See discussions, stats, and author profiles for this publication at: https://www.researchgate.

net/publication/5364679

Determination of ink photoinitiators in packaged beverages by gas

chromatography-mass spectrometry and liquid chromatography-mass
spectrometry

Article  in  Journal of Chromatography A · July 2008

DOI: 10.1016/j.chroma.2008.04.057 · Source: PubMed

CITATIONS READS

56 846

8 authors, including:

Gianni Sagratini Giovanni Caprioli

University of Camerino University of Camerino
147 PUBLICATIONS   1,901 CITATIONS    106 PUBLICATIONS   915 CITATIONS   

SEE PROFILE SEE PROFILE

Dario Giardinà Massimo Ricciutelli

University of Camerino University of Camerino
85 PUBLICATIONS   1,179 CITATIONS    103 PUBLICATIONS   1,477 CITATIONS   

SEE PROFILE SEE PROFILE

Some of the authors of this publication are also working on these related projects:

CLIMATE CHANGES, GRASSLANDS AND LIVESTOCK MANAGEMENT: A MULTIDISCIPLINARY STUDY TO IMPROVE THE SUSTAINABLE DEVELOPMENT OF APENNINES
PASTORAL SYSTEMS (CLIMAPP) View project

Conference: “The quality of Coffee: a never ending research” View project

All content following this page was uploaded by Giovanni Caprioli on 19 June 2018.

The user has requested enhancement of the downloaded file.

 Journal of Chromatography A, 1194 (2008) 213–220

Contents lists available at ScienceDirect

Journal of Chromatography A
journal homepage: www.elsevier.com/locate/chroma

Determination of ink photoinitiators in packaged beverages by gas

chromatography–mass spectrometry and liquid
chromatography–mass spectrometry
Gianni Sagratini, Giovanni Caprioli, Gloria Cristalli, Dario Giardiná, Massimo Ricciutelli,
Rosaria Volpini, Yanting Zuo, Sauro Vittori ∗
Dipartimento di Scienze Chimiche, Facoltà di Farmacia, Università di Camerino,
via S. Agostino 1, 62032 Camerino, Italy

a r t i c l e i n f o a b s t r a c t

Article history: A new analytical method, using gas chromatography–mass spectrometry (GC/MS) and liquid
Received 12 February 2008 chromatography–mass spectrometry (LC/MS) techniques, was developed for the determination in
Received in revised form 17 April 2008 packaged food beverages of five ink photoinitiator residues: 2-isopropylthioxanthone (ITX), ben-
Accepted 21 April 2008
zophenone, 2-ethylhexyl-4-dimethylaminobenzoate (EHDAB), 1-hydroxycyclohexyl-1-phenyl ketone
Available online 25 April 2008
(IRGACURE 184) and ethyl-4-dimethylaminobenzoate (EDAB). Samples were extracted from selected
beverages (milk, fruit juices and wine) and relative packagings, using n-hexane and dichloromethane,
Keywords:
respectively, purified on solid-phase extraction (SPE) silica gel cartridges, and then analyzed in GC/MS
Photoinitiators
ITX
and LC/MS. The recovery percentages, obtained spiking the beverage samples at concentrations of 4
Beverages and 10 ␮g l−1 with a standard mixture of photoinitiators, were in the range 42–108% (milk), 50–84%
Food packaging (wine), and 48–109% (fruit juices). The repeatability of the method was assessed in all cases by the
Gas chromatography–mass spectrometry % of correlation value, that was lower than 19%. The lowest limits of detection (LODs) and limits of
Liquid chromatography–mass spectrometry quantification (LOQs), obtained using GC/MS, were in the range 0.2–1 and 1–5 ␮g l−1 , respectively.
The method was applied to the analysis of forty packaged food beverages (milk, fruit juices and
wine samples). The most significant contamination was that of benzophenone, found in all samples
in a concentration range of 5–217 ␮g l−1 . Its presence was confirmed by an LC/Atmospheric-Pressure
PhotoIonization (APPI)/MS/MS analysis. The photoinitiator (EHDAB) was found in eleven out of forty
beverages in a concentration range of 0.13–0.8 ␮g l−1 . Less important was the ITX contamination,
found in three out of forty samples in a range 0.2–0.24 ␮g l−1 . The work proposes a new method
to analyze ink photoinitiator residues in polycoupled carton packaging and in contained food bever-
ages.
© 2008 Elsevier B.V. All rights reserved.

1. Introduction
The alert for food contamination by photoinitiators, a class
of molecules used in UV-cured inks for printing the surface of
polycoupled carton for beverages [1], arose in Europe in Novem-
ber 2005, when the Italian authorities withdraw from the market
thirty million liters of infants milk, because of contamination with
the photoinitiator 2-isopropylthioxanthone (ITX) [2]. Some weeks
later, a report published by the European Food Safety Agency
(EFSA) stated that the observed level of ITX in some milk products
(27–440 ␮g l−1 ) and fruit juices (5–249 ␮g l−1 ) did not constitute
∗ Corresponding author. Tel.: +39 0737402266; fax: +39 0737637345.
E-mail address: sauro.vittori@unicam.it (S. Vittori).
a real risk for the consumers’ health [3]. However, no fully con-
vincing data on ITX genotoxicity have been published to date. In
fact, whereas contradictory results were displayed in a limited
in vitro study [4], the negative results obtained in two in vivo
studies [5,6] did not confirm the ITX mutagenic effects in exper-
imental animals. Noteworthy, in a recent article Momo et al. [7]
reported that ITX can effect the mobility/rigidity status of bio-
logical membranes by strong interactions with the cellular lipid
bilayer.
Various articles have been published in the last 2 years, pre-
senting different analytical methods to detect ITX in food and in
polycoupled cartons containing food beverages. Most of authors
used LC/MS/MS [8–12], in two cases [8,12] compared to GC/MS,
while others used HPLC/diode-array detection (DAD)/fluorescence
detection (FLD) [13], HPTLC/MS [14], and GC/MS [15] techniques.

0021-9673/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.chroma.2008.04.057
214 G. Sagratini et al. / J. Chromatogr. A 1194 (2008) 213–220
The extraction methods varied from liquid–liquid extraction (LLE)
with conventional organic solvents followed by liquid–liquid par-
tition (LLP) [10] or solid-phase extraction (SPE) [13], to pressurized
liquid extraction (PLE) [9,12], or precipitation by proper reagents
[11].
Besides ITX, residues of other photoinitiators have been
recently found in foods. Thus, in 2005, the 2-ethylhexyl-4-
dimethylaminobenzoate (EHDAB), used as co-initiator in the
polymerization process of UV-inks, was detected as contaminant in
fruit juice and milk samples in concentration ranges of 5–125 ␮g l−1
[3] and 27–134 ␮g l−1 [3] or 8–126 ␮g l−1 [12], respectively.
The benzophenone has been, in the years, one of the most stud-
ied photoinitiators used in the polymerization process producing
plastics, i.e. polyethylene and polypropylene films [16]. The stud-
ies attested various toxicities for benzophenone as carcinogenic
activities in mice and rats [17], utero hypospadias estrogen receptor
mediated [18], and allergic skin reaction [19]. Nevertheless, stud-
ies on benzophenone migration from cartonboard packaging into
the contained food revealed that 72% of the analyzed samples was
contaminated by this compound [20].
Concerning the photoinitiator 1-hydroxycyclohexyl-1-phenyl
ketone (IRGACURE 184), it was only included, together with ITX,
benzophenone and EHDAB, in a study of migration tests for UV-
cured ink printed substrates in aqueous simulants [1], while the
ethyl-4-dimethylaminobenzoate (EDAB) was cited for its use as
UV-inks photoinitiator in a recent report [21].
Taking into account the possible presence of photoinitiator
residues as contaminants of food beverages contained in polycou-
pled cartons, the aim of this work was to set up a new method
to analyze, in food and related packaging at the same time, the five
above indicated photoinitiators, that is ITX, EHDAB, benzophenone,
IRGACURE 184 and EDAB, by using rapid extraction and purification
systems and analyzing the extracts in GC/MS and LC/MS. Chem-
ical structures and properties of the analyzed photoinitiators are
reported in Fig. 1. The developed method was applied to forty real
foods and forty relative packagings by using the GC/MS for ana-
lytes quantification and the LC/MS/MS for benzophenone presence
confirmation.
Fig. 1. Chemical structures and properties of the analyzed photoinitiators.
2. Experimental
2.1. Materials and standards
Photoinitiators, i.e. isopropyl-9H-thioxanthen-9-one (ITX,
C16 H14 OS, molecular weight 254.08, 97%, mixture of 2- and
4-isomers, CAS Nos. 5495-84-1 and 84846-86-0 respectively),
benzophenone (C13 H10 O, molecular weight 182.22, 99% GC, CAS
No. 119-61-9), 1-hydroxycyclohexyl-phenylketone (IRGACURE
184, C16 H13 O2 , molecular weight 204.26, CAS No. 947-19-3),
ethyl-4-dimethylaminobenzoate (EDAB, C11 H15 NO2 , molecular
weight 193.24, >99%, CAS No. 10287-53-3), 2-ethylhexyl-4-
dimethylaminobenzoate (EHDAB, C17 H27 02 N, molecular weight
277.40, 98%, CAS No. 21245-02-3) were supplied by Sigma–Aldrich
(Milano, Italy). Individual stock solutions were prepared by dis-
solving 100 mg of each compound in 100 ml of methanol and
stored in glass-stopper bottles at 4 ◦ C. Standard working solutions,
at various concentrations, were daily prepared by appropriate
dilution with methanol of aliquots of the stock solutions.
Dichloromethane, n-hexane, and ethyl acetate solvents for
residue analysis were supplied by Fluka-Riedel-deHaën (Milan,
Italy). HPLC-grade methanol and sodium sulphate >99% were
supplied by Sigma–Aldrich, HPLC-grade formic acid by Merck
(Darmstadt, Germany), HPLC-grade ammonium acetate by J.T.
Baker B.V. (Deventer, Holland). Deionized water (<8 M cm resis-
tivity) was obtained from the Milli-Q SP Reagent Water System
(Millipore, Bedford, MA, USA). All the solvents and solutions were
filtered through a 0.45 ␮m PTFE filter from Supelco (Bellefonte, PA,
USA) before use. Cartridges Discovery SPE DSC-Si Silica Tube 6 ml,
1 g, was purchased from Supelco.
2.2. Sample collection
Forty packaged beverages (fruit juices, milk, wine), produced by
different food companies, were purchased in different supermar-
kets of the Camerino area. The polycoupled cartons were produced
by three different companies, i.e. Tetrapak, SIG-Combibloc and IPI.
Various typologies of beverages were analyzed for each kind of food.
 G. Sagratini et al. / J. Chromatogr. A 1194 (2008) 213–220 215

Milk based food (13) were: milk chocolate, growth milk, whole milk,
skim milk, partially skim milk, milk added with vitamins, soya milk.
Fruit juices (16) were: apricot, pineapple, apple, strawberry, orange,
multivitaminic juices. Wines (11) were red and white.
Food beverage samples were stored at room temperature before
use. Once opened they were stored into specific food containers at
4 ◦ C and analyzed within 3 days.
2.3. Sample preparation
2.3.1. Food packaging material
The food cartonboard was opened and the liquid food content
was stored in polypropylene food containers (Gamma Test, by Ani-
crin s.r.l., Scorzè, Italy), while the packaging material was gently
washed on the internal side with distilled water and then wiped.
A 10 cm × 5 cm scrap of packaging polycoupled carton was cut in
pieces of 1 cm2 area, then soaked in 50 ml of dichloromethane in
a glass bottle (250 ml) for 24 h. After that, the variously colored
liquids obtained from ink dissolution of extraction solvents were
filtered and separated from carton pieces, then totally evaporated
under vacuum (60 mbar) at 30 ◦ C by a Büchi apparatus (Büchi R200,
Labortechnik, Flawil, Switzerland). The obtained residue was recon-
stituted with 1 ml of n-hexane before purification on a DSC-Si Silica
cartridge (6 ml, 1 g). Firstly, the cartridge was conditioned with
2 × 2 ml of a n-hexane/ethyl acetate mixture (30/70), then the hex-
anic solution was loaded onto the cartridge at a flow rate lower than
0.5 ml min−1 ; after that, the elution was performed using 2 × 2 ml
of a n-hexane/ethyl acetate mixture (30/70) at a flow rate lower
than 0.5 ml min−1 . The eluate was totally evaporated under a nitro-
gen stream and the residue dissolved in 1 ml of a n-hexane/ethyl
acetate mixture (30/70) before the GC/MS analysis.
Extraction recoveries were determined by spiking polycoupled
carton-dichloromethane solution with 10 ␮l of standard pho-
toinitiators mixture at a concentration level corresponding to
0.5 ␮g dm−2 .
2.3.2. Food beverages
Three types of liquid food samples were analyzed: milk, fruit
juices and wine. Before extraction, the apricot juices were centrifu-
gated at 4000 rpm for 15 min, then taking the supernatant. This is to
reduce the presence of insoluble fibers, significantly lowering the
recovery process. The liquid sample (25 ml) was extracted in a glass
separating funnel with 3 × 30 ml of n-hexane, then the organic layer
was dried passing it through a glass funnel containing dry Na2 SO4
and evaporated to dryness under vacuum (60 mbar) at 30 ◦ C. The
extract was reconstituted with 1 ml of n-hexane and then purified
on DSC-Si Silica cartridge (6 ml, 1 g) according to the purification
step used for packaging material, as described in Section 2.3.1. The
eluate was evaporated under a nitrogen stream and the residue was
dissolved in 1 ml of a n-hexane/ethyl acetate mixture (30/70) for the
GC–MS analysis, or in 1 ml of methanol for the HPLC/MS analysis.
The extraction recoveries were determined by spiking the food
beverages with 10 ␮l of standard photoinitiators mixture at con-
centration levels of 4 and 10 ␮g l−1 .
2.4. GC/MS analysis
A gas chromatograph/mass selective detector (GC/MSD) system
(Agilent 6890N equipped with Agilent 5973N, Agilent, Santa Clara,
CA, USA), coupled with an AgilentChem workstation, was used. Sep-
aration was performed on a HP 5 MSI column (30 m × 0.25 mm,
0.25 ␮m film thickness). All injections were split (split ratio was
1:50) and the volume was 5 ␮l. The flow rate (He) was 0.8 ml min−1 .
The injector temperature was 270 ◦ C. The column temperature pro-
gram was from 80 ◦ C (1 min) to 180 ◦ C at 25 ◦ C min−1 , from 180 ◦ C
(0 min) to 300 ◦ C at 10 ◦ C min−1 , then 300 ◦ C for 4 min. Data were
acquired in the electron impact (EI) mode using the selected ion
monitoring (SIM) mode. The SIM ions and time conditions for each
photoinitiator are reported in Table 1.
2.5. LC/DAD, LC/MS and LC/MS/MS analyses
The separation was achieved on an analytical column Luna C18
(250 mm × 4.6 mm I.D., 5 ␮m) from Phenomenex (Chesire, UK). The
mobile phase for LC/DAD and LC/electrospray ionization (ESI)-MS
(single quadrupole) analyses was methanol–water at a flow rate of
0.9 ml min−1 . The solvent composition varied as follows: 0–13 min,
70:30 A:B (v/v); 13–18 min, 95:5 A:B (v/v); 18–30 min, 95:5 A:B
(v/v); 30–35 min, 70:30 A:B (v/v), where A = methanol, B = water.

Table 1
Conditions and transitions used in mass spectrometry experiments

Photoinitiators SIM ion (m/z) Fragmentor (eV) Time window (min)

GC/MS
Benzophenone 105, 182 70 3.50–7.60
IRGACURE 184, EDAB 81, 99, 148, 164, 193 70 7.60–9.00
EHDAB 148, 165, 277 70 9.00–13.01
ITX 239, 254 70 13.01–21.00

LC/MS
Benzophenone 205 [M+Na]+ 50
0
EDAB 216 [M+Na]+ 50
–21.00
IRGACURE 184 227 [M+Na]+ 50

EHDAB 277 [M+Na]+ 90 21.00

ITX 300 [M+Na]+ 90 –35.00

Photoinitiators Transition Cut-off Amplitude Time window (min)

LC/MS/MSa
IRGACURE 184 227.0 61 –
0
Benzophenone 183 → 105 49 0.9
–17.5
EDAB 194 → 166 52 0.9

ITX 255 → 213 69 0.7 17.5

EHDAB 278 → 166 75 0.7 –35
The order of the five photoinitiators was set on the basis of the increasing retention time that each molecule has in the three different chromatographic analyses.
a
In MS/MS experiments (quadrupole ion trap) the width parameter was in all cases equal to 4.0.
216 G. Sagratini et al. / J. Chromatogr. A 1194 (2008) 213–220
The mobile phase for LC/ESI-MS/MS (quadrupole ion trap)
analysis was methanol–water with ammonium acetate and
formic acid at a flow rate of 0.9 ml min−1 . The solvent
composition varied as follows: 0–10 min, 35:65 A:B (v/v);
10–15 min, 0:100 A:B (v/v); 15–25 min, 35:65 A:B (v/v), where
A = 5 mM ammonium acetate in water plus 0.1% formic acid,
B = 5 mM ammonium acetate in methanol plus 0.1% formic
acid.
The mobile phase for LC/APPI-MS/MS (quadrupole ion trap)
analysis was methanol–water (90:10, v/v) at a flow rate of
0.9 ml min−1 in isocratic mode.
LC/DAD and LC/MS studies were performed using a Hewlett-
Packard (Palo Alto, CA, USA) HP-1090 Series II, made from an
autosampler, a binary solvent pump, with a diode-array detector
(DAD) and a mass spectrometer detector (MSD) equipped with an
ESI interface in positive ionization (PI) mode.
LC/DAD analysis was performed monitoring three different
wavelengths: 245 nm for IRGACURE 184, 254 nm for benzophenone
Fig. 2. GC/MS chromatograms of: (A) standard mixture of the analyzed photoinitiators at concentration of 100 ␮g l−1 , (B) sample of milk packaging material contaminated
by benzophenone, and (C) milk sample contaminated by benzophenone, EHDAB and ITX.
and ITX, 310 nm for EDAB and EHDAB. For the step of method devel-
opment and optimization LC-DAD analysis was used.
Optimization of the LC-MS conditions was carried out by vary-
ing them in flow injection analysis (FIA) of the analytes (20 ␮l of a
50 ␮g ml−1 individual standard solutions). The optimized param-
eters of the ESI interface were: vaporizer temperature, 325 ◦ C;
nebulizer gas (nitrogen) pressure, 50 psi; drying gas (nitrogen)
flow rate, 11 ml min−1 ; temperature, 350 ◦ C; capillary voltage,
3500 V. Time scheduled conditions for monitoring photoinitiators
are reported in Table 1.
LC/MS/MS studies were performed using an Agilent 1100 series
(Santa Clara, CA, USA) and MSD Trap SL equipped with an ESI
and APPI sources operating in positive ionization mode. The mass
spectrometer was tuned for each compound, optimizing ioniza-
tion source parameters, voltages of the lenses, and trap conditions
in the ExpertTune mode of the Daltonic Esquire Control soft-
ware, while infusing a standard solution (10 ␮g ml−1 ) via a syringe
pump at a flow rate of 4 ␮l min−1 , which was mixed with the
 G. Sagratini et al. / J. Chromatogr. A 1194 (2008) 213–220 217

mobile phase at 0.5 ml min−1 by means of a T piece. Operating Table 2

Percent recovery and repeatability of the method evaluated in GC–MS on the three
ESI conditions were the same as mentioned before for the single
different kinds of beverages at two fortification levels of 4 and 10 ␮g l−1 (n = 5)
quadrupole, while those for APPI source differ only for nebulizer
gas (nitrogen) pressure (60 psi) and drying gas (nitrogen) flow rate Photoinitiators Concentration (␮g l−1 ) % recovery % CV
(7 ml min−1 ). Milk
The mass spectrometer was run in full scan, and MRM modes. Benzophenone 4 108 15
Positive ions were detected using the standard scan at normal res- 10 106 14

olution (scan speed 10,300 m/z/s; peak with 0.6 FWHM/m/z). The EDAB 4 64 17
trap parameters were set in Ion Charge Control (ICC) using rolling 10 67 12
averaging set at 2 with a target of 20,000, and maximum accumu- EHDAB 4 43 14
lation time of 50 ms at m/z range from 50 to 500 u. The fragments, 10 45 12
the fragmentation and time conditions are reported in Table 1. IRGACURE 184 4 68 14
10 67 10
3. Results and discussion ITX 4 42 18
10 45 15
3.1. Gas chromatography–mass spectrometry and liquid
chromatography–mass spectrometry Wine
Benzophenone 4 61 18
10 59 9
As reported above, the GC/MS analysis was performed in
selected ion monitoring, choosing the most abundant and charac- EDAB 4 50 18
10 52 11
teristic ions for each molecule and setting four time windows to
increase the method sensibility (Table 1). Fig. 2A reports the chro- EHDAB 4 77 13
matogram of a standard mixture of the analyzed photoinitiators, at 10 74 10

a concentration of 100 ␮g l−1 , obtained by GC/MS. IRGACURE 184 4 81 19

Also the LC/MS analysis was performed in SIM conditions, set- 10 84 18
ting two time windows for two groups of compounds. In all cases, ITX 4 63 19
using water/methanol as mobile phase and the electrospray ion- 10 62 17
ization source, we obtained the sodium adduct [M+Na]+ as base
peak for all compounds. Fig. 3 shows the chromatogram of a stan- Fruit juice
Benzophenone 4 85 18
dard mixture of the analyzed photoinitiators, at a concentration of
10 88 12
1000 ␮g l−1 , obtained by LC/MS. The low signal observed for ben-
EDAB 4 109 18
zophenone indicates that the used conditions were not suitable for
10 102 15
an effective ionization of this molecule.
LC/MS/MS experiments were performed subjecting the base EHDAB 4 48 17
10 52 13
peak of the mass spectrum of studied compounds to the first stage
of MS/MS transition. The MS/MS analysis of tested photoinitiators IRGACURE 184 4 88 18
with the ESI interface in positive polarity provided ions for all of 10 91 15

them except for IRGACURE 184 (Table 1). A different compound
ionization was obtained using a mixture of methanol–water con-
taining ammonium acetate (c = 5 mM) and 0.1% (v/v) formic acid, as
mobile phase. Applying these conditions, benzophenone, EDAB and
ITX produced proper molecular ions [M+H]+ (m/z 183, 194, and 255,
respectively) and the relative fragmentation into the product ions
(m/z 105, 166, and 213, respectively). In MS/MS experiments two
time windows were set to monitor the analyzed photoinitiators,
the first from 0 to 17.5 min and including IRGACURE 184, benzophe-
none and EDAB, the second from 17.5 to 35 min and including ITX
and EHDAB.
Fig. 3. HPLC/ESI-MS chromatogram of a standard mixture of the analyzed photoini-
tiators at concentration of 1000 ␮g l−1 .
ITX 4 64 18
10 62 10
3.2. Method validation
Calibration curves of the analyzed compounds were constructed
injecting 20 ␮l of standard solutions at five different concentra-
tions, i.e. 10, 50, 100, 500 and 1000 ␮g l−1 in GC/MS technique, and
100, 300, 500, 1000 and 5000 ␮g l−1 in LC/MS. Five replicates for
each concentration were performed, and the relative standard devi-
ations (RSDs) ranged from 1 to 4.6% for run-to-run precision, and
from 7 to 14% for day-to-day precision. All the calibration curves
of the analyzed photoinitiators showed a correlation coefficient
greater than 0.996.
In the GC/MS packaging analysis, the recoveries obtained by
spiking the polycoupled carton-dichloromethane solution at level
of 0.5 ␮g l−1 with a standard mixture of photoinitiators were in
the range 69–100% for all analyzed compounds, with a percent-
age correlation value (% CV) < 15% (n = 8). The recovery percentages
referred to GC/MS analysis of food, obtained spiking the food bev-
erages (milk, wine and fruit juices) at levels of 4 and 10 ␮g l−1 with
a standard mixture of photoinitiators, are reported in Table 2. The
obtained recoveries for milk, wine and fruit juices samples were
in the range 42–108, 50–84, and 48–109%, respectively. All recov-
ery data were normalized taking into account the percentage of
recovery.
218 G. Sagratini et al. / J. Chromatogr. A 1194 (2008) 213–220

The repeatability of the method, evaluated 5 times on each kind Table 3

Limits of detection (LODs) and limits of quantification (LOQs) obtained for the five
of food, was expressed by % CV, that was lower than 19%. It is inter-
photoinitiators, expressed in ␮g l−1
esting to note that the most lipophilic molecules, ITX and EHDAB
(log P 4.55 and 5.03 respectively), showed the lowest recovery val- Photoinitiator GC/MS HPLC/DAD HPLC/ESI-MS HPLC/ESI-MS/MS
ues in milk samples (42 and 43%), confirming their difficulty to be LOD
extracted from milk, that was the most lipophilic matrix analyzed. Benzophenone 0.2 50 20 50
In Table 3 are reported the limits of detection (LODs) and the EDAB 1 50 10 10
EHDAB 0.2 20 2 2
limits of quantification (LOQs) of the five studied photoinitiators,
IRGACURE 184 1 50 10 100
expressed in ␮g l−1 , calculated in GC/MS, LC/DAD, LC/MS, LC/ESI- ITX 0.2 20 10 5
MS/MS, and, for benzophenone only, in LC/APPI-MS/MS. The lowest
LOQ
LODs and LOQs values were obtained using GC/MS, being in the Benzophenone 1 300 100 200
range 0.2–1 and 1–5 ␮g l−1 , respectively. An important lowering EDAB 5 300 50 50
of benzophenone LOQ value was obtained with the LC/MS/MS by EHDAB 1 100 10 10
changing the ESI source (200 ␮g l−1 ) with the APPI (50 ␮g l−1 ). IRGACURE 184 5 300 50 500
ITX 1 100 50 30

The LOD and LOQ values obtained for benzophenone using the HPLC/MS/MS with
3.3. Analysis of food packaging materials and beverages an APPI source were 10 and 50 ␮g l−1 , respectively.

The analytical data obtained in GC/MS from forty packaging

materials and forty related food beverages are reported in Table 4,
where food type (milk, fruit juice or wine), sample description and
concentrations of photoinitiators found in food beverages and in
relative packagings, expressed in ␮g l−1 and ␮g dm−2 , respectively,
are shown. In addition, a graphical visualization of the relative
photoinitiators contamination of all analyzed foods and packag-
ings is reported in Fig. 4. The data display that all the packaging
materials and beverages contain, at least, one of the monitored
photoinitiators. In particular, benzophenone was found in all pack-
aging materials in a concentration range of 0.2–387 ␮g dm−2 , and
in all beverages in a concentration range of 5–217 ␮g l−1 , with the
highest value observed in a red wine sample. In comparison, the

Table 4
Amount of the analyzed photoinitiators in food beverages and relative packaging materials

Food type Sample description Photoinitiators

Food (␮g l−1 ) Packaging (␮g dm−2 )

Milk 1 Chocolate milk Benzophenone 11.5, EHDAB 0.3 Benzophenone 1.3, EHDAB 0.04, IRGACURE 0.44, ITX 0.026
Milk 2 Growth milk Benzophenone 9.5 Benzophenone 0.8
Milk 3 Skim milk Benzophenone 14, EHDAB 0.8 Benzophenone 1.20, EHDAB 0.2
Milk 4 Skim milk + iron and vitamin C Benzophenone 9.9, EHDAB 0.8 Benzophenone 0.67, EHDAB 0.1
Milk 5 Chocolate milk Benzophenone 14.8, EHDAB 0.2 Benzophenone 1.7, EHDAB 0.1
Milk 6 Partially skim milk Benzophenone 9.3 Benzophenone 3.7
Milk 7 Partially skim milk Benzophenone 11.6 Benzophenone 1.65
Milk 8 Growth milk Benzophenone 10.9, EHDAB 0.5 Benzophenone 2.24, EHDAB 0.043
Milk 9 Chocolate milk Benzophenone 9.98, EHDAB 0.3 Benzophenone 3.75, EHDAB 0.02
Milk 10 Chocolate milk Benzophenone 14.7 Benzophenone 387
Milk 11 Soya milk Benzophenone 5.25, EHDAB 0.13 Benzophenone 17, EHDAB 0.06
Milk 12 Skim milk Benzophenone 12.6 Benzophenone 3.3
Milk 13 Chocolate milk Benzophenone 39 Benzophenone 134, EDAB 6.1

Fruit juice 1 Apricot juice Benzophenone 13, EHDAB 0.5 Benzophenone 1.2, EHDAB 0.05, IRGACURE 0.8
Fruit juice 2 Pineapple juice Benzophenone 5 Benzophenone 0.4
Fruit juice 3 Multivitaminic juice Benzophenone 7.2, EHDAB 0.8 Benzophenone 2, EHDAB 3.8, ITX 0.08
Fruit juice 4 Apple juice Benzophenone 9.9 Benzophenone 5.3
Fruit juice 5 Strawberry juice Benzophenone 8.6 Benzophenone 9.5
Fruit juice 6 Orange juice Benzophenone 15.4 Benzophenone 0.98
Fruit juice 7 Apricot juice Benzophenone 9.58 Benzophenone 1.95
Fruit juice 8 Apricot juice Benzophenone 6.6 Benzophenone 1.6
Fruit juice 9 Orange juice Benzophenone 12.7 Benzophenone 4.28
Fruit juice 10 Multivitaminic juice Benzophenone 8.6 Benzophenone 1.17
Fruit juice 11 Pineapple juice Benzophenone 8.68 Benzophenone 166, EHDAB 0.06
Fruit juice 12 Multivitaminic juice Benzophenone 7.42, EHDAB 0.14, ITX 0.2 Benzophenone 275, EHDAB 0.011, ITX 0.013
Fruit juice 13 Pineapple juice Benzophenone 5.8 Benzophenone 3.4, ITX 0.01
Fruit juice 14 Pineapple juice Benzophenone 90, EHDAB 0.16 Benzophenone 13.5, EHDAB 0.02, ITX 0.04
Fruit juice 15 Multivitaminic juice Benzophenone 41 Benzophenone 333
Fruit juice 16 Pineapple juice Benzophenone 30 Benzophenone 170

Wine 1 Red wine Benzophenone 5.5 Benzophenone 0.2, EHDAB 0.03

Wine 2 Red wine Benzophenone 16 Benzophenone 0.51, ITX 0.03
Wine 3 Red wine Benzophenone 14.9 Benzophenone 2.56
Wine 4 Red wine Benzophenone 10.5 Benzophenone 2.4, IRGACURE 0.18, ITX 0.05
Wine 5 Red wine Benzophenone 9.35 Benzophenone 1.34
Wine 6 White wine Benzophenone 13.2 Benzophenone 0.88
Wine 7 White wine Benzophenone 9.6, ITX 0.2 Benzophenone 2.2, EHDAB 0.04, ITX 0.04
Wine 8 Red wine Benzophenone 6.5, ITX 0.24 Benzophenone 0.89, ITX 0.016
Wine 9 White wine Benzophenone 4.73 Benzophenone 1.89
Wine 10 Red wine Benzophenone 217 Benzophenone 12.6
Wine 11 White wine Benzophenone 87, IRGACURE 1.2 Benzophenone 4.1, IRGACURE 0.13, EHDAB 0.03
 G. Sagratini et al. / J. Chromatogr. A 1194 (2008) 213–220 219

However, the maximum level of benzophenone found in a sam-

ple in this research (red wine, 217 ␮g l−1 ) does not exceed the SML
established by law.
As concerning the food beverages contaminated by the EHDAB
photoinitiator, the data show that mostly (64%) it is present in milk
samples, with a minor percentage in fruit juices (36%), whereas no
case occurred for wine samples. This difference could be related
to the particularly high lipophilic character of EHDAB molecule
(log P = 5.03), that allows it to be easily found in foods containing
high fat level, as milk. Moreover, we found that, in five out of nine
packagings containing ITX, this photoinitiator was present in com-
bination with EHDAB, that can work as a co-initiator, supposing a
possible combination use in the ink polymerization reaction.
Opposite to benzophenone, the photoinitiator EHDAB displayed,
at least in three analyzed samples, an evident correspondence
between the relative quantities found in packagings and in
corresponding contained beverages. Thus, the highest EHDAB con-
centrations, discovered in packaging samples of juice 3, milk 3, and
milk 4 (3.8, 0.2, 0.1 ␮g dm−2 respectively) correspond to the high-
est concentration levels of this photoinitiator in the related foods
(0.8 ␮g l−1 in all three samples).

3.4. Confirmation of benzophenone by LC/APPI-MS/MS

The results obtained from the analysis in GC/MS on forty

packaged beverages show that the photoinitiator benzophenone
is present in all analyzed samples. To confirm these results we
analyzed the same samples using LC/MS/MS, a technique often rec-
ommended, as in case of pesticides [23], to confirm the validity
of results already obtained. First of all, applying the chromato-
Fig. 4. Relative number of packaging and food samples contaminated by photoini-
graphic conditions reported in Section 2.5, we performed the
tiators with respect to the total analyzed.
MS/MS analysis of a standard solution of benzophenone, using ESI
in positive mode as ionization source and monitoring the transition
m/z 183 → 105. Unfortunately, in these conditions benzophenone
tertiary amine EHDAB was present in fifteen packagings and eleven at a concentration of 100 ␮g l−1 gave a broad and small peak, that
food beverages in ranges of 0.01–3.8 ␮g dm−2 and 0.13–0.8 ␮g l−1 , resulted as not suitable for the confirmation of the results obtained
respectively. ITX was found only to a very low extent both in poly- with GC/MS. Consequently, after an unsatisfactory testing of the
coupled cartons (found in 9 samples, with concentration range Atmospheric Pressure Chemical Ionization (APCI) as interface, we
being 0.01–0.08 ␮g dm−2 ) and food beverages (found in 3 samples, changed the ESI with the Atmospheric-Pressure PhotoIonization
concentration range being 0.2–0.24 ␮g l−1 ). This ITX contamination (APPI) source, that is normally utilized to ionize aromatic or pol-
level is lower than that reported in article related to food bever- yaromatic structures [24]. Using a methanol/water mixture (90/10,
ages published in 2007 [10,11,13]. On the other hand, contamination v/v) as mobile phase and monitoring the same transition m/z
by EDAB and IRGACURE 184 was negligible, being detectable in 183 → 105 of the previous LC/ESI-MS/MS analysis, we got an opti-
just one packaging sample in the case of EDAB (6.1 ␮g dm−2 ), and mization of benzophenone signal that, at the same concentration of
four carton samples and one food sample in the case of IRGACURE 100 ␮g l−1 as above, was increased 3 times with respect to analysis
184.
Considering benzophenone contamination, it could derive from
the use of it both as photoinitiator in the outer packaging surface,
and from benzophenone being used as photoinitiator in the pro-
duction of the polyethylene (PE) coating film [16]; the latter, in
polycoupled cartons, is directly in contact with the liquid food. Lit-
erature suggests that benzophenone, because of its low molecular
weight, can easily migrate from the carton to the liquid food [20]
and, in the case of a polycoupled cartons, from the polyethylene film
that is in contact with the beverage. As a consequence, obtained
data do not account for a direct correspondence between ben-
zophenone concentration determined in analyzed beverages and
that found in the relative polycoupled containers.
Among the analyzed photoinitiators, benzophenone is the only
substance having a Specific Migration Limit (SML) set by law
(600 ␮g l−1 ), giving the maximum quantity which is tolerated to
migrate from plastic material used to package food to food itself
Fig. 5. HPLC/APPI-MS/MS chromatograms of (A) wine sample fortified with
[22]. On the contrary, benzophenone and other photoinitiators used
benzophenone at concentration of 100 ␮g l−1 , (B) standard benzophenone at
in UV-cured inks are not regulated, up to date, by European law with concentration of 100 ␮g l−1 , and (C) water sample not contaminated with benzophe-
regards to potential food contamination activity. none.
220 G. Sagratini et al. / J. Chromatogr. A 1194 (2008) 213–220
with ESI source. Moreover, the resulting peak was better defined
and with a higher chromatographic resolution. As a consequence,
using the APPI as ionization source, the LC/MS/MS analysis was used
as a confirmation method to attest the presence of benzophenone in
all packaging and beverage samples previously analyzed by GC/MS.
As an example, in Fig. 5 the overlapping of three chromatograms
obtained by LC/APPI-MS/MS of a wine sample fortified with ben-
zophenone at 100 ␮g l−1 , a standard benzophenone at 100 ␮g l−1 ,
and a water sample not contaminated with benzophenone is
reported. The above results excluded the presence of benzophe-
none in water sample reinforcing the validity of the proposed
method.
4. Conclusions
A new analytical method to detect five different photoinitiators,
used in UV-inks, in packaged beverages, has been developed. An
easy and rapid step of sample preparation allowed to analyze the
purified sample both in GC–MS and LC/MS. The developed method
was applied in the analysis of forty packagings and relative liquid
foods from Italian market, using the GC/MS to quantify all the pho-
toinitiators, and the LC/MS/MS to confirm the results obtained for
benzophenone, which resulted as present in all analyzed samples.
Overall, the results indicated that the food contamination by ITX is,
at present, regressing, while that of benzophenone is more signif-
icant and widely present, even if the found level is under the SML
(600 ␮g l−1 ). Nevertheless, the case of the food photoinitiator con-
tamination, that was bursted in November 2005, had repercussions
on the industry producing packaged liquid foods: many food bev-
erages, in fact, especially the infants milk, are marketed in plastic
bottles instead of polycoupled carton packagings.
Acknowledgements
The authors thank Ms. Flavia Gigli (Department of Chemical Sci-
ences, Università di Camerino, Italy), for helping in GC–MS analysis,
View publication stats
and Dr. Ernesto Corradetti (Agenzia Regionale Protezione Ambiente
Marche, Ascoli Piceno, Italy) and Dr. Giorgio Petrucci (Università
di Firenze, Italy) for their meaningful discussions. This work was
financially supported by Italian Ministry of Research (PRIN 2006).
References
[1] S. Papilloud, D. Baudraz, Prog. Org. Coat. 45 (2002) 231.
[2] 2005 Chronology of Withdrawal of Nestlè and Other Liquid Milks, document
available at www.ibfan.org/site2005/abm/paginas/articles/arch art/416-1.doc.
[3] Opinion of the Scientific Panel of Food Additives, Flavourings, Processing Aids
and Materials in contact with Food on a request from the commission related
to 2-isopropyl thioxanthone (ITX) and 2-ethylhexyl-4-dimethylaminobenzoate
(EHDAB) in food contact materials, EFSA J. 293 (2005) 1 (available at
http://www.efsa.eu.int/science/afc/afc opinions/catindex en.html).
[4] D. Kirkland, M. Aardema, L. Henderson, L. Muller, Mutat. Res. 584 (2005) 1.
[5] B.V. Notox, Notox Project 411885, 5 October 2004.
[6] B.V. Notox, Notox Project 411874, 23 November 2004.
[7] F. Momo, S. Fabris, R. Stevanato, Biophys. Chem. 127 (2007) 36.
[8] M. Bononi, G. Andreoli, F. Tateo, Ind. Bevande XXXV (April) (2006) 126.
[9] G. Sagratini, J. Mañes, D. Giardinà, Y. Picò, J. Agric. Food Chem. 54 (2006) 7947.
[10] C. Sun, S.H. Chan, D. Lu, H.M.W. Lee, B.C. Bloodworth, J. Chromatogr. A 1143
(2007) 162.
[11] R. Bagnati, G. Bianchi, E. Marangon, E. Zuccato, R. Fanelli, E. Diavoli, Rapid
Commun. Mass Spectrom. 21 (2007) 1998.
[12] A. Gil-Vergara, C. Blasco, Y. Picò, Anal. Bioanal. Chem. 389 (2007) 605.
[13] T. Rothenbacher, M. Baumann, D. Fuegel, Food Addit. Contam. 24 (2007) 438.
[14] G. Morlock, W. Schwack, Anal. Bioanal. Chem. 385 (2006) 586.
[15] E. Corradetti, M. Mirti, S. Celani, D. Corradetti, P. Ceccarelli, Boll. Un. Chim. Igien.
57 (2006) 7.
[16] A. Di Gianni, R. Bongiovanni, A. Priola, S. Turri, Int. J. Adhes. Adhes. 24 (2004)
513.
[17] M.C. Rhodes, J.R. Bucher, R.S. Chhabra, Food Chem. Toxicol. 45 (2007) 843.
[18] M.H. Hsieh, E.C. Grantham, B. Liu, R. Macapagal, E. Willingham, L. Baskin, J. Urol.
178 (2007) 1637.
[19] N. Cook, S. Freeman, J. Dermatol. 42 (2001) 257.
[20] W.A.C. Anderson, L. Castle, Food Addit. Contam. 20 (2003) 607.
[21] G. Petrucci, Bollettino Esperti Ambientali 1 (2007) 16.
[22] Commission Directive 2002/72/EC relating to plastics materials and articles
intended to come into contact with foodstuffs, Off. J. Eur. Commun. L220 (2002)
18.
[23] Document No. SANCO/10232/2006, Quality Control Procedures for Pesticides
Residues Analysis, European Union, Brussels, 2006.
[24] N. Itoh, Y. Aoyagi, T. Yarita, J. Chromatogr. A 1131 (2006) 285.