Coupling near infrared spectroscopy and chemometrics for food and drug authentication

1. Coupling near infrared spectroscopy and chemometrics for food and drug authentication Federico Marini

2. Or better…. Marini - WSC8

3. 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

4. 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

5. 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

6. Data set split Marini - WSC8

7. NIR on flour b-glucan Protein content Marini - WSC8

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. Calibration • Best pretreatment: MSC + 2nd derivative • RMSEC=17.63; RMSEP=17.82 Marini - WSC8

15. Classification • Best pretreatment: MSC + 2nd derivative • Classification accuracies: • 96.4% Cont.; 92.5% non cont. (calibr) • 80% cont.; 100% non cont. (test) Marini - WSC8

16. 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

17. Traceability: 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

18. 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. Tracing 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. Marini - WSC8

19. An 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 Marini - WSC8

20. 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

21. PLS-DA on MIR data • Best results with MSC + quadratic bl. • %cc on test set: 85.7% (sabina); 86.7% (other origins) Marini - WSC8

22. 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

23. 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

24. SIMCA on MIR data • Sensitivity decreases in CV. • Best model with d1 (based on geometric average of sens & spec) • Test set: 71.43% (sens.); 73.33% (spec.) Marini - WSC8

25. SIMCA on NIR data • 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.) Marini - WSC8

26. Effect of year (SIMCA NIR) • Best model with det, d1 & MSC+det • Models highly sensitive and specific in cal. &CV • Test set (2010 samples): high specificity, no sensitivity Marini - WSC8

27. Effect of year (Coomans) • If discriminant classification is sought, still a good classification ability could be obtained, notwithstanding the diversity Marini - WSC8

28. 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

29. 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

30. Training/test splitting Marini - WSC8

31. PLS-DA modeling Best model: MSC+detrending Bronte: red India: blue Iran: black Syria: green Turkey: cyan USA: purple Training set: empty Test set: filled Marini - WSC8

32. Predictions Marini - WSC8

33. VIP Marini - WSC8

34. SIMCA • Optimal complexity evaluated as those resulting in the best geometric average between sensitivity and specificity in CV Marini - WSC8

35. SIMCA • Best model: MSC+detrending Marini - WSC8

36. SIMCA models Marini - WSC8

37. Bronte in detail Marini - WSC8

38. 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

39. 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

40. Samples • Preliminary results only on lipid content. Marini - WSC8

41. BiPLS …. • 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 6 intervals 1200 variables 8 intervals 1600 variables 7 intervals 1400 variables Marini - WSC8

42. GA Population of nchromosomes with mgenesP(t) . . . . . a d c b e . . . . . cromosomi 1 . . . . . . . . . . . . . . . . . . . . n evaluation of thefitness of chromosomes generation ranking of chromosomesaccording tofitness cross-over mutation new generation P(t+1) stop Marini - WSC8

43. BiPLS-GA Marini - WSC8

44. 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

45. 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

46. 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

47. Ibuprofen: NIR spectra Marini - WSC8

48. Ibuprofen: calibration & testing • Best pretreatment: MSC + 2nd der • 4 LVs • RMSEC=2.11 • RMSECV= 2.43 • RMSEP=1.71 Marini - WSC8

49. 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

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