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Coupling near infrared spectroscopy and chemometrics for food and drug authentication. Federico Marini. Or better…. Outline. Nutritional quality of cereals Food contamination by mycotoxins Traceability of foodstuff Quantification of nutrients in baby powdered milk - PowerPoint PPT Presentation
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Coupling near infrared spectroscopy and chemometrics for food and drug authentication
Federico Marini
Or better….
Marini - WSC8
Outline
• Nutritional quality of cereals• Food contamination by mycotoxins• Traceability of foodstuff• Quantification of nutrients in baby powdered milk• Determination of ee in drugs
Marini - WSC8
Nutritional quality of cereals
• Oat (Avena sativa) is considered one of the most important grain cereals for human consumption.
• Indeed, oat products are important sources of dietary fiber, β-glucan, good nutritional value proteins, vitamins and other components,which are demonstrated to be beneficial for human health.
• With the aim to assess the nutritive potential value in new naked oat genotypes during breeding work, this study focuses on the possibility of developing a rapid, accurate and precise alternative method for the simultaneous quantification of β-glucan and protein content in naked oat samples.
Marini - WSC8
The data set
• The whole data set comprises 168 naked oat samples from 12 varieties, originally coming from Italy and other European countries and being representative of a large genetic range.
• 166 samples analysed as flour by NIR spectroscopy• 54 samples analyzed as flour by NIT spectroscopy• 168 samples analyzed as whole grain by NIT spectroscopy
• Robust calibration models built by Partial Robust M-Regression
Marini - WSC8
Data set split
Marini - WSC8
NIR on flour -glucan
Marini - WSC8
Protein content
Did we need robust methods?
• The significant amount of weights <1 both for horizontal and vertical outlyingness is an indication that the choice of using robust calibration
Marini - WSC8
Results
• Reflectance measurements on flour seem to be the best experimental setup.
• However, a different test set was used in the three experiments.
• Model building repeated on a common data set.
Marini - WSC8
Comparison of the three spectroscopic setups
• Analysis was carried out only on the 54 samples analyzed by the three different spectroscopic approaches
• Clearer evidence of NIR on flour being the better setup
Marini - WSC8
Outline
• Nutritional quality of cereals• Food contamination by mycotoxins• Traceability of foodstuff• Quantification of nutrients in baby powdered milk• Determination of ee in drugs
Marini - WSC8
Micotoxin
• Micotoxins are products of secondary metabolism of pathogenic fungi
• They are among the highest impact contaminants for cereal cultures.
• Attention was focused on DON (a micotoxin produced by fungi of the species Fusarium)
• EU limit of 1750 ppb for wheat to be considered contaminated.
• NIR-based approach for quantification and/or assessment of the contamination status.
Marini - WSC8
Data set
• More than 150 samples analyzed at least in replicate by NIR and NIT.
• 45 samples left aside as independent test set.
Marini - WSC8
Calibration • Best pretreatment: MSC + 2nd derivative• RMSEC=17.63; RMSEP=17.82
Marini - WSC8
Classification • Best pretreatment: MSC + 2nd derivative• Classification accuracies:
– 96.4% Cont.; 92.5% non cont. (calibr)– 80% cont.; 100% non cont. (test)
Marini - WSC8
Outline
• Nutritional quality of cereals• Food contamination by mycotoxins• Traceability of foodstuff• Quantification of nutrients in baby powdered milk• Determination of ee in drugs
Marini - WSC8
Marini - WSC8
Traceability: introductionTraceability: introduction
• Labelling issues are of increasing concern.
• Growth and promotion of “added value” regional foods such as those produced under “Organic” and “Designated Origin” labels.
• Many labelling claims that relate to perceived added value are rarely supported by analytical data, leaving regulators to rely solely on paper auditing procedures to monitor compliance.
• Need for analytical specifications for labelling issues relating to food origin:– geographical origin,
– production origin
– species origin.
Marini - WSC8
Tracing the origin of foodstuffTracing the origin of foodstuff
• The assessment of the typicalness of a product and its traceability should imply an analytical method to determine the origin of the sample.
• Unfortunately, even if a great host of instrumental analytical techniques are at present under investigation, no one of those can be listed whose results can be directly related to the origin of the samples.
An alternative way to cope with this problem is to use mathematical and statistical methods (chemometrics) to process the results of a set of determination performed on the samples in order to obtain the desired classification.
Marini - WSC8
An example: olive oilAn example: olive oil
• Authentication of the origin of olive oil samples• 57 extra virgin olive oil samples
– 20 from Sabina, Lazio (13 harvested 2009, 7 harvested 2010)– 37 samples of different origin (22 from 2009, 15 from 2010
• MIR and NIR spectra recorded on each sample
Training/test set selection• Duplex algorithm repeated class-wise on each pretreatment separately
(Split ratio: 2/1)• Data selected more than 10 times (out of 15) in test set
Marini - WSC8
PLS-DA on MIR data
• Best results with MSC + quadratic bl.• %cc on test set: 85.7% (sabina); 86.7% (other origins)
Marini - WSC8
PLS-DA on NIR data
• Best results in CV with 4 pretreatments.• %cc on test set (d1): 100% (sabina); 100% (other origins) • %cc on test set (other 3): 100% (sabina); 93.3% (other origins)
Marini - WSC8
Spectral interpretation
• All the spectral regions identified as relevant correspond to significant NIR features: – the bands at around 4450-5000 cm-1, which may be attributed to
combination bands of C=C and C-H stretching vibration of cis unsaturated fatty acids,
– the bands between 5650 and 6000 cm-1, due to the combination bands and first overtone of C-H of methylene of aliphatic groups of oil,
– and those between 7074 and 7180 cm-1, corresponding to C-H combination band of methylene.
Marini - WSC8
SIMCA on MIR data
Marini - WSC8
• Sensitivity decreases in CV.• Best model with d1 (based on geometric average of sens & spec)• Test set: 71.43% (sens.); 73.33% (spec.)
SIMCA on NIR data
Marini - WSC8
• Best model with det & MSC+d1 (based on geometric avg. of sens & spec)• Test set (det): 100% (sens.); 93.33% (spec.)• Test set (MSC+d1): 71.43% (sens.); 86.67% (spec.)
Effect of year (SIMCA NIR)
Marini - WSC8
• Best model with det, d1 & MSC+det• Models highly sensitive and specific in cal. &CV• Test set (2010 samples): high specificity, no sensitivity
Effect of year (Coomans)
Marini - WSC8
• If discriminant classification is sought, still a good classification ability could be obtained, notwithstanding the diversity
A second example: pistachio nuts
• The major pistachio-producing countries of the world are, in order, Iran, USA (California), Turkey, Syria, but to lesser extent, other countries cultivate pistachios as well, between these Italy and India.
• In Italy, only one variety (Bianca) is cultivated mainly in Bronte. • Italian production is very low in comparison to that of Asia and the
USA; however, it is compensated by the very high quality.• Each producing country’s applied tariff rates and national laws on
commodities vary dramatically. Therefore, pistachio variation in quality, food safety (e.g., contamination by aflatoxins), import/export fees, legal implications, and financial concerns makes determining the country of origin for pistachios important to protect the consumers against potential fraud, and there is a need to develop analytical methods to determine their geographical origin.
Marini - WSC8
Samples
• 483 pistachio samples from the 4 main producing countries + Italy and India were analyzed (NIR spectra recorded on both halves of nut and averaged)
Marini - WSC8
Country Nr. of samples
Italy (Bronte) 41
Iran 121
India 41
Syria 40
Turkey 120
USA 120
Training/test splitting
Marini - WSC8
Country Nr. of samples
Italy (Bronte) 22+19
Iran 83+38
India 23+18
Syria 25+15
Turkey 86+34
USA 81+39
PLS-DA modeling
Best model:
MSC+detrending
Marini - WSC8
Bronte India Iran Syria Turkey USA
Calibration 97.48% 96.82% 90.54% 96.47% 95.17% 99.79%
CV 97.32% 95.30% 89.48% 93.97% 93.15% 99.17%
Prediction 95.14% 91.29% 83.59% 93.63% 91.71% 99.19%
Bronte: red
India: blue
Iran: black
Syria: green
Turkey: cyan
USA: purple
Training set: empty
Test set: filled
Predictions
Marini - WSC8
VIP
Marini - WSC8
SIMCA
• Optimal complexity evaluated as those resulting in the best geometric average between sensitivity and specificity in CV
Marini - WSC8
SIMCA
• Best model: MSC+detrending
Marini - WSC8
Class Sensitivity (Cal)
Specificity (Cal)
Sensitivity (CV)
Specificity (CV)
Sensitivity (Pred)
Specificity (Pred)
Bronte 95.45% 95.64% 72.73% 95.30% 89.47% 96.53%
India 100.00% 90.57% 65.22% 93.60% 83.33% 98.62%
Iran 98.80% 68.35% 93.98% 67.93% 92.11% 76.80%
Syria 88.00% 88.81% 88.00% 87.46% 73.33 82.43%
Turkey 95.35% 79.06% 84.88% 80.77% 73.53% 80.62%
USA 93.83% 100.00% 85.19% 100.00% 87.18% 99.19%
SIMCA models
Marini - WSC8
Bronte in detail
Marini - WSC8
Outline
• Nutritional quality of cereals• Food contamination by mycotoxins• Traceability of foodstuff• Quantification of nutrients in baby powdered
milk• Determination of ee in drugs
Marini - WSC8
Quantification of nutrients in powdered milk for babies
• Baby powdered milk is a product based on milk of cows or other animals and/or other ingredients which have been proven to be suitable for infant feeding.
• The nutritional safety and adequacy should be scientifically demonstrated to support normal growth and development of infants.
• In addition to the compositional requirements, other ingredients may be added to ensure that the formulation is suitable as the sole source of nutrition for the infant, or to provide other benefits that are similar to outcomes of populations of breastfed babies.
• The suitability for the particular nutritional uses of infants and the safety of additional compounds added at the chosen levels shall be scientifically demonstrated.
Marini - WSC8
Samples
Marini - WSC8
• Preliminary results only on lipid content.
BiPLS
Marini - WSC8
….
• Spectral region is divided into intervals & PLS models are computed removing one interval at a time.
• Interval whose deletion results in lowest RMSECV is removed.• Procedure is iterated up to the desired number of retained variables
8 intervals1600 variables
7 intervals1400 variables
6 intervals1200 variables
Marini - WSC8
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evaluation of the fitness of chromosomes
ranking of chromosomes according to fitness
cross-over
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new generation P(t+1)
Population of n chromosomes with m genes P(t)
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GA
BiPLS-GA
Marini - WSC8
Method RMSECV
No sel 1.41
BiPLS 0.96
BiPLS-GA 0.90
Outline
• Nutritional quality of cereals• Food contamination by mycotoxins• Traceability of foodstuff• Quantification of nutrients in baby powdered milk• Determination of ee in drugs
Marini - WSC8
Motivation
• Quite often only one enantiomeric form of an IPA is pharmacologically active.
• After cases such that of thalidomide, FDA recommends that the enantiomeric purity of pharmaceutical formulations is checked.
• Present methods:– Polarimetry– HPLC on chiral columns– NMR
• In this study the possiblity of using NIR+chemometrics to predict the enantiomeric excess of an IPA is studied.
Marini - WSC8
Ibuprofen: phase diagram
• Phase diagram confirms that ibuprofen crystalizes as a racemic mixture.
• This suggests that R and S have the same spectrum but both have a different spectrum than the racemate
Marini - WSC8
Ibuprofen: NIR spectra
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Ibuprofen: calibration & testing
• Best pretreatment: MSC + 2nd der• 4 LVs• RMSEC=2.11• RMSECV= 2.43• RMSEP=1.71
Marini - WSC8
Interpretation - VIP
• 4000-4800 cm-1: combination of bands from methylenic C-H and overtones of the bendings
• 6000 cm-1: First overtone of the aromatic C-H stretching and of asymmetric stretching of methyls
Marini - WSC8
Marini - WSC8