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Michele Suman – 24 Febbraio 2011

SPECIFIC MIGRATION OF PLASTICIZERS COMING FROM CLOSURE GASKETS:

THE LC-MS DIRECTION.

M. SumanBarilla SpA – Food Research Labs, Parma, Italy

e-mail: [email protected]


I propose this way….

Presentation Outline

> Gaskets & Plasticizers: a brief overview

> Section A: LC-MS solution for the analysis of the most well knownplasticizer: epoxidized soy bean oil (ESBO)

> Section B: LC-MS new approaches for the multiresidual analysis of plasticisers and derivatives in PVC gaskets and food

> Section C: LC-MS as promising solution also for the new generation of polymeric plasticizers: polyadipates (PA)


Contamination from food packaging ‘ignored’31 August 2006Chemist calls for rigorous investigation of leached compounds

24 agosto 2006CORRIERE SALUTE

Gli scienziati preoccupati per le conseguenze sull'organismo

La plastica è ormai nella catena alimentare Assorbita da pesci di cui poi ci nutriamo. Ma presente anche in contenitori per la conservazione del cibo

STRUMENTIVERSIONE

STAMPABILEI PIU' LETTIINVIA QUESTO

ARTICOLO

http://www.corriere.it/Rubriche/Salute/Medicina/2006/08_Agosto/22/plastica.html

http://www.corriere.it/Rubriche/Salute/Medicina/2006/08_Agosto/22/plastica.html


Introduction

Glass jars with metal twist closures are used for containing of wide range of foods. The inner part of the metal lids is covered by plastisols for ensuring good seal of the closure against the glass rim.

The plasticisers and additives being incorporated in PVC gasket seals have the potential for migration into the food during sterilization and storage. Fatty\Oily food matrices (like for example pesto or sauces with an high

fat content) present high extraction potential towards these plasticizers with respect to other product.

With this technical solution, it is possible to form an airtight seal, which prevents microbiological contamination in sealed jars and bottles adopted to contain food commodities, and provide easy opening.

The plastisols consist of poly(vinyl chloride) (PVC) usually containing 25–45% by weight of plasticisersand additives. Most frequently epoxidised soybean oil (ESBO), dibutyl sebacate (DBS), acetyl tributyl citrate

(ATBC), and finally polyadipates.


Surveys\legal restrictions on plasticizers up to 2008


Gaskets’ composition survey in 2005

Number of lids with gaskets containing the components listed as the most important plasticizers and percent of the total number of lids analyzed;

survey of June 2005

Main plasticizer June 2005 Number of lids % Epoxidized soy bean oil ESBO 104 64Epoxidized linseed oil ELO 12 7.4Di-(2-ethylhexyl) phthalate DEHP 15 9.3Diisononyl phthalate DINP 9 5.6Diisodecyl phthalate DIDP 11 6.8Di-(2-ethylhexyl) adipate DEHA 9 5.6Acetylated partial glycerides acPG 2 1.2Di-isononyl-cyclohexane-1,2-dicarboxylate DINCH 2 1.2

References-Fankhauser-Noti A, Fiselier K, Biedermann S, Biedermann M, Grob K, Armellini F, Rieger K, Skjevrak I (2005) Eur Food Res Technol-Fankhauser-Noti A, Grob K (2005) Trends in Food Science & Technology-Fankhauser-Noti A, Fiselier K, Biedermann-Brem S, Grob K (2005) J Chromatogr A-Biedermann M, Fiselier K, Grob K (2005) J Separation Science-Biedermann-Brem S, Biedermann M, Fiselier K, Grob K (2005) Food Additives and Contaminants


LC-MS solution for the analysis of the most well known plasticizer: epoxidized soy bean oil (ESBO)

Jar rim

Gasket in food contact

Coatedmetal

Corner filledwith oil

Section A


The history started in Summer 2004…- In the early summer 2004 it was noticed that the migration of plasticizers and other additives

from the gaskets into oily normal foods, such as sauces and products in oil, often far exceeded the legal limits.

- Typically, 150-400 mg plasticized PVC is in direct food contact, contains 25-45 % additives (primarily plasticizers), and the transfer easily approaches completeness if an oily food is in contact with the gasket. As a consequence, legal limits were exceeded by far for whole ranges of products.

- A major Swiss distributor estimated the value of his products exceeding the limits to roughly 20 Mio Euros. Extrapolated to the whole of Europe, the annual production of affected foods might be in the order of several thousand millions of Euros.

- In July 2004, the Swiss enforcement authorities stopped the sale of one product in glass jars with ESBO far exceeding 60 mg/kg.


What is ESBO??? ESBO is a modified oil resulting from an epoxidation reaction of soybean oil. Soybean oil consists of a mixture of triglycerides whose average composition inpredominant fatty acids is about 11% palmitic (16:0), 4% stearic (18:0), 23% oleic (18:1),55% linoleic (18:2), and 8% linolenic (18:3).

ESBO is a clear pale yellow viscous liquid of low odour, with a melting point of ~ 4°C.

Its CAS Registry Number is CAS 8013-07-8 EINECS/ELINCS 232-391-0

The plastics Directive (72/2002/EC) contemplate ESBO for food contact use in plastics. Its structural formula can be represented as shown here in figure below


Biological and toxicological data

- ESBO has very low acute toxicity in rats (LD50>5 g/kg bw).

- Repeated exposure studies show slight changes in uterus, liver and kidney weight, and no alteration of blood parameters, in rats fed with diet containing up to 5% ESBO (approx 2.5 g/kg bw/day) for 2 years. The No Observed Adverse Effect Level (NOAEL) was approximately 140 mg/kg bw/day and the Lowest Observed Adverse Effect Level(LOAEL) was approximately 1400 mg/kg bw/day. This figure was used by the UKCommittee on Toxicity to derive, using an uncertainty factor of 100, a TDI of 1 mg/kg bw(DoH, 1995). The same TDI was adopted by the SCF in 1995-96.

- No effect on fertility was observed in rats treated with up to 1g ESBO/kg bw/day. - No evidence of carcinogenicity was seen in rats fed with 2.5 % ESBO in the diet for two years.- No evidence of genotoxicity was obtained in the Ames/Salmonella test.

Viceversa, derivatives of ESBO, such as chlorohydrins (formed by the interaction of ESBO withHCl eliminated from PVC degradation processes) and also cyclic compounds, are at the momentunder investigation to better understand their real migration in food, chemical structure andtoxicological concerns.


HOW MUCH ESBO INSIDE?


The method of Castle et al. extracts the lipids from foodand transesterifies them.

Then the cyclopentanone dioxolanesof the epoxy functions are formed, which enables the detectionof the diepoxy linoleic acid exploiting the GC-MS technique.

The dioxolanes increase the retention,pulling the analyte away from the bulk of the fatty acid

methyl esters.

This method provided good results in numerouslaboratories and has a detection limit in foods of

around several mg/kg.

GC-MS method developed in 1988


• homogenization of the content of the jars (in order to obtain an average result, including also the food adhering to the lid) with UltraTurrax

• an accurately weighed sub-sample (2g) in a vortex mixer with dichloromethane (20 mL) 1 min. • mixture centrifuged at 3000 rpm for 10 min. • filtration through a syringe filter before LC-MS analysis.

“ESBO free” pesto and tomato-based sauces, were appropriately produced in pilot plants and packed without the use of gaskets (the absence of ESBO had been in any case verified apart): they were then used as the blank to calculate matrix-matched LOD and LOQ and to generate calibration curves directly obtained in the matrix itself.

If an “ESBO-free” matrix was not effectively available the selected procedure executed on each single unknown sample was the standard addition method at three different levels at least (in general 30, 60, 90 mg kg-1)

The optimized final method consists of the quantification of the 5 more intense and significant signals which all together represent approximately 90% of the ESBO mixture.

LC-ESI-IonTrap-MS/MS method

BIBLIOGRAPHIC REFERENCESuman, M.; La Tegola, S.; Catellani, D.; Bersellini, U.

“Liquid chromatography – electrospray ionisation – tandem mass spectrometry method for the determination of Epoxidized Soybean Oil in food products”

Journal of Agricultural and Food Chemistry 2005, Vol-53, pp. 9879-9884.


LC-ESI-IonTrap-MS/MS methodC:\Barilla\...\infusione ESBO 10ppm 10/14/2004 04:12:33 PM

RT: 0.06 - 1.51

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)

0

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0.950.870.28 1.381.280.56 1.331.150.410.33 1.020.770.51 0.660.46 0.61 1.10

0.10 0.20

NL:3.24E7m/z= 887.65-1152.49 MS infus io ne ESBO 10ppm

infusione ESBO 10ppm #8-55 RT: 0.20-1.41 AV: 48 NL: 3.63E6T: + c ESI Full ms [ 150.00-2000.00]

860 880 900 920 940 960 980 1000 1020 1040 1060 1080 1100 1120 1140m/z

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998.42

999.32

1011.97985.34

984.45943.30 1012.97971.53 1015.85946.19 1029.74929.49

1057.63915.35886.57 1082.45859.04 1096.46 1129.54 1149.60

Previous scientific works suggested that the natural occurrence of diepoxidised fatty acids and triglycerides are negligible in relation to level arising from migration. The analytical method developed was in fact accordingly

based on measurement of the di-epoxidised fatty acid component (linoleic acid) and thus levels of ESBO recorded in foods using this method can be assumed to originate primarily from ESBO migration


MS/MS parameters for detection of ESBO

ESBO component Parent ion

m/z

Fragments monitored

m/z

Normalized

Collision Energy

%

MI 1012 699 36

MII 998 685 36

MIII 984 685, 671 36

MIV 971 685, 671, 658 37

MV 943 685, 630 37

It has to be evidenced that in the fragmentation pattern of each single component there is the loss of a fragment correspondent to the di-epoxidized linoleic chain (m/z: 313).


ESBO mixture components signals detected by LC-MSSignal m/z Attributions (1) Formula % in the mixture (2)

[MI + Na]+ 1012 (LnLL)ox, (LnLnO)ox C57H96O13 14[MII + Na]+ 998 (LLL)ox, (LnLO)ox, (LnLnS)ox C57H98O12 34[MIII + Na]+ 984 (OLL)ox, (LnOO)ox, (LnLS)ox C57H100O11 25[MIV + Na]+ 971 (LOO)ox, (LLS)ox, (LnOS)ox C57H102O10 14[MV + Na]+ 943 (SOO)ox, (SSL)ox C57H106O8 13

(1) symbols used are referred to the triacylglycerol structure: Ln = linolenic chain; L = linoleic chain; O = oleic chain; S = stearic chain. With the acronym "ox" it is intended that all the triacylglycerol double bonds have been oxidized Attributions reported in bold style are considered the most probable taking into account of the medium composition of natural soybean oil (2) these percentages have been calculated neglecting ESBO mixture minor constituent (which altogether represent approximately 10% of the total)

[M+Na] + clusters for the different epoxidized

tryacylglycerols.

The percentage contribute of each of these peaks has been evaluated according to their

signal intensity.

Example of three ESBO mixture component signals, detected in

Single Reaction Monitoring (SRM) mode and related to a

-pesto sauce extract with an ESBO content of

few mg kg-1 (left)

- the same extract spiked with ESBO standard solution up to

reach a final concentration of 60 mg kg-1 (right).

RT: 5.37 - 51.53 SM: 15G

10 15 20 25 30 35 40 45 50Time (min)

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10024.43

25.92

18.4118.05

19.81

13.49

11.79 14.94

NL: 1.58E6TIC F: + c ESI SRM ms2 [email protected] [ 670.00-672.00, 684.00-686.00] MS Pesto sample 60

NL: 1.12E6TIC F: + c ESI SRM ms2 [email protected] [ 684.00-686.00] MS Pesto sample 60

NL: 4.20E5TIC F: + c ESI SRM ms2 [email protected] [ 698.00-700.00] MS Pesto sample 60

RT: 6.06 - 51.34 SM: 11G

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23.95

21.1125.44

18.2317.90

17.58

19.0216.79 20.05

13.58

13.10

12.8614.42

11.35

NL: 2.22E4TIC F: + c ESI SRM ms2 [email protected] [ 670.00-672.00, 684.00-686.00] MS Pesto sample

NL: 1.83E4TIC F: + c ESI SRM ms2 [email protected] [ 684.00-686.00] MS Pesto sample

NL: 2.75E3TIC F: + c ESI SRM ms2 [email protected] [ 698.00-700.00] MS Pesto sample


Accuracy…

Mean recoveries and relative standard deviation for ESBO mixture component signals, determined by spiking an “ESBO free” pesto sauce up to reach final concentrations of 30 mg kg-1 and 100 mg kg-1

ESBO spiking level:

30 mg kg-1

Mean Recovery

(%)

RSD (%)

(9 determinations)

ESBO component

MI 95.8 7.9

MII 91.3 6.6

MIII 90.8 7.1

MIV 91.5 5.2

MV 92.4 7.6

ESBO spiking level:

100 mg kg-1

Mean Recovery

(%)

RSD (%)

(9 determinations)

ESBO component

MI 101.2 7

MII 94.2 2.1

MIII 92.5 6.5

MIV 97.8 2.6

MV 93.4 6.7


Linearity and Precision…

Matrix-matched calibration curves established in pesto sauce extracts using the LC-ESI-MS/MS method (concentration range: 10 – 100 mg kg-1)

ESBO component Mandel’s testb (F value)

b1 ± sb1b

MI 0.993 1.862 (±0.032) MII 0.996 5.359 (±0.029) MIII 0.995 4.629 (±0.024) MIV 0.997 1.882 (±0.027) MV 0.994 2.797 (±0.016)

a Calibration function: y = b1x b Confidence level at 95%

Repeatability of the LC-MS/MS method calculated on pesto sauce matrix

ESBO component RSD (%) at 15 mg kg-1

RSD (%) at 30 mg kg-1

RSD (%) at 100 mg kg-1

RSD (%)

Overall

MI 8.1 7.9 7.7 7.9 MII 6.9 5.2 2.3 4.8 MIII 7.2 6.7 5.1 6.3 MIV 5.3 4.5 3.4 4.4 MV 6.2 5.8 4.4 5.5


Methods comparison on real samplesESBO concentration data in different food sauce products (from Italian retail markets) obtained by the

traditional GC-MS procedure and compared with correspondent measurements using the present LC-ESI-MS\MS method

Food sauce samples ESBO concentration

LC-MS (mg kg-1)

ESBO concentration

GC-MS (mg kg-1)

Pesto sauce 1 45 58

Pesto sauce 2 14 13

Pesto sauce 4 78 64

Pesto sauce 5 90 83

Tomato-cheese sauce 1 49 67

Tomato-cheese sauce 2 65 79

Tomato-basil sauce 1 13 19

Tomato-cheese-pepper sauce 1 20 26

Tomato-meat sauce 1 16 10


Real application on italian market products

70 food sauces packed in glass jars (pesto and different kinds of tomato-based ones) were analysed for their ESBO contents. These are the levels found:

pesto based sauces 50-80 mg kg -1

tomato-cheese based sauces 40-60 mg kg -1

tomato-basil based sauces 15-30 mg kg -1

tomato-chilli\meat based sauces 10-20 mg kg -1


The future work on plasticizers in PVC gaskets…

Gasket components of concern, with the abbreviations used, the approved toxicological evaluation and the legal status in the EU. SMLs in brackets: SMLs calculated by conventional assumptions from the TDI

and…??

Plasticizer TDI (mg/kg bw) Reference SCF-list EU listed SML (mg/kg) Epoxidized soy bean oil ESBO 1 EFSA 2004 2 2002/72 EC (60) Epoxidized linseed oil ELO SCF-Liste 7 no Diisodecyl phthalate DIDP

Group-TDI, 0.15 SCF, 1999 [8] 2 no (9) Diisononyl phthalate DINP Di(2-ethylhexyl) phthalate DEHP 0.05 SCF, 1994 2 no (3) Di(2-ethylhexyl) adipate DEHA 0.3 SCF, 2000 2 2002/72 EC 18 Dibutyl sebacate DBS 2002/72 ECAcetyl tributyl citrate (Citroflex A) ATBC SCF, 1998/1 [8] 7 no R=5 2-Ethylhexyl palmitate Ehol-16 no 2-Ethylhexyl stearate Ehol-18 no 2-Ethylhexanol ADI, 0.5 JECFA 1993 1 no (30)Diisononyl cyclohexane 1,2 dicarboxylate DINCH noAcetylated mono- and diglycerides acPG 2002/72 ECPolyadipates PA Group-TDI, 0.5 SCF, 1998/2 [9] 2 2004/19 EC 30 (<1000 D)


SectionB

LC-MS new approaches for the multiresidual analysis ofplasticisers and derivatives in PVC gaskets and food

O

OCH3

CH3

ClOHOHCl


LC-MS potentialities…

The simultaneous analysis of some plasticisers and additives in PVC gaskets and food are historically described in scientific literature following the transesterification reaction and gaschromatographic procedures (combined also with GPC clean-up) or suggesting the application of special injectors (e.g. internal thermal desorption ).

New LC-MS instrumentation has the potential capability to face this challenge in an easier and more effectiveness way: reverse-phase liquid chromatography well separates small low polar molecules and then mass spectrometry in single reaction monitoring mode can detect them with high sensitivity and selectivity.


Multiresidual screening of plasticizers by LC-MS:preliminary results (under publication) about gaskets• Preliminary results have been obtained working firstly on gaskets:- dissolving 10 mg of material from the outer region in 2.5 mL THF: the PVC can be then precipitated by adding 5 mL of ethanol (1 night at refrigerated temperature) - 1 ml of the supernatant is finally filtered and opportunely diluted to a final volume of 100 ml withacetonitrile before the LC-MS injection.

• Comparing ESI and APCI interfaces it has been concluded that APCI guarantee better performances as a total compromise in terms of average sensitivity for all the detectableplasticizer molecules in a qualitative screening (with the future perspective of a quantitative measurement): - fewer matrix-ionization problems- higher signal to noise ratios- responses for the major part of these relatively smaller molecular-weight compounds

• Sebacates and phthalates for example can achieve limit of detection lower than 1 ppm .

Among the possible components a special attention is devoted to ESBO mono- and/or polychlorohydrins and other cyclic derivatives….


PVC degradation & ChlorohydrinsMain pathway of PVC degradation involves the elimination of HCl which can react with epoxy groups

of ESBO, leading to the formation of mono-and/or poly-chlorohydrins and/or other cyclic derivatives

Derivatives formation is affected by particular conditions like acidity, temperature, ionic force, etc., and have neither been exhaustively studied yet.

The major chlorohydrins formed are a function of the nature of the unsaturated triglycerides present in soybean oil which have been epoxidised (i.e. mainly 18:2); three main types are shown in the figure here below (as fatty acid methyl esters, isomers not considered):

Methyl diepoxylinoleate (18-2E)

18-E-OHCl

18-2OHCl

O

OCH3

CH3

O O

O

OCH3

CH3

O ClOH

O

OCH3

CH3

ClOHOHCl

Demertzis, P.G., Riganakos, K.A. and Akrida-Demertzi, K., European Polymer Journal 1991,, 27, 231-235.Howell, B.A., Betso, S.R., Metzer, J.A., Smith, P.B., and Debney, M.F., Termochimica Acta, 1990, 166 , 207-218.


Gilbert et al. investigation on chlorohydrins...(~1980)

A first investigation on ESBO reaction products with HCl was executed by Gilbert et al. between 1979-1981 in model experiments…

Sheperd, M.J.; Gilbert, J. European Polymer J. 1981, 17, 285.


Grob et al. investigation on chlorohydrins...(~2004)

Grob et al., K. Mitt. Lebensm. Hyg, 2001-2004

Grob et al.used 1,2-epoxy-octane and 1,2-4,5-diepoxy-pentane as models to study reactions with HCl.

Epoxy-octane formed chlorohydrins which remained stable at typical curing conditions (200°C,15min).

Diepoxy-pentane (methylene-interrupted diepoxide, simulating linoleic acid) formed, in the same conditions, mono and di-chlorohydrins:

- Heating caused also ring formation- Complex mixture of dimers observed: sum of these derivatives below 5% of the converted material.

It was deduced that heating 200°C for 15 min converts 5-15% of the ESBO fatty acids into chloro-containing derivatives: this proportion should also be expected in the migrates.


EFSA opinion (2004 – 2006)

European Food Safety Authority (EFSA) noted (2004 and 2006) that the potential dietary exposure of adults to ESBO is below the TDI of 1mg/kg

Human exposure to ESBO derivatives is expected to follow the same pattern, peaking at between 6 and 12 months of age (levels found in the range 0.02-0.1 mg/kg bw), decreasing significantly afterwards.

There are not enough analytical and toxicological data to express an opinion on the significance for health of such derivatives.

At present there are no official analytical methods available for chlorohydrins determination directly into foodstuffs.

Furthermore, in scientific literature there are only a few indicative results of direct measurements of ESBO derivatives, due to difficulties in developing an analytical strategy for complex food matrices.

Opinion EFSA-Q-2005-219, The EFSA Journal 2006, 332, 1.

http://www.efsa.europa.eu/EFSA/efsa_locale-1178620753812_home.htm


Simoneau et al. investigation on chlorohydrins...(~2005)A weighty GC-MS method optimized for the quantification of ESBO chlorohydrins in baby foods

Chlorohydrins recoveries from spiked samples on the silylated extract were always greater than 90%

The method was tested on a number of baby food products (e.g. fruit, vegetables, cheese and meat). The limit of detection was 7 ppb for 18-OHCl (S/N=4).

Piccinini, P., Valzacchi, S., Simoneau, C. - Proceedings of the 8th Intern. Symposium on Hyphenated Techniques in Chromatography and Hyphenated Chromatographic Analyzers (HTC-8), RSC, KVCV, 4-6 February 2004, Bruges (BE).

Piccinini, P.; Valzacchi, S.; Ezerskis, Z.; Simoneau, C. Proceedings of the 3rd Intern. Symposium on Food Packaging: Ensuring the Safety, Quality and Traceability of Foods, ILSI, 17-19 November 2004, Barcelona (ES).

BABY FOOD EXTRACT

- migration behaviors influenced by fat content, composition, dispersion…)

- chlorohydrins concentrations generally between 50-100 ppb or < 50ppb


OUR PROJECT TARGETSet up of an LC/MS/MS method for the qualitative characterization of ESBO chlorohydrins in closure gaskets and foodstuffs:

SensitiveSelective

PLANNED STEPS1. Production of a chlorohydrins solution by ESBO hydrolysis procedure with HCl with a raw

estimation of its final concentration2. Infusion of the chlorohydrins solution in HPLC/MS/MS to identify the correspondent

fragmentation pattern and optimize experimental mass spectrometry conditions3. Optimization of the chromatographic procedure4. Olive oil spiking with chlorohydrins solution (to reach a concentration around 1-5 mg/kg) to

develop a dedicated extraction procedure (transesterifcation + SPE clean up)5. Evaluation of matrix suppression effects6. Set up of the method on other complex matrices like tomato based \ pesto genovese sauces


Preparation of Chlorohydrins solutionTransesterification

Isooctane extraction

Water(rinsing)Organic fraction

Hydrolysis (HCl 3% in diethyl ether)

Evaporation to dryness + MeOH dissolution

Transesterification was obtained with sodium methoxide in methanol at room temperature.The upper procedure starts from ~100mg ESBO and is based also on these assumption\hypothesis:- Chlorohydrins stability in the reaction conditions applied- Yield of the reaction at least equal to 50%- Linoleic diepoxidized acid derived from ESBO at least equal to 50% w/w

The concentration of this chlorohydrins solution (+ eventual cyclic or polymeric derivatives) could betherefore roughly estimated: a macroscopic and indicative value useful for the following establishment ofadequate experimental conditions in the LC-MS method development.


Ion Trap MS signals characterizationHPLC 1100/MSD Trap Agilent Technologies XCT

MS/MS [M+H]+ ions result to be more selective than the correspondent [M+Na]+ signals and for this reason experimental conditions were then optimized to maximize their intensity (ESI interface)

Parent (m/z)

Daughters (m/z)

[18-OHCl+H]+ 349 331; 295; 263; 277 [18-OHCl+Na]+ 371 335 [18-E-OHCl+ H]+ 363 345; 309; 277; 291 [18-E-OHCl+ Na]+ 385 349 [18-2OHCl+ H]+ 399 381; 349; 345; 309; 291; 277 [18-2OHCl+ Na]+ 421 385; 349; 331

Optimisation of HPLC chromatographic conditionsInstrument: HPLC/MSD Trap 1100 Agilent Technologies XCT. Column type: Zorbax XDB C18 (100 mm x 2,1 mm i.d. x 3,5 mm)Column temperature: 30 °CElution: 0,2 ml/min - gradientMobile phase: A) Ammonium acetate 10 mM B) MeOHChromatographic total run time: 40 minInjection volume: 10 µl


Ion Trap MS signals characterization (HPLC)

1 2

1

2

18-E-OHCl[M+H] +

18-2OHCl [M+H] + 3

45

6

3

4

5

6

18-E-OHCl and 18-2OHCl positional isomers appears to be overlapped (co-elution) For this reason, to reach a better resolution, a more powerful chromatographic solutions has been investigated: UPLC


Optimisation of UPLC chromatographic conditions

Instrument: UPLC/Quattro Premier Waters MicromassColumn type: Acquity UPLCTM BEH C18 (50 mm x 2,1 mm i.d.x 1,7 mm)Column temperature: 30 °CElution: 0,2 ml/min - gradientMobile phase: A) Ammonium acetate 10 mM B) MeOHChromatographic total run time: 15 minInjection volume: 5 µl

esteri metilici r.t. (min)

p.m. (m/z)

[M+H]+ (m/z)

MS/MS (m/z)

18-E-OHCl (I+II) 10,22 362 363 277, 291, 309 18-E-OHCl (III+ IV) 10,66 362 363 277, 291, 309 18-2OHCl (I) 8,95 398 399 277, 291, 309 18-2OHCl (II) 9,12 398 399 277, 291, 309, 345 18-2OHCl (III+ IV) 9,6 398 399 277, 291, 309, 345 18-2OHCl (V+VI) 10,05 398 399 277, 291, 309, 345 18-2OHCl (VII+VIII) 11,13 398 399 277, 291, 309, 345

Tempo (min)

B% Flusso (ml/min)

0 60 0,3 1 60 0,3

7,5 70 0,3 12,5 70 0,3 12,6 100 0,3 13,5 100 0,3 13,6 0 0,3 14,6 0 0,3 14,7 60 0,3

BIBLIOGRAPHIC REFERENCESuman, M.; De Dominicis, E.; Commissati, I.

“Trace Detection of the Chlorohydrins of Epoxidized Soybean Oil in Foodstuffs by ESI-Tandem Quadrupole UPLC/MS/MS ”

Journal of Mass Spectrometry 2010, 45, 996-1002


Optimisation of UPLC chromatographic conditions

4 isomers for “18-E-OHCl chlorohydrin” and 8 isomers for “18-2OHCl” chlorohydrin were identified.

1a 1b

2a

2b

3a 3b4a

4b

5a 5b

6a 6b

18-E-OHCl [M+H]+

18-2OHCl [M+H]+


Analysis of sauces – fats extraction\transeterification

Fats extraction from the sauce:

sample 5-25 gr

acetone/hexane 1:1

stirring/centrifugation

Evaporation to dryness (rotavapor)

Na2SO4 anhydrous

Transesterification:

fat extract 100mg

H2SO4 2% MeOH 105°

transesterified 1 ml

phosphate buffer ; hexane

Na2SO4 anhydrous

stirring/centrifugation


Analysis of sauces – Clean up: chlorohydrins l/l extraction + SPE

Chlorohydrins l/l extraction:

hexane extract 5 ml

Vortex/centrifugation

acetonitrile extract 4 ml

Acetonitrile

Evaporation to dryness (nitrogen stream)

HCOONH4 10 mM/MeOH 40:60

Solid Phase Extraction Clean up:

The aim of the previous and this further sample treatments is to obtain a significant reduction of ions suppression matrix effects in the following LC-MS analysis:

SPE cartridge: C18 500 mg (3 ml)Conditioning: MeOHWashing & Elution: HCOOH/MeOH

(gradient)


Chlorohydrins on Pesto sauces

18-E-OHCl 18-2OHCl

Commercialsauce packed

in jar with cap

Spiking onsauce not

packed in jar with cap

Sauce not packed in jar

with cap


Chlorohydrins on Tomato sauces

18-E-OHCl 18-2OHCl

Commercialsauce packed

in jar with cap

Spiking onsauce not

packed in jar with cap

Sauce not packed in jar

with cap


Analysis of sauces – main results

Different commercial food sauces samples closed in glass jars with twist-off caps were subjected to qualitative determination, resulting positive to the so called “chlorohydrin 18-E-OHCl “(indicative concentration level: 5÷50 ppb)

To demonstrate the effectiveness of this new analytical strategy, the same samples, not previously closed in glass jars with caps, were analyzed, showing complete absence of chlorohydrins

In no samples were the so called “chlorohydrin 18-2OHCl” detectable

Tomato based sauces seems to contain 18-E-OHCl chlorohydrin in a quantity that can be estimated roughly three times higher than that for other high fats content sauces like pesto


SectionC

LC-MS as promising solution also for the new generation of polymeric plasticizers: polyadipates (PA)

(C H2)4

O O

(C H2)4

OO

OC H2

C HC H3OO

C H2

C HC H3 O

(C H2)4

OO

(C H2)4

O O

O(C H2)2

C HC H3OO

(C H2)2

C HC H3 O

O

C H2

C H

C H3

O (C H2)4

O O

OC H2

C HC H3O

(C H2)4

O

O

(C H2)4O

OOC H2

C HC H3

O

O (C H2)2

C H

C H3

O (C H2)4

O O

O(C H2)2

C HC H3O

(C H2)4O

OO(C H2)2

C HC H3

O

(C H2)4

O

O

a) b)

c) d)


Polyadipates: what they are?what they have to fulfill?The demand for new alternative plasticizers is strong and non-toxic polymeric plasticizers seem to be a real appealing potential solution, even if as the molecular weight of the polymeric plasticizer increases, migration is reduced but at the same time the material becomes more difficult to process.

Polyadipates are defined in the following way: “polyesters of 1,2-propanediol and/or 1,3- and/or 1,4-butanediol and/or polypropyleneglycol with adipic acid, which may be end-capped with acetic acid or fatty acids C 12 -C 18 or n-octanol and/or n-decanol” .

Polyadipate group TDI is 0.5 mg kg-1 bwaccording to opinion of SCF (1998). Partial hydrolysis of poly(butylene adipate) and poly(propylene adipate) plasticizerswas observed in contact with simulated gastric conditions: this reaction mainly conduces to the formation of oligomersand not more dangerous monomeric products.A Specific Migration Limit (SML) of 30 mg kg-1 is applicable according to the Directive 2004/19/EC.

This SML is valid only for the fraction with molecular weight below 1000 Da according to the Practical Guide EC 2003. The composition of the material < 1000 Da varies between different commercial polyadipates which can differ in the diol used as linker, in the termination (acid or alcohol based) and in the end-capping (obtainable by exploiting free alcohols, esterification with octanol/decanol, acetylation or acylation with fatty acids).


A first pioneer method to estimate polyadipate migration concentrations in foodstuffs was reported by Castle et al. in 1988, extracting the PA into an acetone/hexane mixture and executing a transmethylation to formdimethyl adipate: a clean-up using size exclusion chromatography (SEC) and a GC-MS quantification based on a range of PA calibration standards permit to complete the procedure.

This approach was later modified (2006) by Biedermann et al. with transesterification to the butyl adipate, applying the SEC step directly on the diluted food. It is unquestionable that these kinds of analytical solutions are effectiveness but reveal numerous drawback, involving multi-step procedures with extraction, clean-up, hydrolysis, esterification, derivatisation, GC measurement and calculations using conversion factors to allow quantification.

Polyadipates: what kind of measurements?

1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 m /z

0 %

2 5 %

5 0 %

7 5 %

1 0 0 %

5 5 9 .9 2 7 e + 7

8 4 3 .0 5 7 e + 7

1 1 3 1 .9 4 2 e + 8

1 5 5 4 .7 6 2 e + 6

1 8 7 9 .1 1 8 e + 7

2 2 7 2 .0 9 1 e + 7 2 9 9

7 .3 5 0 e + 6

3 7 3 5 .2 8 7 e + 7

5 0 1 1 .7 0 9 e + 7

S p e c tru m 1 A1 9 .6 3 0 m in . S c a n : 1 5 3 9 C h a n n e l: M e rg e d Io n : N A R IC : 3 .5 2 9 e + 9 (B C )B P 1 1 3

558M+

a)

b)

5 0 1 0 0 1 5 0 2 0 0 2 5 0 3 0 0 3 5 0 m /z

0 %

2 5 %

5 0 %

7 5 %

1 0 0 %

4 1 4 .9 3 2 e + 7

5 5 1 .3 5 2 e + 8

1 0 1 2 .9 0 0 e + 7

1 1 3 1 .9 1 1 e + 8

1 2 7 9 .4 7 8 e + 7

1 5 3 2 .6 5 0 e + 6

1 8 7 1 .6 6 1 e + 8

2 0 1 2 .4 7 5 e + 7 2 5 2

8 .5 7 2 e + 6 3 1 3

7 .7 0 4 e + 6 3 7 2

8 .9 7 2 e + 6

S p e c tru m 1 A1 4 .8 0 6 m in . S c a n : 1 0 1 8 C h a n n e l: M e rg e d Io n : N A R IC : 1 .9 8 6 e + 9 (B C )B P 1 1 3

0 1 0 0 2 0 0 3 0 0 4 0 0 m /z

0 %

2 5 %

5 0 %

7 5 %

1 0 0 %

S

5 5 1 .1 7 2 e + 9

5 6 2 .2 3 6 e + 8

1 0 9 2 .0 6 6 e + 7

1 1 1 4 .3 1 5 e + 8

1 2 7 8 .9 3 1 e + 8

1 8 3 1 .4 4 6 e + 8

2 0 1 8 .7 8 4 e + 8

2 5 7 9 .2 4 3 e + 7 3 4 1

3 .9 3 0 e + 7 4 0 0

4 .6 7 2 e + 7

S p e c tru m 1 A1 5 .7 2 1 m in . S c a n : 1 1 2 2 C h a n n e l: M e rg e d Io n : N A R IC : 1 .0 3 7 e + 1 0 (B C )B P 5 5

0 1 0 0 2 0 0 3 0 0 4 0 0 5 0 0 6 0 0m /z

0 %

2 5 %

5 0 %

7 5 %

1 0 0 %

4 5 2 .0 8 1 e + 7

5 5 4 .2 2 3 e + 8

1 1 1 1 .7 0 3 e + 8

1 2 8 2 .4 4 3 e + 7

2 0 1 2 .2 2 8 e + 8

2 5 5 5 .0 4 9 e + 7

4 0 1 3 .7 8 3 e + 7 5 2 9

1 .7 7 9 e + 7

S p e c tru m 1 A2 1 .2 8 5 m in . S c a n : 1 7 3 5 C h a n n e l: M e rg e d Io n : N A R IC : 8 .7 8 4 e + 9 (B C )B P 5 5

600M+

c)

d)

(C H 2)4

O O

(C H2)4

OO

OC H2

C HC H3OO

C H2

C HC H3 O

(C H2)4

OO

(C H2)4

O O

O(C H2)2

C HC H3OO

(C H2)2

C HC H3 O

O

C H2

C H

C H 3

O (C H 2)4

O O

OC H2

C HC H 3O

(C H2)4

O

O

(C H2)4O

OOC H 2

C HC H3

O

O (C H2)2

C H

C H 3

O (C H 2)4

O O

O(C H 2)2

C HC H3O

(C H2)4O

OO(C H 2)2

C HC H3

O

(C H2)4

O

O

a) b)

c) d)

Identification of cyclic oligomers : 1,3-butanediol adipate and 1,2-propanediol adipate structural elements

BIBLIOGRAPHIC REFERENCESL. Castle, A.J. Mercer, J. Gilbert, “Gas chromatographic~mass spectrometric determination of adipate based polymeric plasticizers in foods”, Journal of Association of Analytical Chemistry 71(2) (1988b) 394-396.M. Biedermann, K. Grob, „GC method for determining polyadipate plasticizers in foods: Transesterification to dibutyl adipate, conversion to migrating polyadipate”, Chromatographia 64(9/10)

(2006) 543-552.


Polyadipates: a promising way…"Analytical screening of polyadipates and other plasticisers in poly(vinyl

chloride) gasket seals and in fatty food by GC-MS"

The presence of polyadipate plasticizers in commercial PVC gasket seals and in the food can be simultaneously studied. In this 2007 research work polyadipates were found in four caps samples at 16 - 46%. The amount of total PA was below OML (60 mg kg-1) in all eight food samples in which it was detected: the average concentration of PAs found in the food samples were 16.3 mg kg-1.

All positive food samples for total PA showed the concentrations significantly below SML (30 mg kg-1). Even in the cases of 45 - 46 % w/w PA in the gaskets, the concentrations of the substance in the pesto sauces are only 14 - 22 mg kg-1.

In most cases the migration rate of polyadipates in the food demonstrated to be significantly lower in comparison with the other plasticizers.

For all these reasons polyadipates can be considered the most promising new plasticizers(as a substitute for ESBO) in PVC gasket seals.

BIBLIOGRAPHIC REFERENCEEzerskis, Z.; Morkunas, V.; Suman, M.; Simoneau, C.

Analytica Chimica Acta 2007, Vol- 604, pp. 29-38.


PA Screening by LC-TOF-MS

The full scan capability of this technique enables detection of the polyadipates directly, avoiding derivatisation and/or hydrolysis.

This makes the procedure much faster and simpler.Authentic standards of each polyadipate oligomer detected are not available, so quantification would be difficult.

BIBLIOGRAPHIC REFERENCEM. Driffeld, E.L Bradley, N. Harmer, L Castle, S. Klump, P. Mottier, “Determination of polyadipates migrating from lid gaskets of glass jars. Hydrolysis to adipic acid and measurement by LC-MS/MS”, Food Additives and Contaminants 27 (2010) 1487-1495.


LC-MS\MS after PA hydrolysisIn this case the determination in the food simulants is obtained in few minutes by the alkaline hydrolysis of the polyadipates to adipic acid.

Adipic acid is measured using liquid chromatography with tandem mass spectrometric detection (LC-MS/MS) in negative electrospray ionisation mode (IS Adipic Acid d10), avoiding the transesterification and derivatisation steps of previous GC-MS methods.

Any of the polyadipates tested can be used as astandard for the quantification of unknownpolyadipates in gaskets.


A method for the detection of ESBO in complex food matrices by RP LC-ESI-ion trap-MS/MS was developed and validated. The new method demonstrates to be an attractive alternative to the traditional GC-MS procedure, reducing time and costs for what concerns samples and standards preparation steps and limiting the possibility of interference from other compounds present in the matrices.

An analytical approach for chlorohydrins qualitative determination in complex food matrices, based on extraction transesterification SPE was demonstrated.Four positional isomers for 18-E-OHCl and eight for 18-2OHCl were separated and identified by the optimization of either High Performance Liquid Chromatography (HPLC) or UltraPerformance Liquid Chromatography (UPLC) techniques.

LC-MS with high resolution detection or with adequate sample hydrolitic pre-treatments seems promising to afford polyadipates \ polymeric plasticizers monitoring in the future.

LC-MS represents a challenging solution either for single-target or multiresidual analysis of plasticisers and additives in PVC gaskets and also in the correspondent foodstuffs, overcoming interferences related especially to fat composition and content.


Acknowledgements:C. Simoneau, Z. Ežerskis, V. MorkūnasEU Joint Research Centre, Ispra

E. De Dominicis, I. CommissatiChelab R&D Lab

Dante Catellani, Martina Vindigni, Ugo BerselliniBarilla - Food Research Labs



What is ESBO??? Epoxidised soybean oil (ESBO) is used as a plasticiser as well as a stabiliser for plasticspolymers such as polyvinyl chloride (PVC). It is used in particular in closure gaskets for

the metal lids used to seal glass jars and bottles, forming the airtight seal needed to prevent microbiological contamination of baby foods.

There is thus a potential for migration into the food bothduring sterilisation and storage.High migration levels might lead to an intake that

exceeds the existing Tolerable Daily Intake of 1mg/kg bw/day.

In addition, the main pathway of PVC degradation involves the elimination of HCl which can react with ESBO, leading to the formation of chlorohydrins. Thus such compounds

may be present in quantifiable amounts in baby foods and other foods and may be of toxicological concern.

Finally, it has been noticed that, confirming previous work, cyclic compounds arealso formed and that mass balance of disappearing substances versus appearing substances

is not preserved, suggesting that some reaction products are not currently accounted for.


Plasticizers in PVC gaskets: examples of legal restrictions

The EU Scientific Committee on Food specified a tolerable daily intake (TDI) for ESBO of 1 mg kg-1 body weight (bw) (SCF 1999a). A specific migration limit (SML) of 60 mg kg-1 is derived from this assumption.

The TDI for DEHP is 0.05 mg kg-1 bw; for DINP and DIDP group-TDI is 0.15 mg kg-1 bw (SCF 1999b). Specific migration limits (SMLs) of 3 and 9 mg kg-1 for DEHP and for the sum of DINP and DIDP are applicable, respectively.

For DEHA, SML of 18 mg kg-1 is specified according to the Directive 2002/72 (EC 2002).

For polyadipate group-TDI is 0.5 mg kg-1 bw according to opinion of SCF (1998) and SML of 30 mg kg-1 is applicable according to the Directive 2004/19/EC (EC 2004b). This SML is valid only for the fraction with molecular weight below 1000 Da according to the Practical Guide (EC 2003).

For tributyl citrate and di-(2-ethylhexyl) sebacate EU synoptic document (EC 2005) outlines restrictions of 5 mg kg-1 and TDI of 0.05 mg kg-1 bw, respectively.

DBS is considered toxicologically acceptable according to the synoptic document and therefore only OML of 60 mg kg-1 is relevant in food and food simulants.


Opinion of the Scientific Panel on Food Additives,Flavourings, Processing Aids and Materials in Contact with Food (AFC) on a request from the Commission related to the use of Epoxidised soybean oil

in food contact materials (Question N° EFSA-Q-2003-073)adopted on 26 May 2004 by written procedure

…it is therefore recommended to develop a specific migration limit for ESBO in baby foods,derived from the TDI of 1 mg/kg body weight and taking into consideration the amountswhich might be eaten on a daily basis by an infant of 6 months of age, weighing 7.5 kg, fedmainly or exclusively on processed baby foods.

In the absence of adequate toxicological data on ESBO derivatives, no advice can yet begiven on the significance for health of such derivatives in foods. The Panel notes that up to5% of the fatty acids in ESBO in gaskets is converted into derivatives. These derivatives areexpected to migrate at the same rate as ESBO and so their concentration in food could achieveabout 5% that observed for ESBO itself.

Further analytical and toxicological data on ESBO derivatives are needed and a programmeshould be established to that end.