in animal feed Received: 29 June 2007 / Accepted: 20 May Wageningen Academic Publishers

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1 World Mycotoxin Journal, August 2008; 1(3): Wageningen g Academic P u b l i s h e r s Single laboratory validation data and uncertainty estimation of a liquid chromatographic method for determination of aflatoxin B 1 in animal feed M. Nocentini, C. Focardi, S. Ninci and G. Biancalani Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Sezione di Firenze, Via di Castelpulci 43, San Martino alla Palma, Florence, Italy; mila.nocentini@izslt.it Received: 29 June 2007 / Accepted: 20 May Wageningen Academic Publishers Abstract In the present work we have validated an analytical method for the determination of aflatoxin B 1 in dairy compound feeds and peanut meal. The method is based on a preliminary extraction with a mixture of methanol and water followed by a clean-up step with immunoaffinity column. The content of aflatoxin B 1 is then established using reversed-phase high-performance liquid chromatography coupled with fluorescence detection. The method is fully validated by using Certified Reference Materials. Limit of detection is found to be 0.5x10-3 mg/kg. The Bartlett test has shown that the method is affected by a systematic error and as a consequence the results must be corrected for the recovery. The precision is found in all cases lower than those calculated by Horwitz equation. Uncertainty estimation has been performed taking into account the bottom-up approach as stated in the Eurachem/Citac guide. With this method the uncertainty is calculated by the addition of variances for a measurand M when the equation for the calculation of M is based only on multiplications and divisions. The bottom-up approach is a tool which identifies the parameters or working steps with an overly contribution to the total uncertainty, thus offering the chance to improve them. The final uncertainty budget has revealed that the main contributions to global uncertainty are those due to the recovery and to the calibration function. Keywords: aflatoxin B 1, measurement uncertainty, single laboratory validation, animal feed, liquid chromatography 1. Introduction Aflatoxins are a group of naturally occurring toxins produced by moulds such as Aspergillus flavus. When cows ingest feed contaminated with aflatoxin B 1, this is metabolised to aflatoxin M 1 and excreted into the milk. Aflatoxin B 1 is the main hepatocancirogen in animals and it is classified by IARC (2002) as belonging to Group 1 (carcinogenic to humans and animals). Aflatoxins represent a serious risk for animal and human health, and for this reason the European Legislation (Directive 2003/100/CE) defined acceptable limits in the case of animal feeds (EC, 2003). The lower level, equivalent to mg/kg, is fixed in the case of feeds for cows. The Regulation (CE) 401/2006 defined the performance criteria for the analytical methods in the specific case of foods for human consumption (EC, 2006). In the absence of specific legislation concerning analytical performance applied to animal feeds, we adopt the criteria reported in the previous Regulation. The method adopted is based on immunoaffinity column cleanup and liquid chromatography with fluorescence detection after derivatisation with trifluoroacetic acid (Barmark and Larsson, 1994).This method is basically the same of the AOAC Official Method with the exception of the post-column derivatisation system. More recently the AOAC Official Method for aflatoxin B 1 in cattle feed has been reported, which differs from the method adopted by us in the solvent used for the extraction step, i.e. acetone/water instead of methanol/water. The validation data are obtained using Certified Reference Materials (CRM 375, 376 and 264), in accordance with the ISSN print, ISSN online, DOI /WMJ

2 M. Nocentini et al. Eurachem/Citac guide (1998). The expanded uncertainty has been evaluated applying the bottom-up approach as reported in the Eurachem/Citac guide. The aim of this work is to provide a means of checking whether this analytical method correctly meets the legal requirements. 2. Materials and methods Reagents and standards Standard Reference Material of Aflatoxin B 1 (AFB 1 ),was purchased from Sigma with a purity of 99.9%. Deionised water is obtained by a Milli-Q system (Millipore). Acetonitrile and methanol of HPLC grade, and sodium chloride of analytical grade were supplied by J.T. Baker. Trifluoroacetic acid (TFA) was purchased by Merck, and immunoaffinity columns AflaTest WB (G1024) were supplied by Vicam. Glass microfibre filters with 47 mm of diameter and 0.7 µm of retention, were purchased from Sartorius and filter papers with 15 cm of diameter were obtained from Schleicher & Schuell. The Certified Reference Materials CRM 375, CRM 376, CRM 264 used during the validation have been reported in Table 1. CRM 375 and CRM 376, as reported by the Community Bureau of Reference, have been obtained by blending dairy feed with a variety of feed components, such as barley, manioc, citrus pulp, soya and cattle nut. The CRM 264 has been used for the validation even if it is not a component of animal feed. The Standard Reference Material is a solution in methanol of aflatoxin B 1 at concentrations reported in Table 1 and it has been used to prepare standard solutions with proper dilutions. Experimental design Method validation together with uncertainty measurement can provide a way to check whether an analytical method meets the legal requirements. To reach this goal we have used the following methodology with different steps: 1. Applicability and fitness for purpose: they have been evaluated by using different matrices in a concentration range corresponding to legal limits. 2. Specificity: this has been tested by verifying the absence of interferences under the stated conditions for the sample matrix. 3. Calibrations study: linearity of the response function has been checked using a calibration standard by performing three replicate analyses for each standard solution on different days by different operators. The goodness of fit has been evaluated with the plot of the residuals, the F-test and the estimation of the g factor. Sensitivity expressed in terms of limit of detection and quantification has been calculated from the calibration curve. 4. Accuracy study: assessment of constant bias has been derived by applying different statistical tests (Bartlett and t-test) to the values obtained with the Certified Reference Materials at two different concentration levels on different days with different operators. The same data have been used for the estimation of trueness and precision. Trueness has been expressed as recovery and precision in terms of relative standard deviations. The measurement uncertainty has been estimated following the bottom-up approach starting from the equation of measurand and applying the error propagation law. The estimations of the variances were performed from the single laboratory validation data. Standards preparation Due to the toxicity of Aflatoxin B 1 all the necessary safety considerations have been taken in to account when handling this substance. The standard stock solution at the concentration level of 250 ng/ml has been obtained for dilution with methanol of the Standard Reference Materials (Table 1). Working standard solutions at four different concentration levels, 15, 25, 50 and 100 ng/ml, were prepared by evaporation to dryness under nitrogen flow respectively of 60 µl, 100 µl, 200 µl and 400 µl of the stock solution. These standards are subsequently derivatised, with TFA as reported below, before injection into the HPLC instrument. The blank standard solution has been prepared by mixing 50 µl of TFA and 1 ml of acetonitrile:water (10:90, v/v). Sample preparation The sample preparation is that reported by Barmark and Larsson (1994) with fewer modifications. The samples are dried, at 105 C for one night, after which they are ground with a mill and homogenised. 50 g of the samples are weighed to the nearest g directly in a conical Table 1. Reference materials used for method validation. Reference Material Matrix Certified Concentration Certified uncertainty Standard Reference Material (SRM) Solution in methanol µg/ml ±0.345 µg/ml CRM 375 Dairy compound feed <0.001 mg/kg - CRM 376 Dairy compound feed mg/kg ± mg/kg CRM 264 Defatted peanut meal mg/kg ±0.013 mg/kg 248 World Mycotoxin Journal 1 (3)

3 Validation data and uncertainty estimation of a liquid chromatographic method for aflatoxin B 1 flask and 250 ml of methanol:water (70:30, v/v) are added. Then the sample is shaken for 45 min with a shaker plate. After filtration through a paper filter, 30 ml of the filtrate is diluted with 60 ml of water. To obtain a clear solution the sample is passed through a glass microfibre filter. The sample is purified with the immunoaffinity column containing immobilised antibodies against aflatoxin. The column is activated by rinsing with 10 ml of water at a constant flow rate by using a syringe. Just before the column becomes dry, 90 ml of diluted extract is put on the column and eluted at the same flow rate. After washing the column with 10 ml of water, it is dried with air. The AFB 1 is eluted slowly (30 s) with 2 ml of methanol and collected in a conical glass vial. The eluate is evaporated to dryness under a stream of nitrogen and derivatised as reported below. Derivatisation 50 µl of TFA is added to the dry residue: then 1 ml of acetonitrile:water (10:90, v/v) is added and the solution is put in a Vortex mixer for 30 s, after which the tube is left at room temperature for 5 minutes. Then the test solution is injected into a LC column according to the published procedure (Kok, 1994). Equipment and chromatographic condition HPLC was performed with a Perkin Elmer (USA) Series 200 system consisting of a quaternary gradient pump, an autosampler, a fluorescence detector and a degassing system using helium. Chromatographic separation was achieved using a Varian Omnisphere 5, C18 ( mm, 5 µm) reversed phase column, with a guard column of the same type. The mobile phase was constituted by water, acetonitrile and methanol in the ratio 60:20:20 (v/v/v). An isocratic elution was performed at a flow rate of 1 ml/min for a total run time of 20 minutes. The injection volume was 20 µl. The detector was set at an excitation wavelength of 365 nm with an emission wavelength of 455 nm. 3. Results and discussion Method validation The validation data are obtained by using Certified Reference Materials as reported in Table 1. Specificity Figure 1 reports the chromatogram corresponding to the following samples injected in the HPLC: (A) AFB 1 standard solution at a concentration level of 50 ng/ml; (B) the Certified Reference Materials CRM 376; (C) a blank animal feed sample. In the chromatogram (C) there is a peak at the retention time of the AFB 1, the area correlated Detector response to this peak is always less than that corresponding to the limit of detection of the method. This peak is due to the fact that our matrix are never completely free from aflatoxin B 1. No subtraction of the area due to the blank from the concentration of the samples has been done. Linearity AFB Time (min) The calibration curve of AFB 1 is obtained in the range of standard concentrations between 15 and 100 ng/ml. Four calibration points are chosen for a linear regression plot of the total peak area versus the injected amount of AFB 1. The homoscedasticity assumption has been tested by plotting residuals versus concentration and applying the F-test (i.e. test for the significant difference in variance). The suitability of the calibration line has also been checked by the calculation of the quality coefficient g, as defined by Miller (1991) and further by De Beer et al. (2007). The residual plot clearly shows that error is randomly distributed around the concentration axis. The F-test, at 95% of confidence, also reveals no significant difference between variances when the experimental F-value (F exp =23) is compared to the tabled one (F tab =39.165). The g value obtained is equal to 0.002, lower than the threshold value of 0.05 reported by the authors (Miller, 1991). As a consequence the unweighted least square method has been used and the relationship between the response, expressed as peak area, and the concentration of analyte is a straight line in the form of y=a+bc x where a is the intercept and b is the slope of the regression line. Evaluation of systematic errors The Bartlett test applied on data obtained with Certified Reference Materials demonstrated that there is no homogeneity between the variance, so we can conclude (A) (B) (C) Figure 1. HPLC-fluorescence chromatogram of AFB 1 : (A) AFB 1 standard solution 50 ng/ml, (B) CRM 376, (C) blank feed sample. World Mycotoxin Journal 1 (3) 249

4 M. Nocentini et al. that this method shows a systematic error. By applying the statistic test t, as reported in the Eurachem/Citac (2000) we obtained, in the cases of CRM 264 and CRM 376, t-values higher than the t crit for n-1 degree of freedom at 95% of confidence. Consequently in the routine analysis the results must be corrected for the recovery obtained in the same set on spiked or certified reference materials. Trueness and precision Table 2 shows the values of trueness and precision related to the CRM materials. As stated in EC Regulation 401/2006 (EC, 2006) the trueness of the method changes with the analyte concentration in the sample, and is respectively for C<0.001 mg/kg between -50% to +20%, for 0.001<C<0.010 mg/kg between -30% to +10% and for C>0.010 mg/kg between -20% to +10%. If we compared these values with our experimental data of trueness reported in Table 2, it is clear that for the lower concentrations tested our method matches the performance criteria. Instead, for the CRM 264 the trueness exceeds the range permitted, due to the bias of the method. We must bear in mind that the concentration level of this CRM is 10 times higher than the maximum permitted limit. The precision of the method is expressed as relative standard deviation (RSD %); these values are always lower then that obtained from the Horwitz equation (RSD max %). Limit of detection and limit of quantification The limit of detection has been calculated by the regression analysis applied to the calibration curve of the standard solutions. The LOD is equal to mg/ kg, consequently the limit of quantification is equal to mg/kg. Uncertainty estimation For the evaluation of the uncertainty related to the method the bottom-up approach has been used. The measurand equation is as follows: C x V f V ext 100 C(mg/kg) = D 10-3 (1) V e M R Where: M = Mass of the sample (in grams); R = Recovery (%); C x = Sample concentration calculated by the calibration function, V ext = Extraction volume (ml); V f = Derivatisation volume (ml); V e = Elution volume from the immunoaffinity column (ml); D = Dilution factor (if no dilution is applied to the sample this factor is equal to 1 and no uncertainty contribution has to be accounted for). The combined standard uncertainty is obtained by propagation of individual contributions, and it is expressed in terms of relative uncertainty by the following formula: ů(c) = (ů(p)) 2 +(ů(c x )) 2 +(ů(r)) 2 +(ů(m)) 2 +(ů(v)) 2 (2) Where: ů(p) is the contribution referred to repeatability data; ů(c x ) is the contribution derived from calibration; ů(r) is the contribution due to the recovery efficiency of the method; ů(m) is the contribution derived from errors on weighting and ů(v) is the combination of the errors related to the dilution volumes (V ext, V f, V e ). The estimation of uncertainty was structured using an Ishikawa diagram as suggested by Barwick and Ellison (1999). As illustrated in Figure 2, every factor of the equation of the measurand (Equation 1) appears in the diagram. The different contributions to the uncertainty have been determined by the validation data. As an example, the data obtained in the case of CRM 264 is reported. 1. Uncertainty due to the precision. It is expressed as a relative standard deviation obtained from repeatability of the data: ů(p) = s = = (3) CM n ½ ½ where the values of C M, s and n are reported in Table Uncertainty due to the determination of the sample concentration by calibration function. This is given by the combination of uncertainty due to the standard preparations and those due to the assumption of a linear Table 2. Data of trueness and precision obtained for CRM samples. Sample C (mg/kg) n C M (mg/kg) s (mg/kg) Trueness (%) RSD (%) RSD max (%) CRM 375 < CRM CRM C = the certified concentrations; n = number of replicates; C M = the average concentration obtained; s = standard deviation associated; RSD (%) = the relative standard deviation obtained under repeatability condition; RSDmax = 2/3x2 (1-0.5logC) (with C the analyte concentration in g/g). 250 World Mycotoxin Journal 1 (3)

5 Validation data and uncertainty estimation of a liquid chromatographic method for aflatoxin B 1 V e e Dilution, V Pipette Cal p T p Pipette Cal p Rep p T p Glassware Cal T g g V ext f(c x ) Concentration, C x A x A std Glassware Cal T g g Pur SRM C std V f Rep p C SRM C std Aflatoxin B 1 (mg/kg) C x Instrument C CRM C M Method recovery C CRM Cal B Balance C CRM C M Pur CRM Precision, P Recovery, R Mass, M Figure 2. Ishikawa diagram of uncertainty contribution for AFB 1 determination in feed. V contribution due to the volume: V ext = extraction volume; V f = derivatisation volume; V e = elution volume (immunoaffinity cartridge) Cal p = calibration of pipettes; T p = temperature variation; Rep p = repeatability of the erogation of pipettes; Cal g = calibration of glassware; T g = temperature variation. C x contribution due to sample concentration calculated by the calibration function: f(c x ) = calibration function; A x = peak area of AFB 1 in the sample; A std = peak area of AFB 1 in the standard solutions; C std = concentration of AFB 1 in the standard solutions; C SRM = certified concentration of AFB 1 in the SRM; Pur SRM = certified purity of the SRM. P contribution due to the repeatability: C CRM = certified concentration of AFB 1 in the CRM; C M = resulting average concentration obtained by replicated measurements; Instrument = repeatability instrument. R contribution due to the recovery: Pur CRM = certified purity of CRM. M contribution due to the mass of the sample: Cal g =calibration laboratory balance. relationship between the instrumental response versus the analyte concentration (Eurachem/Citac, 2000): ů(c x )= (ů(c std )) 2 +(ů(f(c x ))) 2 = (0.029) 2 +(0.012) 2 = (4) ů(c std ) is a combined relative standard uncertainty which arises from the uncertainty associated with the Reference Material, reported in Table 1, and from the dilutions required for preparing the standard calibrations. Uncertainty of calibration ů(f(c x )), can be estimated by the calibration graph and its standard deviation s(c x ) (Eurachem/Citac, 2000; Miller, 1991). s(c x ) is calculated by the following formula: S y/cx 1 1 (y x Ŷ) 2 s(c x ) = ( ) { + + [ i (C xi Ĉ x ) -2 ]} ½ (5) b m p b 2 concentration C x, while Ŷ is the average of the area values of the standards. Σ i (C xi Ĉ x ) -2 is the variation of the abscissa C xi for each standard and Ĉ x is the average value. By considering the n replicates of sample determination, the uncertainty is given by the formula (6): ů(f(c x )) = Σ i (s(c x ) i )2 (6) n 3. Uncertainty associated with the recovery. The calculation of the relative uncertainty due to the recovery from CRM is based on the following equation (Barwick and Ellison, 1999): ů(r)= s2 +[ů(c CRM )] 2 = (0.057)2 +[0.063] 2 = (7) n C 2 M 13 (0.152)2 where: b is the slope of the calibration curve obtained by regression analysis; m the number of replicates performed for each C x value; s y/cx the standard deviation of the residuals and p the total number of measurement points in the calibration. The term y x is the value associated with the where ů(c CRM ) is the relative uncertainty stated in the certificate of analysis (see Table 1). The values corresponding to C M, s and n are reported in Table 2. World Mycotoxin Journal 1 (3) 251

6 M. Nocentini et al. 4. Uncertainty associated with the mass weight. We have assumed a rectangular distribution of standard deviation stated in the certificate of analysis. 5. Uncertainty associated with the volumes. We have assumed a rectangular distribution of the tolerance given by the manufacturer. The different contributions to uncertainty, as well as the resulting combined standard uncertainty, related to the Certified Reference Materials, CRM 376 and CRM 264, are sumarised in Table 3. The different contributions are schematically represented in the error budget diagram (Figure 3). The contribution to uncertainty related to the mass weight and the volume are negligible compared to the recovery and to the calibration curve for both samples analysed. The final step is to derive an expanded uncertainty (Ů(C)) based on the combined standard uncertainty: Table 3. Relative and combined standard uncertainty calculated for CRM 264 and CRM 376. Different contributions to uncertainty 1 Distribution function CRM 376 CRM 264 ů(r) Gaussian ů(c X ) Gaussian ů(p) Gaussian ů(v) Rectangular ů(m) Rectangular ů(c) ů(r) is the contribution due to the recovery efficiency of the method; ů(c x ) is the contribution derived from calibration; ů(p) is the contribution referred to repeatability data; ů(v) is the combination of the errors related to the dilution volumes (V ext, V f, V e ); ů(m) is the contribution derived from errors on weighing; ů(c) is the combined standard uncertainty. Parameters R Cx P V M CRM 264 CRM Relative uncertainty Figure 3. Error budget diagram for crm 264 and CRM 376. Ů(C) = k ů(c) (8) Where k is the coverage factor. Using a coverage factor of 2, corresponding to the 95 % of probability with the effective degrees of freedom equal to 17, the relative expanded uncertainty is the following: Ů(C) = = 0.15 (9) By using the relative expanded uncertainty we can calculate the expanded uncertainty at the maximum permitted levels stated in the Directive 2003/100/EC for the feed materials (C=0.02 mg/kg) and for the complete feedingstuff for dairy animals (C=0.005 mg/kg) by using the expanded relative uncertainty: U(C) = Ů(C) C = = mg/kg U(C) = Ů(C) C = = mg/kg 4. Conclusion The chromatographic method has been validated by using Certified Reference Materials. The limit of detection of the method has been defined and is equal to 0.5x10 3 mg/kg. The precision of the method complies with the performance criteria reported in the Regulation EC 401/2006 in all cases. The repeatability relative standard deviation (RSD) obtained for the three Certified Reference Materials are lower than that reported for the same method by Barmark and Larsson (1994). By comparing our results with those of AOAC Official Method , we can see that the repeatability values are of the same order of magnitude. The accuracy data obtained show that the method is affected by systematic error and correction for recovery is necessary, in accordance with those reported previously by the authors (Barmark and Larsson, 1994). In each set of routine analyses the recovery must be evaluated analysing spiked or certified reference materials. The new AOAC Official Method shows an improvement in the accuracy since no systematic error occurs. Uncertainty has been evaluated by using the bottom-up approach from the validation data; the major contributions are found to be the recovery factor and the calibration function, the other contributions due to the volume and mass are negligible. Taking into account that our laboratory precision is lower than that reported in the interlaboratory study (Barmark and Larsson, 1994), we could also use the reproducibility standard deviation as an estimate of relative uncertainty of the method, but no significant difference is obtained with those two approaches. In fact the reproducibility standard deviation in corn matrix at the concentration levels of 20 and 30 ng/g are respectively 14.4% and 20.0%. 252 World Mycotoxin Journal 1 (3)

7 Validation data and uncertainty estimation of a liquid chromatographic method for aflatoxin B 1 References Barmark, A.L. and Larsson, K., Immunoaffinity Column Cleanup/Liquid Chromatographic determination of aflatoxins: an interlaboratory study. Journal of AOAC International 77: Barwick, V.J. and Ellison, L.R., Measurement uncertainty: approaches to the evaluation of uncertainty associated with recovery. Analyst 124: De Beer, J.O., De Beer, T.R. and Goeyens, L., Assessment of quality performance parameters for straight line calibration curves related to the spread of the abscissa values around their mean. Analytica Chimica Acta 584: European Commission (EC), Commission Directive 2003/100/ EC of 31 October 2003 amending Annex I to Directive 2002/32/ EC of the European Parliament and of the Council on undesirable substances in animal feed. Official Journal of the European Union L 285: European Commission (EC), Commission Regulation 401/2006 of 23 February 2006 laying down the methods of sampling and analysis for the official control of the levels of mycotoxins in foodstuffs. Official Journal of the European Union L 70: Eurachem/Citac, Quantifying Uncertainty in Analytical Measurement. 2 nd ed. Available at: Eurachem/Citac, The Fitness for Purpose of Analytical methods. A Laboratory Guide to Method Validation and Related Topics edition 1.0 LGC. Available at: International Agency for Research on Cancer (IARC), Monograph on the evaluation of carcinogenic risks to humans. IARC Lyon, France vol. 82, p Kok, W.T., Derivatization reaction for the determination of aflatoxins by liquid chromatography with fluorescence detection. Journal of Chromatography B 659: Miller, J.N., Basic statistical methods for analytical chemistry. Part 2. Calibration and regression methods. A review. Analyst 116: World Mycotoxin Journal 1 (3) 253

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