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1 Prezentare BIALOWIEZA septembrie 2008-final · 1 IFIN-HH National Insitutefor Physics and Nuclear...
Transcript of 1 Prezentare BIALOWIEZA septembrie 2008-final · 1 IFIN-HH National Insitutefor Physics and Nuclear...
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IFIN-HH National Insitute for Physics and Nuclear Engeneering
Multipurpose Irradiation Facility IRASM
A Study on optimization the sample preparation for
gas chromatographic methods used in routine
irradiated foodstuffs detection
M. Virgolici, A.V. Medvedovici, M.M. Manea, M. Cutrubinis,
I.V. Moise, R.M. Georgescu
International Coference on
Recent Developments and Applications of Nuclear Technologies
Bialowieza, Poland
- 15 – 17 September 2008 -
METHODSIFIN-HH
Centrul de Iradieri Tehnologice IRASM
1. Using the Gas Chromatographic Microbial Identification System
(MIDI, Inc.) for fatty acids profiling of extracted fat from irradiated
foods. Enabling Cn-1:0 and Cn-2:1 hydrocarbons (HC) and 2-
alkylcyclobutanones prediction virtually from any fats which contain
fatty acids with chain length ranging from 9 – 20 carbon atoms in
length
2. Structure confirmation of main identified fatty acids by Gas
Chromatography coupled with Mass Spectrometry (GC-MS)
3. Cn-1:0 and Cn-2:1 HC detection using EN 1784 standard and
optimized GC-MS method for identification of irradiated foods that
contain fat
4. 2-Alkylcyclobutanones detection using EN 1785 standard and
optimized GC-MS method for identification of irradiated foods that
contain fat
5. Intercomparison with the ESR method for detection of irradiated
foods which contain bones
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Samples : Chicken, Salmon, Crab and Shrimp aquired from Romanian market
Samples storage: in freezer before and after irradiation
Gamma Irradiation : SVST Co-60 IRASM facility
Irradiation dosses : 0, 2, 7 and 10 kGy
Dosimetry : ECB (STDEV + 2% @ 1σ)
Fatty acids profiles and identification : Sherlock 6.0 Microbial Identification
System (MIDI, Inc. USA) and GC-MS performed on Agilent GC 6890N gas
chromatograph with two channels:
- classical split/splitless injector (S/SL) – Ultra 2 non polar capillary column –
classical Flame ionisation detector (FID) for Sherlock
- programable temperature vaporizer (PTV) CIS 4 (cooled injection systems)
made by Gerstel with Peltier cooling to 10C – HP-5ms non polar capillary
column – coupled with Agilent 5795 inert EI MSD (Mass Spectrometric Detector)
for structural identification using reference substances spectra and NIST 2005
MS Library
Samples and Instrumentation
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HC profiles and identification : performed on Agilent GC 6890N on both
channels (Flame ionisation and MS detection)
2-ACB identification : performed on Agilent GC 6890N on PTV GC-MS
channel
ESR free radical detection: Magnettech MiniScope 200 spectrometerMagnettech MiniScope 200 spectrometer
Samples and Instrumentation
Fatty acids profiling – Sherlock MIS 6.0IFIN-HH
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Methylation
Saponification
NaOH solution water : methanol
HCl
methanolic solution
Pure Lipids
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Liquid – Liquid extraction
Cleanup
Extraction solvents
n-Hexane,
MTBE
NaOH
aqueous solution
Sherlock MIS GC-FID analysis
- injection hot split @ 250C
- i. d. 0,32mm, 25m long non-polar HP Ultra 2 capillary column
- 28K/min from 170C to 288C (~300 fatty acids in a window of 3.2 min)
- fast GC analysis (6min/sample)
- sample preparation time ~2h/15 samples
Fatty acids profiling – Sherlock MIS 6.0
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620:4 w6,9,12,15c
4661231
25
(18:2 w6,9c)18:2 w6,9c/18:0 ante (Linoleic acid)
172146323218:1 w9c (Oleic acid)
618418:1 w7c
13335618:0 (Stearic acid)
417:0
75516:1 w7c/16:1 w6c
30715202116:0 (Palmitic acid)
25114:0 (Myristic acid)
Shrimp
(MIDI)
Crab
(MIDI)
Salmon
(MIDI)
Chicken
(MIDI)
Chicken
(literature)
Fatty acid
FAME profiles
Fatty acids profiling – Sherlock MIS 6.0
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Fatty acids profiling – GC-MS
FA
ME
9:0
FA
ME
10:0
FA
ME
11:0
FA
ME
12:0
FA
ME
13:0
FA
ME
14:0
FA
ME
15:0
FA
ME
16:0
FA
ME
17:0
FA
ME
18:0
FA
ME
19:0
FA
ME
20:0
- GC reference substances : FAME C9:0 – C20:0 with concentrations of 0,01% and 0,005%
- injection: 1ul hot split @ 250C (PTV CIS 4) of same samples prepared by MIS procedures
- i.d. 0.25mm, 30m long non-polar HP-5ms capillary column; const. flow 1,3 ml/min
- 20K/min from 170C to 288C (fast GC analysis: 8min/sample)
Salmon meatIFIN-HH
2-tetradaca-5',8'-
dienylcyclobutanone1,7,10-C16:36,9-C17:212
18:2 w6,9c/18:0 ante
(Linoleic acid)
(2-TDCB)1,7-C16:28-C17:14618:1 w9c (Oleic acid)
C16:2C17:1818:1 w7c
(2-TCB)1-C16:1C17:0318:0 (Stearic acid)
C14:2C15:1516:1 w7c/16:1 w6c
(2-DCB)1-C14:1C15:01516:0 (Palmitic acid)
1-C12:1C13:0514:0 (Myristic acid)
2-ACBCn-2:1Cn-1:0
Composition
%Fatty acid
FA
ME
14:0 FA
ME
16:0
FA
ME
16:1
w7
c/1
6:1
w6
c
FA
ME
18:0
FA
ME
18:1
w9c
FA
ME
18:2
w6
,9c
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Fatty acids profiling – GC-MS
- MSD: transfer line @ 280C, EI source @ 230C, Quad @ 180C, PFTBA tune @ 1.3 ml/min
- for co-eluted peaks, MS spectral deconvolution was made with AMDIS v 2.62/2005
- spectral identification was made by means of reference substances (FAME), retention
time indexes based on Sherlock MIS callibration data, all correlated with MS spectra from
NIST05 Library
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Optimization of EN 1784 HC GC-MS detection method
GC-MS EN 1784 optimized classic chromatographic method:
- inj 1ul @ 250C – hot splitless (1min)
- temperature programme: 10K/min from 50C to 130C; 5K/min to 230C
- MSD detection : EI, SCAN 50-300amu
GC-MS EN 1784 PTV LVI chromatographic method:
- inj 5ul @ 10C – solvent vent @ 4ml/min (1min)
followed by splitless injection @ 300C (1.5min) & PTV cleanup @ 350C
- same temperature programme and MSD parameters
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Optimization of EN 1784 HC GC-MS detection method
10C was selected for minimizing the loss of analites during solvent evaporation in injector
1-C
12:1
C13:0
10C
20C
30C
40C
10C
20C
30C
40C
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Optimization of EN 1784 HC GC-MS detection method
- hot splitless : 1 ul @ 250C (1min)
- Calibration mixture 1ppm
C15:0
1-C
12:1
C13:0
1-C
14:1
1,7
-C16:2
1-C
16:1
C16:0
C17:0
C18:0
IST
DC
20:0
1-C
18:1
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Optimization of EN 1784 HC GC-MS detection method
- Solvent vent temperature optimization : @ 10C & 4ml/min
- Calibration mixture 1ppm
1-C
12:1
C13:0
1-C
14:1
C15:0
1,7
-C16:2
1-C
16:1
C16:0
C17:0
C18:0
IST
DC
20:0
1-C
18:1
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Optimization of EN 1784 HC GC-MS detection method
- solvent vent injection enables at least 10 times smaller concentrations
1UL
HOT SPLITLESS
5UL
SOLVENT VENT
0.1ppm
0.5ppm
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Optimization of EN 1784 HC GC-MS detection methodChicken Meat
2-tetradaca-5',8'-
dienylcyclobutanone1,7,10-C16:36,9-C17:231
18:2 w6,9c/18:0 ante
(acid linoleic)
(2-TDCB)1,7-C16:28-C17:13218:1 w9c (acid oleic)
(2-TCB)1-C16:1C17:0518:0 (acid stearic)
(2-DCB)1-C14:1C15:02016:0 (acid palmitic)
2-ACBCn-2:1Cn-1:0%Fatty acid
HC and 2-ACB
prediction
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1,7
-C16:2
1-C
14:1
C15:0
1,7
,10-C
16:3
1-C
16:1
6,9
-C17:2
8-C
17:1
C17:0
IS C
20:0
Chicken irradiated @ 10kGy
HOT SPLITLESS
Optimization of EN 1784 HC GC-MS detection methodChicken Meat
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1-C
14:1
C15:0
1-C
16:1
6,9
-C17:2
8-C
17:1
C17:0
IS C
20:0
1,7
-C16:2
1,7
,10-C
16:3
Chicken irradiated @ 10kGy
Solvent vent method – same sample
Optimization of EN 1784 HC GC-MS detection methodChicken Meat
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Salmon irradiated @ 10kGy
Solvent vent method
Optimization of EN 1784 HC GC-MS detection methodSalmon Meat
C15:0
1-C
14:1
1-C
12:1
6,9
-C17:2
8-C
17:1
1,7
-C16:2
1,7
,10-C
16:3
IS C
20:0
C17:0
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Optimization of EN 1785 2-ACB GC-MS detection method
GC-MS EN 1785 optimized classic chromatographic method:
- inj 1ul @ 250C – hot splitless (1min)
- temperature programme: 15K/min from 55C to 300C
- MSD detection : EI, SIM of m/z 98 (dwell 50ms) and m/z 112 (dwell 50ms)
GC-MS EN 1785 PTV LVI chromatographic method:
- inj 5ul @ 10C – solvent vent @ 4ml/min (1min)
followed by splitless injection @ 300C (1.5min) & PTV cleanup @ 350C
- same temperature programme and MSD parameters
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Optimization of EN 1785 2-ACB GC-MS detection method
- hot splitless : 1 ul @ 250C (1min)
- 2-dodecylcyclobutanone (2-DCB) 0.25ppm
2-D
CB
IS 2
-CC
H
O
2-cyclohexylcyclobutanone
O
2-dodecylcyclobutanone
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- Solvent vent temperature optimization : @ 10C & 4ml/min
- 2-dodecylcyclobutanone (2-DCB) 0.25ppm (~ six times more sensitive than hot splitless)
2-D
CB
IS 2
-CC
H
O
2-cyclohexylcyclobutanone
O
2-dodecylcyclobutanone
Optimization of EN 1785 2-ACB GC-MS detection method
IFIN-HH
- solvent vent injection enables at least 10 times smaller concentrations
Optimization of EN 1785 2-ACB GC-MS detection method
1UL
HOT SPLITLESS
5UL
SOLVENT VENT
0.025ppm
0.25ppm
better
linearity and sensitivity
Optimization of EN 1785 2-ACB GC-MS detection methodSalmon Meat
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2-D
CB
IS 2
-CC
H
2-T
CB
2-ACB inj hot splitless
MSD: SIM m/z 98 si m/z 112
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Optimization of EN 1785 2-ACB GC-MS detection methodSalmon Meat
2-ACB inj solvent vent – better sensitivity
MSD: SIM m/z 98 si m/z 112
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2-D
CB
IS 2
-CC
H
2-T
CB
2-DCB STRUCTURAL CONFIRMATIONSALMON MEAT
2-DCB NIST05
MSD: SCAN m/z 35 - m/z 300
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Component at scan 1931 (13.795 min) [Model = TIC] in C:\ msdchem\ 1\ DATA\ _LDAI_\ _SELECTIE-CONFERINTE-SEPT-2008\ QC-EN-1785-SOMON-10KGY-SCAN-SVENT.D\ data.ms2-Dodecylcyclobutanone
30 60 90 120 150 180 210 240
0
50
100
50
100
41
41
55
55
69
69
84
84
98
98
112
112
125
125
138
138
153166
166
192
194
207
210
220 238
238
2-TCB
MSD: SCAN m/z 35 - m/z 300
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(ma inlib ) Cyclobutanone, 2-tetradecyl-
50 70 90 110 130 150 170 190 210 230 250 2700
50
100
55
69
84
98
112
124138 152 166 180 194 222 238 248 266
O
(Text File) Component a t scan 2356 (15.161 min) [Model = +98u] in C:\ msdchem\ 1\ DATA\ _LDAI_\ _SELECTIE-CONFERINTE-SEPT-2008\ QC-EN-1785-SOMON-10KGY-SCAN-SVENT.D\ data.ms
30 60 90 120 150 180 210 240 2700
50
100
4155
69 83
98
112
125 140 157 173 193 211 238 266
2-TCB STRUCTURAL CONFIRMATIONSALMON MEAT
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Solvent extraction procedures
Standard extraction procedures involve time and high volumes of solvent:
- Soxhlet extraction : 150 ml/sample – 6hours extraction
Straight forward extraction techniques:
- Ultrasound fat extraction allows smaller extraction time, simple glassware, with the same volume of high purity solvents or smaller
- Direct solvent extraction method (Tewfik 2008) enables 3 times smaller solvent volumes, total reduction to sample preparation to 90 min, and the same results for 2-ACB.
Use of last two techniques could lead to comparable results in more resonabletime per sample and resonable costs.
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Detectie RES os pui
0
10000
20000
30000
40000
50000
60000
329 330 331 332 333 334 335 336 337 338 339
Camp magnetic (mT)
Inte
ns
itate
(u
.a.)
0 kGy 2 kGy 7 kGy 10 kGy
ESR method for detection of irradiated bonesDetection of irradiated Chicken
IFIN-HH
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Detectie RES os somon
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
55000
329 330 331 332 333 334 335 336 337 338 339
Camp magnetic (mT)
Inte
ns
ita
te (
u.a
.)
0 kGy 2 kGy 7 kGy 10 kGy
ESR method for detection of irradiated bonesDetection of irradiated Salmon
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Detectie RES os crab
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
329 330 331 332 333 334 335 336 337 338 339
Camp magnetic (mT)
Inte
sit
ate
(u
.a.)
0 kGy 2 kGy 7 kGy 10 kGy
ESR method for detection of irradiated bonesDetection of irradiated Crab
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Detectie RES os crevete
0
10000
20000
30000
40000
50000
60000
70000
80000
90000
100000
329 330 331 332 333 334 335 336 337 338 339
Camp magnetic (mT)
Inte
ns
ita
te (
u.a
.)
0 kGy 2 kGy 7 kGy 10 kGy
ESR method for detection of irradiated bonesDetection of irradiated Shrimp
ConclusionsIFIN-HH
Centrul de Iradieri Tehnologice IRASM
- Fatty acid profiling using Sherlock MIS method is an efficient way of
determination fatty acid composition of various fats before HC and 2-
ACB detection enabling use of standardized methods EN 1784 and EN
1785 outside of the available validation data
- Use of PTV Cooled Injection Systems can enable detection of smaller
quantities of HC and 2-ACB comparing with classical methods
- The sensitivity improvement of HC and especially 2-ACB detection in
conjunction with more faster extraction methods will enable a larger
number of samples for routine testing, and maybe smaller costs
- ESR method for detection of irradiated foodstuffs on bone samples
can be used for intercomparison purposes with chemical methods for
detection of meat samples
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Thank You for You Attention
Aspecte teoreticeProdusi chimici rezultati in urma iradierii lipidelor
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Posibili produsi de radioliza ai
trigliceridelor
2-ACB
Reactii de formare
.....
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Formarea 2-ACB.
Cele mai comune 2-ACB
prezente in alimente.