02/18/2019
This article is a review of bioanalytical factors that need to be considered before using BLQ data. It is in press in Bioanalysis, scheduled for release this spring. Here is a preview:
Quantification Below the LLOQ in Regulated LC-MS/MS Assays: A Review of Bioanalytical Considerations and Cautions
Jeffrey X. Duggan
JXD Bioanalytics
159 Lakemere Drive, Southbury, CT 06488
Abstract
In response to an earlier workshop covering the pros and cons of quantification below the LLOQ, the author reviews the topics discussed from the bioanalytical standpoint. Important considerations for estimating concentrations below the LLOQ include: method signal-to-noise, baseline shape and condition, close lying interference peaks (especially for protein methods), matrix effect, adsorption and stability of the analyte at low concentrations and carryover. These methodological issues are discussed as possible contributors to inaccuracy in BLQ estimations, and appropriate cautions are provided via examples. A proposed method for the evaluation of BLQ estimations utilizes extended Incurred Sample Reanalysis (ISR)analysis where BLQ samples or spiked simulated samples are analyzed with QCs and standards in addition to those in the original study. Generally, BLQ estimations are discouraged, with the recommendation that any extrapolations should be done in close collaboration between the PK and bioanalytical scientists in consultation with the regulatory agency.
Introduction
Recently, the author participated in an AAPS workshop discussing the pros and cons of releasing plasma concentrations in GLP and GCP studies below the LLOQ (Lower Limit of Quantification) for PK or PD purposes [1]. The discussion at this workshop was quite spirited, and there was great overall interest in the topic. As such, it seemed worthwhile to publish a discussion of the topics presented at this workshop from the bioanalytical point of view, specifically pertaining to LC-MS/MS methods for both small molecules and protein analytes but excluding ligand binding methods. Note that, while many of these bioanalytical considerations can also pertain to metabolites and biomarkers, these discussions are intended for conventionally validated drug analyte molecules.
There are often reasons to seek estimated (calculated concentrations from actual chromatographic run data) concentrations below the LLOQ (BLQ)[2] or extrapolated BLQ concentrations (concentration data projected to a sub-LLOQ concentration via mathematical calculations [3]). These reasons include: obtaining a value for terminal half-life at lower drug doses, determining trough values for extended dosing or low doses, and PK modeling exercises [4,5]. While these methods all seek to obtain usable PK data to support their respective studies, there are many potential bioanalytical pitfalls that must be considered when “extending below the LLOQ” using even the best modeling techniques or direct estimations from existing chromatographic data. Beyond the basic regulatory considerations for reporting values outside the calibration range, there are chemical and bioanalytical issues that can impact the accuracy of reported BLQ values. In this paper we will discuss those issues, using examples where possible.
PK extrapolations /estimations
In many cases extrapolations or estimations of concentrations below the LLOQ are helpful, and in some cases, they are necessary for modeling and interpretive reasons[3-5]. Despite these needs, the following bioanalytical questions need to be answered when extrapolations are considered:
1. What is the relative magnitude of the extrapolations or estimations proposed for a given method and study?
2. What are the bioanalytical factors that come into play to affect values in the desired range below the LLOQ and how can they be critically evaluated?
3. When is it scientifically valid to use extrapolated values or to estimate them from sub-LLOQ chromatograms?
4. What, if any, validation or verification of the extrapolated or estimated values needs to be performed?
Questions must be asked both from a) the interpretive standpoint: what is the purpose and value of the BLQ extrapolation for the PK exercise we wish to perform? and b) from the standpoint of bioanalytical validity: how much extrapolation can be made? Can valid concentrations be estimated at 20%, 50%, 100% or more below the determined assay LLOQ? The answer in terms of question b) can be determined empirically and is likely different for each assay and for each study. It may even differ for individual runs within the same study. Depending upon the assay and circumstances, the extrapolations may also be supportable by validation or verification data (described below).
Here, we will look at some of the important factors that can adversely impact quantification below the LLOQ. All these factors are known to experienced bioanalytical scientists, as they are routinely encountered in various assays; but they have not been collectively considered as they may apply to BLQ extrapolations/estimations. As such, this discussion might be useful to PK scientists and their bioanalytical colleagues who are considering BLQ extrapolations for regulated studies.
regulatory considerations
The regulatory guidances for bioanalytical validation both from FDA [6-8] and EMA [9] clearly specify that the calibration range of an assay is defined by a series of standards prepared in matrix which is bounded by the upper limit of calibration (ULOQ) and the lower limit of calibration (LLOQ). Another series of quality control (QC) samples are prepared at concentrations near the lower, middle and upper ends of the curve and processed along with standards and samples in each analytical run. These standards and QCs qualify the run, determining whether it passes or fails acceptance according to fixed accuracy and precision criteria over 3 runs. As such, the regulatory agencies strictly define the qualified assay range at the time of validation and generally do not support reporting values below the LLOQ. A quote from the 2001 FDA guidance on this subject reads as follows: “Estimation of concentration in unknown samples by extrapolation of standard curves below LLOQ or above the highest standard is not recommended. Instead, the standard curve should be redefined ...” [6], Similarly, the 2013 Draft FDA validation guidance states “Concentrations in unknown samples should not be extrapolated below the LLOQ or above the ULOQ of the standard curve. Instead, the standard curve should be extended and revalidated, or samples with concentrations above the ULOQ should be diluted and reanalyzed. Concentrations below the LLOQ should be reported as zeroes.” [7]. The most recent FDA Guidance states “ Concentrations listed below the LLOQ should be reported as below the LLOQ (BQL)” [8].
The regulators consistently state that extrapolations below the LLOQ should be avoided and that additional validation experiments be performed to verify the performance of the method at any points below the LLOQ if such values are to be used for analysis. Their concerns lie in the possible losses of accuracy, precision and linearity that may occur below the lower limits of the assay range; yet there are certainly situations where an extended concentration below the LLOQ can potentially provide useful data for various PK-related exercises [3-5]. Here we will consider some of the important chemical, physical, and matrix-related forces that have impacted quantification at low analyte levels in our labs over the past years, and we will discuss their potential for disruption of the quantitative properties of bioanalytical assays at sub LLOQ concentrations.
