Simultaneous Screening of Six Families of Antibiotic Residues in Milk Samples by Biochip Multi-array Technology.

Background: Antimicrobial compounds are used in animal husbandry to prevent and treat bacterial diseases and as illegal growth-promoting agents. Due to the excessive and inappropriate use of antibiotics, the antibiotic residues in milk can cause allergic reactions and antibiotic resistance. A rapid biochip-based method for the multi-analyte screening of 6 families of antibiotic residues (quinolones, ceftiofur, florfenicol, streptomycin, tylosin, and tetracyclines) in milk was validated based on Commission Decision 2002/657 and the European guidance for screening methods for veterinary medicinal products. Methods: This methodology allows the 6 antibiotic families to be detected simultaneously, increasing the screening capacity and reducing costs in test settings. The method's applicability was shown by screening 38 UHT cow milk samples taken from Tehran province, IR Iran. Results: The results showed that the positive threshold T was above Fm, and the CCß was below the European Commission's Maximum Residue Limit (MRL) (100 ppb for ceftiofur and tetracycline and 50 ppb for tylosin in milk). Norfloxacin was detected in about 8% of the samples and tylosin in 2.63%. The total antibiotic concentration in UHT cow milk samples was lower than the European Commission's MRL. Conclusions: This study showed that the biochip technique is valid for screening tylosin, ceftiofur, streptomycin, tetracycline, norfloxacin, and florfenicol in milk. It was found that the method was easy, quick, and capable of detecting 6 families of antibiotic residues simultaneously from a single milk sample without sample preparation.

Comparison of a portable Vis-NIR hyperspectral imaging and a snapscan SWIR hyperspectral imaging for evaluation of meat authenticity.

The performance of visible-near infrared hyperspectral imaging (Vis-NIR-HSI) (400-1000 nm) and shortwave infrared hyperspectral imaging (SWIR-HSI) (1116-1670 nm) combined with different classification and regression (linear and non-linear) multivariate methods were assessed for meat authentication. In Vis-NIR-HSI, total accuracies in the prediction set for SVM and ANN-BPN (the best classification models) were 96 and 94 % surpassing the performance of SWIR-HSI with 88 and 89 % accuracy, respectively. In Vis-NIR-HSI, the best-obtained coefficient of determinations for the prediction set (R2p) were 0.99, 0.88, and 0.99 with root mean square error in prediction (RMSEP) of 9, 24 and 4 (%w/w) for pork in beef, pork in lamb and pork in chicken, respectively. In SWIR-HSI, the best-obtained R2p were 0.86, 0.77, and 0.89 with RMSEP of 16, 23 and 15 (%w/w) for pork in beef, pork in lamb and pork in chicken, respectively. The results ascertain that Vis-NIR-HSI coupled with multivariate data analysis has better performance rather than SWIR-HIS.

Performance Evaluation of Biochip Chemiluminescent Immunoassay for Screening Seven Mycotoxins in Wheat Flour Simultaneously.

Background: Wheat grains are susceptible to mycotoxins, toxic natural secondary metabolites generated by certain fungi on agricultural produce in the field during growth, harvest, transportation, or storage. Therefore, wheat flour can be contaminated with mycotoxins, which seriously threaten human health. Methods: A rapid method for screening seven mycotoxins in wheat flour was validated in accordance with Commission Decision 2002/657/EC. With this multi-analytical screening method, 7 prevalent mycotoxins (fumonisin B1, ochratoxin A, aflatoxin G1, deoxynivalenol, T-2 toxin, aflatoxin B1, and zearalenone) can be determined simultaneously. The method's applicability was demonstrated by screening 7 mycotoxins in 39 wheat flour samples collected from different bakeries in Tehran province, Iran. Results: The validation results indicated that for all 7 mycotoxins, the positivity threshold (T) was above the cut-off value (Fm), and no false positive results were obtained for any of the mycotoxins. The screening results of 12 packaged and 27 bulk wheat flour samples indicated that the concentrations of all mentioned mycotoxins were higher than the cut-off (in the relative light unit [RLU]), and all the samples were compliant. Conclusions: The present study revealed that the biochip-based technique is valid for identifying and assessing the levels of 7 mycotoxins in grain samples, such as wheat flour, at the measured validation concentrations. The method was simple, fast, and able to screen 7 mycotoxins simultaneously. The test process of the kit is easy to conduct, and the results are straightforward to interpret.

Health risk assessment of process-related contaminants in bread.

Bread constitutes a popular and even daily component of human diet world-wide, Iran included. However, there are concerns that various processing methods such as frying and baking could result in the production of potentially a source of known carcinogens such as acrylamide (AA) and benzo(a)pyrene (BaP). The present study tried to perform a risk assessment on seven categories of bread consumers, based on age and gender, calculating the Target Hazard Quotient (THQ), Incremental Lifetime Cancer Risk (ILCR), and Margin of Exposure (MOE) related to the dietary intake of AA and BaP. AA and BaP were analyzed in 87 bread samples using LC-MS/MS and GC-MS/MS, respectively. The results indicated that more than 94% (mean concentration: 25.9 ng/g) and about 20% (mean concentration: 1.98 ng/g) of the samples were contaminated with AA and BaP, respectively. The THQ of AA intake through bread consumption for seven categories was in the following decreasing order: semi-industrial Sangak bread of Shiraz (SIS-Sh)> traditional Sangak bread of Shiraz (TS-Sh)> traditional Sangak bread of Tehran (TS-Th)> commercial bread of Tehran (C-Th). The non-neoplastic and neoplastic MOE for AA (˂10,000) indicates a high risk of exposure for all people in Tehran and Shiraz through the consumption of all tested bread. Due to the consumption of TS, SIS, and C bread, the BaP MOE for all people in Tehran and Shiraz was >10 000, which shows a low health risk for consumers. Our findings showed that ILCR for AA in seven classes of people who had TS-Sh and TS-Th was remarkably higher than ILCR for all categories that consumed the C-Th. BaP ILCR for the people who ingested TS-Th, TS-Sh, SIS-Th, and SIS-Sh was about 2-4 times higher than people who had C-Th. This study indicates that bread is the main source of AA and BaP intake in Iran, which might elevate the cancer risk.

A Neglected Challenge in the Analysis of Active Pharmaceutical Substances with Aldehyde Functional Group in Aqueous Matrices.

Aldehydes are compounds that are widely used and popular in organic synthesis due to their high reactivity. This advantage is a disadvantage in medicinal chemistry. Due to the ability of aldehydes to participate in nucleophilic reactions (especially in aqueous biological media) and access to nucleophiles such as amino acids and nucleic acids, drugs with aldehyde functional groups are always used with caution and carefully quantified in biological fluids. Our experience in working on biologically active aldehydes indicates the transformation of these groups of compounds in aqueous or alcoholic solution and thus the failure of analytical methods for their accurate monitoring in such media. Both mass spectrometry and Proton nuclear magnetic resonance spectroscopic findings indicate the reaction of spiramycin with water molecules in an aqueous solution, resulting in the conversion of spiramycin to a new molecule with 18 mass unit difference and thus, the residue amount which is measured and reported based on a mass spectrometries method does not show the correct amount of spiramycin in these samples.

Challenges for the determination of spiramycin in aqueous matrices using LC-MS/MS: evidence for the solvent intrusion on the molecule integrity.

Liquid chromatography-tandem mass spectroscopy (LC-MS/MS) is an accurate and specific technique for drug residue analysis in different matrices. The high specificity and sensitivity of the multiple reaction monitoring (MRM) approach for detecting drugs such as aldehydes, which have the potential to change mass during the sample preparation phase, becomes a drawback during the analysis process. In this study, concerns about the intrusion of solvent molecules into spiramycin's chemical structure as an aldehydic drug as well as the stability of spiramycin in the milk matrix were addressed. Furthermore, the binding sites where the solvent molecules could bind to spiramycin molecules were investigated through nuclear magnetic resonance (NMR) spectroscopy. It was revealed that water, ethanol, and methanol as protic solvents can add to the formyl group of spiramycin molecules during standard solutions preparation while there was no evidence for the addition of acetonitrile and dimethyl sulfoxide (aprotic solvents). In addition, as time passed, the peak area of spiramycin decreased either in the spiked aqueous sample or milk sample while an increase in the peak area of H2O-bound spiramycin was observed. After 96 h, more than 90% of spiramycin was converted to H2O-bound spiramycin. In conclusion, we can propose the use of aprotic solvents for the preparation of spiramycin standard solutions especially when the prepared solutions are not used instantly. Moreover, ion transitions for both spiramycin and its H2O-added form (843.6 m/z to 173.9 m/z and 861.5 m/z to 173.9 m/z, respectively) should be considered for the accurate quantification of spiramycin residue in aqueous samples such as milk.

Comparison of Portable and Benchtop Near-Infrared Spectrometers for the Detection of Citric Acid-adulterated Lime Juice: A Chemometrics Approach.

Background: Since the incidence of food adulteration is rising, finding a rapid, accurate, precise, low-cost, user-friendly, high-throughput, ruggedized, and ideally portable method is valuable to combat food fraud. Near-infrared spectroscopy (NIRS), in combination with a chemometrics-based approach, allows potentially rapid, frequent, and in situ measurements in supply chains. Methods: This study focused on the feasibility of a benchtop Fourier-transformation-NIRS apparatus (FT-NIRS, 1000 - 2500 nm) and a portable short wave NIRS device (SW-NIRS, 740 - 1070 nm) for the discrimination of genuine and citric acid-adulterated lime juice samples in a cost-effective manner following chemometrics study. Results: Principal component analysis (PCA) of the spectral data resulted in a noticeable distinction between genuine and adulterated samples. Wavelengths between 1100 - 1400 nm and 1550 - 1900 nm were found to be more important for the discrimination of samples for the benchtop FT-NIRS data, while variables between 950 - 1050 nm contributed significantly to the discrimination of samples based on the portable SW-NIRS data. Following partial least squares discriminant analysis (PLS-DA) as a discriminant model, standard normal variate (SNV) or multiplicative scatter correction (MSC) transformation of benchtop FT-NIRS data and SNV in combination with the second derivative transformation of portable SW-NIRS data on the training set delivered equal accuracy (94%) in the prediction of the test set. In the soft independent modeling of class analogy (SIMCA) as a class-modeling approach, the overall performances of generated models on the auto-scaled data were 98% and 94.5% for benchtop FT-NIRS and portable SW-NIRS, respectively. Conclusions: As a proof of concept, NIRS technology coupled with appropriate multivariate classification models enables fast detection of citric acid-adulterated lime juices. In addition, the promising results of portable SW-NIRS combined with SIMCA indicated its use as a screening tool for on-site analysis of lime juices at various stages of the food supply chain.

Simultaneous Screening of Nitrofuran Metabolites in Honey Using Biochip Array Technology: Validation Study According to the Decision 2002/657/EC of the European Union.

Background: Although no authorization is available for antibiotics to treat bee diseases, some veterinary compounds are used by beekeepers, and each country sets its own thresholds. Inappropriate and excessive use of these drugs can cause allergic reactions and antibiotic resistance in humans who consume the remaining antibiotic residues in honey and its products. It is, therefore, relevant to monitor the presence of antibiotic residues in this matrix. Objectives: A rapid method for the simultaneous screening of nitrofuran metabolite residues in honey was validated according to Commission Decision 2002/657/EC (C.D 657) and the European guideline for the validation of screening methods for veterinary medicines. Methods: This multi-analytical screening method enables the simultaneous determination of four nitrofuran metabolites [3-amino-2-oxazolidone (AOZ), 3-amino-5-morpholinomethyl-2-oxazolidinone (AMOZ), 1-Aminohydantoin HCl (ADH), and semicarbazide (SEC)] from a single honey sample. Thirty-five honey samples were collected randomly as real samples for screening from Tehran, IR Iran, Germany, and the Netherlands in 2018. Results: For all four antibiotic residues, the positivity threshold T was higher than the cut-off value Fm, and no false-positive results were obtained for three antibiotics (AOZ, AMOZ, and SEC). Detection capabilities (CCß) of all compounds were under the minimum required performance limit (MRPL) authorized by the European Commission (currently 1 µg/kg). The screening results of 15 domestic and 20 imported honey samples showed that the levels of AOZ in 6.66% and 10% of the samples, the level of AMOZ in 13.33% and 0% of the samples, and the level of SEC in 33.33% and 40% of the samples were less than the cut-off ([in relative light units (RLUs)], respectively. Conclusions: This study found that this technique is valid for detecting and quantifying three antibiotic residues in honey samples at the measured validation levels. This method was simple, rapid, and capable of simultaneously screening three nitrofuran metabolites from a single honey sample.

Occurrence of Aflatoxins in Commercial Cereal-based Baby Foods in Iran: A Probabilistic Risk Assessment to Health.

The aim of this study was to assess the occurrence of Aflatoxins (AFs) including B1, B2, G1 and G2 in commercial cereal-based baby foods by HPLC-FLD method in Iran and related risk assessment in three baby age groups (6-12, 12-18, and 18-24 months) using Monte Carlo simulation approach. Results showed an occurrence ranging from 20% to 60% for B1, B2, and G2 aflatoxins, while AFG1 was not detected in any assessed samples. Exposure and risk assessment was estimated to be two groups (aflatoxin B1 and total aflatoxins). The highest estimated dietary exposure to both AFB1 and total AFs was estimated for 6-12 months aged babies, representing 5.81 ng/kg BW/day and 8.55 ng/kg BW/day, respectively. Overall, the margin of exposures to AFB1 and total AFs were lower than 10,000 in all age groups, indicating a health concern about AFB1 and total AFs exposure through cereal-based baby food consumption. High cancer risk for high consumers (P95) of baby food was also estimated in all age groups, calling for immediate intervention due to serious claims that AFB1, is a highly carcinogenic component, causes hepatocellular carcinoma. Risk ranking results indicated the presence of AFB1 is classified as high risk for babies who consume cereal-based foods, which demands the attention of risk managers to reduce or eliminate this risk for the most vulnerable sector of society, whose aged <24 months.

Validation of a Multiclass Method for the Screening of 15 Antibiotic Residues in Milk Using Biochip Multi-array Technology and Its application to Monitor Real Samples.

Veterinary drugs are extensively and legally consumed to treat and prevent disease in chattels but some are also used illegally as growth-stimulating agents. Inappropriate or intensive use of antibiotics can cause allergic reactions and, above all, antibiotic resistance. A multiclass approach for the screening of antimicrobial substances in milk was validated in consonance with Commission Decision 2002/657/EC and to the European guideline for the validation of screening methods for veterinary medicines. This biochip-based approach enables the simultaneous determination of a total of 13 sulphonamide, dapsone and trimethoprim. For monitoring of antibiotic residues, 53 UHT milk samples collected from Tehran, IR Iran were screened applying this technology. The result showed that for all antibiotic residues, the positivity threshold T was much more than the cut-off value Fm. A false positive rate of less than 5% was found for all antibiotics which are satisfactory. All detection capabilities (CCß) were well below the Maximum Residue Level (MRL) set by the European Commission (100 µg/kg for the sum of all sulphonamides and 50 µg/kg for trimethoprim in milk). The screening results of 53 milk samples showed that 71.7% of samples were compliant and all positive samples were below the MRL set by European Commission. This study showed that the biochip-based technique is valid to identify and quantify antibiotic residues in milk at the studied validation levels. The method was rapid, easy, safe, and able to screen 13 sulphonamide, dapsone and trimethoprim from a single milk sample simultaneously with no sample preparation procedure (or just one-step centrifugation).

Exposure to multiple mycotoxins in domestic and imported rice commercially traded in Tehran and possible risk to public health.

Mycotoxins are secondary fungi metabolites that induce acute and chronic toxic effects in humans and animals. In the present study, nine mycotoxins including aflatoxins (AFB1, AFB2, AFG1 and AFG2), fumonisins (FB1 and FB2), Ochratoxin A (OTA), deoxynivalenol (DON), and zearalenone (ZEN) were determined in one hundred rice samples collected from Tehran using high performance liquid chromatography (HPLC) with fluorescence or photodiode array detector. In addition, possible risk to public health was investigated by assessing dietary exposure through rice consumption, the margin of exposure (MOE), respective risk of cancer and hazard index (HI) of the monitored mycotoxins in children and adults. The higher mean levels were determined for DON (102.22 µg.Kg-1), followed by FB1 (85.00 µg.Kg-1). For the rests of mycotoxins the levels did not exceed 20 µg.Kg-1. The estimated AFB1 intake for the adults and children through rice consumption exceeds the safe levels established for both carriers and non-carriers of hepatitis B virus. The mean and median determined exposure levels of OTA, DON ZEN and FB1, were found lower than the Provisional Maximum Tolerable Daily Intake (PMTDI) value for both adults and children of Tehran that consuming domestic and imported rice. The mean HI for adults and median HI for adults and children were below one, and mean HI for children was close to one. All the mean, median and maximum MoE values were <10,000 in adults and children, indicating a risk due to AFB1 exposure through rice consumption in Tehran. In addition, the calculated mean cancer risk in adult and child populations of Tehran were 0.27 and 0.64 cases per year per 105 individuals, respectively, that shows population in Tehran could be at risk of cancer due to AFB1 exposure through rice consumption as calculated. So further studies are necessary for the monitoring mycotoxins in rice and different food products as well as estimating average dietary exposure and cumulative exposure assessment of mycotoxins for main foods in IR Iran.

Overview, consequences, and strategies for overcoming matrix effects in LC-MS analysis: a critical review.

The high-performance liquid chromatography-mass spectrometry (LC-MS) technique is widely applied to routine analysis in many matrices. Despite the enormous application of LC/MS, this technique is subjected to drawbacks called matrix effects (MEs) that could lead to ion suppression or ion enhancement. This phenomenon can exert a deleterious impact on the ionization efficacy of an analyte and subsequently on the important method performance parameters. LC-MS susceptibility to MEs is the main challenge of this technique in the analysis of complex matrices such as biological and food samples. Nowadays, the assessment, estimation, and overcoming of the MEs before developing a method is mandatory in any analysis. Two main approaches including the post-column infusion and post-extraction spike are proposed to determine the degree of MEs. Different strategies can be adopted to reduce or eliminate MEs depending on the complexity of the matrix. This could be done by improving extraction and clean-up methods, changing the type of ionization employed, optimization of liquid chromatography conditions, and using corrective calibration methods. This review article will provide an overview of the MEs as the Achilles heel of the LC-MS technique, the causes of ME occurrence, their consequences, and systemic approaches towards overcoming MEs during LC-MS-based multi-analyte procedures.

Validation of Simultaneous Biochip-based Method for Screening of 3 Beta-Lactam Families Residues in Cow's Milk in Accordance with the European Union Decision 2002/657/EC and its Application on Real Samples.

Illegal and excessive use of veterinary antibiotics as a food additive for growth promotion in livestock can lead to allergic reactions and antibiotic resistance, which is a worldwide concern. A biochip-based semi-quantitative screening method of antimicrobial residues in milk was validated based on Commission Decision 2002/657/EC and the European guideline to validate screening methods for veterinary medicines. This multi-analytical screening method enables to determine of 3 beta-lactams (cefalexin, ampicillin, and cefuroxime) simultaneously. Analysis of 20 blank and 20 spiked milk samples showed that for all 3 antibiotic residues, the positivity threshold T was above cut-off value Fm, and no false-positive results were obtained for all 3 antibiotics. All detection capabilities (CCß) were below Maximum Residue Level (MRL) authorized by European Commission. 47 UHT cow's milk samples collected from Tehran province, IR Iran, were screened, and compliance was found in 100% of samples. This study found that the biochip method is valid to determine antibiotic residues in milk samples at the measured validation levels. The method was fast, simple, and able to simultaneous screen three families of beta-lactams from a single milk sample with almost no sample preparation.

The Feasibility of Two Handheld Spectrometers for Meat Speciation Combined with Chemometric Methods and Its Application for Halal Certification.

Handheld visible-near-infrared (Vis-NIR) and near-infrared (NIR) spectroscopy can be cost-effective, rapid, non-destructive and transportable techniques for identifying meat species and may be valuable for enforcement authorities, retail and consumers. In this study, a handheld Vis-NIR (400-1000 nm) and a handheld NIR (900-1700 nm) spectrometer were applied to discriminate halal meat species from pork (halal certification), as well as speciation of intact and ground lamb, beef, chicken and pork (160 meat samples). Several types of class modeling multivariate approaches were applied. The presented one-class classification (OCC) approach, especially with the Vis-NIR sensor (95-100% correct classification rate), was found to be suitable for the application of halal from non-halal meat-species discrimination. In a discriminant approach, using the Vis-NIR data and support vector machine (SVM) classification, the four meat species tested could be classified with accuracies of 93.4% and 94.7% for ground and intact meat, respectively, while with partial least-squares discriminant analysis (PLS-DA), classification accuracies were 87.4% (ground) and 88.6% (intact). Using the NIR sensor, total accuracies of the SVM models were 88.2% and 81.5% for ground and intact meats, respectively, and PLS-DA classification accuracies were 88.3% (ground) and 80% (intact). We conclude that the Vis-NIR sensor was most successful in the halal certification (OCC approaches) and speciation (discriminant approaches) for both intact and ground meat using SVM.

Novel Application of Near-infrared Spectroscopy and Chemometrics Approach for Detection of Lime Juice Adulteration.

The aim of this study is to investigate the novel application of a handheld near infra-red spectrophotometer coupled with classification methodologies as a screening approach in detection of adulterated lime juices. For this purpose, a miniaturized near infra-red spectrophotometer (Tellspec®) in the spectral range of 900-1700 nm was used. Three diffuse reflectance spectra of 31 pure lime juices were collected from Jahrom, Iran and 25 adulterated juices were acquired. Principal component analysis was almost able to generate two clusters. Partial least square discriminant analysis and k-nearest neighbors algorithms with different spectral preprocessing techniques were applied as predictive models. In the partial least squares discriminant analysis, the most accurate prediction was obtained with SNV transforming. The generated model was able to classify juices with an accuracy of 88% and the Matthew's correlation coefficient value of 0.75 in the external validation set. In the k-NN model, the highest accuracy and Matthew's correlation coefficient in the test set (88% and 0.76, respectively) was obtained with multiplicative signal correction followed by 2nd-order derivative and 5th nearest neighbor. The results of this preliminary study provided promising evidence of the potential of the handheld near infra-red spectrometer and machine learning methods for rapid detection of lime juice adulteration. Since a limited number of the samples were used in the current study, more lime juice samples from a wider range of variability need to be analyzed in order to increase the robustness of the generated models and to confirm the promising results achieved in this study.

Development of a Sensitive and Rapid Method for Determination of Acrylamide in Bread by LC-MS/MS and Analysis of Real Samples in Iran IR.

A new sample preparation procedure and a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method were developed for the quantitative analysis of acrylamide in bread. The method is based on sample extraction in methanol, purification with Carrez solutions and clean- up with Primary Secondary Amine (PSA).The developed method offers an efficient, inexpensive, easy sample preparation and very sensitive procedure for determination of acrylamide in bread. The use of spiked calibration curves for constructing the calibration curve substantially reduced adverse matrix-related effects. Recoveries were between 96 and 105.3%. Good results were obtained with respect to repeatability (RSDs <11%). The limit of detection and quantification of the method was 0.3 and 1 ng/g, respectively, which shows the method is very sensitive. The developed method was used for the determination of acrylamide in 26 traditional bread samples (Sangak) collected from Shiraz. The results showed that about 96% of Sangak bread samples were contaminated with acrylamide that 64.3 and 33.3 of semi-industrial and traditional Sangak bread were higher than benchmark levels (50 µg/kg), respectively. There are a few reports concerning contamination of Sangak bread samples with acrylamide in Iran. Therefore, this method could be used for a comprehensive survey of acrylamide in Sangak bread samples in the country.

Health risk assessment of aflatoxin M1 in infant formula milk in IR Iran.

In this study, two accurate, precise, selective and sensitive methods were developed for determining aflatoxin M1 (AFM1) in infant formula milk using immunoaffinity column clean-up followed by high performance liquid chromatography (HPLC) with fluorescence detection. The validated methods were used for determination of AFM1 in 29 samples of 6 different infant formula milk brands and the risk of AFM1 in infants aged zero to 6 months old was assessed using cancer risk, Margin of Exposure (MOE) and Hazard Index (HI). Only one sample (3.4%) was contaminated with AFM1. Although the results showed that MOE values for the mean and median exposure to AFM1 was <10,000 in infants, the additional cancer risk due to mean and median exposure to AFM1 in infant <6 months were 0.00010 and 0.00012 additional cases per year per 105 individuals, respectively, which indicates no health concern. In addition, HI values for the mean and median exposure to AFM1 for infants were quite below one which indicates no health concern. To the best of our knowledge, this is the first report on risk assessment of AFM1 in infant formula milk consumed by Iranian infants <6 months old, presenting a low risk for the evaluated groups.