Download Soluciones Sigma-Aldrich en el Análisis de Alimentos

Soluciones Sigma-Aldrich en el Análisis
de Alimentos
• Dr. Pedro Gutiérrez
• Sales & Application Specialist. Analytical
I Taller Análisis de Alimentos
sigma-aldrich.com
Córdoba, 20 de junio 2012
El mundo del análisis de alimentos
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Food & Beverages
• Quality Control Food Production
• Analysis & design of flavours
• Leachables from packaging
• Nutritional Facts of Foods
• Trans fatty acid food labelling
• Vitamins in food supplements
• Artifi cial & natural flavours
• Preservatives
• Food Monitoring
• Pesticides, heavy metal ion content in foods
• Micotoxines
• Acrylonitrile, PAHs in fried foods
• Persistant Organic Polutants (POPs)
e.g. dioxins, furans in fish and eggs
• Allergenes
• Adulteration of natural products
• Essential oil composition
• Drinking Water Contaminants
e.g. Pesticides, NO2, Pharmaceuticals
• Alcohol content of wine, spirits
• Natural flavour labelling
• Water content of food e.g. coffee or margarin
2
Agenda
• Nuevas columnas capilares para GC. SLB-IL. Análisis FAMEs
• Ascentis Express (UHPLC virtual). Análisis de plaguicidas por LC-MS
• Supel™ QuE Z-Sep+ de los prepesados tubos de QuEChERS
• SupelMIP™ PAHs. SPE de alta selectividad. Analisis de PAHs en
aceite de oliva.
• Aplicaciónes de la técnica SPME
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Columnas Capilares con Líquidos Iónicos
• Los líquidos iónicos son disolventes que consisten en cationes orgánicos
asociados con aniones, con propiedades deseables como fases
estacionarias de CG.
• Aguantan altas temperaturas
• Casi sin sangrado
• Poco susceptibles al agua y el oxígeno.
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
anion
CF3
O=S=O
NO=S=O
Ionic Liquids description
cation
linkage
N + N
CF3
anion
CF3
cation
N + N
O=S=O
NO=S=O
(-IL100 phase)
• Benefits: not polymers, greater stability, vast chemistry modifications possible
• Dicationic (shown) or polycationic
• Limitless choices exist for cations, linkages, and anions
– Cations: imidazolium (shown), phosphonium, pyrrolidinium, etc.
– Linkage: alkane (shown), polyethylene glycol, etc. of various lengths
– Anion: nTf2- (shown), triflate, etc.
• Cation and/or linkage pendent groups: vinyl (shown), alkyl, phenyl, chiral, etc.
• Drawbacks: new, not in methods, cannot cover non-polar column range
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
CF3
Líquidos iónicos en el analisis de alimentos
Actual applications
•FAMEs IL111
•Flavours IL59
•POP IL 82
•Aromatic IL59 /IL111
Under
development(example)
Alcohols IL60
Flavours IL60
OmegaFAMEs IL60
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
6
GC Column Polarity Scale
Visual Representation
-Octyl
-1
280
350
C
C
°
NonPolar
0
-5
° 360°
C
-20
300
C
-1701
-35
-50
C
C
C
Intermediate Polar
10
-225 PAG PEG
240
220
280
C
C
C
° 280° 300° 310°
°
°
°
Polar
-2330 -2331 -2560
250
275
250
C
C
C
°
°
°
TCEP
145
C
°
Highly Polar
32
65
SLB-IL59
300 C
°
SLB-IL61
290 C
°
Extremely Polar
100
SLB-IL76
270 C
°
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
sigma-aldrich.com/il-gc
SLB-IL82
270 C
°
SLB-IL100 SLB-IL111
230 C
270 C
°
°
Analisis detallado de FAME C18:1
cis/trans usando la columna SLB-IL111
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Especificaciones de la Columna
• Overview
• Application: This extremely polar ionic liquid column was the world’s first
commercial column to rate over 100 on our GC column polarity scale. As such, it has
the most orthogonal selectivity compared to commonly used non-polar and
intermediate polar columns, providing increased selectivity for polar and polarizable
analytes. It should also be considered for use in GCxGC applications. Its
temperature limit of 270oC is very impressive for such an extremely polar column.
• The 100 m version is suitable for detailed cis/trans FAME isomer analysis, and is a
great complementary column to the SP-2560.
• USP Code: None
• Phase: Non-bonded; proprietary
• Temp. Limits: 50 °C to 270 °C (isothermal or programmed)
• Catalog Numbers
• 28925-U, 15 m x 0.10 mm I.D., 0.08 µm
• 28927-U, 30 m x 0.25 mm I.D., 0.20 µm
• 29647-U, 100 m x 0.25 mm I.D., 0.20 µm
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
sigma-aldrich.com/il-gc
SLB-IL111 Column
• Columnas altamente polares (por encima del 100% en la escala de
polaridad) están bien adaptados para la resolución de los analitos
polarizables, tales como compuestos aromáticos y FAMEs insaturados,
debido a su capacidad para ofrecer interacciones dipolo-dipolo inducido.
Pruebas preliminares con una columna de 30 mx 0,25 mm de diámetro, 0,20
m SLB-IL111, mostraron un buen comportamiento de la columna y se decidio
hacer una de 100 m con el fín de hacer una equivalencia de las fases ciano.
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
The first publication in 2010
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
cis, trans FAME Mix Test Conditions
Oven:
168°C Isothermal (until last peak elutes)
Inj.:
250 °C
Det.:
FID, 250 °C
Carrier Gas: hydrogen, 1ml/min constant set @ 168°C
Injection:
1 µL, split, 100:1
Sample:
Various samples, various concentrations
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
SLB-IL111 cis/trans FAME Isomers
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
sigma-aldrich.com/il-gc
Un caso estudiado....
Intralab Validation of the EN 15662 Method for the
Determination of Pesticide Residues Using a FusedCore™ Ascentis® Express RP-Amide HPLC Column by
LC-MS/MS and Clean-up by Dispersive SPE (QuEChERS)
Enio Belotti, Luca Meni, Marco Ruggeri, Water&Life Entratico (BG) Italy and R. Ferrari, Sigma Aldrich
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Experimental:
According EU Guideline The European guideline SANCO/3131
document (Method Validation and Quality Control Procedures for
Pesticide Residues Analysis in Foods and Feeds) the following
different representative fruit and plant origin matrices were choiched:
•Pear: example for high sugar content
•Kiwi: example for low pH
•Salad : example for high chlorophyll content
•Corn meal: example for low water content
A group of 29 compounds (Table 1) was used as a representative group
of different pesticides; these fall into the following categories:
acaricides, insecticides, fungicides, and so forth.
Abamectina B1a
Buprofezin
Cyazofamide
Ethoprofos
Fenpyroximate
Methomyl
Spinosad A e D
Thiametoxam
Acetamiprid
Carbendazim
Cyprodinil
Ethofenprox
Flufenoxuron
Pirimicarb
Spiroxamine
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Aldicarb
Carbofuran
Difenconazol
Fenhexamide
Imidacloprid
Rotenone
Tebufenozide
Azoxystrobin
Clothianidin
Dimethoate
Fenpropimorph
Methiocarb
Setoxydim
Thiabendazole
Preparation of Standard Solution
•As recommended in the EN 15662:2008 method, the results obtained for the analytes to
be identified in the sample extract are compared with those obtained for the pesticides in
the calibration solutions.
•Signal suppression in MS/MS detection can arise from the matrix and other interferences.
To avoid this, the calibration standard solutions were prepared using acetonitrile blank
matrix extracts.
•Although the use of matrix-matched standards is preferred, for a first estimate of the
residue level of pesticides in food, or to show their absence, standard solutions in pure
solvent can be used. They can also be used for quantification if preliminary experiments
indicate that any suppression or enhancement effects experienced do not significantly
affect the results obtained. As soon as relevant residue concentrations are detected, a
more precise determination using matrix-matched standards, or using the standard addition
method, should be used.
NOTE 1. Matrix effects influence the response of target analytes in sample extracts compared to the response of
standard solutions in pure solvent.
NOTE 2. The calibration range should be appropriate to the residue concentrations to be quantified. Thus, it may
be necessary to construct more than one calibration graph.
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Intra-Lab Validation of QuEChERS/HPLC
• Calibration curve for Ethoprophos in extract of salad
• IS: Triphenyl phosphate (acc. EN 15662)
Enio Belotti, Luca Meni, Marco Ruggeri, Water&Life Entratico (BG) Italy and R. Ferrari, SiAl
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Intra-Lab Validation of
QuEChERS/HPLC - Results
Column: Ascentis Express RP Amide, 10 cm x 2.1 mm ID
HPLC: Shimadzu Prominence UFLC XR
MS/MS: Applied Biosystems API 3200
Mobile Phase A: NH4HCO2 in H2O (5 mmol/L, 0.1 % formic acid)
Mobile Phase B: NH4HCO2 in MeOH (5 mmol/L, 0.1 % formic acid)
Temp.: 40 oC
Gradient:
Time (min) Mobile Phase A %
0
95
0.5
90
12
5
15
5
Enio Belotti, Luca Meni, Marco Ruggeri, Water&Life Entratico (BG) Italy and R. Ferrari, SiAl
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Intra-Lab Validation of
QuEChERS/HPLC
The separation of a complex mix of
200 compounds on the
Ascentis Express RP-Amide column
under the same conditions has been
also performed
The results show that the Ascentis
Express RP-Amide
HPLC column is particularly suitable
for the resolution of those
components having a
log Kow < 0,5 that are weakly
retained on a C18 column.
Enio Belotti, Luca Meni, Marco Ruggeri, Water&Life Entratico (BG) Italy and R. Ferrari, SiAl
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Intra-Lab Validation of QuEChERS/HPLC
• New method:
• High recovery
• Reproducibility
• HPLC:
• Resolution
• Robustness
Enio Belotti, Luca Meni, Marco Ruggeri, Water&Life Entratico (BG) Italy and R. Ferrari, SiAl
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
The “Fused Core” Choice
Small Molecules (< 3000 D)
Analytical:
(2.1, 3.0, 4.6 mm ID; 2, 3, 5, 7.5, 10, 15 cm Lenght)
• 150 m2/gram surface area
(comparable to ~225 m2/g
porous particle)
•
•
•
Pore Size: 90 Ångstrom
Pressure Limit: 9000 psi (tested)
pH range: 2-9
•
PHASES AVAILABLE:
1.
C18
C8
HILIC (Silica)
RP Amide
Phenyl Hexyl
Pentafluorophenylpropyl (PFP) F5
2.
3.
4.
5.
6.
Monomeric
I Taller Análisis de Alimentos.
20 de Junio 2012
Córdoba,
Endcapping
Applications:
Analytical: acid, basic, ionic,
apolar compounds
Analysis de PAHs en aceite vegetal
SupelMIP™ SPE methodology
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
SupelMIP™ SPE methodology
Polymer backbone
The MIP binding
site (H-bonding,
HYDROPHOBIC
1.
2.
ionic, Van der
Waals)
3.
O
H
O
O- H
N
H
O
H
4.
O
N
H
H
5.
MIP methodology differs from conventional
SPE methodology
Protocols for reversed phase, ionicexchange etc CANNOT be used
Typically the loading from aquous samples
is non-selective via hydrophobic
interactions
Selectivity is introduced during the
interference wash step with organic
solvents.
Analytes are then retained selectively in
the ’analyte specific sites’ of the MIP
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
H-bonding/ionic
Van der Waals interactions
π – π interactions
Molecular Imprinted Polymer
MIPs son robusto con una alta selectividad al analito.
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Metodología de uso de SupelMIP™
¡Analito limpio
y concentrado!
1. Acondicionado
de la columna
2. Carga de
muestra
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
3. lavado de los
compuestos
interferentes
4. Elución
Recuperación de PAHs en aceite de oliva fortificado.
» Spiked (2 μg/mL)
• Compound
• Fluoranthene
• Benzo(a)anthracene
• Chrysene
• Benzo(b)fluoranthene
• Benzo(k)fluoranthene
• Benzo(a)pyrene
• Indeno(1,2,3-cd)pyrene
• Dibenzo(ah)anthracene
• Benzo(ghi)perylene
Blank (μ
μg/kg)
39.6
3.4
9.4
2.2
1.4
3
2.4
1.8
3.4
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Recovery
48%
65%
70%
82%
84%
87%
95%
82%
87%
Condiciones de trabajo
Procedimiento SupelMIP SPE – PAHs
SPE Cartridge: SupelMIP SPE – PAHs, 50 mg/3 mL (Cat. No.52773-U)
1. Acondicionar el cartucho con 1 mL de ciclohexano
2. Cargar la muestra de aceite
3. Lavar con 1 mL de ciclohexano
4. Eluir con 3 x 1 mL de acetato etilo
5. Evaporar el eluido con corriente de nitrogeno a 40 °C y reconstituir en 0.2 mL de acetato de etilo
• Condiciones GC-MS para análisis de
PAH
• column: SLB™-5ms, 30 m x 0.25 mm I.D., 0.25
μm (Cat. No. 28471-U)
• instrument: Agilent GC-MS
• oven: 60 °C , 25 °C/min. to 275 °C ( 5 min.),
10 °C/min. to 300 °C
• (1 min.)
• flow rate: helium, 2 mL/min., constant
• injector temp.: 300 °C
• MS detection: Autotune + EM offset of 300
• scan range: SIM
• injection: 1 μL, splitless
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Compuestos analizados
Se utiliza como blanco una mezcla de oleinas (mono,di,tri).
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Condiciones de Trabajo. LC-MS (excepto Cloranfenicol)
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
• Evaporate at 50 oC to 0.75 mL.
• Add 0.15 mL acetonitrile and water to total volume of 1.0 mL.
• Filter the sample prior to LC-MS analysis using 0.45 m filter.
μ
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Recuperación de drogas añadidas en el extracto
en blanco.
La diferencia con el 100% es debida a la presencia de efectos de ionización
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Recuperaciones de drogas Veterinarias añadidas
a la muestra.
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Algunos conclusiones de la comparación
•• Supel™ QuE Z-Sep+ es mas activo en la captura de
compuestos de color.
•• Debido a la doble funcionalidad del Supel™ QuE Z-Sep+ se
requiere el uso de ac.acético para evitar la retención de ácidos y
compuestos quelantes. (ejem.ciprofloxacine).
•• Mejor comportamiento de C18 con leche y mejor
comportamiento de Supel™ QuE Z-Sep+ en higado.
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
•SPME
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
SPME
Pierce
Expose
Sample Septum Fiber/Extract
Retract Fiber &
Remove
Pierce GC
Inlet Septum
7131345
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
Expose
Fiber/Desorb
to Column
Retract Fiber &
Remove
7131345
Análisis de la perdida de picor de una salsa de
marisco
Sample:
0.5g aliquots of 1mg phenyl isothiocyanate
(int. std.) in 100g sauce
SPME:
100µm polydimethylsiloxane phase fiber 20min
headspace sampling, 40 C1min desorption
DB-1 phase, 30m x 0.25mm ID, 1µm film
60 C (1min) to 230 C at 4 C/min, hold 5min helium,
30cm/sec (set at 150 C)
MS (m/z = 38-200)
235 C, split/splitless
Column:
Oven:
Det.:
Inj.:
°
°
° °
°
°
1
2
250000
Before
Storage
A
b
u 200000
n
d
a 150000
n
c
e 100000
A
b
u
n 180000
d
a
n
c 120000
e
2
1
60000
50000
0
0
6
8
10
12
14
16
18
20
Figure provided by Dr. A. Harmon, McCormick & Co., Inc., Hunt Valley, MD USA
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
95-0239
After 5 days
°F
Storage at 85°
6
8
10
12
14
16
18
20
Aceite de menta en una barrita de chocolate
Sample:
SPME Fiber:
Extraction:
Desorption:
Column:
4g peppermint cookie bar
100µm PDMS
headspace, 1 min, 45°
°C
5 min at 250°
°C
PTE™-5, 30m x 0.25mm ID,
0.25µm film
Detector: FID, 250°
°C
Injector: splitless (3 min), 250°
°C
3
1
5
1.
2.
3.
4.
5.
Solvent
Internal standard
cis-Menthone
trans-Menthone
Menthol
2
4
0
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
96-0116
4
Mi
n
8
1
2
794-0862
Volátiles en vino blanco por SPME y GC/MS
Sample:
SPME Fiber:
Extraction:
Desorption:
Column:
Detector:
white wine + 25% NaCl
Carboxen™/PDMS
10 min headspace, 40°C
3 min at 290°C
VOCOL™, 30m x 0.25mm ID, 1.5µm film
GC/MS, Quadrapole, m/z = 31-240
5
2
1. Sulfur dioxide
2. Ethanol
3. Methyl formate
4. Acetic acid
5. Ethyl acetate
6. Isobutanol
7. Isopentanol
8. 2-Methyl-1butanol
9. Ethyl butyrate
10. 2,3-Butanediol
11. Hexanol
12. Isoamyl acetate
13. Ethyl hexanoate
16 14. Hexyl acetate
15. Octanoic acid
16. Ethyl octanoate
7
13
8
1
6
3
10
9
4
5
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
98-0377
12
11
1
0
14
Mi
n
1
5
15
2
0
97-0191
Análisis del sabor de la leche por SPME y GC-MS
Análisis de la descomposición de ácidos grasos en la leche por la
exposición a la luz UV resultando algunos aldehídos de olor y sabor
desagradables.
Condiciones del análisis por SPME y GC-MS
Sample: 3g of 2% milk + 10µL internal standard solution (20µg/mL 4-methyl-2pentanone) (9mL GC vial)
SPME Fiber: PDMS/Carboxen™, 75µm film
Extraction: headspace, 15 min with constant stirring at 45°
°C
Desorption: 5 min, 250°
°C
Column: Supel-Q™ PLOT, 30m x 0.32mm ID
Oven: 70°
°C (2 min) to 140°
°C at 6°
°C/min (2 min hold)
then to 220°
°C at 6°
°C/min (5 min hold)
Carrier: helium, 35cm/sec
Inj.: splitless (closed 2 min)
Det.: GC/MS ion trap, m/z = 33-300
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
98-0386
Análisis del sabor de la leche por SPME y GC-MS
Prior to Exposure to Sunlight
1
2
1.
2.
3.
4.
5.
6.
IS
3
Acetone
2-Butanone
3-Methylpentane
Pentanal
Dimethyldisulfide
Hexanal
4-Methyl-2-pentanone (int. std.)
IS
6
4
After 1-Hour Exposure to Sunlight
5
1
0
1
5
2
0
2
5
Mi
n
I Taller Análisis de Alimentos. Córdoba,
20 de Junio 2012
98-0385
3
0
3
5
Chromatogram provided by Ray Marsili,
Dean Foods Technical Center, Rockford,
G00507, 508
IL, USA.
Componentes del aroma del café
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
2-Methyl furan
2-Butanone
2-Pentanone
3-Methyl butanal
2,5-Dimethylfuran
2-Acetyloxy-2propanone
2-Ethyl hexanol
Dimethyldisulfide
Phenol
Hexanal
2-Methyl thiophene
n-Methyl pyrrole
4-Methylphenol
2-Ethyl pyrrole
Pyridine
Pyrazine
Methyl pyrazine
4-Methyl thiazole
3-Hydroxy butanone
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
Dimethyl phenol (isomer)
1,2-Ethanediol, monoacetate 40.
41.
2,5-Dimethylpyrazine
42.
2,3-Dimethylpyrazine
43.
2-Ethylpyrazine
44.
2,6-Dimethylpyrazine
45.
2-Ethyl-6-methylpyrazine
46.
2-Ethyl-5-methylpyrazine
47.
Trimethylpyrazine
48.
2-Ethyl-3-methylpyrazine
49.
2,6-Diethylpyrazine
50.
2-Ethenylpyrazine
2-Ethyl-3,5-dimethylpyrazine 51.
52.
Glycerol
53.
2,3-Diethylpyrazine
2-Ethyl-3,6-dimethylpyrazine 54.
55.
2-Furancarboxaldehyde
56.
2-Isopropenylpyrazine
3,5-Diethyl-2-methylpyrazine
57.
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
98-0401
Furfural formate
2-Furonyl ethanone
Methyl benzoylformate
Furanmethanol acetate
5-Methyl-2-furancarboxaldehyde
Furanmethanol proprionate
Furfanyl furan
Pyridine methanol
2-Methyl-5-propenylpyrazine
Furanmethanol
3-Ethyl-4-methyl-2,5-furandione
Pyrazinecarboxamide
2-Ethyl-3-hydroxy-4H pyran-4-one
1-(2-Furanylmethyl)-pyrrole
2-Methoxyphenol
1-(1H-pyrrole-2-yl)-ethanone
4-Ethyl-2-methoxy phenol
3-Phenylpropenal or
2-Methylbenzofuran
3,5-Dimethylbenzoic acid
Granos de café por SPME
Sample:
SPME Fiber:
Extraction:
Desorption:
5g coffee grounds in 40mL vial
DVB/Carboxen™/PDMS (StableFlex™ Fiber)
headspace, 30 min at 65°C
270°C for 5 min
Column: SUPELCOWAX™ 10, 30m x 0.25mm x 0.25µm film
Oven: 40°C (5 min) to 230°C at 4°C/min
Inj.: splitless/split, closed 0.5 min, 270°C, with 0.75mm liner
Det.: ion trap mass spectrometer, m/z = 30-350 at 0.6 sec/scan
Selected ions used for quantitation.
36
17
23
32
22
24 26
33
21
34
39
40
27
28
16
42
29
35
15
1
34
2 56
4
14
7 8 9 101213
11
8
1
2
19
18 20
1
6
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012
98-0397
31
25
38
57
48
43
49
52
53
55
56
51
46
54
50
37 41 44 45 47
30
2
0
Mi
n
2
4
28
3
2
36
40
I Taller Análisis de Alimentos. Córdoba, 20 de Junio 2012