Scheilla Vitorino Carvalho de Souza

Detection of several common adulterants in raw milk by MID-infrared spectroscopy and one-class and multi-class multivariate strategies

A sequential strategy was proposed to detect adulterants in milk using a mid-infrared spectroscopy and soft independent modelling of class analogy technique. Models were set with low target levels of adulterations including formaldehyde (0.074g.L-1), hydrogen peroxide (21.0g.L-1), bicarbonate (4.0g.L-1), carbonate (4.0g.L-1), chloride (5.0g.L-1), citrate (6.5g.L-1), hydroxide (4.0g.L-1), hypochlorite (0.2g.L-1), starch (5.0g.L-1), sucrose (5.4g.L-1) and water (150g.L-1). In the first step, a one-class model was developed with unadulterated samples, providing 93.1% sensitivity. Four poorly assigned adulterants were discarded for the following step (multi-class modelling). Then, in the second step, a multi-class model, which considered unadulterated and formaldehyde-, hydrogen peroxide-, citrate-, hydroxide- and starch-adulterated samples was implemented, providing 82% correct classifications, 17% inconclusive classifications and 1% misclassifications. The proposed strategy was considered efficient as a screening approach since it would reduce the number of samples subjected to confirmatory analysis, time, costs and errors.

An appropriate and systematized procedure for validating qualitative methods: its application in the detection of sulfonamide residues in raw milk.

The lack of well-established references for the validation of qualitative analyses and the increasing demand for reliable binary responses were the main motivating factors for this study. A detailed procedure for single-laboratory validation of qualitative methods is proposed. The experimental design and the tools for data analysis were based on the theoretical background, as well as the aspects of efficiency, convenience and simplicity. Four experimental steps were defined, as follows: (i) preliminary tests for the determination of the concentration range, (ii) a study of the rates, unreliability region, detection limit, and the accordance and concordance values, (iii) a study of the selectivity in the presence of known interferences, and (iv) a study of robustness. The applicability of the procedure was demonstrated by the validation of a qualitative commercial kit for detecting sulfonamide residues in raw milk using both the visual and instrumental reading techniques. Reliability rates of 100% were obtained for the blank samples. For the samples spiked with sulfamethazine at 10.8 and 108 μg L(-1) and with sulfadimethoxine or sulfathiazole at 10 and 100 μg L(-1), the reliability rates ranged from 93.3 to 100%. Selectivity was demonstrated using trimethoprim as a potential interferent. The method was considered robust for the factors of the temperature (54 and 58°C) and time (6 and 10 min) for incubating the test strips. The estimated detection limits and unreliability regions confirmed the suitability of the kit for this purpose, based on the legislated residue limits.

Eficiência de um kit de ELISA na detecção e quantificação de aflatoxina M1 em leite e investigação da ocorrência no estado de Minas Gerais

Procedures for intralaboratory validation of an ELISA kit were adopted to verify its efficiency in aflatoxin M1 detection and quantification in milk. Assays were accomplished with standard solutions provided with the kit, spiked samples and naturally contaminated samples. The mean recovery values obtained for the kit standard solutions were between 85.6 and 114.8%, with coefficient of variation ranging from 11.6 to 23.1%. Working with spiked samples, a concentration range for quantitative analysis (0.019 to 0.090µg/L) was defined. The occurrence of aflatoxin M1 in 110 milk samples from the State of Minas Gerais was investigated. Five of the 27 positive samples in the screening for ELISA were confirmed by thin layer chromatography.

Determination of main fruits in adulterated nectars by ATR-FTIR spectroscopy combined with multivariate calibration and variable selection methods

Grape, orange, peach and passion fruit nectars were formulated and adulterated by dilution with syrup, apple and cashew juices at 10 levels for each adulterant. Attenuated total reflectance Fourier transform mid infrared (ATR-FTIR) spectra were obtained. Partial least squares (PLS) multivariate calibration models allied to different variable selection methods, such as interval partial least squares (iPLS), ordered predictors selection (OPS) and genetic algorithm (GA), were used to quantify the main fruits. PLS improved by iPLS-OPS variable selection showed the highest predictive capacity to quantify the main fruit contents. The selected variables in the final models varied from 72 to 100; the root mean square errors of prediction were estimated from 0.5 to 2.6%; the correlation coefficients of prediction ranged from 0.948 to 0.990; and, the mean relative errors of prediction varied from 3.0 to 6.7%. All of the developed models were validated.

Otimização e validação de método para determinação de resíduos de oxitetraciclina, tetraciclina e clortetraciclina em leite por cromatografia líquida de alta eficiência

Um metodo para determinacao de residuos de oxitetraciclina, tetraciclina e clortetraciclina em leite por cromatografia liquida de alta eficiencia foi otimizado e validado. Ensaios intralaboratoriais empregando solucoes padrao, brancos de amostras e amostras adicionadas de solucoes padrao dos antimicrobianos foram realizados para avaliacao da linearidade, seletividade, exatidao, precisao, limites de deteccao e de quantificacao do metodo. As amostras foram homogeneizadas, tratadas com solucao tampao de succinato de sodio pH 4,0 e centrifugadas, descartando-se o precipitado. As tetraciclinas foram removidas do sobrenadante por quelacao com ions cobre reversivelmente ligados a resina quelante e eluidas com tampao McIlvaine-EDTA pH 4,0. As medias de recuperacao para as tetraciclinas foram de 76,2 a 104,9% com coeficiente de variacao na faixa de 1,4 a 18,9%. Limites de deteccao de 10, 15 e 40ng/mL e limites de quantificacao de 10, 20 e 50ng/mL foram determinados para oxitetraciclina, tetraciclina e clortetraciclina, respectivamente.

Determining performance parameters in qualitative multivariate methods using probability of detection (POD) curves. Case study: Two common milk adulterants

A strategy for determining performance parameters of two-class multivariate qualitative methods was proposed. As case study, multivariate classification methods based on mid-infrared (MIR) spectroscopy coupled with the soft independent modelling of class analogy (SIMCA) technique for detection of hydrogen peroxide and formaldehyde in milk were developed. From the outputs (positive/negative/inconclusive) of the samples, which were unadulterated and adulterated at target value, the main performance parameters were obtained. Sensitivity and specificity values for the unadulterated and adulterated classes were satisfactory. Inconclusive ratios 12% and 21%, respectively, for hydrogen peroxide and formaldehyde were obtained. To evaluate the performance parameters related to concentration, Probability of Detection (POD) curves were established, estimating the decision limit, the capacity of detection and the unreliability region. When inconclusive outputs were obtained, two additional concentration limits were defined: the decision limit with inconclusive outputs and the detection capability with inconclusive outputs. The POD curves showed that for concentrations below 3.7gL-1 of hydrogen peroxide and close to zero of formaldehyde, the chance of giving a positive output (adulterated sample) was lower than 5%. For concentrations at or above 11.3gL-1 of hydrogen peroxide and 10mgL-1 of formaldehyde, the probability of giving a negative output was also lower than 5%.

Production and evaluation of a reference material for moisture, ash, and total fat mass fractions, and titratable acidity in whole milk powder

Given the importance of reference materials (RM) in the structuring and maintenance of quality management systems for food analysis laboratories, RM were produced for the measurement of moisture, ash, total fat, and titratable acidity in whole milk powder. Two types of packaging and three storage temperatures were evaluated. The RM were considered homogeneous. The materials that were packaged in the pouches and those packaged in the amber glass bottles with screw caps (stored at room temperature) were not stable for moisture. Nevertheless, considering the four measurands, the amber glass bottles under refrigeration and freezing temperatures were the best packaging and storage conditions, respectively. Homogeneity and stability studies were carried out according to the ISO Guide 35, IUPAC harmonized protocol, and ISO 13528, considering the potential applicability of the produced materials as certified reference materials or even as RM for use in proficiency testing schemes. For homogeneity, despite the different criteria, there was agreement between the results. Considering the stability, the agreement depended on the evaluation of the regression assumptions, which is not required by the ISO Guide 35. The contribution of uncertainty associated with homogeneity was greater than that related to stability. The results indicated that the produced materials, i.e., the candidate certified RMs, were suitable for assessing trueness and could be submitted to further inter-laboratory characterization.

Performance improvement and single laboratory validation of classical qualitative methods for the detection of adulterants in milk: starch, chlorides and sucrose

Milk stands out among the foods that are subject to adulteration. Several studies have shown that milk adulteration is a reality in many countries, despite the existing inspection routines. Furthermore, there is an aggravating factor in that there are no reports in the literature concerning the validation of the classical qualitative methods that are commonly used to detect fraud in milk and that are described in the legislation. In the present study, a novel validation approach was applied on classical qualitative methods related to the detection of the density restoratives starch, chlorides and sucrose in raw milk, considering the official and modified versions. Rates, unreliability regions, detection limits, accordance, concordance, selectivity and robustness were estimated. Although simple, the proposed modifications significantly increased the efficiency of the methods, generating a lower frequency of errors or false results and a reduction in the detection limits and uncertainty ranges, consequently enhancing the capability of detecting adulterations.

Accelerated solvent extraction method for the quantification of polycyclic aromatic hydrocarbons in cocoa beans by gas chromatography–mass spectrometry

An accelerated solvent extraction (ASE) procedure for use with gas chromatography-mass spectrometry (GC-MS) was optimized for the determination of eight polycyclic aromatic hydrocarbons (PAHs) in cocoa beans. Plackett-Burman and rotatable central composite design (RCCD) indicated that three variables affected the recoveries of PAHs during the extraction and purification steps: agitation time in the second liquid-liquid partition, weight of silica gel in the column, and volume of hexane for PAH elution from the column. After obtaining the optimal conditions, a single laboratory method validation was performed. Linearity was demonstrated for benzo[a]pyrene in the concentration range from 0.5 to 8.0mgkg-1 of sample, corresponding to 1.25-20.0μgkg-1 of cocoa on a fat basis. For the other analytes, linearity was observed from 0.75 to 8.0μgkg-1 of sample (1.88-20.0μgkg-1 of cocoa on a fat basis). Significant matrix effects were found for chrysene and benzo[b]fluoranthene. The precision of the method was verified with relative standard deviations (RSDs) ranging from 2.57 to 14.13% and from 4.36 to 19.77% under repeatability and intermediate precision conditions, respectively. The average recoveries of the eight PAHs ranged from 74.99 to 109.73%. These parameters, limits and measurement uncertainties met the performance criteria established by European Union regulations, except for the theoretical limit of detection for chrysene. The method was applied to the analysis of samples of Brazilian cocoa beans, and only one sample was found to have a PAH content above the maximum limit defined by the European Union legislation. This optimized and validated method is intended to be used as part of the official Brazilian monitoring programs investigating contaminants and residues in food.

Detection of adulterants in grape nectars by attenuated total reflectance Fourier-transform mid-infrared spectroscopy and multivariate classification strategies

There is no any doubt about the importance of food fraud control, as it has implications in food safety and in consumer health. Focusing on fruit beverages, some types of adulterations have been detected more frequently, such as substitution with less expensive fruits. A methodology based on attenuated total reflectance Fourier-transform mid-infrared spectroscopy (ATR-FTIR) and multivariate classification was applied to detect whether grape nectars were adulterated by substitution with apple juice or cashew juice. A total of 126 samples were obtained and analyzed. Two strategies were proposed: one-class and multiclass approaches. Soft independent modeling of class analogy (SIMCA), partial least squares discriminant analysis (PLS-DA) and partial least squares density modeling (PLS-DM) were used to build the models. Among them, PLS-DA presented the best performance with a sensitivity and specificity of nearly 100%. The multiclass strategy was preferred if the adulterants to be studied are known because it provides additional information.