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CHROMA-355083; No. of Pages 9 ARTICLE IN PRESS

Journal of Chromatography A, xxx (2014) xxxxxx

Contents lists available at ScienceDirect

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

Review

Youden test application in robustness assays during method

validation
Eftichia Karageorgou, Victoria Samanidou
Laboratory of Analytical Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece

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

Article history: Analytical method validation is a vital step following method development for ensuring reliable and
Received 6 November 2013 accurate method performance. Among examined gures of merit, robustness/ruggedness study allows
Received in revised form 15 January 2014 us to test performance characteristics of the analytical process when operating conditions are altered
Accepted 17 January 2014
either deliberately or not. This study yields useful information, being a fundamental part of method
Available online xxx
validation. Since many experiments are required, this step is high demanding in time and consumables.
In order to avoid the difcult task of performing too many experiments the Youden test which makes use
Keywords:
of fractional factorial designs and has been proved to be a very effective approach. The main advantage
Method validation
Robustness
of Youden test is the fact that it keeps the required time and effort to a minimum, since only a limited
Ruggedness number of determinations have to be made, using combinations of the chosen investigated factors. Typical
Youden test applications of this robustness test found in literature covering a wide variety of sample matrices are
briey discussed in this review.
2014 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.1. Food analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.2. Biouids and pharmaceutical analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
1.3. Environmental analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
2. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
Conict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. Introduction

According to the International Organization for Standardization, Method validation has been studied extensively in the literature,
method validation is dened as conrmation through examina- as well as in several guidelines from regulatory agencies, including
tion and provision of objective evidence that the requirements for Eurachem [2], the International Union of Pure and Applied Chem-
a specied intended use or application are fullled [1]. istry [3], International Conference on Harmonization (ICH) [4], the
The scientic applicability of an analytical method is required U.S. Food and Drug Administration [5] and the European Commis-
in terms of validation procedures not only in new method devel- sion Legislation through Commission Decision 2002/657/EC [6].
opment, but for established method revision, methods used in The ofcial guidelines do not present a validation experi-
different laboratories, the employment of different analysts or ment sequence because the optimal sequence may depend on the
instrumentation, and for demonstration of equivalence between method itself and the application. For quantitative analytical HPLC
two different methods as well [2]. methods, however, the following sequence has proven to be useful
(Fig. 1):

Corresponding author. Tel.: +30 2310997698; fax: +30 2310997719. 1. Selectivity

E-mail address: samanidu@chem.auth.gr (V. Samanidou). 2. Linearity and range

http://dx.doi.org/10.1016/j.chroma.2014.01.050
0021-9673/ 2014 Elsevier B.V. All rights reserved.

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Table 1
Potential factors to be examined in the robustness testing of main separation
techniques.

Separation technique Factors

HPLC Mobile phase constituents

Mobile phase pH
Organic modier %
Buffer concentration, salt concentrations or
ionic strength
Concentration of additives (ion pairing agents)
Flow rate
Column temperature
Gradient elution: initial mobile phase
composition slope of the gradient
Column stationary phase
Column manufacturer
Wavelength of detection

Gas chromatography (GC) Injector temperature

Fig. 1. The main factors examined during a method validation procedure.
3. Precision (repeatability and intermediate precision)
4. Accuracy
5. Detection limit
6. Quantitation limit
7. Robustness/ruggedness
8. Stability [4].
The knowledge of the robustness/ruggedness of analytical pro-
cesses is an essential feature in the analytical validation [7].
Robustness is dened as the susceptibility of an analytical method
to changes in experimental conditions which can be expressed as
a list of the sample materials, analytes, storage conditions, envi-
ronmental and/or sample preparation conditions under which the
method can be applied as presented or with specied minor mod-
ications. For all experimental conditions which could in practice
be subject to uctuation (e.g. stability of reagents, composition of
the sample, pH, temperature) any variations which could affect the
analytical result should be indicated. Robustness and ruggedness
are closely related terms often used interchangeably in the litera-
ture [6]. Although there is a confusion between the terms due to
their similarity, ruggedness is an older term replaced by intermedi-
ate precision which is a measure showing how well is the method
performing under normal conditions from lab to lab, instrument to
instrument and analyst to analyst. Robustness is the measure show-
ing the performance of the method when small deliberate changes
are made to the normal conditions [8].
Robustness testing is performed at the end of development or
the beginning of validation. When method robustness is found to
be acceptable, the method can be further validated, and when the
validation results are regarded as satisfactory, it can be routinely
applied. Otherwise, the method should be adapted or redeveloped
[9].
A robustness study examines the alteration of factors impor-
tant for the analytical procedure. In experiments to study the main
effects of factors, screening designs are used. Plackett and Burman
developed such designs which are suitable only when the inter-
actions are negligible or when considering a key set of dominant
factors, since their designs cannot deal with factor interactions [10].
The basic idea of Plackett and Burman designs in brief, is described
as follows. The strategy is based on a landmark procedure sug-
gested by Youden in 1967: (a) identify the inuential factors; (b) for
each factor, dene the nominal and the extreme values expected in
routine work and encode them as follows: nominal value = 0, high
value = +1 and low value = 1; (c) arrange the experimental design
by using a two level 27-4 fractional PlackettBurman design; and
Column temperature
Detector temperature
Temperature program (initial and nal
temperature)
Slope of the temperature Gradient
Gas ow-rate
Split or splitless conditions
Liner type
Column manufacturer
Column stationary phase
Capillary electrophoresis (CE) Electrolyte concentration
Buffer pH
Concentration of additives
Temperature
Applied voltage
Sample injection time
Sample concentration
Rinse times
Wavelength of detection
(d) perform the experiments in random order on a control sample
with analyte concentration halfway in the concentration range of
the method scope. Youden selected a 27-4 PlackettBurman design
because it enabled the study of up to seven factors in eight experi-
ments. The eight runs are split into two groups of four runs on the
basis of levels +1 or 1. The effect of every factor is estimated as
the difference of the mean result obtained at the level +1 from that
obtained at the level 1. Once effects have been estimated, to deter-
mine whether variations have a signicant effect on the result, a sig-
nicance t-test is used. The t-value is compared with the 95% con-
dence level two-tailed tabulated value with the degrees of freedom
coming from the precision study for each concentration [11].
Youden and Steiner in 1975, introduced a useful robustness test
to analytical chemistry [12]. These tests examine, within one single
laboratory, the susceptibility of analytical methods against small
changes in the method parameters using experimental designs.
Such preliminary tests are less expensive than collaborative studies
and allow adjusting the method before it is studied collaboratively
[13].
Youden robustness test can be performed either during the
optimization of the separation technique used, or during the opti-
mization of sample preparation. Various factors important for the
analytical procedure can be investigated. Tables 1 and 2 summarize
the most important separation and sample preparation techniques
used, as well as they give examples for the potential factors that
can be used for the robustness study.
Other approved methods may be used at this point. The Youden
approach, however, keeps the required time and effort to a
minimum. This robustness test is a fractional factorial design. Inter-
actions between the different factors cannot be detected [6].
The basic idea is not to study one alteration at a time but to
introduce several variations at once. As an example, let A, B, C, D, E,

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Table 2 statistical treatment, like method-performance studies (collabo-

Potential factors to be examined in the robustness testing of common sample prepa-
rative trials), laboratory-performance studies (prociency tests),
ration techniques.
collaborative bias evaluation, inter-laboratory evaluation of to-be
Sample preparation technique Factors standard methods as well as certication studies for reference
SPE Sorbent type materials. Furthermore, new robust statistics and graphical meth-
Sorbent manufacturer ods were taken into account [13].
Sorbent mass Accuracy proles of chemical assays were introduced and
Sample mass or volume
derived from the uncertainty of the analytical result. Gonzalez et al.
Wash solvent
Elution solvent in 2006 explained the calculation of accuracy proles, based on the
Evaporation temperature estimation of the measurement uncertainty of the analytical assay
pH of sample from validation data. For the sake of illustration, a case study deal-
pH of buffer constituents in solvents
ing with the spectrouorimetric determination of quinine in tonic
MSPD Sorbent type water is explained in detail [14]. Also in 2007, the same authors
Sorbent manufacturer gave more information on the topic, through a detailed step-by-
Sorbent mass
step guide to analytical method validation, considering the most
pH of sample
pH of buffer constituents in solvents relevant procedures for checking the quality parameters of ana-
Sonication time lytical methods. Using a holistic approach, authors also explained
Evaporation temperature the estimation of measurement uncertainty and accuracy proles,
Wash solvent
which they discussed in terms of accreditation requirements and
Elution solvent
Sample mass or volume
predened acceptability limits [10].
Dejaegher et al. in their tutorial in 2009, introduced the appli-
cation of experimental designs in separation science. Method
F, G denote the nominal values for seven different factors that could optimization is often divided into screening and optimization
inuence the results, if their nominal values are changed slightly. phases. In the rst step, many factors, potentially affecting the sep-
Let their alternative values be denoted by the corresponding lower aration, are tested to identify those with the largest effects. These
case letters a, b, c, d, e, f and g. This results in 27 or 128 different are then further examined in an optimization phase, to determine
possible combinations. It is possible to choose a subset of eight of the best separation conditions. During the rst phase of method
these combinations that have a balance between capital and small optimization and during robustness testing, screening designs are
letters (Table 3). Eight determinations have to be made, which will usually applied, whereas during the second phase of optimization,
use a combination of the chosen factors (AG). The results of the response-surface designs are often used. In this tutorial, the differ-
determinations are shown in Table 3 as sz [6]. ent steps in the application of both types of design are explained
After the comparison of the averages of the capital letters (AA and elaborated with some examples [9].
to AG) with the averages of their corresponding small letters (Aa In 2013 Cuadros-Rodrguez et al. discussed a dual general pro-
to Ag), the effect of a factor is evaluated, when the difference is cedure for checking robustness/ruggedness in both intrinsic and
signicant larger than the differences of the other factors. Standard extrinsic validation. Inertia study is introduced to designate
deviation of the differences Di (SDi ) is calculated according to the this methodology in extrinsic validation, whereas precision and
equation: trueness tests are proposed inside the inertia study. Several exper-

  D2 
imental designs, depending on the goal of robustness/ruggedness
i study and the type of variables (quantitative or qualitative) are
SDi = 2
7 considered [7].
In the eld of pharmaceutical analysis one review is published
When SDi is signicantly larger than the standard deviation of in 2013 trying to assist in the planning of validation procedures in
the method carried out under within-laboratory reproducibility quantitative high-performance liquid chromatographic methods.
conditions it is a foregone conclusion that all factors together have Authors also discuss relevant approaches of various parameters.
an effect on the result, even if every single factor does not show a Finally, this article provides several up-to-date examples, which
signicant inuence and that the method is not sufciently robust are useful as an introduction to analytical validation for practical
against the chosen modications [6]. applications either in academic research or the industrial sector
Various review studies are available in the literature referring [15].
to the usefulness of validation process and Youden robustness Scope of this review article is to present characteristic appli-
test in analytical chemistry. In year 2000 Hund et al. dis- cations of Youden test, during the validation process of analytical
cussed various inter-laboratory studies performed with different methods in the eld of food, biouids and pharmaceutical as well
aims and consequently require different evaluation methods and as environmental analysis.

Table 3 1.1. Food analysis

Robustness tested by applying seven small but deliberate changes in the operating
parameters. A liquid chromatographic method with tandem mass spectro-
Parameter Experiment number metric (LCMS/MS) detection and identication is proposed for the
determination of chloramphenicol (CAP) in honey. After a prelimi-
1 2 3 4 5 6 7 8
nary dissolution in water, samples were extracted with a mixture of
A/a A A A A a a a a dichloromethane/acetone and evaporated to dryness; the follow-
B/b B B b b B B b b ing clean up was carried out on an octadecyl SPE cartridge. Method
C/c C c C c C c C c
D/d D D d d d d D D
ruggedness was estimated by means of the Youden robustness
E/e E e E e e E e E test. Seven different variables chosen within the entire analyti-
F/f F f f F F f f F cal process were taken into account in the evaluation of method
G/g G g g G g G G g ruggedness: dichloromethane concentration in the extraction mix-
Observed results s t u v w x y z
ture, extract evaporation temperature, extract evaporation mode,

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buffer pH, SPE cartridge elution mode, SPE cartridge elution vol-
ume, ammonium acetate concentration. The experimental design
was planned with the aim of identifying the critical points in the
procedure. The effect of each factor was estimated according to the
Youden approach. As the obtained value was less than the standard
deviation of the within-laboratory reproducibility, it was demon-
strated that all selected factors together do not signicantly affect
the analytical performance. Besides this general result, the inu-
ence of each variable on method robustness was also evaluated by
applying the t-test and no factor had a signicant effect on rugged-
ness. The ruggedness test demonstrated that the performance of the
method was not inuenced by minor changes in the key analytical
variables [16].
A conrmatory method based on high-performance liquid
chromatographytandem mass spectrometry (HPLCMS/MS), for
the simultaneous determination of lincomycin and ve macrolide
antibiotics in honey, was developed. The analytes were extracted,
and cleaned-up by a single solid-phase extraction step on OASIS
HLB column. Validation of the method performed according to
European Commission Decision 2002/657/EC: linearity, specicity,
decision limit, detection capability, repeatability, reproducibility,
recovery and ruggedness. The robustness test was conducted by
the Youden procedure cited by Commission Decision. Seven vari-
ables were chosen and deliberately altered: the volume of dilution
buffer, pH and molarity of dilution buffer, the methanol percent-
age during the washing steps of the SPE purication, the ammonia
solution percentage in the elution solvent, the SPE elution volume,
and the evaporation temperature of the solvents in the nal extract.
The results indicate that the method is robust and minor but still
signicant uctuations in the operative parameters that can occur
during the routine application of the method have no signicant
effect on its performance characteristics [17].
An experimental design developed by Youden and Steiner was
applied to micro-fermentation experiments with two different
grape musts, in order to verify the impact of several parameters on
the fermentation course and on avor development. The follow-
ing seven parameters were studied: turbidity of the must, yeast
strain, temperature of the alcoholic fermentation, de-acidication
of the must, suspension of the lees, presence of oxygen and nitrogen
supplementation. The inuence of each parameter on the fermen-
tation process is determined by calculating the mean value and the
absolute differences between the two variants. Results indicated
the positive effects of a higher fermentation temperature on the
development of 3-mercaptohexanol, an important contributor to
the characteristic aroma of the Petite Arvine wine, whereas results
obtained with the vinication trials conrm previous studies, thus
proving the correctness of the experimental design [18].
Anethole was determined in aniseed drinks by high per-
formance liquid chromatography (HPLC). A C18 column and a
methanolwater (80:20, v/v) mobile phase with isocratic elution
were used. UV detection at 257 nm was carried out. Validation
was performed according to the EURACHEM guidelines. In order to
establish the robustness of the proposed method, the inuence of
four factors was studied, column temperature, % of methanol in the
mobile phase, ow rate and wavelength of detection. Robustness
study was performed according to Youden approach. Furthermore,
an experimental design was performed by using a two level 27-4 fac-
torial PlackettBurman design. There are no signicant differences
between the results obtained when factors have the maximum
and minimum values. Therefore, these factors do not affect to the
method development so that the robustness of the method can be
assessed [19].
An HPLC method with uorimetric detection was developed for
the determination of aatoxin M1 (AFM1) in milk. Validation was
performed according to European Decision 2002/657/EC includ-
ing selectivity, recovery, precision, decision limit (CC), detection
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capability (CC) and robustness. Robustness was evaluated through
the fractional factorial Youden design by selecting seven factors as
variables for Youden test: milk thermal treatment (raw or pasteur-
ized), evaporation temperature, storage at +4 C for 1 day, eluate
ltration, immunoafnity cartridge lot, and lot and commercial
company of methanol and acetonitrile. Results indicated that the
method was not affected by slight variations of some critical factors
as pre-treatment and clean-up of milk samples, thermal treatment
and different storage conditions, as well as by major changes val-
ued in terms of milk produced by different species (buffalo, goat
and sheep) [20].
A GCMS method for the determination of anabolic steroids
used as growth promoting agents in piglet feed samples has been
developed and validated according to the Commission Decision
2002/657/EC criteria. The robustness was evaluated using the
Youden test. For this purpose, minor changes of seven variables in
the sample preparation procedure were chosen: operator, leach-
ing volume, saponication-volume, saponication-temperature,
liquidliquid extraction (LLE) volume, evaporation temperature of
the nal extract, and derivatization-time. Following the experi-
mental plan, results indicate that Saponication-temperature is
the parameter with the biggest inuence on the robustness of the
method. Moreover, all the chosen factors together do not signi-
cantly affect the method performances [21].
The same group of authors developed an improved sample
preparation procedure for the determination of 17 steroids (cor-
ticoids and androgenic anabolic steroids) in feed samples. This
procedure is based on two reported LCUV methods. Validation was
performed meeting European Legislation criteria. Robustness of the
method was carried out using the Youden test described in the
Directive 2002/657/EC. The variables chosen were: the analyst, lix-
iviation volume, several SPE parameters (cartridges age, ultrapure
water pH wash, % MeOH wash, elution volume), and nal evap-
oration temperature. The obtained results indicate SPE % MeOH
wash is the parameter with the larger inuence, whereas all the
chosen factors together do not signicantly affect the method per-
formances [22].
A precise method for the determination of 10 sulphonamide
antibiotics in egg by liquid chromatographytandem mass spec-
trometry (LCMS/MS) has been developed. Drugs were extracted
using a mixture of dichloromethane/acetone (50:50, v/v), acidi-
ed with acetic acid and then cleaned-up on a cation-exchange
solid-phase extraction (SPE) cartridge. The method was validated
according to European Community Guidelines, whereas robust-
ness was estimated according to the Youden test. The chosen
investigated factors are: dichloromethane in the extraction, addi-
tion of acetic acid, SPE sulphonic cartridge, acetic acid for SPE
conditioning, ammonia for SPE elution, SPE elution volume and
ammonium acetate in the mobile phase. The results arising from the
eight relevant robustness experiments indicate a decrease in sul-
famethoxazole recovery rates when the acetic acid concentration is
at low level. The uctuations introduced in the current parameter
were in the range between 50% around the nominal value (5%)
instead of the common 10% and so they represent a heavy mod-
ication in the analytical procedure. In fact, these variations can
hardly be included in the typical denition of minor changes and
consequently it cannot be inferred that the variable into account
has a signicant effect on method ruggedness, so that the method
itself is not able to withstand minor uctuations in the operative
parameters that can occur during its routine application [23].
A sensitive multiresidue method is developed for the simul-
taneous determination of tropane alkaloids in grains and seeds
(wheat, rye, maize, soybean, linseed). The analytes were sepa-
rated by isocratic HPLC and detected using a triple quadrupole
mass spectrometer with electrospray ionization (ESI). For a fast
and effective clean-up procedure for oily matrices such as soybean
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and linseed, matrix solid phase dispersion (MSPD) C18 material
was used to remove co-extracted non-polar components. Method
performance is presented in terms of linearity, specity, selectiv-
ity, accuracy, precision and ruggedness. In this study, the Youden
approach, a fractional factorial design, to determine any critical
points was employed. The factors examined were: extraction pH,
centrifuge speed, evaporation temperature, buffer concentration,
column temperature and mobile phase ow. The test conrmed
that the method was not signicantly inuenced by the combined
minor variations introduced and can be considered acceptably
robust [24].
In this study, a validated HPLC method with uorescence detec-
tion for the simultaneous quantication of hydroxytyrosol and
tyrosol in red wines is presented. The validation of the analytical
method was based on selectivity, linearity, robustness, detection
and quantication limits, repeatability, and recovery. A fractional
factorial experimental design including percentage of pH, modier
in solvents, ow rate, equilibrating time, percentage of ethanol in
the sample and column temperature on the peak area for hydroxy-
tyrosol and tyrosol, was performed to analyze the inuence of these
six different conditions on analysis. Fractional factorial design was
chosen instead of full factorial design to reduce the total number of
necessary experiments. Non signicant differences were found in
the resulting peak areas for both peaks. Therefore, the developed
method was proven to be robust in the assayed experimental con-
ditions for the six studied variables. The nal optimized method
allowed the analysis of 30 nonpretreated Spanish red wines to
evaluate their hydroxytyrosol and tyrosol contents [25].
For the quantitative determination of polyphosphates in prod-
ucts of animal origin (meat, dairy and sh products), an ion
chromatography with suppressed conductivity detection method
was developed. The method validation, performed according to
Regulation 882/2004/EC and Decision 657/2002/EC in terms of lin-
earity, specicity, precision, recovery, detection and quantication
limits and ruggedness. Method robustness was tested by using
the Youden experimental design, performed for different prod-
ucts of animal origin: cooked ham, wurstel, processed cheese and
surimi. The seven factors used as Youden test variables were the
matrix and six ctitious factors; therefore, the experimental design
requires eight independent experiments, four with the validation
matrix (cooked ham) and four with each alternative matrix. Results
indicated that the matrix variation has no effect on the analytical
performances, and, consequently, the method is also applicable to
wurstel, processed cheese and surimi samples [26].
Authors have had a thorough research on the presence of
antimicrobials agents in milk, food of animal origin. They have
developed various multi-class conrmatory methods by liquid
chromatography, using analytical columns of conventional and
novel technology [2730]. Validation was fully performed accord-
ing European Decision 2202/657/EC criteria in terms of specicity,
linearity response, trueness, precision (repeatability and between-
day reproducibility), decision limit (CCa), detection capability
(CCb), and ruggedness. In the eld of sample preparation, an
innovative ultrasound-assisted MSPD approach together with new
sorbent materials was used, and all target analytes were well
resolved from complex milk matrix.
In the rst method, both penicillins and amphenicols were
studied as described. The robustness of this method was assessed
according to the Youden and Steiner approach. The basic idea is
instead of studying one alteration at a time, several variations are
introduced at once. This was performed by adopting the experi-
mental design described in Decision 2002/657/EC and tested by
the introduction of seven small but deliberate changes in the
operating parameters (variables) during sample preparation pro-
cess and by the consequent assessment of their inuence on the
method results. The changes were: mass of Strata sorbent, mass of
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5
QuEChERS sorbent, sonication time, milk volume, volume of wash
solvent, volume of methanol, and volume of ACN. The results indi-
cate that the factor that has less inuence is the sonication time.
However sonication is considered as absolutely necessary during
the pre-treatment procedure because it ensures efcient contact
between the solid and the extractant. Change in sorbent mass inu-
ences signicantly either in positive or negative way ve out of
seven analytes. Furthermore, the developed method was proven to
be robust in the assayed experimental conditions for the sum of
studied variables [27].
In the second method 12 -lactam antibiotics, (four penicillins
and eight cephalosporins) were effectively extracted and deter-
mined from milk matrix. Method ruggedness was estimated for
minor changes by means of the seven parameters/eight exper-
iments design, also known as the Youden and Steiner test. The
inuence of following parameters on the results were tested: mass
of Oasis sorbent, mass of QuEChERS sorbent, sonication time, wash
solvent volume, amount of acetone in wash solvent which is water,
eluent volume (MeOH), and eluent volume (ACN). From the results
arising, the factor that has the minor inuence is the mass of Oasis
sorbent. On the other hand the most important parameter is the vol-
ume of methanol. Any change of this factor during the elution step
inuences signicantly either in positive or negative way ve out
of twelve examined antibiotics. Less negative or positive inuence
on target analytes have factors of the mass of QuEChERS sorbent
and eluent volume ACN, whereas all the chosen factors together do
not signicantly affect the method performances [28].
A sensitive and selective conrmatory method for milk-residue
analysis of ten quinolones and eight cephalosporins by LCMS/MS
has been developed in the third method. The Youden approach was
used for the ruggedness test of the method. The following parame-
ters were tested: mass of Plexa sorbent, mass of QuEChERS sorbent,
sonication time, percentage of acetone in wash solvent, volume of
wash solvent which is water, volume of methanol, and volume of
ACN. From the examined factors the mass of Plexa sorbent is the
one with the major inuence on most of the analytes. Factors of
less inuence appear to be the mass of QuEChERS sorbent and son-
ication time, while the volume of solvents in washing and elution
steps has the minor inuence. The method proved to be robust for
all investigated factors together [29].
In the last method tetracycline antibiotics were examined.
Target analytes were determined by an accurate and sensitive chro-
matographic analytical method. Method robustness was estimated
for minor changes by means of the Youden and Steiner test. Seven
different factors were chosen, because of their possible critical
inuence. These included sonication time, sorbent material, evap-
oration temperature, milk volume, concentration of oxalic acid,
volume of elution solvent, and nature of elution solvent. Evapora-
tion temperature inuences in a negative way all compounds and
especially OTC. This effect was expected as tetracyclines are tem-
perature sensitive. The nature of organic solvent at elution step
inuences positively all tetracyclines and the most TC in favor of
acetonitrile. Moreover, all the chosen factors together do not sig-
nicantly affect the method performances [30].
1.2. Biouids and pharmaceutical analysis
For the determination of captopril in tablet dosage forms, a
high performance liquid chromatorgaphic (HPLC) procedure using
indirect photometric detection was developed. A low capacity
anion-exchange column was used with potassium phthalate as the
mobile phase marker and indirect detection at 280 nm. The chro-
matographic conditions were optimized using the Box and Behnken
factorial experimental design. The mobile phase related factors
including pH, ow rate, marker concentration, % organic modi-
er, along with the column temperature, detector wavelength and
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column age were chosen as the seven variables for Youden and
Steiners robustness test. The effect of each variable was investi-
gated at two levels. A total of eight experiments were conducted
in order to identify variables that may have to be controlled in
order to obtain accurate assay results. Results showed that the
method is robust. Using the developed method, commercially avail-
able captopril tablets were assayed and the results were found to
be comparable with those obtained by the compendial method
[31].
A capillary gas chromatographic method with ame ionization
detection (FID) has been developed for the analysis of tamoxifen,
anastrozole, and letrozole in their pharmaceutical preparations,
drugs used in advanced breast cancer, using clomipramine as
the internal standard in order to achieve quantication. A test
of the ruggedness of this method was carried out using the
PlackettBurman fractional design with a matrix of fteen exper-
iments. Factors chosen were: column head pressure, time for
the splitless step, temperature for the splitless step, injector
temperature, detector temperature, injected volume, nal oven
temperature. The mean effect of each variable is the average differ-
ence between observations made at the extreme levels and those
made at the optimal level. Mean effects and standard errors were
calculated using the procedures described by Youden and Steiner.
Taking into account the results when the selected operating fac-
tors were tested using PlackettBurman experimental design and
YoudenSteiner statistical analysis, this method has proved to be
rugged for all the variations tested in this study. The only critical
values are, in some cases, the injected volume and the nal oven
temperature, as was to be expected [32].
In this paper, a capillary zone electrophoresis method is used
for the separation of six antidepressants used for the treatment
of mental illness. Optimum conditions for their separation were
investigated. A robustness test of the proposed method was per-
formed using the fractional factorial model of PlackettBurman,
requiring 15 experiments, in which the inuence of seven factors
at three different levels was tested on different electrophoretic
results: efciency; resolution; and corrected peak area, whereas
statistical evaluation was performed by Youden and Steiners
method. The main interaction effects are produced by injection
time over the corrected peak areas and by the ionic strength of the
electrolyte over efciency. However, the ruggedness obtained in all
cases allows use of this method by different laboratories, analysts,
or instruments without any appreciable error [33].
Paroxetine and three metabolites have been determined at
clinical levels in human urine by a validated nonaqueous capil-
lary electrophoresis (NACE) method. Prior to NACE determination,
the samples were puried and enriched by an extraction-
preconcentration step with a preconditioned C18 cartridge and
eluting the compounds with methanol. Good results were obtained
for different aspects including stability of the solutions, linearity,
accuracy, and precision. Fractional factorial designs developed by
Plackett and Burman with a matrix of 15 experiments were used
for a ruggedness test of the method, based on balanced incomplete
blocks according to procedures described by Youden and Steiner.
The variables selected in three value levels in this study were: (A)
voltage, (B) injection time, (C) concentration of NH4 OAc, (D) tem-
perature of the separation, (E) % ACN, (F) % acetic acid and (G) time
of rinse with electrolyte. The mean value of each variable is the
average difference between observation made at the extreme lev-
els and those made at the optimal level. Mean effects and standard
errors were calculated using the procedures described by Youden
and Steiner. The main interaction effects over paroxetine area are
produced by injection time and voltage, so it is important to work
with an internal standard. Results indicated that this analytical
method for measuring paroxetine and its metabolites has proved
to be rugged to all the variations tested [34].
Please cite this article in press as: E. Karageorgou, V. Samanidou, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.01.050
A chiral capillary electrophoresis (CE) method has been
developed allowing the enantiomeric separation of racemic
citalopram (R-() and S-(+) citalopram) using chiral selector
as carboxymethyl--cyclodrextrin (CM--CD). The inuence of
chemical and instrumental parameters on the separation such as
cyclodextrin and buffer concentrations, buffer pH, voltage, injec-
tion pressure was investigated, so good chiral separation of the
racemic mixture was achieved in less than 4 min. A robustness
test was performed using the PlackettBurman fractional design
using a matrix of 15 experiments for seven factors (internal
parameters) with a statistical treatment suggested by Youden and
Steiner. Factors chosen in this study were: pH phosphate buffer,
buffer concentration, cyclodextrin concentration, voltage ramp,
injection time, injection pressure, time of rinsed with electrolyte.
Results showed that the most critical factors in this developed
electrophoretic procedure could be the decrease of cyclodextrin
concentration below its optimal value and the variation of volt-
age ramp and injection pressure too. Method proved to be robust
for all the variations tested in this study for all the operating fac-
tors on every studied electrophoretic parameter. Applicability of
the method has been proved in the analysis of four pharmaceutical
formulations [35].
Five antidepressant drugs (uoxetine, uvoxamine, citalopram,
sertraline and paroxetine) were determined by a sensitive capil-
lary GCMS method. For GC separation were investigated, ow
rate, column head pressure, injector temperature, injection split-
less conditions and oven temperature program. MS detection
was performed in SIM mode to increase the sensitivity. The
PlackettBurman fractional design based on balanced incomplete
blocks was employed for the robust test of the method, using a
design for seven factors and 15 experiments. Youden and Steiner
approach was used for the statistical analysis. Investigated factors
were: Injection temperature, splitless temperature, splitless time,
EM voltage, oven time program and the two different oven tem-
perature programs. Results indicated that splitless temperature is
the most critical factor (but still robust) from all examined ones.
Finally, the method was applied to the analysis of these antide-
pressants in nearly all their pharmaceutical formulations, obtaining
high recoveries [36].
An extraction method, using a polypropylene membrane sup-
porting dihexyl ether (three-phase hollow ber-based liquid phase
microextraction followed by a HPLCDAD determination) was used
for the analysis of three pharmaceuticals (salicylic acid (SAC),
ibuprofen (IBU) and diclofenac (DIC)). The extraction procedure is
controlled by the donor/acceptor pH and the extraction time (fac-
tors). A full factorial design for three factors and two levels involving
eight experiments (23 ), has been used to determine the effect and
importance of the mentioned variables on the nal result. The
effects were differential quantities that are related to the response
change as a result of changes in one or more factors/variables. From
the results arises, that the main factor is the acceptor pH for all the
analytes. The method has been successfully applied to their deter-
mination in human urine and the results obtained demonstrated
that could also be applied to the determination of the correspond-
ing metabolites [37].
In the next example, the robustness of a chromatographic
method applied to quantify lumefantrine in raw material sam-
ples was assessed using Youdens test. Seven analytical parameters
were selected and small variations were induced in the nominal
values of the method. Then, eight runs were performed aiming to
determine the inuence of each parameter in the nal result. The
seven analytical parameters employed were: methanol concentra-
tion in mobile phase, mobile phase pH, column temperature, mobile
phase ow rate, column supplier, methanol supplier, chromato-
graph model. The chromatographic method proved to be highly
robust regarding the lumefantrine content. Some parameters such
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as mobile phase ow rate and methanol supplier presented low
inuence in the evaluated factors of the chromatographic method.
Youdens test showed to be a simple and feasible procedure to
evaluate the robustness of chromatographic methods [38].
In this work, a three phase hollow ber-based liquid phase
microextraction (HF-LPME) combined with an HPLC procedure
using diode array and uorescence detection has been developed
for the determination of eight uoroquinolones. The robustness
study is based on a landmark procedure suggested by Youden. Fac-
tors investigated were pH values, for donor and for the acceptor
phase. Stirring time is not considered as a variable for robustness
study due to its high optimum value and the fact that variations in
the order of minutes do not have signicant effects in the extraction
efciency. Results obtained that t values calculated for each factor
are lower than the tabulated one, so the procedure can be con-
sidered robust against the considered factors for all the analyzed
compounds. The proposed method was applied to the determina-
tion of the analytes in bovine urine and in environmental water
samples (surface, tap and wastewater) [39].
1.3. Environmental analysis
Edgell et al. in the early 1990s used Youden test in their vali-
dation studies on ofcial USEPA (United States Environmental
Protection Agency) methods. Firstly, an interlaboratory method
validation study was conducted on EPA Method 531.1. This method
refers to the measurement of N-Methylcarbamoyloximes and
N-Methylcarbamates in water by HPLC with post column deriva-
tization, to determine the precision and mean recovery of 10
carbamate pesticide compounds in reagent water and in nished
drinking waters. The study was based on Youdens plan for col-
laborative tests of analytical methods. Samples were spiked with
10 carbamate pesticides at 6 concentration levels, as 3 Youden
pairs. Eight laboratories analyzed the samples. Results were ana-
lyzed using an EPA computer program, which measured precision
and recovery for each of the 10 compounds and compared the per-
formance of the method between water types. The method was
acceptable for all analytes tested, whereas the results for the pooled
drinking waters were not signicantly different from the results for
the reagent waters [40].
A joint U.S. Environmental Protection Agency/AOAC interlab-
oratory method validation study was conducted on EPA Method
507. The method refers to the determination of nitrogen and
phosphorus pesticides in nished drinking water by Gas chro-
matography with a nitrogen-phosphorus detector, to determine
the mean recovery and precision for analyses of 45 nitrogen- or
phosphorus-containing pesticides in reagent water and nished
drinking waters. The study was based on Youdens nonreplicate
plan for collaborative tests of analytical methods. Like in the
previous method, samples were spiked with pesticides at 6 con-
centration levels, prepared as 3 Youden pairs. Ten laboratories
extracted the spiked waters with organic solvents and analyzed
an aliquot of each extract by GC. Results were analyzed using an
EPA computer program, which measured recovery and precision
for each of the 45 pesticides and compared the performance of
the method between water types. The ofcial method found to
be acceptable for all analytes tested except merphos, which ther-
mally decomposed in the injection port of the gas chromatograph
[41].
A joint EPA-AOAC interlaboratory method validation study
was conducted on USEPA National Pesticide Survey Method 6,
which refers to the determination of ethylene thiourea (ETU) in
nished drinking water by Gas chromatography with a nitrogen-
phosphorus detector. The purpose of the study was to determine
and compare the mean recoveries and precision for determina-
tion of ETU in reagent water and nished drinking waters. As
Please cite this article in press as: E. Karageorgou, V. Samanidou, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.01.050
7
described before, study design was based on Youdens nonrepli-
cate plan for collaborative tests of analytical methods. Samples
were spiked with ETU at 6 concentrations levels, prepared as 3
Youden pairs. Water was extracted by passing the sample through
an absorbent matrix type tube, from which ETU was recovered with
methylene chloride, and nally an aliquot of each extract is ana-
lyzed by GC. In the study participated twelve laboratories. Data
were analyzed using a USEPA computer program, which measured
recovery and precision for ETU and compared the performance
of the method between the two water types tested. Results for
the water matrixes showed no statistically signicant differences
[42].an interlaboratory method validation study was conducted
on EPA Method 515.1, referring to the determination of chlori-
nated acids in water by gas chromatography with an electron
capture detector. This method is one of the 6 pesticide meth-
ods developed for the USEPA National Pesticide Survey (NPS).
Method recovery and precision for analyses of sub-ppb to low-ppb
concentrations of chlorinated acids were determined in reagent
water and nished drinking waters. Target analytes included the
12 pesticides that were quantitatively measured in the National
Pesticide Survey (bentazon, 2,4-D, 2,4-DB, 3,5-dichlorobenzoic
acid, DCPA-diacid, dicamba, dichlorprop, 5-hydroxydicamba, pen-
tachlorophenol, picloram, 2,4,5-T, and 2,4,5-TP) and 5 pesticides
(aciuorfen, chloramben, dalapon, dinoseb, and 4-nitrophenol)
that were only qualitatively assessed in the National Pesticide Sur-
vey because of recognized method imprecision. The study design
was based on Youdens nonreplicate plan for collaborative tests of
analytical methods. The waters were spiked with all chlorinated
acids, each at 6 concentration levels, prepared as 3 Youden pairs.
Eight laboratories analyzed the water samples. Statistical analy-
sis performed with a USEPA computer program, which measured
recovery and precision for each of the 17 compounds and compared
the performance of the method between water types. The of-
cial method proved acceptable for the 12 NPS analytes recovered
quantitatively. Results for the 5 other NPS analytes were impre-
cise so these compounds are not included in the adopted method
[43].
In order to test for the presence of low pharmaceuticals in the
environment highly sensitive and selective analytical procedures
are required. Hollow ber-based liquid-phase microextraction (HF-
LPME) is a relatively new technique used in analytical chemistry
for sample pre-treatment that offers high selectivity and sensitiv-
ity compared to most traditional extraction techniques. This paper
describes an extraction method using a polypropylene membrane
supporting dihexyl ether for the analysis of three pharmaceuticals,
salicylic acid (SAC), ibuprofen (IBU) and diclofenac (DIC) in raw
and treated wastewaters followed by a HPLC/MSMS determina-
tion. A full factorial design on the experimental conditions for the
HF-LPME extraction for three factors and two levels involving eight
experiments (23 ), has been used to determine the effect and impor-
tance of the mentioned variables on the nal result. Factors tested
are the donor pH, acceptor pH and stirring time. As it can be seen,
the main factor is the acceptor pH for all the analytes. For DIC and
IBU, an increase in the acceptor pH leads to better results in the
extraction, while in the case of SAC leads to a decrease in the sig-
nal; so this pH value is a critical parameter. Stirring time seemed
to be the less critical factor and always with a positive effect on
the extraction procedure. From the results obtained it is possible
to consider the acceptor pH as a critical factor that must be care-
fully controlled, while the extraction procedure can be considered
a robust extraction procedure [44].
The same group of authors, developed an electromembrane
extraction method, combined with a HPLC procedure using diode
array and uorescence detection for the determination of six widely
used non-steroidal anti-inammatory drugs (salicylic acid, ketoro-
lac, ketoprofen, naproxen, diclofenac and ibuprofen) in wastewater
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8 E. Karageorgou, V. Samanidou / J. Chromatogr. A xxx (2014) xxxxxx
samples. The drugs were extracted from aqueous sample solu-
tions, through a supported liquid membrane. The proposed method
was successfully applied to urban wastewaters, whereas excel-
lent selectivity was demonstrated as no interfering peaks were
detected. The robustness study of the developed method was based
on the procedure suggested by Youden. A design matrix with two
factors in eight experiments was used where the +1 and 1 levels
corresponded to slight modications in the more critical variables:
pH values of donor and acceptor phases and stirring time. A sig-
nicance t-test was used to determine whether variations have a
signicant effect on the result. The t values were compared with
the corresponding critical t values (n = 4) at 5% signicance level
and three degrees of freedom. The results obtained showed that the
procedure can be considered robust against the considered factors
for all the analyzed compounds [45].
A three phase-hollow ber-based liquid phase microextraction
combined with a HPLC procedure using diode array and uo-
rescence detection has been developed from the same group of
authors in the two previous methods, for the determination of
four widely used sulfonamides: sulfadiazine, sulfamerazine, sul-
famethazine, sulfamethoxazole and their main metabolites. The
proposed method was applied to the determination of the analytes
in environmental water samples (surface, tap and wastewater).
Robustness study performed based on the procedure suggested by
Youden, exactly in the same way as described in [44,45]. Factors
tested were pH values (4.5 and 3.5 for donor phase and 12.5 and
11.5 for the acceptor phase). From the results obtained, method
can be considered robust against examined factors for all target
analytes [46].
The occurrence of cyanobacterial blooms and the production
of toxins as Cylindrospermopsin (CYN), in aquatic environments
were increasing in many regions of the world due to progressive
eutrophication of water bodies. In this work, an analytical method
has been developed an optimized for the determination of CYN
from Aphanizomenon ovalisporum cultures. The analytical proce-
dure is based on solvent extraction followed by a purication step
with graphitized cartridges and CYN quantication by LCMS/MS.
The extraction and purication steps were optimized using a two-
level full factorial design with replications according to Youden
approach. The three factors considered, relative to the SPE proce-
dure, were: the batch of the graphitized carbon cartridges, the ow
rate of the sample through the cartridge, and the nal redissolved
water volume after SPE treatment, which permits its validation. The
effect of each considered factor was estimated as the difference
of the mean result obtained at the high level from that obtained
at the low level. Once effects have been estimated, to determine
whether variations have a signicant effect on the results, a signif-
icance t-test is used, and the t-values were compared with the 95%
condence level with the degrees of freedom from the precision
study for each concentration. Finally, the present method can be
considered as robust against the three factors (at the levels xed in
the study) [47].
In the last example, a new sample preparation procedure is
described, for the determination of aminopolycarboxylic acids in
river water. The procedure consists of the solid-phase extraction of
the aminopolycaroxyllic acids on activated charcoal cartridges after
increasing the ionic strength and acidifying the sample. Chromato-
graphic technique used was GC with mass spectrometric detection.
The robustness of the method was evaluated by analyzing data
from sample preparation procedure, after checking seven vari-
ables according to Youden and Steiners robustness test. Factors
tested were: pH, NaCl mass, charcoal mass, eluent volume, reagent
volume, derivatization reaction time and derivatization reaction
temperature. Results indicated that the proposed method was
robust against all examined variables, and for all target analytes
[48].
Please cite this article in press as: E. Karageorgou, V. Samanidou, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.01.050
2. Conclusions
The robustness/ruggedness study during method validation
yields useful information concerning the performance of the
method if minor changes in operating parameters occur unex-
pectedly. Since these changes may happen either during sample
preparation or during analysis, a high number of experiments seem
to be necessary, taking into consideration the most important
parameters. A useful test to avoid the difcult task of perform-
ing too many experiments can be the Youden approach which is a
fractional factorial design. By this approach the number of experi-
ments is limited, however we have to keep in mind that interactions
between the different factors cannot be detected. Till now this use-
ful tool has been applied in many methods covering a wide range
of applications.
Conict of interest
Authors have declared no conict of interest.
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Please cite this article in press as: E. Karageorgou, V. Samanidou, J. Chromatogr. A (2014), http://dx.doi.org/10.1016/j.chroma.2014.01.050