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Why am I getting less than 100% recovery after solid phase extraction?
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Mike Isbad
Why am I getting less than 100% recovery after solid phase extraction?
Hi, Please remember that SPE extraction recovery depends on many factors including many manual steps (if you make SPE manually). Sometimes even if sorbent and its content (mass) are optimal for your analyte concentration volume and phys-chem then other steps could not be optimal. This is why very often analyte rest could be present (after the procedure) still in sorbent bed or in washing eluent. Generally, if you do not check every step (impact of volume/time/concentration or type of eluent for all other steps) but simply applicate method from scientific paper etc then a random search of the problem could be very long. Of course, the question is what kind of recovery you have 30% or 90% if 30 then probably you have some serious problem in the procedure or a few minor which finally gives huge losses. Finally please remember that nobody expects in extraction procedure like SPE that you have 100% of recovery it's not aim per se. The aim is the repeatable level of extraction .
Its industrial regulation but the same are used in "pure science"
"The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent"
Hi, Please remember that SPE extraction recovery depends on many factors including many manual steps (if you make SPE manually). Sometimes even if sorbent and its content (mass) are optimal for your analyte concentration volume and phys-chem then other steps could not be optimal. This is why very often analyte rest could be present (after the procedure) still in sorbent bed or in washing eluent. Generally, if you do not check every step (impact of volume/time/concentration or type of eluent for all other steps) but simply applicate method from scientific paper etc then a random search of the problem could be very long. Of course, the question is what kind of recovery you have 30% or 90% if 30 then probably you have some serious problem in the procedure or a few minor which finally gives huge losses. Finally please remember that nobody expects in extraction procedure like SPE that you have 100% of recovery it's not aim per se. The aim is the repeatable level of extraction .
Its industrial regulation but the same are used in "pure science"
"The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent"
Hi, Please remember that SPE extraction recovery depends on many factors including many manual steps (if you make SPE manually). Sometimes even if sorbent and its content (mass) are optimal for your analyte concentration volume and phys-chem then other steps could not be optimal. This is why very often analyte rest could be present (after the procedure) still in sorbent bed or in washing eluent. Generally, if you do not check every step (impact of volume/time/concentration or type of eluent for all other steps) but simply applicate method from scientific paper etc then a random search of the problem could be very long. Of course, the question is what kind of recovery you have 30% or 90% if 30 then probably you have some serious problem in the procedure or a few minor which finally gives huge losses. Finally please remember that nobody expects in extraction procedure like SPE that you have 100% of recovery it's not aim per se. The aim is the repeatable level of extraction .
Its industrial regulation but the same are used in "pure science"
"The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent"
Hi, Please remember that SPE extraction recovery depends on many factors including many manual steps (if you make SPE manually). Sometimes even if sorbent and its content (mass) are optimal for your analyte concentration volume and phys-chem then other steps could not be optimal. This is why very often analyte rest could be present (after the procedure) still in sorbent bed or in washing eluent. Generally, if you do not check every step (impact of volume/time/concentration or type of eluent for all other steps) but simply applicate method from scientific paper etc then a random search of the problem could be very long. Of course, the question is what kind of recovery you have 30% or 90% if 30 then probably you have some serious problem in the procedure or a few minor which finally gives huge losses. Finally please remember that nobody expects in extraction procedure like SPE that you have 100% of recovery it's not aim per se. The aim is the repeatable level of extraction .
Its industrial regulation but the same are used in "pure science"
"The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent"
Check each step of the SPE for identifying the point of the major loss...Probably loading in ACN with %100ACN will conclude as flow though without or very less binding. Reduce the hydrophobicity of the loading buffer to give permission to diazepam which is able to interact with c18 if the SPE you used is c18. Even you design the perfect experiment, it is nearly impossible to get 100% recovery when you are performing SPE. As I indicated, collect each fraction after loading, washing, and elution and see where the compound is. Afterward, carefully optimize the experiment to improve retain or elution strategy. Be careful your equilibration composition should be the same or very similar to get the best trap of the target. In this example, they are very different and not convenient.
Check each step of the SPE for identifying the point of the major loss...Probably loading in ACN with %100ACN will conclude as flow though without or very less binding. Reduce the hydrophobicity of the loading buffer to give permission to diazepam which is able to interact with c18 if the SPE you used is c18. Even you design the perfect experiment, it is nearly impossible to get 100% recovery when you are performing SPE. As I indicated, collect each fraction after loading, washing, and elution and see where the compound is. Afterward, carefully optimize the experiment to improve retain or elution strategy. Be careful your equilibration composition should be the same or very similar to get the best trap of the target. In this example, they are very different and not convenient.
Hi,
Please remember that SPE extraction recovery depends on many factors including many manual steps (if you make SPE manually). Sometimes even if sorbent and its content (mass) are optimal for your analyte concentration volume and phys-chem then other steps could not be optimal. This is why very often analyte rest could be present (after the procedure) still in sorbent bed or in washing eluent. Generally, if you do not check every step (impact of volume/time/concentration or type of eluent for all other steps) but simply applicate method from scientific paper etc then a random search of the problem could be very long. Of course, the question is what kind of recovery you have 30% or 90% if 30 then probably you have some serious problem in the procedure or a few minor which finally gives huge losses.
Finally please remember that nobody expects in extraction procedure like SPE that you have 100% of recovery it's not aim per se. The aim is the repeatable level of extraction .
Its industrial regulation but the same are used in "pure science"
"The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent"
https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf
"Recovery of the analyte does not need to be 100%, but the extent of recovery of an analyte and of the IS (if used) should be consistent."
https://www.ema.europa.eu/en/documents/scientific-guideline/draft-ich-guideline-m10-bioanalytical-method-validation-step-2b_en.pdf
Best regards
Tomasz
Hi,
Please remember that SPE extraction recovery depends on many factors including many manual steps (if you make SPE manually). Sometimes even if sorbent and its content (mass) are optimal for your analyte concentration volume and phys-chem then other steps could not be optimal. This is why very often analyte rest could be present (after the procedure) still in sorbent bed or in washing eluent. Generally, if you do not check every step (impact of volume/time/concentration or type of eluent for all other steps) but simply applicate method from scientific paper etc then a random search of the problem could be very long. Of course, the question is what kind of recovery you have 30% or 90% if 30 then probably you have some serious problem in the procedure or a few minor which finally gives huge losses.
Finally please remember that nobody expects in extraction procedure like SPE that you have 100% of recovery it's not aim per se. The aim is the repeatable level of extraction .
Its industrial regulation but the same are used in "pure science"
"The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent"
https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf
"Recovery of the analyte does not need to be 100%, but the extent of recovery of an analyte and of the IS (if used) should be consistent."
https://www.ema.europa.eu/en/documents/scientific-guideline/draft-ich-guideline-m10-bioanalytical-method-validation-step-2b_en.pdf
Best regards
Tomasz
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Hi,
Please remember that SPE extraction recovery depends on many factors including many manual steps (if you make SPE manually). Sometimes even if sorbent and its content (mass) are optimal for your analyte concentration volume and phys-chem then other steps could not be optimal. This is why very often analyte rest could be present (after the procedure) still in sorbent bed or in washing eluent. Generally, if you do not check every step (impact of volume/time/concentration or type of eluent for all other steps) but simply applicate method from scientific paper etc then a random search of the problem could be very long. Of course, the question is what kind of recovery you have 30% or 90% if 30 then probably you have some serious problem in the procedure or a few minor which finally gives huge losses.
Finally please remember that nobody expects in extraction procedure like SPE that you have 100% of recovery it's not aim per se. The aim is the repeatable level of extraction .
Its industrial regulation but the same are used in "pure science"
"The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent"
https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf
"Recovery of the analyte does not need to be 100%, but the extent of recovery of an analyte and of the IS (if used) should be consistent."
https://www.ema.europa.eu/en/documents/scientific-guideline/draft-ich-guideline-m10-bioanalytical-method-validation-step-2b_en.pdf
Best regards
Tomasz
Hi,
Please remember that SPE extraction recovery depends on many factors including many manual steps (if you make SPE manually). Sometimes even if sorbent and its content (mass) are optimal for your analyte concentration volume and phys-chem then other steps could not be optimal. This is why very often analyte rest could be present (after the procedure) still in sorbent bed or in washing eluent. Generally, if you do not check every step (impact of volume/time/concentration or type of eluent for all other steps) but simply applicate method from scientific paper etc then a random search of the problem could be very long. Of course, the question is what kind of recovery you have 30% or 90% if 30 then probably you have some serious problem in the procedure or a few minor which finally gives huge losses.
Finally please remember that nobody expects in extraction procedure like SPE that you have 100% of recovery it's not aim per se. The aim is the repeatable level of extraction .
Its industrial regulation but the same are used in "pure science"
"The sponsor should optimize the recovery of the analyte to ensure that the extraction is efficient and reproducible. Recovery need not be 100 percent"
https://www.fda.gov/files/drugs/published/Bioanalytical-Method-Validation-Guidance-for-Industry.pdf
"Recovery of the analyte does not need to be 100%, but the extent of recovery of an analyte and of the IS (if used) should be consistent."
https://www.ema.europa.eu/en/documents/scientific-guideline/draft-ich-guideline-m10-bioanalytical-method-validation-step-2b_en.pdf
Best regards
Tomasz
More
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Check each step of the SPE for identifying the point of the major loss...Probably loading in ACN with %100ACN will conclude as flow though without or very less binding. Reduce the hydrophobicity of the loading buffer to give permission to diazepam which is able to interact with c18 if the SPE you used is c18. Even you design the perfect experiment, it is nearly impossible to get 100% recovery when you are performing SPE. As I indicated, collect each fraction after loading, washing, and elution and see where the compound is. Afterward, carefully optimize the experiment to improve retain or elution strategy. Be careful your equilibration composition should be the same or very similar to get the best trap of the target. In this example, they are very different and not convenient.
emir
Check each step of the SPE for identifying the point of the major loss...Probably loading in ACN with %100ACN will conclude as flow though without or very less binding. Reduce the hydrophobicity of the loading buffer to give permission to diazepam which is able to interact with c18 if the SPE you used is c18. Even you design the perfect experiment, it is nearly impossible to get 100% recovery when you are performing SPE. As I indicated, collect each fraction after loading, washing, and elution and see where the compound is. Afterward, carefully optimize the experiment to improve retain or elution strategy. Be careful your equilibration composition should be the same or very similar to get the best trap of the target. In this example, they are very different and not convenient.
emir
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Dear Aulia Zulfa
What is the SPE sorbent?
Cheers
Detlef.
Dear Aulia Zulfa
What is the SPE sorbent?
Cheers
Detlef.
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Thank you for replaying
Dear Detlef;
The sorbent is a molecular imprinted polymer with methacrylic acid as the monomer
Thank you for replaying
Dear Detlef;
The sorbent is a molecular imprinted polymer with methacrylic acid as the monomer
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