IMPROVEMENTS ON LENGTH OF RIPENING DIFFERENCIATION OF ILDRØD PIGEON (Malus domestica) APPLES BY USING GC-MS AROMA PROFILE AND PARAFAC2

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1 IMPROVEMENTS ON LENGTH OF RIPENING DIFFERENCIATION OF ILDRØD PIGEON (Malus domestica) APPLES BY USING GC-MS AROMA PROFILE AND PARAFAC2 José Manuel Amigo*, Marta Popielarz, Raquel Callejón, M.L. Morales, A.M. Troncoso, T.B. Toldam-Andersen, M.A. Petersen *Dept. Food Quality and Technology U. Copenhagen, Denmark

2 Danish apples ILdrød Pigeon (Malus domestica) Red colored Middle size Intense and characteristic aroma Very popular in traditional Danish cuisine cakes æbleflæsk (pork and apples) pastry 2

3 Danish apples ILdrød Pigeon (Malus domestica) Very popular in Christmas time It is important that apples arrive in perfect full aroma quality time to the customers 3

4 Danish apples ILdrød Pigeon (Malus domestica) What is the route of the apples before arriving to the market? 1) Harvesting 2) Cold storage (1.5 o C) 3) Ripening Storage at room temperature To increase the aroma and the flavor To obtain the best quality for customers 4

5 Target To study the aroma profile of danish apples being ripened during different time intervals To study the new appearing components as well as the degradation products 6

6 Ripening to increase the aroma There is no sure method of determining a ripe apple other than taste but taste is subjective It is not subjective Intensity (a.u.) Aroma chromatographic profile x elution time (scans.) GC-MS analysis (SCAN mode) Dozens of analytes Major components ethyl butanoate, ethyl 2- methylbutanoate, 2-methylbutyl acetate, hexyl acetate, hexyl propanoate, etc Minor components The size is not important!!! (in aroma profiles) 5

7 Experimental 36 apples divided into 3 groups (12 apples each). Ripening time 5 days 8 days 15 days N 2 1) Pressing to obtain 10 ml of juice 2) Dynamic headspace 3) GC-MS analysis of the desorbed aroma in SCAN mode 7

8 Data Analysis Solve several problems before integration of peak: INTEGRATION 9 x 10 6 Peak shifts Not a problem 8 7 Intensity (a.u.) Low S/N ratio Baseline drift Complicated No alternative elution time (scans.) Severe overlapping Complicated Selective ions Deconvolution TIME CONSUMING 8

9 PARAFAC2 analysis on GC-MS data PCA Classification of apples Improve in resolution of peaks Detect and identify new analytes 9

10 PARAFAC2 analysis on GC-MS data Dividing the chromatogram in 26 blocks and modeling with PARAFAC2 Intensity (a.u.) Elution time (a.u.) 10

11 PARAFAC2 analysis on GC-MS data For all the intervals: 1) Check the number of components Heuristic approach based on: Explained variance with number of components Residuals 2) Nonnegativity constraint in all the models 3) Integration of all the obtained peaks to perform PCA 11

12 Results: PARAFAC2 modeled peaks Intensity (a.u.) x raw data Intensity (a.u.) 10 5 x 10 5 PARAFAC2 result 5 different peaks 2-propamine Heptane Ethyl-eter N-penthyl-alcohol Scan number Scan number Pentane x 10 4 raw data x 10 4 PARAFAC2 result Intensity (a.u.) Intensity (a.u.) 2 1 We could model rests of other peaks!! Scan number Scan number 12

13 Results: PARAFAC2 modeled peaks Intensity (a.u.) x 10 5 raw data Scan number Intensity (a.u.) x 10 4 PARAFAC2 result Scan number Baseline modeling!! x 10 4 raw data x 10 4 PARAFAC2 result 10 Intensity (a.u.) Intensity (a.u.) 2 1 Almost embedded peaks Scan number Scan number 13

14 Results: PARAFAC2 new analyte detection 50 analytes identified by using the obtained mass profile x 10 5 PARAFAC2 result NH2 Intensity (a.u.) Scan number Intesity (a.u.) O OH m/z fragments Checked with ChemStation (similarities higher than 0.850) 14

15 Results: PCA of PARAFAC2 peaks and relationship 5 days 8 days 15 days Samples/Scores Plot of matrix2d 8 6 Scores on PC 2 (17.10%) Scores on PC 1 (23.81%) 15

16 Results: PCA of PARAFAC2 peaks and relationship Samples/Scores Plot of matrix2d 0.4 Variables/Loadings Plot for matrix2d Scores on PC 2 (17.10%) Loadings on PC 2 (17.10%) Scores on PC 1 (23.81%) 0 5 days 8 days 15 days propanamine propanol Butiric acid Butanoic acid 2 methyl, methyl ester ethanol 2-butanol propanol Propanenitrile 1-pentanol Butanal AA propil ester 2-Methylbutanoic acid pentane ehtyl-eter Acetic Acid propanal 1-butanol 2-hexyl acetate 2-propanone 2-methyl-butanal Butyl buterate 1-hexanol heptane Cyclohexyl isothiocyanate AA hexyl ester heptene methanesulfonic anhidre cycolhexane, 1,3,5-trimethyl Tricloromethane 2-hexenal farnacine Hexanoic acid Ethyl-Acetate benzene Pentanal neo-penthyl-alcohol ethylcyclohexane hexanal 3-Methylbutanoic acid Trend to transform alcohol in acids 4-methyl-1pentanol 1-octanol Loadings on PC 1 (23.81%) 16

17 Conclusions PARAFAC2 has been able to model artifacts in the dataset Retention time shifts Overlapping peaks Baseline drift Typical chromatographic problems have been overcome by using the combination of both techniques Further PCA demonstrated differences between different ripening times The working methodology has demonstrated its applicability Dividing the chromatogram + PARAFAC2 + PCA = SAVE HUMAN TIME IMPROVEMENT OF CHROMATOGRAPHIC INTEGRATION 17

18 SURVEY Most of you already knew, or suspected that PARAFAC2 would work You use 3-way methods very often I wanted to know what chromatographists think about three-way analysis 5 research groups 4 countries SURVEY 8 researchers 1 professor 2 assistant professors 2 PhD Some questions 3 PhD students (3rd year) 18

19 SURVEY 5 different groups, 8 researchers MAINLY focused on Chromatography 1) How many Chromatographic systems do you have? 2) How often do you use them? HPLC-UV-Fluor. HPLC-MS GC-FID GC-MS Continuously without interruption 3) Do you know wether you can measure the complete spectral range for each retention time? 4) Do you correct the baseline drift? INDIRECTLY NO, NEVER YES, I KNOW YES, BUT I DON T USE IT IF IT IS NECESSARY 19

20 SURVEY 5 different groups, 8 researchers MAINLY focused on Chromatography 6) How do you integrate peaks? Manually? Automatically? How do you solve the overlapping problems? MANUALLY. IF OVERLAPPING I LOOK FOR SPECIFIC ION MANUALLY. DECONVOLUTION FOR SLIGHT OVERLAPPING. FOR SEVERE OVERLAPPING, OPTIMIZATION BUT IT REQUIRES MUCH TIME!!! 7) Do you know if there is any multivariate routine or peak purity in your software? YES, BUT I DON T KNOW HOW IT WORKS YES NO IDEA 20

21 SURVEY 5 different groups, 8 researchers MAINLY focused on Chromatography 8) Do you know any multivariate technique? If you know any of them, do you use them? PCA AND LDA SOME OF THEM (PCA, LDA, ) CLUSTERS NO ANOVA ONLY PCA 9) Do you know three-way techniques? Have you ever heard about PARAFAC, PARAFAC2, GRAM, TLD? I HAVE HEARD ABOUT PARAFAC, BUT NO IDEA HOW TO USE IT NO 21

22 SURVEY 1982 First application of Curve Resolution in Chromatography 1987 First application of three way in Chromatography 22

23 SURVEY So.MY IMPRESSION IS THAT We have demonstrated that three-way techniques are VERY useful to solve problems in chromatography We do not care about integration and other artifacts We are able to SAVE HUMAN TIME COMPUTING TIME!!! BUT SOMEHOW CHROMATOGRAPHISTS ARE NOT AWARE OF THE BENEFITS OF THREE-WAY TECHNIQUES 23

24 SURVEY So. What can we do to solve this problem? Let me share with you some of my thoughts 24

25 SURVEY Publishing one nice paper about chemometrics in chromatography 1st reviewer Suitable without changes Excellent idea Extremely important CHEMOMETRICIAN FRIEND Possibly suitable with major changes 2nd reviewer This paper will only have an impact if it is read by chromatographers and Anal. Chemist IT MUST BE SPOKEN THE SAME LANGUAGE 25

26 SURVEY LANGUAGE Matrix APPLES Matrix 2D array Retention time shifts Not trilinear structure 26

27 SURVEY DO YOU THING THAT THREE WAY ANALYSIS COULD BE EASILY IMPLEMENTED IN COMMERCIAL SOFTWARE?

28 SURVEY DO YOU THINK THAT EXCLUSIVE SOFTWARES COULD BE USEFUL? You don t need to be Engineer to drive a car Language!!! Overlapping problem Check retention time shifts Check baseline Initial estimation LOAD DATA CHECK NO COMP PARAFAC PARAFAC2 MCR-ALS PCA SELECT INTERVAL SELECT COMPONENTS 28

29 SURVEY WHAT IS YOUR OPPINION ABOUT INCLUDING DATA ANALYSIS IN BACHELOR DEGREES???? DO WE NEED MORE TUTORIAL PAPERS? One of the responses: I have no idea how to use your MATLAB tricks 29

30 CONCLUSIONS WHAT CAN WE DO TO SOLVE THIS ISSUE? It requires some efforts FROM BOTH SIDES BY THE WAY ANY QUESTION ABOUT APPLES? THANK YOU!!! And remember: One apple a day, keeps the doctor away!! 30