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Dear Members,
I am a new one for ELISA GLP bioanalytical methods. I have few queries on ELISA
GLP Bioanalytical Method parameters.
1. Masking: During Method Validation and Study sample analysis the
calibration standards should be in duplicate in each run. Can we mask one of the
standard, if we analyze in duplicate? What is the criteria for masking? How many
individual standards can mask as per GLP? What is the criteria for masking of
QCs?
2. Selectivity: The acceptance criteria for LC-MS based methods is the
response at RT in blank is less than 20% of the LLOQ for the analyte. But in
case of ELISA methods as per EMEA guidelines, the accuracy should be within 20%
(25% at the LLOQ) of the nominal spiked concentration in at least 80% of the
matrices evaluated; is there any criteria for response (Absorbance) of blank
matrix verses LLOQ like LC-MS method?
3. Dilution linearity of study samples: if the study sample is serially
diluted and the calculated concentration of all serially diluted samples (or
more than one dilution) falls within the CC range, which one has to be reported?
(Mean off all dilutions? or only one dilution? If individual which one to be
reported?)
Thanking you all.
Regards
Akena Venkatesham, Ph D
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Dear Venkatesham,
1. Masking: During Method Validation and Study sample analysis the
calibration standards should be in duplicate in each run. Can we mask one of the
standard, if we analyze in duplicate? What is the criteria for masking? How many
individual standards can mask as per GLP? What is the criteria for masking of
QCs?
Response : It is always preferable to use in singlicate if your method is
supporting to analyze CC and QC and Subject samples in singlicate, unless there
is high variability of analyte etc. Regarding masking of standards, the accepted
replicate should be included in the calibration, however many standards of
singlicate failures indicates the inaccuracy of the method.
2. Selectivity: The acceptance criteria for LC-MS based methods is the
response at RT in blank is less than 20% of the LLOQ for the analyte. But in
case of ELISA methods as per EMEA guidelines, the accuracy should be within 20%
(25% at the LLOQ) of the nominal spiked concentration in at least 80% of the
matrices evaluated; is there any criteria for response (Absorbance) of blank
matrix verses LLOQ like LC-MS method?
Response: There is no such generalised criteria for absorbance since it may not
be linear with concentration always.
3. Dilution linearity of study samples: if the study sample is serially
diluted and the calculated concentration of all serially diluted samples (or
more than one dilution) falls within the CC range, which one has to be reported?
(Mean off all dilutions? or only one dilution? If individual which one to be
reported?
Response: Dilution linearity of study sample experiment shall be conducted in
method validation. For study samples which were diluted (more than one
dilution), report the least diluted study sample.
Thanks and Regards
Santosh Kumar Manthri
Research scientist
Bioanalytical Research Department,
Synapse Labs Pvt. Ltd,Dear Colleagues,
Is it required to keep the integration parameters like smoothing, noise
threshold, area threshold of P&A-01 of method validation throughout the
validation and throughout the sample analysis?
Best Regards
Narayana Kumar
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Dear Narayana kumar,
Is it required to keep the integration parameters like smoothing, noise
threshold, area threshold of P&A-01 of method validation throughout the
validation and throughout the sample analysis?
Response: As per latest industrial practice, it is required ,
Please find below is the latest ANVISA recommendations for the same.
"The Bioequivalence Coordination, in order to clarify some procedures of sample
analysis and to avoid repeated requests, recommends regarding the analytical
phase:
- The integration parameter smooth must be as low as possible;
- Smooth of 5 or higher needs to be justified and the CRO should send
chromatograms with and without smoothing. The dwell time and signal/noise ratio
should also be informed;
- Smooth, if used, must be consistent across the validation and sample
analysis. Hence, when validated, it should be used for all the batches for
consistency.
Regards,
General Office of Drugs
Brazilian Health Surveillance Agency
e-mail: bioequivalencia.-at-.anvisa.gov.br"
Thanks and Regards
Santosh Kumar Manthri
Research scientist
Bioanalytical Research Department,
Synapse Labs Pvt. Ltd,
Majestic Plaza, S.No.21/5,
Nr. Nyati Empire,
Kharadi-Mundhwa Bypass,
Kharadi, Pune-411014
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>> 1. Masking: During Method Validation and Study sample analysis the
>> calibration standards should be in duplicate in each run. Can we mask one of the
>>
>> standard, if we analyze in duplicate? What is the criteria for masking? How many
>>
>> individual standards can mask as per GLP? What is the criteria for masking of
>> QCs?
> Response : It is always preferable to use in singlicate if your method is
> supporting to analyze CC and QC and Subject samples in singlicate, unless there
> is high variability of analyte etc. Regarding masking of standards, the accepted
> replicate should be included in the calibration, however many standards of
> singlicate failures indicates the inaccuracy of the method.
How many measures are used for each point? If one delete the point , if two
delete both. There must be at least six surviving points for the working range
of your curve or 75% of the working curve points. Should include the lloq and
uloq. Some will allow loss of either so long as 1) there are still six points
and 2) the Lqc/ hqc are bracketed by remaining calibrators. An additional
constraint is that failed points should not be adjacent. If three
determinations are made for each sample, calibrator and qc there is more
flexibility permitting rejection of one of the three points. The analsisy and
acceptance/rejection must be clear
>> 2. Selectivity: The acceptance criteria for LC-MS based methods is the
>> response at RT in blank is less than 20% of the LLOQ for the analyte. But in
>> case of ELISA methods as per EMEA guidelines, the accuracy should be within 20%
>> (25% at the LLOQ) of the nominal spiked concentration in at least 80% of the
>> matrices evaluated; is there any criteria for response (Absorbance) of blank
>> matrix verses LLOQ like LC-MS method?
> Response: There is no such generalised criteria for absorbance since it may not
> be linear with concentration alway
There is none per se but it is Reasonable to require that the matrix blank
should be lower (higher for displacement assay) than the lloq LCMs uses noise
not absolute response, since the absolute response can vary in LCMs
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