Yam, Kit L. (ed.). The Wiley encyclopedia of packaging technology

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BIBLIOGRAPHY

‘‘Ampuls and Vials,’’ R. P. Abendroth and J. E. Lisi, in The Wiley

Encyclopedia of Packaging Technology, 1st ed., Wiley, New

York, Owens-Illinois, Inc, 1986, pp. 16–20.

1. The United States Pharmacopeia XXII, and the National

Formulary, The United States Pharmacopeial Convention,

Inc., Rockville, MD, 1990.

ANALYTICAL METHODS FOR FOOD

PACKAGING AND SHELF LIFE STUDIES

CRISTINA NERIN

Aragon Institute of Engineering

Research (I3A), CPS;

University of Zaragoza, Dept.

Analytical Chemistry, Zaragoza,

Spain

INTRODUCTION

According to the Encyclopedia of Packaging Technology,

shelf life is the time after production and packaging that a

product remains acceptable under deﬁned environmental

Figure 6. Tube converting for vial manufacture.

Figure 5. Vial contour-forming sequence.

38 ANALYTICAL METHODS FOR FOOD PACKAGING AND SHELF LIFE STUDIES

conditions. To establish this period of time is nowadays

required for every food product, not only for commercial

reasons but also for safety reasons, as all foods deteriorate

with time. The main causes for these deteriorative pro-

cesses are as follows:

1. Oxidation processes during which the color fades

and may give way to a brownish appearance,

rancid tastes, and odors and the food becomes

unacceptable.

2. Microbiological, which is the consequence of the

micro-organisms growth, either molds, bacteria or

yeasts, some of them pathogens and dangerous for

human beings. Often together with the micro-organ-

isms, new compounds appear that are responsible

for the bad odors or off ﬂavors.

3. Water losses, which is the consequence of the aging

that affect the texture, hardness, appearance, and

the taste.

However, although the causes of the limitation of the

shelf life were known, the analysis of the macroscopic

effects usually is not enough to establish the limits, and

more and more other parameters are required to establish

the end of the shelf life.

Besides, the evaluation of the deadline cannot be sub-

jective, just as a global measurement, and analytical

procedures are needed to standardize the parameters

used for establishing the deadline limits.

The 21st century is characterized by globalization. In

the food area, this means to have the food packaged,

because in these conditions, the transport, storage, logis-

tic, and distribution chain can reach distant markets, and

a global commercialization can take place. Foodstuffs

themselves cannot stand for long time, and new technol-

ogies such as vacuum packaging and modiﬁed atmosphere

packaging, combined with the use of high barrier materi-

als and active packaging, can extend the shelf life of

the food inside the package. Emerging technologies like

intelligent packaging are then proposed to show that the

packaged food is still good and safe.

But the challenge is how to measure the shelf life

of food. When developing the packaging material or

the packaging system, there are different materials,

parameters, and variables to modify and to measure.

Figure 1 shows a scheme in which the material, the

food, and the internal atmosphere play an important

role in the shelf-life deﬁnition and testing. Interactions

between them as well as absolute measurements have to

be carried out to study the shelf life. According to this

process and depending on the type of samples that

we have, different parameters and different analytical

methods will be used. This chapter deals with the main

analytical methods, which can be useful for shelf-life

evaluation, taking into account that the main purpose in

this task is to have an objective and independent series of

methods. Avoiding the subjective and personal skills is the

main way to measure the key parameters. There is not

an exhaustive list of analytical methods, and only those

considered as the most appropriate or most common in

this type of studies are referred to.

As Figure 1 shows, the food, internal atmosphere, and

packaging material should be tested to establish the shelf

life. The food qualities have to remain constant and be as

good as possible and the most similar to the fresh food or

just-made food. These qualities involve color, taste, hard-

ness, texture, volatile compounds, odor, water content,

chemical contaminants, absence of microbiological con-

tamination, and freshness, among others.

The internal atmosphere means the analysis of gases

such as oxygen, carbon dioxide, and other permanent

gases and vapors, such as water, volatile compounds

transferred by the packaging material, or those released

by the food.

Finally, the packaging material, which involves the

testing of the permeability properties, the analysis of

likely contaminants as potential migrants, the migration

tests to ensure that the material is safe for being in

Figure 1. Scheme of the food packaging to measure

the shelf life.

ANALYTICAL METHODS FOR FOOD PACKAGING AND SHELF LIFE STUDIES 39

contact with the food, and the sorption properties of the

material to guarantee that the material does not affect

the quality of food, by scalping aromas or by trapping the

compounds responsible for the color, ﬂavor, or other food

attributes.

In all cases, the ﬁrst key point is the selection of the

parameters to be measured in a quantitative manner for

the shelf-life studies, and the second point is the selection

of the analytical technique and the procedure to measure

the changes, if any, in the whole system.

THE ANALYTICAL PROCESS

Figure 2 shows a scheme of the analytical process. Three

main steps can be distinguished: the ‘‘sampling,’’ the

‘‘sample treatment,’’ and the ‘‘ﬁnal analysis.’’ A short

comment of the main procedures involved in each step

will be described below.

THE SAMPLING STEP

Usually, this is the ﬁrst step, as the sample has to be taken

for analysis. Some critical requirements should be fol-

lowed to be sure that the ﬁnal analysis represents a real

diagnostic of the sample. The three points mentioned in

the Figure 2 have to be decided by the analyst, and such a

decision should be based on scientiﬁc parameters. The

ﬁrst step, the sample size, seems to be obvious, as if low

concentrations are expected for the analytes, higher

amount of sample should be taken. However, the sample

size will condition the second step of the analytical

procedure, which is the sample treatment, and it will

affect the handling, time, and price of the analysis. How-

ever, if the sample size is small the representativeness of

the sample is in danger. Then, to decide about this

parameter, it is recommended to think about the detection

limit of the ﬁnal analytical technique in which the vari-

able under study will be measured as well as the expected

concentration of the analyte after the test. Sometimes,

only differences between the concentration before and

after the shelf-life studies are going to be measured

instead of absolute values. In this case, the sensitivity is

higher, as the absolute value measured is higher than the

difference itself. When absolute values have to be mea-

sured, for example, the concentration of an analyte formed

during the shelf life study, the expected value can be

extremely low, and thus higher amount of sample is

needed.

Representativeness

This point is critical and affects any kind of analysis. It is

close-related with the homogeneity of the sample. With

food samples, the lack of homogeneity can let us take

discriminant portions of food for the analysis, and ob-

viously in this case the diagnostic is wrong. To avoid

this problem, a previous homogenization is recommended.

Sample size is also conditioned here, as with small sample

size there is a high risk of discrimination, which ruins the

analysis. All parts of the food should be taken and mixed

together to reproduce the real situation as much as

possible. Interactions between the different components

in the food sample is also very important, what requires

that all the components were represented in the sample

taken for analysis.

Homogeneity

The problem of homogeneity is more common in food

analysis, where the samples can be macroscopically homo-

geneous but nonhomogeneous at microscopic level. Differ-

ent devices are nowadays available to homogenize the

samples. Mills, blenders, mixers, and similar machines

Figure 2. The analytical process.

40 ANALYTICAL METHODS FOR FOOD PACKAGING AND SHELF LIFE STUDIES

are common in any laboratory. Liquid and solid portions

are mixed together if they are present in the food sample.

Then, an homogeneous sample is produced, usually

smashed and ﬂuid enough to be sure that any portion of

it has the same composition.

SAMPLE TREATMENT

The sample treatment involves the sample dry, the ex-

traction and the clean-up steps. To dry the sample is not

always required before the analysis, although the values

should be always referred to the dry sample for compar-

ison. Sometimes, an aqueous extraction is going to be

applied to the sample, and then there is no point to remove

the water. Anyway, it is important to measure the water

content in a different aliquot from that used for the sample

analysis.

However, the extraction and the clean up steps are

usually the bottleneck of the analytical process (1). It is in

fact the area in which more analytical development has

taken place in the last decade, and there are several

reasons for that. Let us go to explain more in detail the

newest extraction techniques that can be applied for shelf-

life studies.

It is true that the shelf-life studies require sensitive

analytical methods for measuring low concentrations of

compounds that are formed either during the shelf life of a

foodstuff or disappear as long as the time goes on. Alter-

natively, the packaging has to be measured, and then low

concentrations of compounds are also analyzed either in

the packaging material or in the atmosphere inside the

packaging. In any case, the analytes have to be isolated

from the matrix, the interferences have to be removed as

efﬁciently as possible, and simultaneously the concentra-

tion of the analyte in the ﬁnal solution that will be

analyzed has to be as high as possible. This way, the

detection and quantiﬁcation limits will be surpassed, and

no limitations concerning the sensitivity will be present.

The classic extraction procedures involve the liquid–

liquid extraction, in which the liquid sample to be ex-

tracted is shaken with a small volume of a nonmiscible

liquid-extracting phase, nonsoluble with the sample, in

which the analytes will be efﬁciently dissolved and then

extracted from the matrix. It is obvious that for an

aqueous matrix, the extracting agent will be an organic,

and vice versa. To gain sensitivity using this extraction

procedure, the volume of extracting agent can be reduced

as much as possible, but in any case, handling low

volumes of liquids and putting them in intimate contact

with a much higher volume of the liquid sample is

difﬁcult, and the risk of losing efﬁciency in the process is

high. Another disadvantage of this classic extraction is the

use of high volumes of organic solvents, which are toxic,

dangereous, and not environmental friendly.

Also included in the classic extraction panorama are

the solid–liquid extraction techniques or better known as

‘‘leakage,’’ which are usually from a solid matrix that can

be either a foodstuff or a packaging material. Among

them, Soxhlet extraction is the most common. Its high

efﬁciency is based on the fact that it is a continuous

extraction in which new solvent is continuously put in

contact with the sample while maintaining the total

volume of solvent. As the partition constant is reached

each time, the efﬁciency can be high without increasing

the total volume of solvent but increasing the total mass of

the analyte extracted. Although it is a good and well-

recognized method, it takes time, usually from 6 to 20 h,

and it requires a high amount of solvent.

An improvement of the classic extraction is the accel-

erated solvent extraction (ASE), which consists of carrying

out the extraction at high temperature and high pressure.

The increase of temperature increases the extraction

coefﬁcient and then the extraction efﬁciency, but when

increasing the temperature, the solvents used as extract-

ing agents are evaporated and they would be in vapor

phase. To avoid the evaporation and maintain the solvent

in liquid phase, a high pressure is applied, and thus the

extraction is accelerated both in time and in efﬁciency.

When the analytes are dissolved in a liquid solvent

either in aqueous solution or organic solvent, the solid-

phase extraction (SPE) is the most common technique to

isolate and concentrate the analytes and to remove the

interferences from the matrix. SPE is also useful in the

cleanup step, which is usually applied to the sample after

any extraction to remove the interferences. The solid

phase usually is inside a cartridge, ready to use, and

commercially available, and the nature of this solid phase

can vary from the current stationary phases based on

polymeric sorbents to modiﬁed silica such as C18, pure

adsorbents such as alumina or active charcoal to ionic

exchange, which are both cationic and anionic phases. Its

versatility and ease of use make SPE one of the most

common techniques in any analytical procedure in which

the isolation of analytes or concentration steps and

cleanup are required.

But without a doubt, the trends in extraction systems

drive us to the microextraction techniques in which a few

drops of solvent, if any, are employed as extracting agents

while maintaining the efﬁciency and having short periods

of extraction.

For these reasons, new microextraction techniques

have been developed to avoid the mentioned problems.

The most important ones will be described in this chapter.

Solid-Phase Microextraction (SPME)

SPME was proposed for the ﬁrst time by Arthur and

Pawliszyn in early 1990s (2), and it has been used more

and more widely in sample preparation. SPME is based on

the

partitioning of analytes

between a coated ﬁber and a

sample. The coated ﬁber consists of a small fused silica rod

covered with a thin layer of a sorbing material, which acts

as stationary phase. When exposed to the vapor phase

above a solution (head space sampling) or by direct

immersion in the solution, this ﬁber enters a mass-trans-

fer process driven by the second law of thermodynamics,

according to which the chemical potential of each com-

pound should be equal throughout the system. The che-

mical species will cross the interface until their

concentrations are such that their corresponding partial

molar free energies are the same in all parts of the system

ANALYTICAL METHODS FOR FOOD PACKAGING AND SHELF LIFE STUDIES 41

formed by the ﬁber and the sample. After the coated ﬁber

is exposed to the sample for a given period, it is inserted

into the injection port of a chromatograph to release the

analyte. In gas chromatography (GC), it is carried out by

thermal desorption, whereas in high-performance liquid

chromatography (HPLC) it is accomplished by dissolution

and injection with the elution solvent (3, 4). In both

approaches, all the substances eventually reach the ana-

lytical instrument detector to produce a trace in which

there is no large solvent peak. Although a small amount of

analyte is transferred, it is sufﬁcient to produce a sig-

niﬁcant analytical signal in modern detectors. The SPME

uses a capillary ﬁber on which the stationary phase is

coated or chemically bonded; therefore, it is a solventless

technique. Usually, only the analytes and related mole-

cules are trapped on the ﬁber, and for this reason most of

the interferences remain in the matrix and do not affect

the ﬁnal analysis (5). This technique is useful in both

modes, headspace, and total immersion, and its high

sensitivity, based on the high concentration rates that

the analytes have on the capillary ﬁber are without a

doubt their main advantages. There is no limitation for

either of the nature of samples, as both packaging materi-

als and foodstuffs can be analyzed (6–16).

Liquid Microextraction Techniques

Using liquid phases for extraction from liquid matrices,

several microextraction techniques have been recently

developed and applied to food packaging and shelf-life

studies. Among them we can point out the single drop

microextraction (SDME), the liquid–liquid–phase micro-

extraction (LLPME), and the hollow ﬁber liquid phase

microextraction (HFLPME).

SDME. SDME was ﬁrst introduced by Liu and Das-

gupta (17), but it is felt that the works by Jeannot and

Cantwell (18, 19), who studied its kinetics and mass

transfer model, and by He and Lee (20), who gave the

technique for much of today’s aspect, provided the basics

needed to consider it as an independent technique. Many

applications have been introduced since then (21) and are

summarized in many devoted reviews (22–25). It consists

of the partition of the analytes between two nonmiscible

solvents, but in this case only one drop of the extracting

solvent hanging from the tip of a syringe is used. Once the

extraction is ﬁnished, the drop is withdrawn into the

syringe and directly injected into the gas chromatograph

for the ﬁnal analysis. It can be considered as solventless

technique as well, as the amount of solvent is negligible.

Also, most of the matrix interferences are avoided in both

exposure modes, as only the compounds soluble in the

drop of solvent, either in headspace mode or in total

immersion in the sample solution are extracted. There is

no doubt that this technique is useful in many applica-

tions and allows the analysis at high sensitivity level. The

drawback of this technique is the fall of the single drop

during the sampling, which requires the procedure to

start again with a new sample, as the presence of the

lost drop of solvent in the same vial affect the extraction

greatly. In such a case, the analytes are simultaneously

extracted in both drops, although only one is analyzed,

which is why a new sampling in a different vial is

required.

Liquid–Liquid Microextraction. Several approaches have

been recently proposed to increase the extraction efﬁ-

ciency using only a few drops of organic solvent. Among

them, those that employ a few microliters of solvent

directly added to the aqueous liquid sample are useful

when using liquid samples, and no particulate matter or

slurries are involved. The supernatant solvent is recov-

ered after the extraction and analyzed. For quantitative

purposes, the use of an internal standard is compulsory to

guarantee the quanitative results, as the handling of such

a low volumes of solvents is always difﬁcult.

HFLPME. Another improvement of the SDME that

eliminates its main disadvantage is the use of polymeric

membranes as support of the small volumes of solvent.

Few microlitres can be swallowed inside the pores of a

semipermeable polypropylene capillary tube, and then

this hollow capillary can be used for extraction, which

allows high concentration factors (26, 27). When organic

compounds in aqueous solution have to be analyzed,

HFLPME is the appropriate technique for sampling and

extraction, using for instance, an organic solvent as

extracting agent inside the hollow ﬁber, being the sample

outside (see Figure 3). This technique has been recently

automatized and proposed for migration studies from

packaging materials (27).

Several studies have been carried out in the last 3 years

dealing with different approaches of this technique, and

there are several options in which up to three liquid

phases can be involved to extract and concentrate ionic

analytes from complex matrices (28–30).

TOTAL DISSOLUTION

When the analyte to be measured cannot be directly

extracted, the total dissolution of the sample is required.

This problem often happens when dealing with plastic

samples, in which the analytes are inside the matrix or

even linked to it. For example, if the shelf life of a new

Figure 3. Scheme of the HFLPME extraction from a liquid

sample.

42 ANALYTICAL METHODS FOR FOOD PACKAGING AND SHELF LIFE STUDIES

active packaging material is under study, then the remain-

ing active agents in the material should be analyzed. If it is

a plastic material, then it may be difﬁcult to apply an

efﬁcient extraction, as the recoveries are commonly low.

Thus, the total dissolution is preferred. The solvent and

conditions used for the total extraction, have to be opti-

mized. If a synthetic polymer is dissolved, the polymer

itself has to be removed from the solution, as the com-

pounds of interest are the small molecules, no the large

polymeric chains. The addition of methanol, or in general

a solvent in which the polymer is insoluble, is usually

enough to precipitate the polymer, and another ﬁltration

removes the polymer from the solution (31). Obviously, the

compounds of interest have to be soluble in the solvents

used in the precipitation step and they have to be free

from the polymer, which means that no chemical bonds

exist between the polymer and the compounds of interest.

Large volumes of organic solvents are obtained, and

additional concentration and cleanup steps are usually

required to cope with the ﬁnal analysis. Anyway, only a

few times the total dissolution is required in shelf-life

studies but often is necessary for the packaging analysis.

Final Analysis

The ﬁnal analysis means the identiﬁcation and quantiﬁ-

cation of the compounds of interest. The analytical tech-

nique to be used depends on several variables as follows:

. The chemical structure and properties of the

analytes

. The concentration level in which the analytes are

expected to be in the sample

. The information required

For volatile compounds, the most appropriate techni-

que is GC. Depending on the information required, differ-

ent detectors can be used, such as follows: (a) ﬂame

ionization detector (FID), which is ideal for organic com-

pounds with carbon atoms, as the response is based on the

carbon atoms number; (b) the electron capture detector

(ECD), which is very sensitive for electronegative atoms

such as halogens and oxygen; (c) the nitrogen-phosphor-

ous detector (NPD) with a high sensitivity for the com-

pounds that contain these atoms; and (d) the thermal

conductivity detector (TCD), which is only sensitive for

those compounds that have a thermal conductivity very

different from that of the carrier used as background or

reference, usually the hydrogen. That means that this

detector is mainly used for permanent gases, such as

nitrogen, carbon dioxide, and so on. and it is no sensitive

for other compounds. None of the mentioned detectors can

be used for identiﬁcation purposes if the nature of the

analyte is unknown, as only using the standards, what

means the pure compounds analyzed exactly in the same

conditions as the sample, the compounds could be identi-

ﬁed. For identiﬁcation purposes, the mass spectrometry

(MS) coupled to GC is the right technique. The molecule is

broken by electronic impact that produces positive ions,

which are the characteristic mass fragments of each

molecule that constitue the mass spectrum. Then, this

mass spectrum of each compound is obtained and com-

pared with the mass spectra contained in the MS library.

The software of this hyphenated technique (GC-MS) give

as the list of compounds with the most similar spectra and

is the analyst who decides which one can be present in the

sample, according to the matching parameters provided

by the software.

Two different systems for MS detectors can be chose:

the quadruple and the ion trap. For quantitative purposes,

it is generally recommended the quadrupole, whereas for

qualitative objectives the ion trap is more appropriate, as

it pemits to have the MS

fragmentation. This means that

once the molecule is fragmented and the characteristic

masses are known, the analyst can select some of these

characteristic masses and apply a new fragmentation.

This operation can be repeated several times. Even the

isomers or quiral compounds, which are very similar

between them, have differences in some of the fragmenta-

tion proﬁle; these differences can be used for unequivocal

identiﬁcation. Recent advances on the MS detectors have

launched in the market new instruments with novel

systems that increase the sensitivity of the MS detectors.

Combinations of MS-MS, triple quadrupole (TQ) or time of

ﬂight (TOF) techniques are now available. These analy-

tical tools allow the identiﬁcation of unknowns and permit

as well the quantitation of the compounds at low level of

concentration. The selection of the right technique de-

pends on the sample, the analyte, and the information

required from the sample.

There are also several options for injecting the com-

pounds into the GC. The head space (HS) technique is

useful for the analysis of volatile compounds. It consists of

injecting the vapor in equilibrium with the sample at a

ﬁxed temperature. The sample is thermostatized, and only

a fraction of the vapor in equilibrium is taken for the

analysis. This technique can be used in either manual or

automatic modes, but the reproducibility is much higher

in the latter. As only vapor is introduced into the GC, most

of the interferences from the matrix remain in the sample,

either in liquid or solid state, and the sample treatment is

considerably simpliﬁed. For this reason, this technique is

applied to a wide series of samples and volatile analytes.

The scarce handling and time consuming required make it

appropriate for the analysis of volatile compounds.

An improvement of this technique is the dynamic HS,

also called purge and trap (P&T), which is coupled to the

GC. The advantage is that in the dynamic system, the

analytes are continuously purged from the sample using

an inert gas and trapped on a solid adsorbent, which is

thermally desorbed to introduce the analytes directly into

the GC. This technique is the most sensitive for volatile

compounds, as the total mass of the analytes present in

the sample is introduced into the analytical system and

arrives at the detector (31, 32). To have an idea of the high

sensitivity of P&T-GC-MS, we can compare the real mass

of analytes at the detector in this case with that of liquid

injection in GC-MS. For example, 10 mL of sample con-

taining 1 ng/mL of a volatile analyte are analyzed by both

direct injection into GC-MS and HS-GC-MS. If 1 mLis

injected in the former case, 1 pg arrives at the detector.

However, in P&T-GC-MS, the total mass of the same

ANALYTICAL METHODS FOR FOOD PACKAGING AND SHELF LIFE STUDIES 43

volatile compound contained in the 10 mL of sample

arrives at the detector, which is 10 ng of analyte. This

amount is 10,000 times higher in the detector. To have the

equivalent mass at the detector in both cases, the concen-

tration of the analyte in the sample should be 0.1 pg/mL,

that is 0.1 ppt. However, the main drawback of this

technique is also its high sensitivity, as it is very difﬁcult

to have a blank sample, and often interfering compounds

appear and overlap the peaks of interest. Another problem

is the calibration plot for quantitative purposes, because

in this system the equilibrium is not reached, as the

volatile compounds that would be in equilibrium in the

vapor phase are continuously removed from the vapor and

trapped on the solid trap. Then, the calibration plot should

be prepared exactly in the same conditions as those used

for the sample.

Another interesting approach for injecting the volatile

or semivolatile compounds into the GC column, apart from

the injection of liquid solutions, is the SPME injection.

Nowadays, there are commercially available automatic

injectors for SPME, HS, P&T, and of course for liquid

injections into the GC.

The analysis of non/volatile compounds is usually

carried out by liquid chromatography (LC), in which the

compounds present in the sample are separated. Several

detectors can be used; the most common ones are ultra-

violet-visible spectrometry (UV-VIS), molecular ﬂuores-

cence spectrometry (Fl), refraction index (RI), and MS.

UV-VIS is the most general and common one coupled to

HPLC, whereas Fl is more selective as only ﬂuorescent

compounds can be detected. RI is usually applied to

analyze sugars, wheras MS can be applied to any com-

pound with a molecular mass higher than 50 units of mass

and able to be ionized in the ionization step of the LC-MS.

As in GC, the separation takes place in the chromato-

graphic column where the stationary phase exerts an

interaction with the compounds. Depending on the nature

of this stationary phase, either partition, ionic exchange,

or size exclusion can be the main process between the

compounds (the analytes) and the column. The analyst

has to take an important decision and choose the right LC

column, according to the analyte and the sample. Also

other conditions such as the size of the column and the

mobile phase in each analytical procedure have to be

optimized in each case. Recent developments to increase

the resolution in LC launched in the market new instru-

ments in which a higher pressure, more narrow LC

columns, and lower particle size in the LC columns occurs.

This is the case of the new systems of ultrahigh perfor-

mance, such as ultrahigh performance liquid chromato-

graphy (UPLC), which increase the resolution and

considerably reduce the time of analysis compared with

the normal HPLC. The UPLC-UV analysis is not in real

time but it can last only a few minutes to have the whole

chromatogram with more than 20 compounds (33).

When using MS as detector, one of the main decisions to

take is the ionization step. This is a critical step in which

the analytes are transformed into ions, either positive or

negative ions, which can be driven to the MS detector in

which they are separated according to their mass and then

counted (abundance). The ionization step in LC is

commonly a soft ionization that mainly produces the

molecular ion. The interface between the ionization step

and the MS analyzer has been the most difﬁcult part of the

development of this hyphenated technique of LC-MS. This

is because the ionization step is applied to the liquid

sample injected into the LC and the nonvolatile solvent,

usually water, methanol-water, or acetonitrile, which has

to be removed efﬁciently to get the high vaccum required

for MS, to avoid the saturation of the MS detector, and to

remove the interferences associated to the solvents. Two

different ionization devices and techniques are the most

common in current LC-MS, and they are the Electrospray

(EI) and the atmospheric pressure chemical ionization

(ApCI), although more and more the EI is gaining impor-

tance, as most problems are solved using the EI.

During the last 10 years, new approaches dealing with

LC-MS have been launched to the market to increase the

sensitivity and reproducibility, and to widen the type of

analytes that can be analyzed. Also, the instrumental

equipments are nowadays more friendly for the users

and easier to handle.

Also, the advanced MS techniques such as the TOF

supply new tools for the identiﬁcation of unknowns, using

the exact mass values. Without a doubt the identiﬁcation

of unknown compounds is the most challenging area, and

new and more sophisticated techniques are required for

this purpose. Artifacts formed during the analysis, degra-

dation compounds caused by the interaction between the

components in the packaging materials or in the food, or

degradation compounds coming from the additives used in

the food or from the packaging materials in contact with

the food, which include the presence of compounds non

intentionally added, pose new analytical problems and

make the analyst face a real challenge in food packaging

and shelf-life studies. To help with the identiﬁcation, MS-

MS can be used. As was mentioned above, this consists of

identifying the characteristic ion (MS) and then applying

the fragmentation to this ion to break this fragment. The

breakage of each fragment is speciﬁc and is of great help to

identify the compounds. Triple quadrupole instruments in

which the ﬁrst fragments (parent ions) obtained in the

ﬁrst quadrupole goes through the second quadrupole in

which a gas, usually argon, enters and crash into the

fragments, causing the second fragmentation (daughters)

of each ion, which are analyzed in the third quadrupole.

These systems are also available in LC-MS and are useful

for identiﬁcation purposes, although the sensitivity using

the triple quadrupole is lower than that obtained when

using only the ﬁrst quadrupole.

Recently double-dimension chromatography has been

developed.

This is a

new hyphenated technique in which

two different chromatographic separations using two col-

umns of different polarity and different size each, con-

nected in series, are applied to the same sample. Usually,

the ﬁrst column is of normal size, for example 30 m in GC,

and the second one is short, about 1.5 m. The ﬁnal detec-

tion can be FID or even better the MS or its different MS

options. This double dimension can be also used in liquid

chromatography. In this case, the use of for example a

size exclusion column as the ﬁrst one to eliminate the

polymers, proteins, or in general the macromolecular

44 ANALYTICAL METHODS FOR FOOD PACKAGING AND SHELF LIFE STUDIES

compounds, and a reverse phase (C18 or similar) as second

dimension can be a good alternative for analyzing in one

single run the compounds of interest without the inter-

ferences from the matrix (34).

A different approach from chromatography is electro-

phoresis, where the compounds are separated based on

the application of an electric ﬁeld. Then, the ionic com-

pounds move through the liquid solvent according to their

mass and their charge. Although gel electrophoresis is old

and novery sensitive, the new capillary electrophoresis

(CE) has been shown as a good technique for ionic organic

molecules, such as drugs, proteins, amino acids, and so on,

as these compounds can be easily transformed into ionic

ones just controlling the pH. This technique can be also

used in shelf-life studies, but its frequency is not as high

as the chromatographic techniques. The new hyphenated

technique CE-MS can be one alternative in future studies.

OTHER ANALYTICAL PROCEDURES

Figures 4, 5, and 6 summarize the most common techni-

ques for shelf-life studies. Besides the analytical techni-

ques mentioned above, the optical techniques, such as UV-

Vis spectrophotometry or infrarred spectrometry (IR) are

useful. The former is used to measure the color and color

changes (VIS) occurred in the food or in the packaging

materials either in absorption or in reﬂexion modes. The

latter (IR) is used to identify the packaging materials or

organic components. The most recent advances in near

infrared (NIR) allow also to quantify some components in

not complex samples, mainly in packaging materials more

than in food.

Also, the combination of different techniques provide

an useful an important information in the shelf-life stu-

dies (35) and can be observed as a powerful tool for having

as much information as possible about the state of the

packaging material, the food inside the packaging, and the

internal atmosphere between them.

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• Microbiology

• Compounds released

Spectrophotometry

Reflexion spectrometry

Colorimetry (CIE-LAB)

In vitro tests

In vivo tests

Cell culture

PCR

SEM

Volatile compounds

Degradation compounds

Food artifacts

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Figure 6. Testing the packaging.

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Figure 5. Testing the internal atmosphere.

ANALYTICAL METHODS FOR FOOD PACKAGING AND SHELF LIFE STUDIES 45

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ANTI-COUNTERFEITING PACKAGING

STEVE WERBLOW

Steve Werblow Communications

Ashland, OR

Counterfeiting is a huge business, threatening not only

sales revenue, but also the health and safety of consumers

who may end up with adulterated food or medicine, unsafe

engine parts, or other dangerous fake goods. Liability for

those counterfeit products, as well as the negative pub-

licity that can befall a company whose brand has been

compromised by piracy, can have devastating impacts on

business.

In the United States, the Brand Protection Alliance

asserts that counterfeiting and piracy cost the U.S. econ-

omy $200 to $250 billion per year. The Organization for

Economic Co-Operation and Development in Europe cal-

culated that international trade alone in counterfeit goods

represented an additional $200 billion in 2005; the Inter-

national Chamber of Commerce (ICC) estimates the

total counterfeit economy at more than three times that

amount. In fact, in the middle of the ﬁrst decade of the

new millenium, ICC ﬁgured the counterfeiting industry

represented about 10% of world trade, equivalent to the

12th largest economy in the world.

Counterfeit products range from cheap bootleg videos

to phony bottles of wine sold for thousands of dollars to

fake aircraft parts.

Fortunately, label and packaging technology can help

deter or thwart counterfeiters. Printing techniques, ma-

terials such as substrates and inks, and high-tech sensors

can all play a role in protecting brand integrity.

OVERT AND COVERT

The ﬁrst decision to make in an effort to protect a brand

from counterfeiting is to determine whether the effort

should be visible or invisible to the counterfeiter.

A highly visible, or overt, tactic can warn off potential

counterfeiters, much as a ﬂashing alarm light does in an

expensive car. An adept thief could break into the vehicle

and disable the alarm, but is more likely to consider

saving the time and hassle by moving on to an unprotected

vehicle.

Overt measures can also communicate to consumers. It

may put them on guard against counterfeit products. It

may reinforce a sales message that says, in essence, ‘‘this

product is so desirable, people want to counterfeit it.’’ And

it may also reassure customers that the product they are

buying is well protected.

By contrast, covert measures tend to be secret and

unobtrusive, generally revealed only through close obser-

vation or with special readers or detectors. The theory

behind covert tactics is often that if counterfeiters do not

know what the authentication features are, they are less

likely to fake them.

Many anti-counterfeiting efforts rely on a combination

of overt and covert methods. It’s like protecting your home

with a good lock, a loud dog, and a burglar alarm.

TACTICS AND MATERIALS

The U.S. $20 bill introduced in 1998 illustrates the use of a

variety of anti-counterfeiting features, all of which may be

employed in the manufacture of labels and packaging.

Rare paper, embedded ﬁbers and strips, watermarks,

46 ANTI-COUNTERFEITING PACKAGING

sophisticated printing, special inks—each feature makes

the bill difﬁcult to reproduce and easy to authenticate.

Special Paper

The U.S. currency anti-counterfeiting effort begins with

cotton/linen paper manufactured speciﬁcally for the U.S.

Bureau of Engraving and Printing for the production of

currency. A swipe with an iodine pen is an easy authenti-

cation measure: Unlike wood-based paper, which contain

starches that turn black in the presence of iodine, the

linen/cotton blend will not show a dark line where the pen

left its mark.

In addition to its unique composition, the banknote

paper includes embedded red and blue ﬁbers, so average

paper stocks cannot be substituted for the real thing.

Papers with embedded ﬁbers or planchettes—in-

clusions that look like confetti or tiny dots—are widely

available as label stocks. Even the use of expensive or

unusual substrates, such as textured stock or parchment,

may serve as a mild deterrent to counterfeiters.

Watermarks

Watermarks are a classic anti-counterfeiting feature found

in the U.S. notes as well as currency and other important

documents around the world. A watermark is created

during the manufacture of the paper: While the paper is

still wet, it is pressed with a cylinder roll bearing an image

in relief. The raised areas on the roll compress ﬁbers in the

paper, making the paper thinner in those areas. Thinner

paper transmits more light, allowing the watermark to

‘‘appear’’ when light is shined through the note.

Embossing

Embossing a document, label, or package—creating a

raised ﬁgure or pattern on the surface using a die, heat,

or varnish—can be used to deter counterfeiters. Emboss-

ing and texture are increasingly important as scanners

and printers improve. Though new imaging technologies

are better than ever at reproducing color and print quality,

they cannot reproduce the texture of an embossed surface.

Reactive Inks

Inks formulated to react to stimuli by changing color

challenge counterfeiters and can be relatively easy for

consumers to recognize. The $20 bill features an optically

variable ink (OVI) that appears to shift from green to

black as light hits it from different angles. Thermochro-

matic inks, which change color in response to changes in

temperature, can be activated when a shopper puts her

thumb on a label. Other inks may ﬂuoresce in the presence

of ultraviolet (UV) light or change colors when exposed to

speciﬁc chemicals.

Inks with highly speciﬁc spectral signatures can be

authenticated with photospectrometers, even when they

appear no different from counterfeiters’ inks to the naked

eye. Inks provide a good illustration of the difference

between overt and covert technology. Thermochromatic

inks are overt—the authenticating feature can easily be

seen by the public. Measuring the photospectral signature

of an ink requires a specialized reader and the knowledge

that the feature is included in the package, making it a

covert technology.

Taggants

Inks may also be impregnated with taggants, microscopic

markers that can be detected with various decoders. For

instance, a microscope can reveal cell-sized particles that

feature a company’s logo. A hand-held reader wand can

detect speciﬁc reﬂection, refraction or electromagnetic

properties of taggants in ink. One Australian wine produ-

cer even blended DNA from a grape vine into its label ink

as a very unique taggant.

Printing Techniques

High-quality printing is a classic anti-counterfeiting mea-

sure. Microprinting, or the creation of extremely tiny

characters such as the words ‘‘United States of America’’

around Andrew Jackson’s portrait on the U.S. $20 bill,

cannot be readily achieved by most small printers or

replicated on inkjets in a home counterfeiting operation.

Similarly, printing elaborate scrollwork and other ultra-

ﬁne geometric patterns—called guilloche printing—can

challenge counterfeiters. Some anti-counterfeiting compa-

nies offer sophisticated guilloche patterns that reveal a

signal word, such as ‘‘VOID,’’ if a protected document is

photocopied. Digital ‘‘watermarks’’ can be embedded in

graphics and detected by scanners.

Holograms

A technology that got its start in the 1940s, holograms

have long been used to authenticate valuable items such

as credit cards, electronic parts, software, and even

clothing.

There are two types of holograms. Surface-relief, or

rainbow transmission, holograms are the familiar rain-

bow-colored images that are common in packaging be-

cause of their low cost and minimal thickness. Surface-

relief holograms are embossed onto thermoplastic ﬁlm

backed with aluminum. Lippmann holograms—created

by capturing wave interference patterns on special photo-

graphic materials—offer the illusion of greater depth and

clarity, are more difﬁcult to produce, and can include

multiple frames, microprinting, and extremely ﬁne details

that can aid in authentication.

Holograms are interesting and attractive enough to

capture the eye of consumers, which offers some overt

brand protection, but the relative ease of counterfeiting

surface-relief holograms themselves mean that the most

effective security features on a good hologram are more

covert.

A variant on holograms, latent image technology, uses a

polarizer to reveal a very sharp, difﬁcult-to-counterfeit

image.

Electronic