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Should internal standards be added before or after SPE?
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+ Solid phase extraction
+ Pharma
+ Water analysis
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Khadijah Khan
Should internal standards be added before or after SPE?
As indicated by the above discussion the nomenclature around 'internal standards' is not clear. Different people call these compounds different things based on when they are added in the analytical process. "Internal Standards" can be added as late as immediately before injection - in which case they are sometimes called "injection standards". In general if your internal stds are going to be used to correct for extraction and clean-up recoveries then they should be added as early in the process as possible. If the internal Standard is being used only to provide a reference for instrumental detection variability they can be added immediately before injection. In some analyses (e.g. dioxin) multiple "internal standards" or standard mixtures are added at various points in the analytical process to account for all possible sources of variability.
As indicated by the above discussion the nomenclature around 'internal standards' is not clear. Different people call these compounds different things based on when they are added in the analytical process. "Internal Standards" can be added as late as immediately before injection - in which case they are sometimes called "injection standards". In general if your internal stds are going to be used to correct for extraction and clean-up recoveries then they should be added as early in the process as possible. If the internal Standard is being used only to provide a reference for instrumental detection variability they can be added immediately before injection. In some analyses (e.g. dioxin) multiple "internal standards" or standard mixtures are added at various points in the analytical process to account for all possible sources of variability.
if you are using HPLC-MS/MS you should work with isotope labelled internal standards so you can perform isotope dilution measurements, which will increase precision and correctness considerably. If your r2 in your calibration is not good something is wrong either with the instrument or the calibration solutions.
if you are using HPLC-MS/MS you should work with isotope labelled internal standards so you can perform isotope dilution measurements, which will increase precision and correctness considerably. If your r2 in your calibration is not good something is wrong either with the instrument or the calibration solutions.
terbutylazine is a pesticide used worldwide, it is as unlabelled compound not among the best possible choices. Generally internal standards can be used to controll procedure (procedural IS) and should be then added at the earleist possible stade or as calibration IS (possibly just added before injection). It is obvious that the procedural IS controlls considerably more possible errors and uncertainties than the calibration IS
terbutylazine is a pesticide used worldwide, it is as unlabelled compound not among the best possible choices. Generally internal standards can be used to controll procedure (procedural IS) and should be then added at the earleist possible stade or as calibration IS (possibly just added before injection). It is obvious that the procedural IS controlls considerably more possible errors and uncertainties than the calibration IS
Once again thank you for your answers. Meanwhile, our group has already come up with a calibration curve (before SPE) with a range of 5ppb-1000ppb. All equations for standards have been developed already and r^2 of the analytes are greater than .99. Also, predictability of the analyte concentration is very near with that of the expected concentration. Everything seems okay but the calibration curve after SPE. We are using LC-MS/MS (Agilent QQQ) for our analysis. are internal standards/labelled compounds really necessary?
Once again thank you for your answers. Meanwhile, our group has already come up with a calibration curve (before SPE) with a range of 5ppb-1000ppb. All equations for standards have been developed already and r^2 of the analytes are greater than .99. Also, predictability of the analyte concentration is very near with that of the expected concentration. Everything seems okay but the calibration curve after SPE. We are using LC-MS/MS (Agilent QQQ) for our analysis. are internal standards/labelled compounds really necessary?
If You have different area readings it means something is wrong with the SPE procedure in my opinion. Try to change the sorbent and during elution, hold first portion of Your eluting agent in the sorbent for few minutes to be sure that all analytes and IS will desorb from the sorbent. And control the pH of Your samples. However, I strongly suggest to use some deuterated or C13 matched IS similar to Your analytes.
If You have different area readings it means something is wrong with the SPE procedure in my opinion. Try to change the sorbent and during elution, hold first portion of Your eluting agent in the sorbent for few minutes to be sure that all analytes and IS will desorb from the sorbent. And control the pH of Your samples. However, I strongly suggest to use some deuterated or C13 matched IS similar to Your analytes.
My brief answer to the question is, yes, internal standards should be added before the SPE extraction; i.e., the internal standards should undergo the same sample preparation procedure as analytes being assayed.
My brief answer to the question is, yes, internal standards should be added before the SPE extraction; i.e., the internal standards should undergo the same sample preparation procedure as analytes being assayed.
I suggest a step wise approach. First check the reproducibility of the determination of your target compounds in your final SPE eluant at around the concentration expected in your final injection sample. This will tell you if your instrumental method is OK. Then put the same compounds in “pure water” through the SPE add your internal standard afterwards. Do several runs and check the reproducibility of your extractions, you could usefully add candidate surrogates along with the analytes. Then do the same but spike your analytes into river water, ideally a sample with undetectable analyte concentrations. Again do this repeatedly and check the reproducibility. Then take a good hard look at the data and you may be able to see where things are going wrong. You could also check the river water samples to see if there is any overlap with analytes or internal standard from interferents in the river water that is not being excluded by the selectivity of your MS detector (unlikely but worth a look). Finally are you sure that you are not just working too close to the detection limit? As the LOD is approached the relative precision degrades significantly and this could really throw out your calibration curves to the extent that you cannot even get a reasonable fit.
I suggest a step wise approach. First check the reproducibility of the determination of your target compounds in your final SPE eluant at around the concentration expected in your final injection sample. This will tell you if your instrumental method is OK. Then put the same compounds in “pure water” through the SPE add your internal standard afterwards. Do several runs and check the reproducibility of your extractions, you could usefully add candidate surrogates along with the analytes. Then do the same but spike your analytes into river water, ideally a sample with undetectable analyte concentrations. Again do this repeatedly and check the reproducibility. Then take a good hard look at the data and you may be able to see where things are going wrong. You could also check the river water samples to see if there is any overlap with analytes or internal standard from interferents in the river water that is not being excluded by the selectivity of your MS detector (unlikely but worth a look). Finally are you sure that you are not just working too close to the detection limit? As the LOD is approached the relative precision degrades significantly and this could really throw out your calibration curves to the extent that you cannot even get a reasonable fit.
If you add the IS after SPE you are only going to correct for instrumental fluctuations. Consequently, it is always better to add it from the very beggining of the analytical procedure. In this case, it is known as a "surrogate". The only drawack about adding it prior to sample analysis is that you'll need a higher ammount of IS. I hope that this helps you.
If you add the IS after SPE you are only going to correct for instrumental fluctuations. Consequently, it is always better to add it from the very beggining of the analytical procedure. In this case, it is known as a "surrogate". The only drawack about adding it prior to sample analysis is that you'll need a higher ammount of IS. I hope that this helps you.
Depends on your choice of absorbents in the SPE catridges/centrifuge tubes. If working with PSA (Primary Secondary Amines), Internal standard should be added after SPE. There is likeliness of absorption of internal standard (especially the TPP) by PSA which would affect the quantifications. This is especially applicable where measurements on Internal Standard are involved in final calculations.
Depends on your choice of absorbents in the SPE catridges/centrifuge tubes. If working with PSA (Primary Secondary Amines), Internal standard should be added after SPE. There is likeliness of absorption of internal standard (especially the TPP) by PSA which would affect the quantifications. This is especially applicable where measurements on Internal Standard are involved in final calculations.
As indicated by the above discussion the nomenclature around 'internal standards' is not clear. Different people call these compounds different things based on when they are added in the analytical process. "Internal Standards" can be added as late as immediately before injection - in which case they are sometimes called "injection standards". In general if your internal stds are going to be used to correct for extraction and clean-up recoveries then they should be added as early in the process as possible. If the internal Standard is being used only to provide a reference for instrumental detection variability they can be added immediately before injection. In some analyses (e.g. dioxin) multiple "internal standards" or standard mixtures are added at various points in the analytical process to account for all possible sources of variability.
As indicated by the above discussion the nomenclature around 'internal standards' is not clear. Different people call these compounds different things based on when they are added in the analytical process. "Internal Standards" can be added as late as immediately before injection - in which case they are sometimes called "injection standards". In general if your internal stds are going to be used to correct for extraction and clean-up recoveries then they should be added as early in the process as possible. If the internal Standard is being used only to provide a reference for instrumental detection variability they can be added immediately before injection. In some analyses (e.g. dioxin) multiple "internal standards" or standard mixtures are added at various points in the analytical process to account for all possible sources of variability.
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After SPE
After SPE
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if you are using HPLC-MS/MS you should work with isotope labelled internal standards so you can perform isotope dilution measurements, which will increase precision and correctness considerably.
If your r2 in your calibration is not good something is wrong either with the instrument or the calibration solutions.
if you are using HPLC-MS/MS you should work with isotope labelled internal standards so you can perform isotope dilution measurements, which will increase precision and correctness considerably.
If your r2 in your calibration is not good something is wrong either with the instrument or the calibration solutions.
More
VOTE
terbutylazine is a pesticide used worldwide, it is as unlabelled compound not among the best possible choices.
Generally internal standards can be used to controll procedure (procedural IS) and should be then added at the earleist possible stade or as calibration IS (possibly just added before injection).
It is obvious that the procedural IS controlls considerably more possible errors and uncertainties than the calibration IS
terbutylazine is a pesticide used worldwide, it is as unlabelled compound not among the best possible choices.
Generally internal standards can be used to controll procedure (procedural IS) and should be then added at the earleist possible stade or as calibration IS (possibly just added before injection).
It is obvious that the procedural IS controlls considerably more possible errors and uncertainties than the calibration IS
More
VOTE
Spiking / recovery experiment or addition of internal standard must be before SPE.
Spiking / recovery experiment or addition of internal standard must be before SPE.
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VOTE
Once again thank you for your answers. Meanwhile, our group has already come up with a calibration curve (before SPE) with a range of 5ppb-1000ppb. All equations for standards have been developed already and r^2 of the analytes are greater than .99. Also, predictability of the analyte concentration is very near with that of the expected concentration. Everything seems okay but the calibration curve after SPE. We are using LC-MS/MS (Agilent QQQ) for our analysis. are internal standards/labelled compounds really necessary?
Once again thank you for your answers. Meanwhile, our group has already come up with a calibration curve (before SPE) with a range of 5ppb-1000ppb. All equations for standards have been developed already and r^2 of the analytes are greater than .99. Also, predictability of the analyte concentration is very near with that of the expected concentration. Everything seems okay but the calibration curve after SPE. We are using LC-MS/MS (Agilent QQQ) for our analysis. are internal standards/labelled compounds really necessary?
More
VOTE
If You have different area readings it means something is wrong with the SPE procedure in my opinion. Try to change the sorbent and during elution, hold first portion of Your eluting agent in the sorbent for few minutes to be sure that all analytes and IS will desorb from the sorbent. And control the pH of Your samples. However, I strongly suggest to use some deuterated or C13 matched IS similar to Your analytes.
If You have different area readings it means something is wrong with the SPE procedure in my opinion. Try to change the sorbent and during elution, hold first portion of Your eluting agent in the sorbent for few minutes to be sure that all analytes and IS will desorb from the sorbent. And control the pH of Your samples. However, I strongly suggest to use some deuterated or C13 matched IS similar to Your analytes.
More
VOTE
My brief answer to the question is, yes, internal standards should be added before the SPE extraction; i.e., the internal standards should undergo the same sample preparation procedure as analytes being assayed.
My brief answer to the question is, yes, internal standards should be added before the SPE extraction; i.e., the internal standards should undergo the same sample preparation procedure as analytes being assayed.
More
VOTE
Spiking / recovery exp or addn of internal std must be after SPE. AND should be compared with calibration after SPE.
Spiking / recovery exp or addn of internal std must be after SPE. AND should be compared with calibration after SPE.
More
VOTE
I suggest a step wise approach. First check the reproducibility of the determination of your target compounds in your final SPE eluant at around the concentration expected in your final injection sample. This will tell you if your instrumental method is OK. Then put the same compounds in “pure water” through the SPE add your internal standard afterwards. Do several runs and check the reproducibility of your extractions, you could usefully add candidate surrogates along with the analytes. Then do the same but spike your analytes into river water, ideally a sample with undetectable analyte concentrations. Again do this repeatedly and check the reproducibility. Then take a good hard look at the data and you may be able to see where things are going wrong. You could also check the river water samples to see if there is any overlap with analytes or internal standard from interferents in the river water that is not being excluded by the selectivity of your MS detector (unlikely but worth a look). Finally are you sure that you are not just working too close to the detection limit? As the LOD is approached the relative precision degrades significantly and this could really throw out your calibration curves to the extent that you cannot even get a reasonable fit.
I suggest a step wise approach. First check the reproducibility of the determination of your target compounds in your final SPE eluant at around the concentration expected in your final injection sample. This will tell you if your instrumental method is OK. Then put the same compounds in “pure water” through the SPE add your internal standard afterwards. Do several runs and check the reproducibility of your extractions, you could usefully add candidate surrogates along with the analytes. Then do the same but spike your analytes into river water, ideally a sample with undetectable analyte concentrations. Again do this repeatedly and check the reproducibility. Then take a good hard look at the data and you may be able to see where things are going wrong. You could also check the river water samples to see if there is any overlap with analytes or internal standard from interferents in the river water that is not being excluded by the selectivity of your MS detector (unlikely but worth a look). Finally are you sure that you are not just working too close to the detection limit? As the LOD is approached the relative precision degrades significantly and this could really throw out your calibration curves to the extent that you cannot even get a reasonable fit.
More
VOTE
If you add the IS after SPE you are only going to correct for instrumental fluctuations. Consequently, it is always better to add it from the very beggining of the analytical procedure. In this case, it is known as a "surrogate".
The only drawack about adding it prior to sample analysis is that you'll need a higher ammount of IS.
I hope that this helps you.
If you add the IS after SPE you are only going to correct for instrumental fluctuations. Consequently, it is always better to add it from the very beggining of the analytical procedure. In this case, it is known as a "surrogate".
The only drawack about adding it prior to sample analysis is that you'll need a higher ammount of IS.
I hope that this helps you.
More
VOTE
Depends on your choice of absorbents in the SPE catridges/centrifuge tubes. If working with PSA (Primary Secondary Amines), Internal standard should be added after SPE. There is likeliness of absorption of internal standard (especially the TPP) by PSA which would affect the quantifications. This is especially applicable where measurements on Internal Standard are involved in final calculations.
Depends on your choice of absorbents in the SPE catridges/centrifuge tubes. If working with PSA (Primary Secondary Amines), Internal standard should be added after SPE. There is likeliness of absorption of internal standard (especially the TPP) by PSA which would affect the quantifications. This is especially applicable where measurements on Internal Standard are involved in final calculations.
More
VOTE