PRINCIPLES OF METHOD VALIDATION AND QUALITY CONTROL:COMPLIANCE WITH THE NEW EU CRITERIA FOR BIOANALYTICALSCREENING OF FEED AND FOOD
Haedrich J1*, Hoogenboom LAP2, Eppe G3, Goeyens L4, Elskens M5, Malagocki P6, Scippo ML3, Vanderperren
H7, Windal I8, Kotz A1, Denison MS9, Malisch R1
1

European Union Reference Laboratory for Dioxins and PCBs in Feed and Food, D-79114 Freiburg, Germany;
RIKILT, Wageningen UR, P.O. Box 230, 6700AE Wageningen, The Netherlands; 3Université Liege, CART,
Faculty of Veterinay Medicine, Department of Food Sciences, Laboratory of Food Analysis, B-4000 Liège,Belgium; 4Life and Chemistry Office, Julius Caesarlaan 106, 1150 Brussels, Belgium; 5Vrije UniversiteitBrussel, Department of Analytical & Environmental Chemistry, Brussels, Belgium; 6National Veterinary
Research Institute, 24-100 Pulawy, Poland; 7Federal Agency Safety Food Chain, B-3080 Tervuren, Belgium;
8Rue du Tivoli 6, B-5030 Gembloux, Belgium; 9University of California Davis, Department of Environmental
Toxicology, Davis, CA 95616, USA2Introduction
For more than a decade, bioanalytical screening methods have effectively been applied in European official
control of feed and food samples for elevated levels of polychlorinated dibenzo-p-dioxins (PCDDs), dibenzofurans (PCDFs) and dioxin-like PCBs (dl-PCBs). Since requirements for application of these methods were first
implemented in 20021,2, experience has grown in bioassay laboratories resulting in revised criteria3,4,5,6 adopted
by EU-legislation7,8 in 2012. Each laboratory must demonstrate fitness-for-purpose of the bioanalytical method
applied and observe specified run acceptance criteria while performing screening analysis. A total of 27 out of
78 requirements center around validation procedures, while a similar number of criteria were set each for quality
control and evaluation of bioanalytical results. A particular focus is on cut-off concentrations for decisions ofsample compliance with regulatory limits9,10. This paper outlines principles of bioanalytical method validationand quality control.
Materials and methods
Validation – a two-step procedure
Bioanalytical screening methods are performed to identify samples with significant levels of PCDD/Fs and dlPCBs. By comparing the screening result expressed in bioanalytical equivalents (BEQs) to a cut-off level preestablished during validation, samples are classified as compliant or suspected to be noncompliant, requiringanalysis by a confirmatory method. The cut-off is based on the respective EU maximum level (ML) or actionlevel (AL), while taking into account GC/HRMS measurement uncertainty and the variability of bioanalyticalresults. It strongly depends on matrix properties and congener patterns, which may vary widely, even among
samples of the same matrix. Therefore, the relationship between bioanalytical and GC/HRMS results (in TEQs)
has to be established in a two-step validation procedure:(1) Initial Validation: Basic method performance is evaluated by a formal validation process using spiked blanksamples for calibration to prove the fitness-for-purpose of the method for each matrix/matrix group of interest.
Variability of congener patterns and matrix properties such as lipid contents leading to more variability of resultsin routine screening are not taken into account. From the calibration data, an initial cut-off level is established.
(2) Re-validation: Method performance and the relationship between BEQs and TEQs shall be re-evaluated after
the method was applied for some time in routine screening, by matrix-matched calibration experiments involving
samples both compliant and noncompliant from GC/HRMS confirmatory analysis, to account for variability incongener patterns and matrix properties. Based on the within-laboratory reproducibility achieved with thesesamples, the bioassay cut-off is re-evaluated.
Continuous quality control
Being closely associated with validation, extensive quality control is the third pillar in ensuring reliable methodperformance and results. QC measures include, but are not limited to: careful selection of suitable reference

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samples, inclusion of a procedure (or matrix) blank and a recovery control (reference) sample in each series,
correction of results from unknown samples for procedure blank and apparent recovery, monitoring of QC dataover time in QC charts and evaluation of QC results, GC/HRMS confirmation of all suspected samples and of 2
to 10% of the samples declared compliant, and continuous successful participation in interlaboratory proficiency
test (PT) studies. Laboratories applying bioanalytical methods for official purposes therefore require a close
cooperation with laboratories using a confirmatory method, for method validation and on-going quality control.
A QC database shall be established for each sample matrix/matrix group of interest, containing: (1) GC/HRMS
results of confirmed suspected and compliant samples (minimum of 20 samples per matrix), to be used in (re-)
evaluation of: the BEQ-TEQ relationship, method performance (re-validation), cut-off concentrations and actual
false-compliant rates; (2) results from inter-laboratory PT studies < 2x ML (optional). Re-evaluation of results
collected in the database shall be performed whenever sufficient new data for a certain sample matrix have been
included, or for any other relevant reason (e.g. if sample results with unusual congener patterns were included,etc.), being an on-going process.
Method calibration
Initial validation: Confirmed blank samples are spiked around the respective level of interest, generating 6 seriesof samples e.g. with concentrations 0x, 0.5x, 1x and 2x ML, for each relevant sample matrix. Use of spiked
instead of incurred materials ensures that the cell response is not significantly influenced by co-extracted nonregulated AhR-agonists but is mainly due to Ah-receptor active dl-compounds present in the sample extract.
Samples are submitted to extraction, clean-up and measurement in 6 consecutive series, providing within-lab
reproducibility conditions for the 6 repetitions on each level.
Re-Validation: Calibration experiments are performed for each relevant sample matrix involving a minimum of
20 confirmed routine samples contaminated in a range from the GC/HRMS-LOQ up to approx. 2x the level ofinterest, taken from the QC database. Samples are submitted to extraction, clean-up and measurement.
Linear regression: Bioanalytical results (BEQ) are plotted vs GC/HRMS results (TEQ), and the calibration(regression) line and its 95%-prediction interval are calculated.
Bioassay cut-off concentrations
In GC/HRMS confirmatory analysis, only those samples exceeding the ML after correction for measurement
uncertainty UDL (e.g. 15%) will be considered noncompliant. The bioassay cut-off is therefore based on theGC/HRMS decision limit (DL) and is calculated as lower endpoint of the distribution of bioanalytical results(RSDR<25%, see below) corresponding to DL (figures 1 and 2). Mathematically, the cut-off is obtained from thelower band of the one-sided prediction interval at xDL, as the BEQ-level above which 95% of the area under theGaussian normal distribution curve of the response variables corresponding to x DL are located:
Bioassay cut-off = y DL  sy,x * t α,f=m-2

1/ n 1/ m  (xDL x)2/ Q xx,

with Qxx =m2
 (x  x)ij 1,

m: number of calibration experiments, n: number of replicates, y DL: mean y-value from n repetitions at xDL, sy,x:
residual standard deviation, tα,f=m-2 (α = 5%, one-sided): student factor, the 95% level of confidence implying afalse-compliant rate < 5%. With this approach, cut-off levels may be estimated both from initial and from revalidation data. Bioassay cut-offs may in practice often be close to 2/3 of the ML, i.e. they may be correspondingto the current AL (figure 2). Alternative methods for establishing cut-off concentrations are also available3,4
however these do not provide full information on method performance as available from calibration experiments.
Restrictions to cut-off concentrations
During initial validation, the within-laboratory reproducibility (RSDR) of the screening method may be below
25% leading to a cut-off (in BEQ) close to or even above the permitted maximum level (in TEQ). In those cases,a modified (lower) cut-off must be calculated based on a RSDR of 25%, to account for increased variability ofsample results expected in routine, due to the large variety of matrix properties and congener patterns potentiallypresent in unknown samples. Only this modified cut-off may then be applied in routine screening to avoid falsecompliant results. Cut-off concentrations should be re-evaluated in a re-validation procedure as soon as aminimum of 20 confirmed sample results covering a range up to approximately 2xML have become available.
Bioassay cut-off levels are matrix/matrix group-related and to be applied during screening only to results of
those sample matrices they were previously established for.

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BioanalyticalResults (BEQ)

Initial Validation
Spiking Experiments
4 levels, 6 repetitions each

BEQDL

Regression Line

with 95%-Prediction Interval
Cut-off Value
BEQAL
ß = 5%+ UDL

ActionLevel

Max. Decision
Level Limit (DL)GC/HRMS Results (TEQ)

Figure 1. Initial validation: Exemplary calibration data (spiked samples), regression line, 95% prediction interval
and preliminary bioassay cut-off concentration. UDL: GC/HRMS expanded measurement uncertainty at DL
Bioanalytical
Results (BEQ)

Re-Validation
Calibration Experiments> 20 confirmed samples

BEQDLRegression Linewith 95%-Prediction Interval
Cut-off Value
BEQAL
ß = 5%+ UDLAction
LevelMax. Decision
Level Limit (DL)GC/HRMS Results (TEQ)

Figure 2. Re-validation: Exemplary calibration data (confirmed samples), regression line, 95% prediction
interval and bioassay cut-off concentration. UDL: GC/HRMS expanded measurement uncertainty at DL
Results and discussion
Verification of method performance criteria
Bioanalytical data included in method calibration during initial and re-validation must comply with the new run
acceptance criteria. Restrictions related to assay performance (triplicate-CVs, standard curve, assay workingrange) apply. An estimate of TEQ-levels may be reported in BEQs only if they are at least by a factor of three
above the procedure blanks. Estimated levels in unknown and recovery control samples must be corrected for theprocedure (or matrix) blank value. Subsequently, sample results are corrected for the recovery estimated fromthe recovery control sample. For this, apparent recoveries must be within required ranges. Several required
method performance criteria may then be verified from the calibration data sets acquired during initial validation

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195and re-validation, while some more general information on method performance may also be obtained:
Results from initial validation: Parameters of 6 individual calibration lines for each sample series (slope orsensitivity, y-intercept or mean matrix blank, coefficient of determination R 2), mean, recovery (corrected) andprecision (RSDR) on each concentration level.
Results from initial and re-validation: Parameters of the overall calibration line (slope/sensitivity of the method,y-intercept/mean matrix blank value, R2), calibrated range, center of mass, variability of bioanalytical data: therelative residual standard deviation may be used (instead of RSD R,) at any concentration within the calibrated
range, correspondence between BEQ and TEQ results, cut-off level at the GC/HRMS-DL (based on ML, or on
any other TEQ concentration), bioassay limit of detection (LOD) e.g. as in DIN 32645:2008. It may further be ofinterest to compare performance characteristics obtained from initial and from re-validation.
Actual false-compliant and false-noncompliant rates: Actual α- and ß- errors may be assessed by applying thecut-off concentration estimated during initial validation to the confirmed compliant and non-compliant results
collected and/or used in re-validation. The fraction of false-compliant results must be below 5%. The fraction offalse-noncompliant results may be calculated based on both the number of all screened samples and the numberof samples suspected to be noncompliant.
After it could be demonstrated that the achieved performance meets all legal requirements, the bioanalyticalmethod is ready for use in screening of feed and/or food samples according to European legislation.
Acknowledgements
We would like to thank the European Commission for the financial support of the work of the European Union
Reference Laboratory for Dioxins and PCBs in Feed and Food (EU-RL), Freiburg, Germany
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