I think Martin's post raises an interesting point and highlights that existing white papers/guidances may not be always scientifically applicable (especially for new modalities). We had an analyte where the expected systemic concentration was BLQ very soon after dosing. In that case, A) the large majority of samples were BLQ, meaning identifying samples for ISR with 3xLLOQ would be mean selecting and reanalyzing ALL the samples with detectable concentrations (which doesn't make sense) and B) the BLQ result was in fact the scientifically important result. So why not perform ISR on the BLQ samples? A related issue can happen with incurred sample stability. For a "free" analyte (e.g., target) that is undetectable in the presence of a binding partner (e.g., drug), we want the incurred stability sample to also return an undetectable result. In both cases, it is more important to ensure the assay returns a BLQ result reproducibly than a detectable result. Unfortunately, while analyzing BLQ ISR samples may be scientifically justifiable, Health Authority reviewers will likely want BA labs to follow the letter of the guidance.
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Michael Partridge
Senior Director
Regeneron Pharmaceuticals
Tarrytown NY
[email protected]Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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Original Message:
Sent: 02-24-2026 15:16
From: Joleen White
Subject: Inclusion of samples below LLOQ in ISR for PK assays?
Dear Martin,
While of course it is ideal to have samples across the whole study, the 3xLLOQ minimum concentration is a practical consideration for performing the statistical analysis. The requirement to include some Cmax is to ensure that the full quantitative range is covered, and the requirement for including elimination phase is also for catching any deviations due to metabolite presence in addition to covering the full quantitative range.
The 3x LLOQ comes from a practical limitation that as you approach assay LLOQ, the variability increases and the risk of a large individual sample deviation leading to a BLQ and therefore unquantifiable %difference increases. So while you could look at concordance, it isn't mathematically helpful. Please note that samples between LLOQ and 3xLLOQ are still in the denominator when calculating the total number of quantifiable results.
This 3xLLOQ is a general guideline, not a regulation. If you have a lot of results below 3xLLOQ, you may want to set a lower threshold. I published an ISR case study for a cell-based assay with half of the numerical results below 3xLLOQ so set the selection criterion to 2xLLOQ to expand the sample set to select from from 1/2 to 2/3 of the quantifiable results. DOI:10.4155/bio.15.47
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Joleen White Ph.D.
AAPS 2026 National Biotechnology Conference Track Chair
Bioanalytical 101 Course Development
Senior Advisor
BioData Solutions LLC
[email protected]
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
Original Message:
Sent: 02-06-2026 10:32
From: Martin Roberge
Subject: Inclusion of samples below LLOQ in ISR for PK assays?
What is your interpretation of the ICH M10 guidance on selection of samples for ISR which states: "... Therefore, it is recommended that the samples for ISR be chosen around the maximum concentration (Cmax) and some in the elimination phase. Additionally, the samples chosen should be representative of the whole study." Also, theGCC white paper from Sangster et al., 2012 (Bioanalysis 4(14) states that "Only samples with concentrations at least three-times the LLOQ concentration should be selected."
Therefore do we need to repeat analysis of samples resulting below LLOQ since these samples are representative of the whole study (although not possible to calculate a % difference)?
Thanks
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Martin Roberge
Director Development and Innovation
Cerba Research
Laval QC
[email protected]
Disclaimer: Opinions expressed are solely my own and do not express the views or opinions of my employer.
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