the ion you choose represents the same part of both molecules.

[Peter Apps]

If you are using isotope labelled standards, then you must be using MS for detection (no other detector can discriminate between analyte and standard). You can use the MS spectra (or MWs) to find which peaks are your analytes.

If you are not using an MS, your senior has purchased an expensive option that will not work any better than the cheaper unlabelled compounds.

Peter

[Camisotro]

If you are using isotope labelled standards, then you must be using MS for detection (no other detector can discriminate between analyte and standard). You can use the MS spectra (or MWs) to find which peaks are your analytes.

If you are not using an MS, your senior has purchased an expensive option that will not work any better than the cheaper unlabelled compounds.

Peter

The problem is not the inability to discriminate between one and the other. The issue is that the sensitivity of the isotopic standard can't be truly relied upon to equal the sensitivity of the genuine target without having at least some access to an analytical standard of the genuine target.

[Peter Apps]

Hi Camistro

Are there any detectors besides MS that can discriminate between isotope-labelled and native compounds ?, and if there are, is there a difference in response between labelled and unlabelled ?.

One of the OP's problems is that he cannot find his analytes among the other peaks on the chromatograms. If he has an MS he can use it to solve that problem. If he does not have an MS then what is the point of having labelled, as opposed to unlabelled, standards ? I suppose that there might be an argument that isotope labelling provides the best possible match to the behaviour of the analyte during sample prep, and so provides the best possible internal standard, but it is still necessary to discriminate between analyte and standard peaks.

Peter

[Camisotro]

Hi Camistro

Are there any detectors besides MS that can discriminate between isotope-labelled and native compounds ?, and if there are, is there a difference in response between labelled and unlabelled ?.

One of the OP's problems is that he cannot find his analytes among the other peaks on the chromatograms. If he has an MS he can use it to solve that problem. If he does not have an MS then what is the point of having labelled, as opposed to unlabelled, standards ? I suppose that there might be an argument that isotope labelling provides the best possible match to the behaviour of the analyte during sample prep, and so provides the best possible internal standard, but it is still necessary to discriminate between analyte and standard peaks.

Peter

Peter, I am not really sure what you are getting at here. Yes it has been established the OP is using an MS. Non-MS detectors are unlikely to benefit from labelled standards.

Labelled standards *in combination with unlabelled standards* are the best way to correct for matrix effects in LC-MS. You spike a known amount of the labelled standards into each sample and calibration as an internal standard, and then calibrate using the signal ratio of unlabelled over labelled. This is because you can typically expect the labelled standard to have as identical as possible behaviour through the entire extraction and detection process.

But the signal of a labelled standard *in the absence of unlabelled standards* cannot be expected to have the correct signal necessary to quantify unknown samples. E.g. if the quant ion for deuterated isovalerylglycine produces 1000 counts per ppb, you cannot expect the parallel quant ion for isovalerylglycine to produce 1000 counts per ppb.

[Peter Apps]

Hi Camistro

Are there any detectors besides MS that can discriminate between isotope-labelled and native compounds ?, and if there are, is there a difference in response between labelled and unlabelled ?.

One of the OP's problems is that he cannot find his analytes among the other peaks on the chromatograms. If he has an MS he can use it to solve that problem. If he does not have an MS then what is the point of having labelled, as opposed to unlabelled, standards ? I suppose that there might be an argument that isotope labelling provides the best possible match to the behaviour of the analyte during sample prep, and so provides the best possible internal standard, but it is still necessary to discriminate between analyte and standard peaks.

Peter

Peter, I am not really sure what you are getting at here. Yes it has been established the OP is using an MS Really, which post was that in ? The use of MS is an assumption based only on the OP's supervisor having purchased labelled standards. Non-MS detectors are unlikely to benefit from labelled standards I completely agree, but any benefit of their having an MS is neatly cancelled by their not having unlabelled standards with which to calibrate the analysis.

Labelled standards *in combination with unlabelled standards* are the best way to correct for matrix effects in LC-MS. You spike a known amount of the labelled standards into each sample and calibration as an internal standard, and then calibrate using the signal ratio of unlabelled over labelled. This is because you can typically expect the labelled standard to have as identical as possible behaviour through the entire extraction and detection process.

But the signal of a labelled standard *in the absence of unlabelled standards* cannot be expected to have the correct signal necessary to quantify unknown samples. E.g. if the quant ion for deuterated isovalerylglycine produces 1000 counts per ppb, you cannot expect the parallel quant ion for isovalerylglycine to produce 1000 counts per ppb. I agree, but given the particular circumstances of this enquiry, it could be that the errors due to the different ionisation efficiencies of labelled and unlabelled molecules might be less than the differences in matrix effects for peaks eluting at different times.

How do you suggest that the OP locates the analytes on the chromatogram ?, which was the problem that I was seeking to address.

Peter