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Validation of analytical methods based on mass spectrometric detection according to the “2002/657/EC” European decision: guideline and application

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Validation of analytical methods based on mass spectrometric detection according to the “2002/657/EC” European decision: guideline and application

Jean-Philippe Antignac∗ , Bruno Le Bizec, Fabrice Monteau, François Andre

Laboratoire d’étude des résidus et contaminants dans les aliments (LABERCA), Ecole Nationale Vétérinaire de Nantes, BP 50707, F-44307 Nantes Cedex 3, France Received 27 June 2002; received in revised form 3 October 2002; accepted 22 October 2002

Abstract The purpose of the present paper is to present an interpretation of the concepts introduced in the new 2002/657/EC European decision and to propose a practical guideline dedicated to the validation of analytical methods based on mass spectrometry. Considering both the statistical signiﬁcance of the results and practical aspects, the minimal number of assays permitting a satisfying validation to be achieved appeared to be 45 for qualitative methods and 55 for quantitative methods. The parameters validated with this protocol are speciﬁcity, sensitivity, linearity, decision limit (CCα), repeatability, detection capability (CCβ) and recovery. It is proposed to estimate these parameters on the basis of the most intense (or unique) ion for screening methods and on the basis of the “critical ion” (less intense ion permitting the unambiguous identiﬁcation of the analyte according to the required number of identiﬁcation points) for conﬁrmatory methods. An application of this guideline is presented and discussed, through the validation of a liquid chromatography-tandem mass spectrometric (LC–MS/MS) method dedicated to the determination of the corticosteroid triamcinolone acetonide (Tri Acn) in meat samples. © 2002 Elsevier Science B.V. All rights reserved.

Keywords: Validation; 2002/657/EC; Mass spectrometry; Decision limit (CCα); Detection capability (CCβ)

1. Introduction Validation of analytical methods dedicated to the measurement of a physico-chemical parameter or property relative to a substance, material or system is not a recent problem. When developing a theory or a technical process, scientists have always tried to demonstrate the validity of their work using original experimental methodology or a more conventional data set, for example some repetitions of the measure∗ Corresponding author. Tel.: +33-2-40-68-77-66; fax: +33-2-40-68-78-78. E-mail address: laberca@vet-nantes.fr (J.-P. Antignac).

ment in identical and/or different experimental conditions. Nevertheless, the diversity of the application ﬁelds, validation procedures, estimated parameters, as well as terms and concepts deﬁnitions, led to a lack of objective quality criteria and harmonization, causing great difﬁculty in comparing methods or results. Moreover, the exponential development of applied mathematics, statistics and informatics as well as theoretical considerations from several expert groups has introduced much in the way of normalization, researching and universal concepts. The two main work axes have been on one side the deﬁnition of pertinent “quality indicators” of an analytical method, and on the other side their associated calculation modalities.

0003-2670/03/$ – see front matter © 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 3 - 2 6 7 0 ( 0 2 ) 0 1 3 7 9 - X

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This approach permitted real progress but some difﬁculties remained, linked both to the existence of several ofﬁcial institutions (ISO, CODEX, IUPAC, etc.) each with their own speciﬁcations, and to the difﬁculty of changing some practical habit in laboratories. The best illustrative example are the concepts limit of detection (LOD) and limit of identiﬁcation (LOI). These two parameters are probably the most widely applied to estimate method performance in terms of sensitivity. However, the nature of the measured signal, the calculation mode (practical approach based on the signal-to-noise ratio or theoretical approach based on the noise amplitude of blank samples), the unambiguous identiﬁcation criteria, as well as the statistical signiﬁcance (number of repetitions, tolerance and conﬁdence level) are not systematically well deﬁned, even though important literature has been dedicated to these concepts [1–6]. At the European level, an important working group was mandated to propose a decision, now referenced 2002/657/EC,1 to harmonize the characterization and the validation procedure of analytical methods performance. This decision provides rules on how methods are to be used in the testing of ofﬁcial samples according to Article 15, paragraph 1, second sentence of the Council Directive 96/23/EC, and common criteria for the interpretation of analytical results of ofﬁcial control laboratories for samples taken according to the same Directive. This decision shall not be applicable where for certain substances more particular rules have been laid down in Community Legislation. The application ﬁeld of this reference document is analysis of biological matrices for residue and contaminants, including organic and mineral substances, forbidden and regulated substances, based on qualitative and quantitative methods, screening and conﬁrmation analysis. One merit of such a document is to extend some general concepts to a very large panel of detection techniques, and to propose a common backbone for the validation of the corresponding analytical methods. The best example could be the notion of identiﬁcation points for conﬁrmatory analysis [7] and the introduction of the decision limit (CCα) and detection capability (CCβ) concepts to replace LOD and LOI. However,

1 2002/657/EC Commission Decision of 12 August 2002. “Implementing Council Directive 96/23/EC concerning the performance of analytical methods and the interpretation of results”.

this very complete and complex document is not always very easy to read for the analyst, and the need for a practical “ﬁt-for-purpose” guideline clearly appeared both for the reference and application laboratories. In this context, the purpose of the present paper was to extract from the original reference document only the necessary theoretical and practical recommendations for the validation of analytical methods dedicated to the analysis (qualitative/quantitative, screening/conﬁrmation) of residues and contaminants (forbidden/regulated) in biological matrices. Only mass spectrometry was considered as the main detection technique for use in this domain. First, the practical meaning of the decision limit and detection capability concepts will be presented. Secondly, a practical validation guideline will be proposed, which was built considering a discussed compromise between statistical signiﬁcance and practical aspects. Finally, an application of this guideline will be presented and discussed, corresponding to the liquid chromatography-tandem mass spectrometry (LC–MS/MS) analysis of the corticosteroid ﬂuocinolone acetonide residues in meat samples.

2. Decision limit and detection capability In the 2002/657/EC European decision, the decision limit (CCα) was deﬁned as “the limit at and above which it can be concluded with an error probability of α that a sample is non-compliant”, and the detection capability (CCβ) as “the smallest content of the substance that may be detected, identiﬁed and/or quantiﬁed in a sample with an error probability of β”. In fact, these concepts had already been introduced in the ISO/11843-1 normative document [8], to propose a method ﬁxing a limit from which a system can be declared different from its basic state (Fig. 1). In the present case, the system is a diagnostic ion chromatogram of the target analyte, and the basic state corresponds to this ion chromatogram for a blank sample (forbidden substances) or for a sample containing the analyte at the MRL concentration (regulated compounds). As shown in Fig. 2, the measured value for characterizing the system is the analyte signal amplitude (noise or analyte peak height), expressed relative to the internal standard signal amplitude. In practice, two main sources have to be considered to explain

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Fig. 1. Deﬁnition of CCα and CCβ according to the ISO/11943 normative document.

the variability observed for the recorded signal; one from the noise (mainly due to the matrix effect, depending of the method speciﬁcity) and one from the measurement (depending of the method sensitivity and repeatability). CCα and CCβ permits these two different factors to be characterized. The signal associated with CCα corresponds to a maximal noise amplitude (forbidden substances) or to a maximal signal amplitude for a sample containing the analyte at the MRL concentration (regulated substances), i.e. the amplitude that can only be exceeded in α% of the cases. This signal can be considered as an horizontal line drawn on the ion chromatogram to materialize a limit below which the observed signal can be declared as not statistically different from the noise (forbidden substances) or not statistically higher than the one induced by a sample containing the analyte at the MRL concentration (regulated

substances). In other words, when the recorded signal is lower than CCα, the sample can be declared compliant (analyte absent or present at a concentration lower than the MRL) with a conﬁdence level of (1 − α). CCβ is the critical real measured concentration above which it can be concluded that the analyte is unambiguously present (forbidden substances) or present at a concentration unambiguously higher than the MRL (regulated substances). In other word, supposing the analysis of a sample (1) giving a signal higher than the decision limit and (2) leading to an estimated concentration C, this sample can be declared non-compliant (analyte present or present at a concentration higher than the MRL) if C ≥ CCβ, with a conﬁdence level of (1 − β). For samples giving a signal higher than the decision limit but leading to an estimated concentration

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Fig. 2. Signal to consider for blank sample (upper) and for sample containing the analyte (lower).

between CCα and CCβ, high suspicion can be generated, but from a statistical point of view, the result remains unclassiﬁed. Depending on political decisions, protection of producers, industrials or consumers, the sample would be declared compliant or not compliant. A second possibility should be to use an alternative method if available and/or increase the concentration of the extract. In theory, CCα and CCβ can be calculated for each different signal characterizing the analyte. However, some additional considerations are needed depending on the screening or conﬁrmatory purpose of the

method. For screening methods, e.g. mass spectrometry based on the most intense ion, or any other screening detection technique, these values can only be estimated for the unique diagnostic signal, and consequently are not subject to further identiﬁcation criteria requirements. For conﬁrmatory methods, i.e. mainly mass spectrometry based on several ions permitting a minimal identiﬁcation score (3 or 4 depending on the substance) to be achieved, These values have to be preferably estimated for the more “critical ion” (in general the less intense one), and only after having veriﬁed that all the identiﬁcation criteria are

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in the correct tolerance range (relative retention time, different ion ratio). 3. Proposed validation protocol 3.1. Qualitative and/or forbidden substances Our objective was to ﬁnd a compromise between the 2002/657/EC decision requirements, an optimized organization for the assays to use the same experiments to validate several parameters, and some practical aspects and limitations linked to a laboratory activity. Finally, it was assumed that 45 assays should permit a satisfactory validation if conducted as follows: ﬁve concentration levels for calibration, 2× 10 blank samples, and 2× 10 spiked samples. It is recommended to divide these assays into ﬁve series conducted on separate days. The analysis of at least 20 blank samples, from different origins to check the ruggedness of the method, permits the speciﬁcity to be evaluated through the average (µN ) and standard deviation (σ N ) of the noise amplitude, expressed relative to the internal standard signal amplitude. The calibration should preferably be realized on a pool of the 20 previously analyzed blank samples and include at least ﬁve fortiﬁcation levels, using in addition the previously estimated noise average (µN ) as a forced intercept. The total number of six concentration levels appears to satisfy for many authors, and for the 2002/657/EC decision, to well deﬁne a calibration graph, but some discussions still remain concerning the choice of these levels as well as their equidistant or non-equidistant separation. Our position was not to use systematically equidistant levels, but on one side to verify the linearity of the concentration domain globally used in practice (for example 0–100 ng ml−1 ) and on the other side to give more importance to the low concentrations (for example 0–5–10–15–50–100 ng ml−1 ), because this part of the graph has a strong inﬂuence on the CCα and CCβ values. Finally, this calibration graph permits linearity to be evaluated through the regression coefﬁcient (R2 ) and the sensitivity through the slope of the ﬁtted graph (a). Then, a and σ N permit the decision limit (CCα) to be calculated considering the equation of the calibration graph (Eq. (1)), where I is the signal amplitude and C the

concentration, and the deﬁnition of CCα (Eq. (2)), the combination leading to the expression given by Eq. (3). ICCα =µN +aCCα ICCα =µN +2.33σN CCα= 2.33σN a (1) (2) (3)

An alternative method consists of using the standard error of the ﬁtted calibration graph interpolated intercept (σ e ) instead of the average of the noise amplitude (σ N ). The advantage of this second approach would be to avoid the analysis of the 20 blank samples and to require only several (at least three) repetitions at each fortiﬁcation level. Nevertheless, the interpolated intercept appears strongly sensitive to the linearity, to the considered fortiﬁcation levels and to the repeatability of each three assays series. Consequently, some interpretation difﬁculties can arise with this method in the case of negative interpolated intercept or standard error artiﬁcially underor over-estimated. For example, a linearity better than 0.9999 due to the inﬂuence of one particular high fortiﬁcation level and an apparent high repeatability can lead to a very poor intercept standard error and ﬁnally to a largely underestimated CCα value. This explains the choice made at present to consider the real measured noise variability value (σ N ) and to force the ﬁtted calibration graph by the corresponding average (µN ). However, it is recognized that this very critical point will merit further discussion and practical testing. The analysis of 20 spiked samples permits the repeatability to be estimated through the standard deviation of the signal amplitude (σ s ). Ideally, the fortiﬁcation level used should be exactly CCβ. In practice, this concentration is estimated during the method development. Typically, a concentration inducing a signal-to-noise ratio of ca. 6 can be used. In order to minimize this estimation error, consideration of the signal relative standard deviation ((R.S.D.)s ) is preferable to the standard deviation (σ s ). Finally, σ N , a, and (R.S.D.)s permit to the detection capability (CCβ) to be calculated, considering the calibration equation (Eq. (4)), where I is the signal amplitude and C the concentration, and the deﬁnition of CCβ (Eq. (5)), the combination of these

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two formula leading to the ﬁnal expression given by Eq. (6). ICCβ = µN + 2.33σN + 1.64σCCβ = µN + 2.33σN + 1.64(R.S.D.)s ICCβ CCβ = 2.33σN + 1.64µN (R.S.D.)s a[1 − 1.64(R.S.D.)s ] (4) (5)

3.2. Quantitative methods and/or regulated substances It was assumed that 55 assays should permit a satisfying validation if conducted as follows. Calibration should be realized using real extracted samples when possible and include blank samples and at least ﬁve fortiﬁcation levels around the critical value (MRL) and blank samples. The minimal number of assays should be 20 replicates at the MRL concentration, 10 replicates at 0.5MRL and 1.5MRL, and 5 replicates under 0.5MRL and above 1.5MRL as well as for blank samples. These assays authorize the evaluation of the linearity around the critical value through the regression coefﬁcient (R2 ), the sensitivity through the slope of the ﬁtted graph (a), and the intercept corresponding to the noise amplitude average obtained on the basis of the ﬁve blank sample analyses (µN ). The 20 spiked samples at the MRL concentration give access to the repeatability at this concentration level through the average (µMRL ) and standard deviation (σ MRL ) of the signal amplitude. Finally, a, µN , µMRL and σ MRL permit the decision limit CCα to be calculated considering the calibration equation (Eq. (6)), were I is the signal amplitude and C the concentration, and the deﬁnition of CCα (Eq. (7)), the combination of these two formula leading to the ﬁnal expression given by Eq. (8). ICCα = µN + aCCα ICCα = µMRL + 1.64σMRL CCα = µMRL −µN + 1.64σMRL a (6) (7) (8)

this concentration is estimated during method development (concentration inducing a signal-to-noise ratio >6) and considering the relative standard deviation of the signal ((R.S.D.)s ) rather than its standard deviation (σ s ) to minimize the estimation error on the concentration used for fortiﬁcation. Finally, a, µN , µMRL , σ MRL and (R.S.D.)s permit the detection capability (CCβ) to be calculated, considering the calibration equation (Eq. (9)), were I is the signal amplitude and C the concentration, and the deﬁnition of CCβ (Eq. (10)), the combination of these two formula leading to the ﬁnal expression given by the Eq. (11). ICCβ = µN + aCCβ ICCβ = µMRL + 1.64σMRL + 1.64σCCβ = µMRL + 1.64σMRL + 1.64(R.S.D.)s ICCβ (10) CCβ= µMRL − µN + 1.64σMRL + 1.64µN (R.S.D.)s a[1 − 1.64(R.S.D.)s ] (11) (9)

4. Application example The analytical method presented to illustrate the proposed validation procedure concerns the analysis for the corticosteroid triamcinolone acetonide (Tri Acn) in meat samples. The extraction and puriﬁcation method was presented elsewhere [9]. The technique was liquid chromatography-tandem mass spectrometry using negative electrospray ionization and MRM acquisition using two transitions. The analyte response was always related to the internal standard response (IS: triamcinolone acétonide-d6, added at 5 ng ml−1 in each sample). The results are summarized in Table 1. The speciﬁcity, in terms of absence of interfering compounds appearing on the ion chromatograms (Fig. 3) and noise variability, was greatly improved by the use of MS/MS but clearly also by the efﬁcient puriﬁcation process. The response linearity was found to be satisfactory with a R2 value >0.99 for the two transitions (Fig. 4). The sensitivity ratio between the two calibration slopes (0.82) perfectly reﬂected the intensity ratio between the two product ions (0.85 ± 0.04, n = 20). For the spiked samples, all the required

The 10 spiked samples at the 1.5MRL concentration to the estimation of the repeatability at this concentration level through the standard deviation of the signal amplitude (σ s ). Ideally, the fortiﬁcation level used should be exactly CCβ. As previously discussed,

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Fig. 3. Triamcinolone acetonide diagnostic ion chromatograms for a blank meat sample (left) and for a 150 ng ml−1 spiked meat sample (right).

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Fig. 4. Calibration graphs determined on the basis of triamcinolone acetonide meat hair samples for the two diagnostic MRM transitions.

Fig. 5. Test of the identiﬁcation criteria requirements for the spiked samples used for the method validation.

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Table 1 Validation results for the method dedicated to the LC–MS/MS determinations of the corticosteroid triamcinolone acetonide (Tri Acn) in meat samples (15 g) Process (0) Diagnostic signals (1) Blank samples (2) Calibration curve Parametera Analyte (Tri Can) Internal standard (Tri Can-d6) Speciﬁcity (µN ; σ N ) Concentration levels Linearity (R2 ) Sensitivity (a) (ng ml−1 ) 0.9985 0.2141 Signal 1 (more intense) 493 > 413 499 > 419 0.0122; 0.006 0.0124; 0.007 0.15; 0.3; 1.5; 3; 7.5 0.9984 0.1748 1.50 5.7 0.4 4.6 96.6 0.07 0.08 100 0.10 0.11 5.8 Signal 2 (less intense) 493 > 375

(3) Spiked samples

Concentration level (ng ml−1 ) Repeatability (R.S.D.) Signal (%) RRT (%) Ratio (%) Recovery (%) CCα (ng ml−1 ) CCβ (ng ml−1 )

(4) Critical limits

a

R2 , coefﬁcient of determination; a, slope of the ﬁtted curve; (µN ; σ N ), average and standard deviation of the noise; R.S.D., relative standard deviation; RRT, relative retention time.

identiﬁcation criteria were successfully tested using a self-made automatic diagnostic ﬁle (Fig. 5) and the repeatability of the two signals appeared very satisfying (5.7 and 5.8%, respectively). The decision limit and the detection capability were calculated for each signal (Table 1). However, it was assumed that the values with main practical interest were the decision limit estimated on the basis of the more intense transition (the objective at this level is to detect the analyte) and the detection capability estimated on the basis of the less intense transition (the objective at this level is to identify unambiguously the analyte). The obtained corresponding values (CCα = 70 ppt) and detection capability (CCβ = 110 ppt) appeared very satisfying and coherent with the observation of the ion chromatograms in term of signal-to-noise ratio as shown in Fig. 3.

5. Conclusion The main objective for the presentation of this validation guideline was to underline the necessary requirements of the 2002/657/EC European decision for

its application in the ﬁeld of drugs and contaminant residue analysis by mass spectrometry. It appeared that the minimal number of assays to achieve this requirements is 45 for qualitative methods and 55 for quantitative methods, if organized and applied as described. Of course, additional assays can be included especially when recovery yield performances are needed. Moreover, a continuous survey using quality test systems as control charts remains as a necessary tool to prevent any deviation of the method performance. This practical methodology was established taking into account a good compromise between statistical signiﬁcance of the results and practical aspects, with the objective to give a more “ﬁt-for-purpose” protocol as possible. However, some important points remains to be deﬁned and harmonized, such as an ofﬁcial position on the decision to give a quantitative result regarding the method CCα and CCβ. In conclusion, we think that a large diffusion and utilization of the detection capability concept should be encouraged. Indeed, it can represent an elegant alternative to highly complex uncertainty calculations. The counterpart is the necessity of highly improved analytical methods, particularly in term of speciﬁcity, for comfortable application.

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J.-P. Antignac et al. / Analytica Chimica Acta 483 (2003) 325–334 [6] H. Van der Voet, J.A.H. Van Rhijn, H.J. Van de Wiel, Anal. Chim. Acta 391 (1999) 159–171. [7] F. André, K. De Wasch, H.F. De Brabander, S. Impens, L. Stolker, L. van Ginkel, R. Stephany, R. Schilt, D. Courtheyn, Y. Bonnaire, P. Fürst, P. Gowik, G. Kennedy, T. Kuhn, J.P. Moretain, M. Sauer, Trends Anal. Chem. 20 (2001) 435– 445. [8] ISO/11843-1, Capability of Detection (Part 1): Terms and Deﬁnitions, International Organization for Standardization, 1997. [9] J.P. Antignac, B. Le Bizec, F. Monteau, F. Poulain, F. André, J. Chromatogr. B 757 (2001) 11–19.

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[1] L.A. Currie, Anal. Chim. Acta 391 (1999) 127–134. [2] L.A. Currie, Anal. Chim. Acta 391 (1999) 105–126. [3] M. Feinberg, N. Raguènès, Anal. Chim. Acta 391 (1999) 239– 252. [4] P. Hubert, P. Chiap, J. Crommen, B. Boulanger, E. Chapuzet, N. Mercier, S. Bervoas-Martin, P. Chevalier, D. Grandjean, P. Lagorce, M. Lallier, M.C. Laparra, M. Laurentie, J.C. Nivet, Anal. Chim. Acta 391 (1999) 135–148. [5] A. Maroto, J. Riu, R. Boqué, F.X. Rius, Anal. Chim. Acta 391 (1999) 173–185.

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