ANTON AMBRÓZY* and JÁN LABUDA MULTI-LAYER SYSTEMS FOR FUNCTIONAL AND ...

111 Pages·2012·3.85 MB·English

Chem. Listy 106, s248s249 (2012) ACP 2012 – Súčasný stav a perspektívy analytickej chémie v praxi Posters
MULTI-LAYER SYSTEMS FOR FUNCTIONAL AND DETECTION DESIGN
OF ELECTROCHEMICAL BIOSENSORS
ANTON AMBRÓZY* and JÁN LABUDA tions and damage to DNA in media with simple matrices
like buffer solution or simple, mostly aqueous extracts1–5.
Institute of Analytical Chemistry, Faculty of Chemical and Among examples of the multilayer applications, am-
Food Technology, Slovak University of Technology in Bra- perometric DNA biosensors designed as DNA films on a
tislava, Radlinského 9, 812 37 Bratislava, Slovakia cellulose nitrate layer and chitosan layer for the determina-
[e-mail] tion of auto-antibodies6 and detection of damage to DNA7,
respectively, can be mentioned. Layered films of enzyme
and DNA were shown to be able of in vitro biochemical
The current task of chemical analysis is small quanti- transformation of chemicals and of testing of potential
ties detection of new substances, often in increasingly toxic chemicals like styrene8. Other layered films of poly
complex matrix of the sample. Meeting this objective re- (styrene-sulfonate), poly(diallyldimethylammonium) and
quires constantly demanding experimental technique ena- dsDNA on the pyrolytic graphite electrode were used at
bling highly efficient separation and sensitive, eventually the biosensor for the detection of damage to natural DNA9.
selective detection of individual components of the mix- Layer-by-layer films of DNA and glucose oxidase were
ture. Using of sensitive and selective chemical biosensors used for specific formation of DNA cleavage agents like
in combination with simple measuring equipments repre- hydrogen peroxide and hydroxyl radicals within the DNA
sents an alternative procedure for solving specific tasks, layer10. Very thin (10–100 nm) films of polymers (based
especially in small testing laboratories. on phenol, phenol derivatives, phenylenediamines,
Biosensors based on surface-modified electrodes suc- overoxidized or electroinactive polypyrrole) were used to
cessfully extend possibilities of electroanalytical instru- design amperometric enzyme biosensors. The film thick-
ments. The biosensor represents an important element of ness was controlled by self-limited growth. The permselec-
the measurement system that includes a biological compo- tive properties of the films can be utilized for the elimina-
nent and an electrode as the physico-chemical transducer tion of electrochemical interference11–13. Other examples
and ensures certain degree of selectivity in analysis of of a control of the detection capability, selectivity,
samples with complex matrices. Currently there are many biocompatibility and other properties of enzyme and DNA
instrumental methods for the determination of trace con- amperometric biosensors will be presented in this
centrations of elements, species and compounds in com- contribution.
plex mixtures. Their disadvantages are time demand, high
price and inability to be applied to large number of sam- This work was supported by the Scientific Grant
ples in short time. The use of specifically designed biosen- Agency VEGA of the Slovak Republic (Project No.
sors appears to be a suitable alternative. 1/0182/11) and the European Fund for Regional Develop-
In practice, amperometric biosensors are the most ment (ITMS project code 26240220072).
commonly used type of devices. Chemically modified
electrode as a functional part of the biosensor represents REFERENCES
from constructive views conventional electrode made from
carbon, carbon paste or inert metal and appropriately 1. Labuda J., Brett A. M. O., Evtugyn G., Fojta M.,
modified by system of thin layer(s) that is (are) enabled for Mascini M., Ozsoz M., Palchetti I., Paleček E., Wang
selective determination of an analyte. This rational chemi- J.: Pure Appl. Chem. 82, 1161 (2010).
cal modification markedly ensures chemical, electrochemi- 2. Labuda J., Vyskocil V.: in: Encyclopedia of Applied
cal, transport and other properties of the electrode. One of Electrochemistry, DNA/Electrode Interface, (Savinell
the serious problems associated with using of amperomet- R. F., Ota K., Kreysa G., ed.). Springer, 2011. URL:
ric biosensors in complex matrices are interferences http://www.springerreference.com/index/
caused by electroactive as well as surface active species. chapterdoi/10.1007/303710 (accessed 2 December,
Multilayer systems used at the construction of biosensors 2011).
are trying to achieve high selectivity and eliminate inter- 3. Labuda J.: in: Nucleic Acid Biosensors for
ferences. We focus on the use of polymeric films applied Environmental Pollution Monitoring, (Mascini M.,
to the biosensor surface in terms of selectivity enhance- Palchetti I., ed.). pp. 121–140. Royal Society of
ment, interference elimination and biocompatibility im- Chemistry, London 2011.
provement, particularly at enzyme and DNA biosensors. 4. Vyskocil V., Labuda J., Barek J.: Anal. Bioanal.
DNA biosensors represent rather new and specific Chem. 397, 233 (2010).
group of chemical sensors directed to the investigation of 5. Ziyatdinova G., Labuda J.: Anal. Methods 3, 2777
reactivity of surface attached DNA. Till now, they have (2011).
been used for investigation of DNA association interac- 6. Babkina S. S., Ulakhovich N. A., Zyavkina Yu. I.:
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Chem. Listy 106, s248s249 (2012) ACP 2012 – Súčasný stav a perspektívy analytickej chémie v praxi Posters
Anal. Chim. Acta 502, 23 (2004).
7. Galandová J., Ziyatdinova G., Labuda J.: Anal. Sci.
24, 711 (2008).
8. Magweru A., Wang B., Rusling J. F.: Anal. Chem. 76,
5557 (2004).
9. Zhang Y., Zhang H., Hu N.: Biosensors
Bioelectronics 23, 1077 (2008).
10. Zu Y., Liu H., Zhang Y., Hu N.: Electrochim. Acta
54, 2706 (2009).
11. Galandová J., Labuda J.: Chem. Pap. 63, 1 (2009).
12. Zajoncová L., Pospíšková K.: Chem. Listy 103, 291
(2009).
13. Dongyue L., Jianbo J., Jianguo W.: Talanta 83, 332
(2010).
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DEVELOPMENT, VALIDATION AND APPLICATION OF METHOD BASED ON
QUECHERS-DISPERSIVE LIQUID-LIQUID MICROEXTRACTION AND GC-MS
FOR THE DETERMINATION OF PESTICIDES IN ACID FRUIT SAMPLES
MÁRIA ANDRAŠČÍKOVÁa, SVETLANA than water jointly with a dispersive solvent with high mis-
HROUZKOVÁa*, and SARA C. CUNHAb cibility in both extractant and water phases, in order to
form a cloudy solution consisting of small droplets of ex-

traction solvent which are dispersed throughout the aque-
a Department of Analytical Chemistry, Faculty of
ous phase. In consequence of the very large surface area
Chemical and Food Technology Slovak University of
formed between the two phases, hydrophobic solutes are
Technology in Bratislava, Radlinského 9, 812 37,
rapidly and efficiently enriched in the extraction solvent
Bratislava, Slovak Republic, b REQUIMTE, Department
and, after centrifugation, they can be determined in the
Chemical Sciences, Laboratory of Bromatology and
phase settled at the bottom of the tube5.
Hydrology, Faculty of Pharmacy, University of Porto, Rua
The aim of this study was to develop a sample prepa-
Aníbal Cunha 164, 4099-030 Porto, Portugal
ration method that combines QuEChERS and dispersive
[e-mail]
liquid-liquid mikroextraction. The effect of several extrac-
tion parameters, such as selection of extractive solvent and
its volume and volume of dispersive solvent has been
A pesticide is defined as any substance or mixture of tested. Extract acquired after liquid-liquid extraction by
substances intended for preventing, destroying, or control- QuEChERS method was used as dispersive solvent.
ling any pest, including vectors of human or animal Analyses were performed by gas chromatography coupled
disease, unwanted species of plants or animals that cause with mass spectrometry using selective ion monitoring
harm during the production, processing, storage, transport, mode. Good results of linearity, limits of detection and
or marketing of food and wood as well as wood products, limits of quantification were acquired by proposed sample
or animal feedstuff, or which may be administered to ani- preparation method. Satisfactory recoveries at three
mals for the control of insects, arachnids, or other pest in studied concentration levels were obtained. Developed
or on their bodies1. The use of pesticides in food produc- method was applied to the analysis of real orange samples.
tion has provided numerous benefits in terms of increasing Only one out of eleven samples was without positive
production and quality but on the other hand pesticide resi- findings of pesticide residues above limit of detection.
dues are of concern in food safety because they are poten-
tial health hazards and ubiquitous contaminants in the en- This research was supported by the Scientific Grant
vironment. Therefore, the analytical determination of these Agency (VEGA, project No. 1/0647/11).
compounds is absolutely necessary and for diverse rea-
sons, such as the large number of substances applied for REFERENCES
this purpose (over 800) and their widespread use in a vari-
ety of crops, cumbersome2. 1. FAO. International Code of Conduct on the
The key step of analytical procedure for determina- Distribution and Use of Pesticides, p. 28. Food and
tion of pesticide residues is the pretreatment of the sample Agriculture Organization of the United Nations,
to isolate interesting compounds from the matrix using ap- Rome: 1986.
propriate and efficient methods3. Recently, a new microex- 2. Soler C., James K. J., Picó Y.: J. Chromatogr., A
traction method, dispersive liquid–liquid microextraction 1157, 73 (2007).
(DLLME), has been developed by Assai and co-workers4 3. Blasco C., Font G., Picó Y.: J. Chromatogr., A 970,
as an efficient sample preparation and preconcentration 201 (2002).
method. Essentially, DLLME consists in the rapid addition 4. Rezaee M., Assadi Y., Hosseini M.-R. M., Aghaee E.,
to an aqueous sample contained in a conical test tube of Ahmadi F., Berijani S.: J. Chromatogr., A 1116, 1
a mixture of two selected solvents, few microliters of (2006).
a water-immiscible extraction solvent with high density 5. Cunha S. C., Fernandes J. O.: Talanta 83, 117 (2010).
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APPLICATION OF SOME CHEMICAL METHODS TO THE INVESTIGATION
OF METAL COMPLEX-DNA INTERACTIONS
LUCIA ANDREZÁLOVÁ* and ZDEŇKA bound ligand can couple with the  orbital of the base
ĎURAČKOVÁ pairs due to the decrease of the →* transition energy,
which results in batochromic shift in DNA spectrum1,2.

After binding to DNA, the transition metal complexes
Institute of Medical Chemistry, Biochemistry and Clinical
can oxidatively modify DNA bases and/or the sugar moie-
Biochemistry, Faculty of Medicine, Comenius University
ty and thus induce DNA strand breaks. Very sensitive and
in Bratislava, Sasinkova 2, 811 08 Bratislava, Slovakia
reliable method for detecting DNA damage at the cellular
[e-mail]
level is single-cell gel electrophoresis (SCGE, or the comet

assay)3. The cells, such as isolated peripheral human lym-
phocytes or cultured cells, are embedded in agarose on
Many small molecules which are bound to DNA and a microscope slide, lysed with detergent and treated with
cleave DNA duplex are effective pharmaceutical agents, high salt concentration. DNA strand breaks allow DNA to
especially in cancer therapy. DNA has a number of sites in extend from lysed and salt-extracted nuclei, or nucleoids,
which molecule can be bound: between two base pairs to form a comet-like tail on alkaline electrophoresis. The
(intercalation), in the minor or major grooves and on the amount of DNA in the tail reflects the number of relaxed
outside of the helix. Upon binding to DNA, small mole- loops and, therefore, the number of breaks in DNA.
cules are stabilized through a series of weak interactions Comets from undamaged cells have tightly packed super-
such as -stacking interactions of aromatic heterocyclic coiled DNA and no tail. The comet assay is not the only
groups between the base pairs, hydrogen bonds and van way to measure oxidative DNA damage, but it is also one
der Waals interactions in the case of binding to the DNA of the most sensitive and accurate way, being relatively
helix groove. free of artefacts4.
Circular dichroism, fluorescence emission spectrosco-
py and electronic absorption spectroscopy are widely ap- This work was supported by the Scientific Grant
plied to determine the binding characteristics of metal Agency VEGA of the Slovak Republic, the project No.
complexes with DNA. The metal complex – DNA interac- 1/1133/11.
tion can be detected by UV-Vis absorption spectroscopy
by measuring changes in the absorption properties of the REFERENCES
metal complex (a drug) or the DNA molecules. The ab-
sorption spectrum of DNA exhibits a broad band in the 1. Firdaus F., Fatma K., Azam M., Khan S. N., Khan A.
UV region with a maximum at 260 nm. Both, hyper- U., Shakir M.: Spectrochim. Acta, A 72, 591 (2009).
chromic and hypochromic effects are the spectral features 2. Khan T. A., Naseem S., Khan S. N., Khan A. U.,
of DNA concerning to its double helical structure. Hypo- Shakir M.: Specrochim. Acta, A 73, 622 (2009).
chromism indicates that the DNA – metal complex binding 3. Collins A. R., Dušinská M., Franklin M., Somorovská
mode is electrostatic interaction or intercalation which can M., Petrovská H., Duthie S., Fillion L., Panayiotidis
stabilize the DNA duplex and hyperchromism means M., Rašlová K., Vaughan N.: Environ. Mol. Mutagen.
a breakage of the secondary structure of DNA. After in- 30, 139 (1997).
teraction with the base pairs of DNA, the * orbital of the 4. Collins A. R.: Mutat. Res. 681, 24 (2009).
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Chem. Listy 106, s252s256 (2012) ACP 2012 – Súčasný stav a perspektívy analytickej chémie v praxi Posters
VALIDATION OF HPLC-ESI-MS-MS METHOD FOR ACRYLAMIDE
DETERMINATION IN BAKERY PRODUCTS (COMPARISON OF SIMPLE
AND IMPROVED ELECTROSPRAY IONIZATION)
ALENA BEDNÁRIKOVÁ* and ZUZANA sensitivity; c) a powerful technique for the quantitative
analysis. Improvement of the ion source assembly declared
CIESAROVÁ
by manufactures to produce dramatic gains in sensitivity is

patent-protected9.
VUP Food Research Institute, Department of Chemistry
Quantification is performed by adding different kinds
and Food Analysis, Priemyselná 4, SK-824 75 Bratislava,
of internal standards2–8. An isotope-labelled acrylamide
Slovak Republic
(e.g. 13C -acrylamide or d -acrylamide) is usually recom-
[e-mail] 3 3
mended to be used for improving the repeatability of the

whole procedure which is important for the complex matrix

of the sample where extraction yields could be varied
Summary
strongly.

This paper presents validation of simple LC-ESI-MS-
An effective sample preparation procedure was estab-
MS method for acrylamide determination in bakery
lished for the determination of acrylamide in bakery
products and selected validation parameters were used to
products by a liquid chromatography-tandem mass spec-
compare two types of ESI.
trometry. The method entails extraction of acrylamide with

acidified water followed by cleanup with ethyl acetate.
Experimental
The chromatographic separations were performed on a oc-

tadecyl silica column with 1% methanol in water as the
Chemicals and reagents
mobile phase. The present study is focused on the com-

parison of two LC-MS-MS systems using simple or im-
Acrylamide (purity 99 %), ethyl acetate (HPLC-
proved electrospray ionization. The limit of quantification
grade) and methanol (HPLC-grade) were purchased from
(LOQ) was 2 ng mL–1 for simple ESI and 0.4 ng mL–1 for
Sigma-Aldrich (Steinheim, Germany). 2,2,3-d -acrylamide
improved ESI which represent 25 ng g–1 and 5 ng g–1, re- 3
(purity 98 %) was obtained from Cambridge Isotope
spectively, in real bakery products.
Laboratories (Maryland, USA). Glacial acetic acid,

potassium hexacyanoferrate trihydrate and zinc sulphate
Introduction
heptahydrate were analytical grade and obtained from

Merck (Darmstadt, Germany). Deionised water was
Since the first mentioning of acrylamide, a toxic and
purified with a purification system PUR1TY Select (HP,
potentially carcinogenic chemical, in heat-treated food1,
Oxon, UK). Nylon syringe filters 0.45 m were purchased
several research groups have been participated in the de-
from Millipore (Billerica, MA, USA).
velopment of rapid and reliable analytical methods for its
Stock solution of acrylamide (0.5 mg mL–1) and d -
quantification in a large variety of foodstuffs and were re- 3
viewed by several authors2–6. Most of the published acrylamide (0.2 mg mL–1) were each prepared by
dissolving the compounds in deionised water and stored at
methods are based on either gas chromatography – mass
4 °C. Working solution was prepared daily by appropriate
spectrometry (GC-MS) or high performance liquid chro-
dilution of the stock solution.
matography – tandem mass spectrometry (HPLC-MS/MS).
Carrez I solution was prepared by dissolving 15 g of
Current applied methods of acrylamide determination in
potassium hexacyanoferrate trihydrate in 100 mL of water,
foods typically include several sample preparation steps
and Carrez II solution was prepared by dissolving 30 g of
(impurity removal, analyte extraction, centrifugation, clean
zinc sulphate heptahydrate in 100 mL of water.
-up, pre-concentration) due to the complexity of food sam-

ples and low levels of acrylamide. On the other hand new
Sample preparation
challenges can emerge (e.g. extraction solvents, time and

temperature, de-fatting, sample particle size etc.). Some of
them have been studied in detail by Petersson et al.7 and A finely ground or homogenized dry sample (1.000 g)
Goldmann et al.8. was weighed into a 10 mL centrifuge tube with a cap, and
50 L of the internal standard d -acrylamide (500 ng) and
Acrylamide does not show any specific wavelength 3
9 mL of diluted acetic acid (0.1 %, v/v) were added. After
absorption maxima and this fact complicates its detection.
shaking by a vortex mixer for 30 s, the mixture was soni-
For detection of acrylamide after LC separation, tandem
mass spectrometry using electrospray in positive mode is cated for 5 min. Then, 500 L of Carrez I solution and
the most frequent method of choice2–8. LC-MS/MS, 500 L of Carrez II solution were added and shaked for
working in multiple reaction monitoring (MRM) mode, in 1 min. After that, the mixture was centrifuged at 8720 g
which the transition from a precursor ion to a product ion for 10 min and a clear supernatant was obtained.
is monitored, has some advantages: a) high selectivity; b) A volume of 5 mL of the clear supernatant was trans-
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Chem. Listy 106, s252s256 (2012) ACP 2012 – Súčasný stav a perspektívy analytickej chémie v praxi Posters
ferred to a separatory funnel and extracted with ethyl ace- dissolved in water. Concentrations were between limit of
tate (3 x 5 mL). The combined organic layers were evapo- quantification (LOQ) to 200 ng mL–1. Quantification was
rated at 35 °C to dryness. The residue was dissolved in 1 performed by comparison of the peak area ratio of acryla-
mL of diluted acetic acid and filtered through a 0.45 m mide with internal standard, d3-acrylamide (50 ng mL–1),
pore size nylon syringe filter prior LC analysis. monitored using the MRM transitions at m/z 72 → 55
(acrylamide) and 75 → 58 (d3-acrylamide).
HPLC-MS-MS analysis HPLC system, the ionization interface and the mass
spectrometric detector were controlled and data were ana-
The quantification of acrylamide was performed by lysed by MassHunter software (Rev.B.01.03, Agilent, Palo
HPLC-MS-MS with positive electrospray ionization Alto, USA).
(ESI+) using two different devices of triple quadrupole
mass spectrometers (Agilent 6410 and 6460 Triple Quad Results and discussion
detector, Agilent Technologies, Palo Alto, USA) coupled
to two HPLC systems Agilent 1200 and 1260 series con- Setting of MS-MS conditions
sisting of a binary pump, a vacuum degasser, an au-
tosampler, and a thermostated column compartment. The Electrospray ionization is a sensitive technique that is
analytical separation was performed on Atlantis dC18 used for the analysis and identification of small molecules.
column (100 mm × 2.1 mm, 1.8 m particle size; Waters, Manufacturers try to achieve/enhance sensitivity in ESI-
Milford, MA, USA) using isocratic mixture of 1 % of MS by improving the desolvation and spatial focusing of
methanol and 0.2 % of glacial acetic acid in water at flow ions9. Thus, we can compare results obtained by two types
rate 0.4 mL min–1 at ambient temperature. The sample of constructed ion sources with optimized setting parame-
volume injected in both systems (LC-QQQ6410 and LC- ters which are shown in Table I. LC-MS/MS working in
QQQ6460) was 20 L and 10 L, respectively. MRM has a high selectivity, therefore the relevant MRM
Acrylamide was identified using multiple reaction transitions for acrylamide and internal standard were se-
monitoring (MRM) experiments which are based on in- lected and optimized. Acrylamide gives the precursor ion
source generation of the protonated molecular ions of with m/z ~ 72 assigned to its pseudo-molecular ion [M+H]+
acrylamide and the internal standard (d -acrylamide), as and fragment ions with m/z 55 (probably formed by am-
3
well as a collision-induced production of specific fragment monia elimination of precursor ion [M+H-NH ]+) and two
3
ions. Instrumental parameters used for the acrylamide complementary fragment ions with m/z 44 and 27. The
analysis in the ESI+ mode are summarized in Table I. first transition at m/z 72 → 55 has been selected for quanti-
Acrylamide was quantified using a linear calibration fying because it shows a relatively high intensity. The se-
curve established with a standard solution of acrylamide cond transition at m/z 72 → 27 has been used for verifica-
Table I
Instrumental parameters used for acrylamide analysis using electrospray ionization in positive mode by two types of ion
sources (where AA = acrylamide)
Source parameter QQQ 6410 QQQ 6460
Specification ESI JetStream
Gas temperature [°C] 350 300
Drying gas (N ) flow [L min–1] 10 10
2
Nebulizer pressure [psi] 50 60
Capillary voltage [kV] 2.5 3.5
Sheath gas heater – 250
Sheath gas flow – 11
Fragmentor [V] 45 50
Dwell [ms] 50 50
Cell accelerator [V] – 4
Acquisition parameters AA d -AA AA d -AA
3 3
Precursor ion m/z 72 75 72 75
Product ion m/z 55 27 58 30 55 27 58 30
Collision energy [V] 6 14 6 14 10 18 10 18
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Chem. Listy 106, s252s256 (2012) ACP 2012 – Súčasný stav a perspektívy analytickej chémie v praxi Posters
tion of acrylamide presence by calculating ratio of quali- tion of acrylamide. Whereas, our choice was Atlantis dC18
fier response to quantifier response. In any given MS/MS column and aqueous 0.2% acetic acid containing 1%
conditions, the relative ratio of acrylamide qualifier to methanol as the mobile phase, as well.
quantifier is 12 % calculated for the standard solution and Hoenicke et al.10 published a method for reliable
± 20 % of uncertainty of presented value is a satisfactory quantification of acrylamide in complex matrices as well
range in real matrices. as in lower concentrations (up to 5 ng g–1) based only on
Internal standard (d3-acrylamide) gives the precursor liquid-liquid extraction with ethyl acetate. They used water
ion with m/z ~ 75 and fragment ions with m/z 58, 44 and extraction at 60 °C followed by removing the fat layer
30, respectively. The relative ratio of d3-acrylamide quali- with iso-hexane. Clear aqueous phase obtained after cen-
fier to quantifier is 16 % calculated for standard solution. trifugation was extracted twice with ethyl acetate. The
combined organic phases were reduced to 1 % of original
Sample treatment volume and analyzed directly by GC-MS/MS. According
to other published results7,8 we slightly change the above
Numerous methods have been developed to determine mentioned procedure as follows: a) extraction with acidi-
acrylamide in different heat-treated food samples. Most re- fied water at ambient temperature; b) skip removing the fat
searchers use reverse-phase chromatography2–8 with mo- layer; c) centrifugation of the sample at higher rpm; d) re-
bile phase containing predominantly aqueous 0.1% acetic ducing the extraction amount of ethyl acetate while ex-
acid or 0.05% formic acid for the chromatographic separa- tracting 3-times; e) evaporation of ethyl acetate to dryness
Table II
Comparison of single and multi-stage aqueous extractions of acrylamide (SD = standard deviation, n = 6)
Aqueous extraction Area AA ± SD Area d -AA ± SD Ratio (AA/d -AA) ± Lord’s test
3 3
(stage) SD (u = 0.250)
krit
1st 4327 ± 492 7318 ± 728 0.591 ± 0.028 –
2nd 1126 ± 119 1906 ± 207 0.591 ± 0.018 u =0.003
12
3rd 385 ± 41 653 ± 70 0.590 ± 0.020 u =0.010
13
merged (1st + 2nd) 2914 ± 58 4955 ± 140 0.588 ± 0.026 u =0.039
1M
Table III
Comparison of analytical characteristics of the proposed methodology for the determination of acrylamide (linear calibra-
tion equation (y= a.x + b) was calculated; where x represents relative concentration of acrylamide vs. internal standard; y
represents relative peak area of acrylamide vs.d -acrylamide; s, s represent standard deviations of calculated parameters
3 a b
of linear regression)
LC-QQQ 6410 LC-QQQ 6460
Retention time, min 2.2 2.2
Aqueous calibration
b -intercept of regression equation, ± s –0.0005 ± 0.0021 0.00102 ± 0.00066
b
a -slope of regression equation, ± s 0.893 ± 0.011 0.9812 ± 0.0012
a
Correlation coefficient, R2 0.9994 0.9998
Limit of detection (LOD), ng mL–1 0.5 0.1
Limit of quantification (LOQ), ng mL–1 2.0 0.4
Matrix calibration
Spiked levels, ng g–1 25, 50, 100, 250 and 500 12.5, 25, 50, 100 and 250
b -intercept of regression equation, ± s 0.148 ± 0.005 0.0552 ± 0.0011
b
a -slope of regression equation, ± s 0.885 ± 0.011 0.9841 ± 0.0012
a
Correlation coefficient, R2 0.998 0.9994
Limit of quantification (LOQ), ng g–1 25.0 5.0
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A B
Fig. 1. Overlaid extracted MRM chromatograms of acrylamide (transition at m/z 72 → 55) in standard aqueous solution
(concentration level 1 ng mL–1; dotted line) and in crisp bread (in case A – determined below LOQ, in case B – determined 28 ng g–1;
solid line) in two LC-MS-MS systems (A/ HPLC- ESI-QQQ6410 – simple ESI+; B/HPLC-ESI- QQQ 6460 – improved ESI+)
and dissolution in 1 mL of acidified water. were spiked at five different levels of acrylamide standard
In this study, cereal based matrices were used as the and matrix calibration curves were estimated. Similar ap-
test material for resolving a problem how many extraction proach for the estimation of LOQ was applied in real ma-
stages are required for acrylamide determination. The in- trices. The obtained results are shown in Table III.
ternal standard d -acrylamide (500 ng) was added to one The concentration of acrylamide in crisp bread A and
3
gram of ground sample and extracted three times with B calculated from aqueous calibration were 175±10 ng g–1
acidified water. There were six replicates in each stage in and 27.6±0.9 ng g–1, respectively, and estimated from ma-
order to calculate the relative responses of acrylamide area trix calibration were 167±14 ng g–1 and 28.1±1.0 ng g–1,
and internal standard (Table II). Although the ratios of are- respectively. Comparing results obtained from aqueous
as in particular stages were similar, a considerable de- and matrix calibration curves it was concluded that the
crease of areas was observed, which is in good agreement preparation of aqueous calibration of acrylamide with in-
with Gökmen et al.11, who claimed that at least two extrac- ternal standard is usable for its quantification in real matri-
tion steps were required for complete extraction of acryla- ces.
mide from cereal and potato based food products. The di- However, the changed volume of the sample injection
lution caused by triple extraction was not advantageous for (from 20 µL to 10 µL) was not taken into account, it is
our purposes. What is more, due to the use of internal clear that LOD and LOQ of the method were reduced 5-
standard, a single extraction proved sufficient. According times only by using the improved technology of ionization.
to the presented results, our sample preparation procedure Fig. 1 shows overlaid MRM chromatograms of the
consists of one step aqueous extraction with protein pre- acrylamide standard solution (at concentration level 1
cipitation followed by simple ethyl acetate extraction and ng mL–1) and acrylamide determined in crisp bread
then again dissolving in water. (approximately 25 ng g–1) measured by the two HPLC-MS
-MS systems.
Method performance comparison on two LC-MS
systems Analysis of test food materials

To check performance of the developed analysis Moreover, the accuracy of the method was observed
method of acrylamide, parameters such as limit of detec- in analyses of different test materials in three consecutive
tion (LOD), limit of quantification (LOQ) and linearity days. Six independent measurements of the test materials
were calculated (see Table III). Quantification was per- supplied from the Food and Environment Research Agen-
formed on the basis of calibration line of the peak area ra- cy (Fera, York, UK) were carried out each day which re-
tio against the concentration ratio of acrylamide with inter- sulted in average acrylamide concentration of 79±15 g kg–1
nal standard, d -acrylamide. The calibration curves for the in crisp bread (FAPAS test material T3018, satisfactory
3
determination of acrylamide in water were linear over the range of 57–145 g kg–1); 288±20 g kg–1 and 315±20
range of relevant LOQ to 200 ng mL–1 with 50 ng mL–1 of g kg–1 in crisp bread (FAPAS test material T3025,
d3-acrylamide and correlation coefficients (r2) obtained satisfactory range of 200–444 g kg–1) and 1180±180
were ≥ 0.995. Limit of detection (LOD) and limit of quan- g kg–1 in biscuit (FAPAS test material T3019,
tification (LOQ) were calculated from linear regression by satisfactory range of 867–1644 g kg–1) (Table IV). Our
following equations: LOD = 3* (s /a) and LOQ = 10* (s /
b b method is sufficiently robust and has been successfully
a), respectively.
used for acrylamide determination in materials such as
Two real samples of crisp bread (marked as A, B) cereal based fried or baked products12,13.
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Chem. Listy 106, s252s256 (2012) ACP 2012 – Súčasný stav a perspektívy analytickej chémie v praxi Posters
Table IV
Accuracy and repeatability of acrylamide determination
AA (ng/g) determined
FAPAS test materials by LC –QQQ 6410 by LC – QQQ 6460
T3018 (crisp bread) 1st day 83 ± 5 –
Mean value 101 ng g–1 2nd day 91 ± 5 –
Satisfactory range: 3rd day 72 ± 5 –
(57–145) ng g–1 average 79 ± 15 –
T3019 (biscuit) 1st day 1288 ± 60 –
Mean value 1256 ng g–1 2nd day 1264 ± 60 –
Satisfactory range: 3rd day 1058 ± 60 –
(867–1644) ng g–1 average 1180 ± 180 –
T3025 (crisp bread) 1st day 289 ± 5 309 ± 6
Mean value 322 ng g–1 2nd day 298 ± 10 330 ± 9
Satisfactory range: 3rd day 278 ± 7 305 ± 6
(200–444) ng g–1 average 288 ± 20 315 ± 20
Note: Test materials T3018 and T3019 are analyzed in years 2008 and 2009
Conclusion 4. Zhang Y., Zhang G.,Zhang Y.: J. Chromatogr. 1075, 1
(2005).
The results presented in this study demonstrate that 5. Keramat J., LeBail A., Prost C., Soltanizadeh N.:
LC-ESI-MS/MS performed in both of two devices is Food Bioprocess Technol. 4, 340 (2011).
a suitable technique for the quantitative analysis of 6. Tekkeli S.E.K., Őnal C., Őnal A..: Food Anal. Method
acrylamide in such complex matrices as cereals products. 5, 29 (2012).
The obtained data demonstrate that LOD and LOQ by im- 7. Petersson E., Rosen J., Turner Ch., Danielsson R.,
proved electrospray ionization technology were reduced 5- Hellenäs K.E.: Anal. Chim. Acta 557, 287 (2006).
times. 8. Goldmann T., Perisset A., Bertholet M. C., Stadler R.
H., Petersson E. V., Hellenäs K. E.: Food Addit. Con-
This work was supported by the Agency of the Minis- tam. 23, 437 (2006).
try of Education of the Slovak Republic for the Structural 9. Hoenicke K., Gatermann R., Harder W., Hartig L.:
Funds of the EU under the contract No. 26240120024 and Anal. Chim. Acta 520, 207 (2004).
2624020050. 10. Agilent – Technical Note 5990-3494 en-Io CMS.pdf;
http://www.agilent.com/chem/
REFERENCES 11. Gökmen V., Morales F. J., Ataç B., Serpen A., Arri-
bas-Lorenzo G.: J. Food Compos. Anal. 22, 142
1. Tareke E., Rydberg P., Karlsson P., Eriksson S., (2009).
Törnquist M.: J. Agric. Food Chem. 50, 4998 (2002). 12. Ciesarova Z., Kukurova K., Bednarikova A., Morales
2. Wenzl T., de la Calle B., Anklam E.: Food Addit. F. J.: J. Food Nutr. Res. 48, 20 (2009).
Contam. 20, 885 (2003). 13. Kukurova K., Morales F.J., Bednarikova A., Ciesa-
3. Castle L., Eriksson S.: J. AOAC Int. 88, 274 (2005). rova Z.: Mol. Nutr. Food Res. 53, 1532 (2009).
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Chem. Listy 106, s257s258 (2012) ACP 2012 – Súčasný stav a perspektívy analytickej chémie v praxi Posters
METODIKA SPME-GC-MS ANALÝZY PRCHAVEJ FRAKCIE OVOCNÝCH DŽÚSOV
VERONIKA GRIGEROVÁ a EVA ako je 1-hexanol a 1-butanol je možné vysvetliť výskytom
v glykozidických väzbách, z ktorých sú uvoľňované pri
BENICKÁ*
tepelnom spracovaní v kyslom prostredí. Hlavnou zložkou

v koncentrovaných džúsoch je furfural.
Ústav analytickej chémie, Fakulta chemickej a potra-
U rôznych džúsov je rozdiel v množstve vonných
vinárskej technológie STU v Bratislave, Radlinského 9,
zlúčenín. Môže to byť spôsobené druhom ovocia,
812 37 Bratislava, Slovensko
pasterizáciou, umiestňovaním do fliaš, balením
[e-mail]
a skladovaním4. Prchavé zlúčeniny ovplyvňujú senzorické

vlastnosti čerstvého ovocia a pripravených ovocných

produktov. Chuť a vôňa ovocia sú tvorené komplexom
Práca je zameraná na analýzu prchavých zlúčenín
skupín chemických zlúčenín ako sú aldehydy, alkoholy,
v ovocných džúsoch, ktoré vo veľkej miere ovplyvňujú ich
ketóny, estery, laktóny, terpény a iné. Informácia
senzorické vlastnosti. Prchavé látky podieľajúce sa na
o aromatickom profile prchavých látok nám umožňuje
tvorbe aromatického profilu môžeme zaradiť medzi
rozlíšiť prírodné džúsy od nápojov s umelými sladidlami.
chemické zlúčeniny, ako sú aldehydy, ketóny, alkoholy,
Koncentrácia týchto prchavých zlúčenín je veľmi nízka
estery, laktóny, terpény. Podľa literatúry boli použité rôzne
(g l–1). Ovplyvňujú ju dva faktory: poľnohospodársky
izolačné a separačné metódy, ako SPE, SPME, SBSE
(druh, klimatologické podmienky, stupeň zrelosti)
a priama extrakcia kvapalina-kvapalina. K následnej
a technologický (podmienky pri zbere úrody, skladovanie
separácií sa využívalo spojenie plynovej chromatografie
a podmienky spracovania).
s hmotnostnou spektroskopiou, výsledky analýzy však
Z porovnania získaných informácií je zrejmé, že
výrazne ovplyvňuje výber vhodnej izolačnej metódy.
zloženie džúsov jednotlivých druhov ovocia je vo veľkej
Aromatický profil jednotlivých džúsov sa líšil v závislosti
miere ovplyvnené druhom, podmienkami skladovania
od druhu ovocia, použitých metód izolácie a separácie, ako
a spôsobom spracovania ovocia a šťavy. Veľký vplyv na
aj spôsobu spracovania, uskladnenia. Informácie
stanovené množstvo prchavých zlúčenín mal aj spôsob
o profilovom zložení džúsov umožňujú rozlíšenie medzi
izolácie a samotná separácia látok.
pravými organickými džúsmi a džúsmi s prídavkom
Enzymatické hnednutie je nežiadúcim procesom
umelých sladidiel.
spôsobujúcim znižovanie kvality, kde kľúčovými
Ovocné džúsy sú produktmi spracovaného ovocia.
enzýmami sú polyfenolové oxidázy. Tieto enzýmy sú
Analýza umožňuje identifikáciu a stanovenie zastúpených
prítomné vo všetkých rastlinách, vo väčšom množstve sa
vonných a nevonných prchavých zlúčenín, ktoré vplývajú
vyskytujú v jablkách, hruškách, mangu a i. Prebieha
priamo na senzorické vlastnosti produktov. Senzorické
oxidácia fenolového substrátu polyfenolovou oxidázou na
vlastnosti vonných zlúčenín, obzvlášť ich hraničné
reaktívny o-chinón a následne dochádza k polymerizáciu
vnímanie, sa môže meniť v závislosti od kombinácie
o-chinónu na pigment hnedého sfarbenia. Enzymatické
prchavých zlúčenín1,2. Potrebné je poznať profil prchavých
hnednutie nemá vplyv iba na sfarbenie, ale aj na výživné
zlúčenín. Vonné vlastnosti štiav sú ovplyvnené fyzikálnym
a iné senzorické vlastnosti, vrátane vône, chuti.
stavom, chemickými vlastnosťami prchavých zlúčenín,
V potravinárskom priemysle sa používa 5 metód na
interakciami medzi nimi počas spracovania a skladovania.
kontrolovanie týchto enzymatických procesov. Tepelné
Tieto potravinové komodity obsahujú vysoké
spracovanie, kontrola pH, vylučovanie kyslíka (spôsobuje
množstvo zlúčenín, avšak len niekoľko z nich môžeme
zníženie produkcie prchavých látok), používanie
zaradiť medzi látky zodpovedné za ich charakteristický
prírodných aditív, používanie činidiel s protihnednúcim
vonný a chuťový profil produktov. Každý druh ovocia
účinkom. Najpoužívanejším činidlom je kyselina
môžeme charakterizovať viac ako 100 prchavými
askorbová, ktorá redukuje o-chinón na pôvodnú fenolovú
zlúčeninami, v závislosti od ich stupňa zrelosti. Vonné
zlúčeninu skôr než nastane vytvorenie pigmentu.
zlúčeniny charakteristické pre citrusové plody ako sú
Nevýhodou je, že kyselina askorbová má negatívny vplyv
pomaranče a citróny sú monoterpény, seskviterpény a ich
na zloženie vonných zlúčenín v jablkových džúsoch,
deriváty. Pre ostatné necitrusové druhy ovocia sú
dochádza k zmene koncentrácií niektorých prchavých
charakteristické estery, aldehydy, napr. pre banány,
zlúčenín zodpovedných za aromatický profil5.
jahody, jablká3. Podľa literatúry bolo HRGC-MS metódou
Proces pasterizácie je založený najmä na tepelnej
v džúsoch identifikovaných 80 zlúčenín. V pôvodnom
energii, na redukciu mikroorganizmov sa používajú aj
džúse prevládali 1-hexanol, 1-butanol, E-2-hexenal, E-2-
metódy ako je použitie vysokého tlaku a pulzné elektrické
-hexenol a butylester kyseliny octovej. V koncentrovaných
pole, vďaka ktorým nedochádza k zhoršeniu kvality
jablkových džúsoch sa nachádzala väčšina prchavých
spôsobenej teplom. Spracovanie oxidom uhličitým
zlúčenín typických pre jablká iba v stopových
prebieha pri nižšej teplote, a tým dochádza k menšiemu
koncentráciách. Prítomnosť malého množstva alkoholov
s257