Analytica Chimica Acta 642 (2009) 193–205

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Analytica Chimica Acta

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Identification and quantification of ciprofloxacin in urine through

excitation-emission fluorescence and three-way PARAFAC calibration
M.C. Ortiz a,∗ , L.A. Sarabia b , M.S. Sánchez b , D. Giménez a,1
a
Department of Chemistry, University of Burgos, Faculty of Sciences, Plaza Misael Bañuelos s/n. 09001 Burgos, Spain
b
Department of Mathematics and Computation, University of Burgos, Faculty of Sciences, Plaza Misael Bañuelos s/n. 09001 Burgos, Spain

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

Article history: Due to the second-order advantage, calibration models based on parallel factor analysis (PARAFAC)
Received 31 July 2008 decomposition of three-way data are becoming important in routine analysis.
Accepted 15 January 2009 This work studies the possibility of fitting PARAFAC models with excitation-emission fluorescence data
Available online 23 January 2009
for the determination of ciprofloxacin in human urine. The finally chosen PARAFAC decomposition is built
with calibration samples spiked with ciprofloxacin, and with other series of urine samples that were also
Keywords:
spiked. One of the series of samples has also another drug because the patient was taking mesalazine.
Ciprofloxacin
The mesalazine is a fluorescent substance that interferes with the ciprofloxacin. Finally, the procedure is
Parallel factor analysis
Urine
applied to samples of a patient who was being treated with ciprofloxacin.
Fluorescence The trueness has been established by the regression “predicted concentration versus added concentra-
tion”. The recovery factor is 88.3% for ciprofloxacin in urine, and the mean of the absolute value of the
relative errors is 4.2% for 46 test samples.
The multivariate sensitivity of the fit calibration model is evaluated by a regression between the loadings
of PARAFAC linked to ciprofloxacin versus the true concentration in spiked samples. The multivariate
capability of discrimination is near 8 ␮g L−1 when the probabilities of false non-compliance and false
compliance are fixed at 5%.
© 2009 Elsevier B.V. All rights reserved.

1. Introduction analysis (PARAFAC), allow quantification of an analyte, even when

The fluorescent properties of substances have been widely
exploited for analytical purposes due to the good capability of
detection of fluorescence techniques. However, the selectivity is
often reduced because of broad spectral overlap or in the presence
of matrix interferences. Nevertheless, fluorescence detection com-
bined with Chemometrics may change this situation. One of the
possible strategies consists of collecting the excitation-emission
fluorescence matrix (EEM) and analyzing it using second-order
calibration methods. It is well established that this strategy is
an attractive alternative of immense potentiality in the field of
biomedical analysis [1] because, besides the low detection limits,
it has low cost, the procedure makes the quantification possible
even if the biological sample interferes with the fluorescence of the
analyte of interest and, generally, pretreatment of the samples is
not necessary. In addition, several second-order calibration meth-
ods such as the procedure proposed here based on parallel factor
∗ Corresponding author.
E-mail address: mcortiz@ubu.es (M.C. Ortiz).
1
Present adress: Laboratorio de Salud Pública, Servicio Territorial de Sanidad y
Bienestar Social de Ávila, C/ San Juan de la Cruz, 28, CP 05001 ÁVILA, Spain.
unknown (that means non-calibrated) interferences are present
(the second-order advantage). All these characteristics have caused
that both the applications of the EEM linked with second-order cal-
ibration methods and the research of new calibration methods for
n-way data, have an increasing interest [2].
In general, better performance characteristics are obtained with
second-order signals and calibration based on partial least squares.
For example, the excitation-emission fluorescence spectra of mix-
tures of 10 polycyclic aromatic hydrocarbons (PAHs) have been
analyzed using different multivariate calibration procedures (two-
and three-way partial least squares regression, n-PLS, and PARAFAC)
by Beltrán et al. [3] for the determination of samples of tap and
mineral waters spiked with all these PAHs. The best results were
obtained with the application of a 3-PLS.
More recently, Divya and Mishra [4] obtain the same results
in the quantitative determination of kerosene fraction present
in diesel by means of excitation-emission fluorescence matrix
together with PARAFAC and n-way partial least squares regression.
Again, n-PLS is the method which gives better results compared to
PARAFAC.
However, it must be remembered that n-PLS does not have the
second-order advantage, so that in multi-fluorophoric mixtures a
n-PLS calibration can be nonviable if the matrix has analytes that

0003-2670/$ – see front matter © 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2009.01.040
194 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205
modify the size and the form of the fluorescence signal. This hap-
pens in the determination of tetracycline in milk by fluorescence
spectrophotometry because the presence of some interferents as
Ca(II), K(I), P(V), Fe(III), Na(I), Zn(II) and Mg(II) at levels of concentra-
tion that are usually in milk modify both the shape and magnitude
of the fluorimetric signals of tetracycline [5]. In this case it is not
possible to calibrate neither with n-PLS nor with a PARAFAC-based
standard addition method. Nevertheless, this last method works
well when the matrix effect is small [6]. The procedure followed in
[7] to obtain satisfactory results in the determination of tetracycline
in milk is a kind of “double calibration”, by spiking tetracycline in
increasing levels of whey from deproteinized milk. Then, a suitable
PARAFAC-based calibration model with the recorded EEM is built
for tetracycline determination.
There are also problems when the matrix in which the fluo-
rophore is present acts as quencher. In this case, Rodríguez et al. [8]
show that it is not possible to use the standard addition method,
but by varying the level of quencher it is possible to quantify the
analyte of interest by a PARAFAC model of four ways. This was made
following the model for the quenching effect proposed by Leurgans
and Ross [9] in the field of Biology.
The second-order advantage of the PARAFAC-based calibrations,
as it extracts of unique way the factor linked to the analyte, allows
the use of calibration samples from different days. In that way, the
inter-day variability is incorporated into the residuals. This has been
done in Ref. [10] with excitation-emission data.
Andersen and Bro present a deep investigation and practical
description of how to apply PARAFAC modelling to fluorescence
excitation-emission measurements [11]. Also, a review of recent
chemical applications of PARAFAC can be seen in Refs. [12,13,2]. The
PARAFAC-based calibration model can be used with experimental
data provided by other analytical techniques, a recent review in the
case of chromatographic data can be seen in Ref. [14].
In order to maintain the second-order advantage, new pro-
cedures for n-PLS calibration have been designed. Recently in
Ref. [15] lanthanide-sensitized luminescence excitation-time decay
matrices were modelled by PARAFAC and n-PLS with residual
bilinearization (n-PLS/RBL). The results indicate a slightly better
predictive ability of the newly introduced n-PLS/RBL procedure
over standard PARAFAC. Similar result has been reported by
Valderrama and Poppi [16] in the quantification of mixtures of
propranolol enantiomers in the pure form and in the pharmaceuti-
cal preparations. The excitation-emission fluorescence surface and
second-order multivariate calibration such as bilinear least squares
(BLLS) and PARAFAC were used in the model development. BLLS
performed better than PARAFAC in this problem.
An alternative to the intrinsically three-way methods can be
found in [17] where Tauler presented a generalization of the
family of bilinear methods known as Multivariate Curve Resolution-
Alternating Least Squares (MCR-ALS) to three-way data sets
including the possibility of imposing the trilinearity as a constraint.
MCR-ALS has been used for analyzing excitation-emission matri-
ces, particularly when the concentration direction does not follow
a global patterned shape, which is different to a calibration exper-
iment. In that sense, MCR-ALS has proven useful for analyzing
hyperspectral and multispectral images of biological specimens,
particularly by means confocal fluorescence microscopy, because
it can operate with little or no a priori information about the emit-
ting species, making it appropriate when multiple fluorophores
are present [18]. In quantification tasks, MCR-ALS and excitation-
emission fluorescence has been used in Ref. [19] to quantify
triphenyltin in seawater samples solving a matrix effect. Several
papers have been published comparing the three-way procedures
based on MCR-ALS with those ones based on PARAFAC [20–23]. The
reader interested is referred to the recent revision by de Juan and
Tauler [24] to see the theory under MCR as well as applications.
With samples of human urine, Espinosa-Mansilla et al. [25]
exploit the second-order advantage in untreated samples, by cou-
pling photoinduced fluorescence that generate three-way data and
the multiway analysis of the photoproducts obtained from UV
irradiation of three fluoroquinolones, enoxacin, norfloxacin and
ofloxacin, in urine samples at biological levels without sample pre-
treatments.
A spectrofluorimetric method has been developed by Muñoz
de la Peña et al. [26] for the quantitative determination of the
anti-inflammatory flufenamic and meclofenamic acids in urine
samples. The method is based on second-order multivariate cali-
bration applying PARAFAC in the standard addition mode.
Also, the determination of ciprofloxacin in human urine has
been used as test field for the second-order advantage in bilin-
ear least squares, BLLS, and PARAFAC [27]. There, it is shown that
there are differences when modeling the spectral profile at pH 6
because an equilibrium occurs between two forms having distinct
fluorescent properties. In this case, PARAFAC performs better. How-
ever, although the performance characteristics are similar for both
methods, the joint confidence region for the slope and intercept
of the regression line fitted between the predicted concentration
versus the true concentration in test samples has smaller surface
for BLLS than for PARAFAC. From a chemometrics point of view,
in the present paper, attention is focused in the performance of
PARAFAC when considering that the matrix where the ciprofloxacin
is determined is varying: urine from different people and presence
of another fluorophore such as the mesalazine.
The most usual methods of analysis to determine ciprofloxacin
in urine are HPLC with different detectors, diode array [28,29],
mass [30] or fluorescence [31,32]. The specificity of the fluorescence
could be improved [33] by using flow injection and chemilumi-
nescence [34], by derivatization of the sample with lanthanides
[35] and other metals [36]. The solid substrate room temperature
phosphorescence (SS-RTP) is another alternative [37].
Espinosa-Mansilla et al. [38] optimized the determination by
HPLC with photoinduced fluorimetric detection of four quinolones.
For ciprofloxacin they obtained a capability of detection, CC␤, equal
to 6.3 ␮g L−1 with both probabilities of false negative and false pos-
itive fixed at 5%.
Ciprofloxacin is a synthetic fluoroquinolone that has Gram(+)
and Gram(−) antibacterial activity and is used in the treatment of
bacterial infections in humans and animals. In addition, the Food
and Drug Administration has recommended its use for postexpo-
sure inhalational antrax [39]. It should be monitored in body fluids
because it has some contraindications and in many occasions it is
administered together with other drugs. Its main excretion path-
way is urinary, and it is estimated that the concentration in urine
surpass 300 mg L−1 if the determination is made 4 h after dosing
[40]. Therefore, taken into account that for the present work the
urine is diluted 1000 or 2000 times, the capability of discrimina-
tion of the proposed method is shown for nominal concentration
of 200 ␮g L−1 , that is, the quantity that is expected to be found
between 12 and 24 h after dosing. The capability of discrimination
refers to the quantity of the ciprofloxacin that can be distinguished
(discriminated) from 200 ␮g L−1 at given probabilities of both false
non-compliance and false compliance.
2. Theory
In this section, basic theoretical properties about PARAFAC and
the capability of discrimination are given. It starts by analyzing the
similarity between the trilinear PARAFAC model and the physical
model for fluorescence. Then, there is a brief description of the steps
to be followed to make a calibration model based on PARAFAC and
n-way data. Finally, there is a summary of the concept and com-
 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205
putation of the capability of discrimination with n-way data by
assuring both the probabilities of false non-compliance and false
compliance. We restrict the discussions to the determination of
ciprofloxacin in complex matrices using excitation-emission fluo-
rescence. Also, some performance characteristics for the procedure
are established.
2.1. PARAFAC
PARAFAC is, to a certain extent, a generalization of principal
component analysis (PCA) to a n-way data set X. For the case of
three-way data, the data cube or tensor, X of dimension I × J × K, is
decomposed into triads or components [41,42]. Each component
consists of three loading vectors. Let xijk denote the element in
position i,j,k in matrix X. Then the structural model of PARAFAC
is expressed as follows:

F
xijk = aif bjf ckf + eijk , i = 1, . . . , I; j = 1, . . . , J; k = 1, . . . , K
f =1
where F is the number of factors, aif , bjf and ckf are the elements of
the three loading vectors and eijk denotes the residual error. When
experimental data follow the model in Eq. (1), it is said that the data
are trilinear.
In fluorescence spectroscopy, for diluted solutions, the amount
of light emission measured is separately linear in the number of
photons absorbed and in the fraction of photons absorbed that lead
to emission at a specific wavelength. Therefore, when sample i-th,
i = 1, . . ., I, containing F fluorophores is illuminated with light at
wavelength ex , j = 1, . . ., J, and the consequent emission of light is
j is similar to the calibration samples, Q and T2 statistics are
measured at wavelength em k
, k = 1, . . ., K then, in theory, we have
the following trilinear equation for the fluorescence intensity, xijk :

F
xijk = aif bjf ckf , i = 1, . . . , I; j = 1, . . . , J; k = 1, . . . , K
f =1
where aif is the concentration of fluorophore f in the i-th sample, bjf
is the relative absorption (extinction coefficient) of fluorophore f at
wavelength ex j
and ckf is the relative emission at wavelength em k
. a PARAFAC decomposition only with the calibration samples, to
It can be observed that the model of Eq. (1) corresponds to the
physical model for fluorescence in Eq. (2). Therefore, if the experi-
mental data are trilinear and there are F fluorescent components in
the samples, the model of F components can be used to estimate: (i)
the extinction coefficients for each analyte f at all wavelengths, i.e.,
the excitation profile bf = (b1f , b2f , . . ., bJf ) or excitation spectrum;
(ii) the relative emission at all wavelengths, i.e., the emission pro-
file cf = (c1f , c2f , . . ., cKf ) or emission spectrum of each analyte; and
(iii) the relative concentration of every analyte in all samples, i.e.,
the sample profile af = (a1f , a2f , . . ., aIf ). Vectors bf and cf are usually
normalized to unit length.
When the data are trilinear, the PARAFAC decomposition pro-
vides unique profiles estimations [43]. This means that, provided
that the rank of the PARAFAC model corresponds to the number
of fluorescent compounds, each PARAFAC component, f, can be
assigned to a compound and the loadings (afi , bfj , and cfk ) can be
interpreted as an estimation of the relative concentrations of the
analytes, excitation spectra and emission spectra respectively for
this compound. Therefore, only if the correct number of compo-
nents is chosen, the true underlying spectra will be found. To select
this number of components, usual criteria are the core consistency
diagnostic (CORCONDIA) and the split-half analysis, among others.
For details, consult Section 5.4 of Ref. [44]
195
2.2. PARAFAC-based calibration
A calibration model for three-way data coming from fluores-
cence and based on a PARAFAC decomposition consists basically of
the following steps:
(i) For each sample, record the EEM (excitation-emission matrix)
of dimension J × K, where J is the number of excitation wave-
lengths and K that of emission wavelengths. Using these data,
if a trilinear model as the one in Eq. (1) is assumed, build the
three-way array, X, made up by the calibration samples at I
levels of concentration of the analyte.
(ii) Compute the PARAFAC decomposition. This gives the spectral
and the sample profiles of the analytes. The absence of anoma-
lies in the spectral or sample profiles is assessed by means of
the T2 and Q statistics.
(iii) Identify the factor, f0 , which corresponds to the analyte of inter-
est.
(iv) The loadings (coordinates) on the sample profile of the
analyte of interest identified in the previous step af0 =
(a1f0 , a2f0 , . . . , aIf0 ) are then regressed versus the added con-
(1) centration to obtain the calibration curve.
(v) Using the concentration values calculated by the calibration
model in step (iv) some performance characteristics (trueness,
intermediate precision and linear range) are established by
means of a new regression model fitted between the concen-
tration obtained with the PARAFAC decomposition versus the
true concentration.
(vi) Determine the concentration of a test sample. To do it,
the excitation-emission matrix of the test sample should be
recorded and added to the array X to carry out the joint
PARAFAC decomposition. To guarantee that the test sample
also used. Both values in the three profiles must be below the
corresponding critical values established at the corresponding
confidence level (usually 95%).
(2) The procedure explained in the previous steps (to merge the EEM
for calibration and test samples and compute a PARAFAC decompo-
sition) is the method currently used in the majority of second-order
prediction work. This procedure will be coded along the text as
model 1. As alternative, another approach consists of calculating
obtain the vectors bf and cf , f = 1, . . ., F, and then using these fac-
tors on test samples to estimate their sample loadings (we will
call this procedure as model 2). There are other procedures, five
of them have been studied by Rinnan et al. [45] who, by using an
index of goodness of prediction, showed that with simulated data,
model 1 is the best procedure, although this is not so with EEM
experimental data. Also, better results are obtained with model 1
when evaluating the capability of detection with three-way chro-
matographic data [46]. In the prediction stage for model 2, it is
necessary to consider more factors than those obtained with the
calibration samples in order to get unbiased predictions of the con-
centration of the analytes. This implies the need of doing another
PARAFAC decomposition. With model 1, a new PARAFAC decom-
position should be computed each time that new test samples are
available. This may imply changes in the fitted calibration line (step
(iv)). However, these changes should be small because in step (iii)
the PARAFAC factor linked to the analyte has been unequivocally
identified, therefore, the possible interferents in the test samples
would appear as new factors that should not affect the determina-
tion of the concentration by means of the calibration curve in step
(iv) [11,47].
In the univariate regression used in steps (iv) and (v) outliers
must be detected. For this task, the Least Median Squares (LMS)
196 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205
regression [48] has proven to be useful [49]. Once the outliers have
been eliminated, a Least Squares (LS) regression is made. Further-
more, the hypotheses implicitly assumed for a LS regression [50]
(normality, homoscedasticity and independence of residuals) must
be verified, and the lack of fit should also be checked.
2.3. Capability of discrimination
Given a nominal concentration, in order to know the behaviour
of an analytical procedure in samples with similar concentrations,
the minimum discriminable concentration (or multivariate sensi-
tivity) is defined as the smallest concentration of the analyte in a
sample which can be distinguished, with probability 1 − ˇ, from the
nominal value. This definition generalises the concept of minimum
detectable net concentration established by ISO norm 11843 that is
restricted to the case in which the nominal concentration is zero.
Given an analytical procedure with a well-established detectable
net concentration (capability of detection in IUPAC recommenda-
tion [51] or CC␤ in EU regulation [52]), it may not be possible to
discriminate this same concentration when the procedure is used
in samples with a much higher nominal concentration. For this rea-
son, the capability of discrimination is a criterion for the selection
of an analytical procedure when it is going to be used to deter-
mine concentrations well above its detection limit (for example,
when a drug or toxic residue has a permitted limit, PL). The capa-
bility of discrimination is established as a hypothesis test based on
the data of a calibration model carried out in a range of concen-
trations which contains the nominal value. For a n-way calibration
model it is computed (step (v) of procedure in Section 2.2) from the
linear regression “concentration calculated by the fitted model” ver-
sus “true concentration” which is formally written by the following
equation:
y = a + bx + ε
where ε is a random variable which is supposed to be independently
normally distributed with mean zero and variance  2 constant for
all x in the working range.
The estimation of the model in Eq. (3) fitted by least squares is
y = â + b̂x
and the (common) variance is estimated as
1 N 2
ˆ 2 = (yn − ŷn )
N−2 n=1
where N denotes the number of calibration samples.
Under the assumed distributional hypothesis, the calculated
concentration ŷ, at a concentration x of analyte, is a Student’s t-
distribution with (N − 2) degrees of freedom, whose mean and
variance are [50]:
E(ŷ) = â + b̂x  centration |x − x0 | at the nominal level x0 . The procedure is
1 1 (x − x̄)2
Var (ŷ) = ˆ 2
R
+
N
+ N 2
= ˆ 2 wx2
n=1
(xn − x̄)
where R is the number of determinations (replicates) on the test
sample and x̄ is the mean of the N concentrations, xn , of the calibra-
tion samples.
Let x0 be the nominal concentration for which one wants to study
the capability of discrimination of the analytical procedure. For-
mally, the compliance of the procedure is stated as x = x0 whereas if
x =/ x0 , the procedure is not compliant. Explicitly, the capability of
discrimination of the procedure demands that non-compliance be
assured for all x =/ x0 . Evaluation will be done using the following
Neyman–Pearson test:
(i) Null hypothesis, H0 : the true concentration in the sample is x0 ,
that is, x = x0 .
(ii) Alternative hypothesis, Ha : the sample concentration is not x0 ,
that is, x =/ x0 .
(iii) Significance level or probability of type I error: ˛, the
probability of rejecting H0 when H0 is true, ˛ = pr{false non-
compliance}.
(iv) Probability of type II error: ˇ, the probability of accepting H0
when H0 is false, thus, ˇ = pr{false compliance}.
The decision to accept or reject the compliance of the procedure
must be established on the basis of the response variable y (the con-
centration found by means of the application of a PARAFAC-based
calibration model to the analytical signal recorded for a sample with
true concentration x). The critical region of the test can be written
as follows:
CR = {|y − y0 | > yc } (7)
where yc is the critical value to asses that “the concentration of
the sample under consideration is not different from the nominal
value” and the non-compliance or compliance probabilities are the
conditional ones:
(8)
Applying Eq. (6) to x0 and expanding Eq. (8) the following holds
ŷ0 = â + b̂x0
(9)
yc = w
ˆ x0 t˛/2
(3) where t˛/2 is the critical value of a Student t with N − 2 degrees
of freedom at level ˛/2, and wx0 is defined in Eq. (6). Note that
it is related to the number of calibration samples, the number of
replicates made at the nominal concentration, and the distribution
of the concentration of the calibration samples with respect to their
mean.
Therefore, ˇ is a function of x, that is, it depends on how much
(4)
the true concentration x differs from the nominal concentration x0 ,
i.e.:
ˇ(x) = pr{t(x ) ≤ t˛/2 } − pr{t(x ) ≤ −t˛/2 } (10)
(5)
where t(x ) is a non-central Student’s t-distribution with (N − 2)
degrees of freedom, an estimated mean ŷ − ŷ0 and a parameter of
non-centrality estimated by means of
b̂|x − x0 |
x = (11)
w
ˆ x0
Eq. (11) allows evaluation of the minimum discriminable con-
(6) presented in Fig. 1. More details about this procedure as well as
its theoretical foundation can be consulted in Ref. [53].
3. Experimental
3.1. Chemicals and reagents
All chemicals were of analytical reagent grade, and solvents
were HPLC-grade. Ciprofloxacin was obtained as a research sam-
ple from Bayer (Leverkusen, Germany). Glacial acetic acid and
sodium hydroxide were obtained from Merck (Darmstadt, Ger-
many). Deionized water was obtained by the Milli-Q Gradient A10
water purification system of Millipore (Bedford, MA, USA).
 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205 197

Fig. 1. Schema of the computation of the multivariate sensitivity by using the regression “concentration calculated by PARAFAC versus true concentration”. See the text for
the definitions.

Working samples were prepared just before being measured.
Standard samples were prepared in deionized water, adding the
corresponding ciprofloxacin concentration plus a buffer solution
acetic acid/sodium acetate to adjust the pH to 4.0.
3.2. Experimental procedure
Table 1 shows the details of the series of samples used during the
work. The first series is made by standard samples of water spiked
with 8 different levels of ciprofloxacin between 100 and 240 ␮g L−1
each 20 ␮g L−1 (two replicates in each case).
Series 2, 3, 4 and 5 are samples of human urine spiked with
the same quantities of ciprofloxacin. In series 2, 3 and 4, the urine
belongs to a young man, an old man and a woman, respectively. The
fifth series corresponds to a patient who was taking mesalazine.
After each series, a blank was measured; samples number 17, 34,
51, 68 and 85 in Table 1. The urine was diluted in water in series 2,
3, 4 and 5 in proportion 1:1000.
Finally, a new series (series 6) was prepared. The series is made
up by 8 levels of concentration of ciprofloxacin added in water from
100 to 240 ␮g L−1 in steps of 20 ␮g L−1 . Also, a blank was measured
and there are two replicates of each sample. The 17 standard sam-
ples were used to determine the concentration of ciprofloxacin in
urine from a patient who was orally taking the drug (ciprofloxacin).
The urine of this patient (series 7 in Table 1) was measured on two
different days with a dilution proportion of the urine of 1:2000.
These urine samples were stored at room temperature and mea-
sured two times with three days of difference.
Different excitation-emission matrices are shown in Fig. 2. The
sub-graphs on the left, Fig. 2(a–c), are for one component: Fig. 2(a)
is from a sample of 240 ␮g L−1 of ciprofloxacin added in water;
Fig. 2(b) corresponds to a sample of diluted urine (1:1000) and
Fig. 2(c) is the EEM of a sample of pure mesalazine in water.
The sub-graphs of the right on its part, Fig. 2(d–e), shows the
interferences: Fig. 2(d) is from a sample of the same quantity of
ciprofloxacin but added in urine and the last one, Fig. 2(e), corre-
sponds to the same concentration of ciprofloxacin in urine 4 (which
contains mesalazine). It is clear that the spectra of the urine and the
mesalazine are overlapped with the one of the ciprofloxacin.
3.3. Instrumentation and software
Fluorometric assays were carried out at room temperature on
a PerkinElmer LS50-B Luminescence Spectrometer equipped with
a xenon discharge lamp and a gated photomultiplier. Intensities
were recorded at emission wavelengths between 350 and 550 nm
(every 1 nm) at different excitation wavelengths between 240
and 310 nm (every 5 nm). Excitation and emission monochroma-
tor slit-widths were both set to 4 nm and scan speed was set at
1500 nm min−1 .
The FL WinLab software (PerkinElmer) was used for measure-
ments, the data were imported to MatlabTM using the INCA software
[54] that also inserts missing values into the matrix in the wave-
lengths that correspond to the Rayleigh effect. The PLS Toolbox for
Matlab [55] was employed for PARAFAC and PLS calculations. Data
were analyzed using STATGRAPHICS [56] for fitting and validating
regressions, PROGRESS [48] was used to do the robust least median

Table 1
Characteristics of the series of samples analyzed with indication of the case-study where they are utilized and their use for calibration or test. The asterisk indicates that the
corresponding sample has been included in the PARAFAC decomposition.

Series Case Type of matrix Number of sample Use of samples

1 2 3 4 1st replicate 2nd replicate Blank

1 * * * Water 1–8 9–16 17 Calibration

2 * * Urine 1 (young man) 18–25 26–33 34 Test
3 * * Urine 2 (old man) 35–42 43–50 51 Test
4 * * Urine 3 (woman) 52–59 60–67 68 Test
5 * Urine 4 (patient taking mesalazine) 69–76 77–84 85 Test
6 * Water 1–8 9–16 17 Calibration
7 * Urine 5 (patient taking ciprofloxacin) 18–23 Test
198 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205
Fig. 2. Excitation-emission landscape of a sample containing: (a) ciprofloxacin 240 ␮g L−1 , (b) diluted urine (1:1000), (c) mesalazine, (d) ciprofloxacin, 240 ␮g L−1 , in urine,
(e) ciprofloxacin, 240 ␮g L−1 , in urine with mesalazine.
of squares (LMS) regression and remove the outliers. NWAYDET is
a home-made program to evaluate the multivariate sensitivity.
4. Results and discussion
The results are discussed for four different cases. In Case 1, the
determination of ciprofloxacin added in human urine is tackled;
in Case 2 also the ciprofloxacin added in human urine is deter-
mined but this time the urine is from a patient who was being
treated with mesalazine that acts as an unknown interferent. In
both cases, model 1 has been used (to merge calibration and test
samples to build the PARAFAC decomposition). On the contrary,
with the same excitation-emission matrices, in Case 3 model 2
has been used, that is, the PARAFAC decomposition is made only
with the calibration samples (water spiked with ciprofloxacin).
Finally, after the estimation of the performance characteristics of
the proposed method, it is applied in Case 4 to the determination
of ciprofloxacin in human urine of a patient who was orally taking
the ciprofloxacin.
The data have never been preprocessed along the analyses
shown in this paper. No constrains have either been imposed in
deriving the PARAFAC model.
4.1. Case 1
The purpose in this case is to determine ciprofloxacin in human
urine without the extraction step prior to the determination.
The samples used are from series 1, 2, 3 and 4 of Table 1, thus
17 calibration (standards in water) samples and 51 real sam-
ples of human urine. In that way, the data cube is made by
68 samples by 201 intensities of the emission spectra by the
15 intensities of the digitalized excitation spectra. Therefore, the
PARAFAC decomposition has been applied to the tensor X which is
68 × 201 × 15.
 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205
Fig. 3. PARAFAC spectral loadings for Case 1 in: (a) emission profile; (b) excitation
profile. The continuous blue line is identified as the ciprofloxacin and the discon-
tinuous green line is identified as the urine. (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of the article.)
When validating the regression line (step (iv) of Section 2.2)
between the loading of the 16 samples spiked with ciprofloxacin
versus the true concentration, two outlier samples were detected by
using the LMS regression. Then, the PARAFAC decomposition was
recalculated without them, so that the data tensor is 66 × 201 × 15.
Two factors have been considered, with CORCONDIA (core
consistency) equal to 99.99% and explained variance 99.8%. The
loadings in the emission and excitation profiles are drawn in Fig. 3.
For the identification of these factors, the correlation between the
PARAFAC spectral loadings and the experimental spectra of urine
and ciprofloxacin has been used. For the ciprofloxacin, the cor-
relation coefficients were 0.999 and 0.997 for the emission and
excitation profiles respectively, whereas for the urine they were
0.993 and 0.995 respectively. Consequently, the two factors are
identified as the ciprofloxacin and the urine. They have been repre-
sented in blue continuous line and dashed green line, respectively,
in both Fig. 3(a) for the emission profile and Fig. 3(b) for the excita-
tion profile.
Fig. 4 shows the loadings in the sample profile. For the samples in
series 1, the loadings linked to the urine (green crosses) are null and
those corresponding to the ciprofloxacin (blue circles) reproduce
the pattern of the ciprofloxacin added to the calibration samples.
The first replicate of samples corresponds to indices from 1 to 8,
and the second replicate corresponds to samples with indices 9–16.
199
Fig. 4. Loadings in the sample profile versus sample index in the PARAFAC decom-
position of Case 1. Blue circles () are the ciprofloxacin loadings. Green crosses (+)
are the urine loadings. Letters a, b, c and d correspond to the blank sample of each
series. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of the article.)
Although samples 15 and 16 are outliers and thus they have not
been used in deriving the PARAFAC model, their indices appear in
Fig. 4 to maintain the initial numbering in Section 3.2 and Table 1.
Sample 17, pointed at in Fig. 4 with an ‘a’, is the blank determined
in water. Its loadings on the ciprofloxacin (124) and on the urine
appear superimposed.
In series 2, the Q and T2 statistics for sample 22 gave values
greater than the critical values at 95% confidence level, and thus its
loadings do not appear in the figure (step (vi) of Section 2.2) but its
index has also been maintained in the abscissa axis.
The last sample of each series (samples 34, 51 and 68, high-
lighted in Fig. 4 as b, c, and d respectively) is always the blank.
For these samples, the loadings on the factor identified as the
ciprofloxacin are, in practice, null (329, 199 and 164 respectively).
However, their loadings (green crosses) on the factor identified as
the urine coincide with those of the rest of the spiked samples in
the same series. In each of the three series (2, 3 and 4) the load-
ings associated with the ciprofloxacin (blue circles) increase with
the quantity added in each replicate and the loadings associated
with the urine remain constant in each series although they differ
from one series to another, as should be expected because the urine
belongs to different people.
By only using the samples of series 1 (without outliers and
without the blank) a calibration line, similar to the one in Eq. (3),
“sample profile loadings versus true concentration” is fitted and
validated. Thus, x is the concentration of added ciprofloxacin and
y is the loading that the same sample has in the factor linked to
the ciprofloxacin in the PARAFAC model. The resulting calibration
model is y = 206.2 + 31.4x, with ˆ = 62.8 (Eq. (5)) and coefficient of
determination equal to 0.998. The regression line is significant (p-
value less than 10−4 ) and there is not lack of fit (p-value of the test
equal to 0.89). Fig. 5 shows the data and the calibration line.
With this calibration line, the mean of the absolute value of the
relative errors in calibration with the 14 spiked samples is 0.9%.
Additionally, when the regression line is applied to predict the
concentration of the 47 remaining samples of urine series 2, 3
and 4 (without blanks and without outliers), the predicted con-
centrations obtained are always less than and proportional to the
concentration of the added ciprofloxacin. This makes computation
of the recovery necessary in the urine samples. To do it, we fit, sep-
arately for each of the series 2, 3 and 4, the regression lines between
the concentration of ciprofloxacin obtained with the calibration
200 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205
Fig. 5. Case 1, regression line between sample profile loadings linked to ciprofloxacin
and the added concentration of ciprofloxacin for series 1.
line and the one added. All the regression models are significant,
without the lack of fit and only sample 64 of series 4 should be
considered as outlier according to the residuals of a LMS regres-
sion although the values of the Q and T2 statistics are both less than
the corresponding critical values. However, its loading on the factor
linked to the ciprofloxacin (see Fig. 4) does not correspond to the
added concentration, thus, it is an error in the preparation of the
sample.
The three intercepts of the regression lines are significantly null
and their slopes are 0.85, 0.88 and 0.91 respectively that, further,
are significantly equal (p-value equal to 0.42 when testing the null
hypothesis “the three slopes are equal” against the two-sided alter-
native). Then, the ‘joint’ regression line (fit with the samples of the
three series) has intercept significantly null (p-value = 0.315) and
the estimated slope is 0.8827, that means that one should admit
that the recovery factor for the urine samples is 88.3% indepen-
dently of the type of urine. By using this recovery factor for the 46
test samples, the mean of the absolute value of the relative errors
is 4.2%.
4.2. Case 2
In this case the samples of urine from a patient that was taking
mesalazine were also included, series 5 in Table 1. In that way, we
study the possible effect on the PARAFAC decomposition of an (in
general) unknown analyte in the samples.
Hence, to the data cube of the previous Case 1 we add the 17
samples of series 5 the last of them being the blank. The same sam-
ples (number 15, 16 and 22) that were removed in Case 1, show
inadequacy here, thus, they were also not considered here. Conse-
quently, the tensor X now is 82 × 201 × 15. In this new PARAFAC
decomposition, three factors were needed, with CORCONDIA equal
to 98.5% and explained variance 99.9%.
The identification of these factors is again made by computing
the correlation between the PARAFAC spectral loadings for the three
factors, Fig. 6, sub-graphs (a) and (b), and the experimental spectra
of urine, ciprofloxacin and mesalazine, Fig. 6, sub-graphs (c) and (d)
for emission and excitation respectively.
Table 2 contains the correlations between factors 1, 2 and 3 and
the true spectra of the analytes present in the sample. We can see
that these factors have correlations greater than 0.98 with the urine,
the ciprofloxacin and the mesalazine respectively while the rest of
correlations remain smaller. Consequently, factor 1 is identified as
the urine, factor 2 as the ciprofloxacin and factor 3 as the ‘inter-
Table 2
Case 2, correlation between each factor of the PARAFAC decomposition and the true
values. In profile 1 the correlation is with the true values of the concentration of
ciprofloxacin. In Profiles 2 and 3, the correlation is with the emission or excitation
experimental spectra of ciprofloxacin, urine and mesalazine. In bold, the maximum
correlation between the experimental data and the PARAFAC factor.
Profile Factora Correlation
Ciprofloxacin Urine Mesalazine
1 (sample) 1 (+) 0.898
2 () 0.999
3 () 0.716
2 (emission) 1 (- -) −0.484 0.984 −0.170
2 (—) 0.999 −0.352 0.854
3 (. . .) 0.868 −0.034 0.999
3 (excitation) 1 (- -) 0.885 0.994 −0.793
2 (—) 0.998 0.865 −0.936
3 (. . .) −0.936 −0.800 0.997
a
The symbols are the same as in Figs. 6 and 7.
ferent’ (mesalazine) and as such are represented in Fig. 6(a) and
(b).
Fig. 7 shows the loadings of the PARAFAC decomposition in the
sample profile (profile 1) separately for the factors linked to the
ciprofloxacin, Fig. 7(a), to the urine, Fig. 7(b), and to the mesalazine,
Fig. 7(c). In Fig. 7(c) we can see that the loadings corresponding to
mesalazine are null for the first four series (until sample 68) and
clearly non-null for series 5 with constant value for all the samples
from 69 to 85. Fig. 7(b) shows that the loadings linked to urine are
constant although different within each series and they are null
for the samples of series 1 (water spiked with the ciprofloxacin).
Finally, Fig. 7(a) shows that the loadings on the factor identified as
the ciprofloxacin increase in each series (as in Fig. 4) according to the
amount of ciprofloxacin added in each sample. This is so because of
the second-order property that implies that the factor linked to the
ciprofloxacin does not change when adding new samples (series 5)
not even if a new analyte is present in the samples. This new analyte
is the mesalazine in this case which is also fluorescent in the same
(excitation-emission) range as the analyte of interest. The blank
of series 5, sample 85, has loading practically null (260.4) on the
ciprofloxacin, Fig. 7(a), whereas the loadings on the urine (Fig. 7(b)
and on the mesalazine (Fig. 7(c)) are equal to the loadings of the
rest of samples in the same series. Finally, note that the loadings on
the ciprofloxacin (Fig. 7(a)) of the blanks of series 1–4 are 110, 393,
244 and 150 respectively, which are similar to the ones obtained in
Case 1.
The calibration line (sample profile loadings versus true concen-
tration) fit with the 15 spiked samples of series 1 is y = 221.2 + 31.3x
with ˆ = 63.8 (Eq. (5)) and coefficient of determination equal to
0.998. The regression model is significant (p-value less than 10−4 )
and there is not lack of fit (p-value 0.89). When comparing this
regression line with the one obtained for Case 1, both are similar,
as should be expected due to the second-order advantage.
As in Case 1, the amount of ciprofloxacin predicted with the
regression line is less than and proportional to the amount added.
For each of the series 2, 3, 4 and 5, the individual regression lines
“predicted concentration of ciprofloxacin versus added concentra-
tion” are significant, and there is not lack of fit in any of them. All the
samples “in prediction” have excitation-emission spectra admis-
sible according to the Q and T2 statistics at 95% confidence level.
The sample number 64 should again be considered outlier when
applying the LMS regression.
Also, in the four regression lines the independent terms are
significantly null and the slopes are 0.84, 0.87, 0.90 and 0.88
respectively. Moreover, these four slopes should be considered sig-
nificantly equal (p-value 0.27). Therefore, fitting the joint regression
line with the data of the four series, the intercept is also signifi-
 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205 201

Fig. 6. Loadings of the PARAFAC decomposition for Case 2 in the (a) emission profile, (b) excitation profile. Experimental spectra of ciprofloxacin, urine and mesalazine in:
(c) emission, (d) excitation. The first factor (urine) is represented with a dashed green line (- -). The second factor, linked to ciprofloxacin, is represented with solid blue line
(—). The third factor (mesalazine) is represented with dotted red line (. . .). (For interpretation of the references to color in this figure legend, the reader is referred to the web
version of the article.)

cantly equal to zero (p-value 0.07), there is not lack of fit (p-value
0.49) and the common estimated slope is 0.8547, thus, the recovery
of the ciprofloxacin for the urine samples is 85.5% independently
of the kind of urine and of the presence of mesalazine. By using
this recovery factor with the test samples (N = 63), without blanks
and without outliers, the mean of the absolute value of the relative
errors is 5.8%.
4.3. Case 3
In this case, we use model 2 to compute the PARAFAC decompo-
sition, that is, it is computed only with the tensor made up by the
17 calibration samples (see Table 1). Samples 15 and 16 are again
outliers as in Cases 1 and 2 and thus, they are not considered.
Only one factor is needed, that explains 99.9% of the variance.
The correlation coefficient between the obtained excitation and
emission profiles and the true spectra of the ciprofloxacin is 0.998
and 0.999 respectively. If, instead, we would have considered two
factors, the obtained emission profile cannot be even considered a
spectrum.
Fig. 8 shows the loadings in the sample profile of the calibra-
tion samples (series 1) as well as the values estimated for the test
samples (series 2–5, from sample number 18 to 85). These load-
ings are comparable to the ones obtained for the factor linked to
the ciprofloxacin in Figs. 4 and 7(a) in the sense that, qualitatively,
the pattern is similar although the values are larger. This is clearly
seen in the blanks with loadings 1118, 784 and 546 for series 2, 3
and 4 respectively, more than three times the values in Case 1 and
Case 2. For the samples with mesalazine (series 5) the blank has a
loading of 10,390, that is, forty times greater than the one obtained
when model 1 was applied (Case 2). On the contrary, the loading
of the blank of series 1 (point a in Fig. 8) is 138, similar to the ones
obtained for the same sample in Cases 1 and 2.
The calibration line “sample profile loadings versus added
concentration” fitted with the 15 spiked samples of series 1 is
y = 231.2 + 31.5x with ˆ = 61.3 (Eq. (5)) and coefficient of determi-
nation equal to 0.998. The regression model is significant (p-value
less than 10−4 ) and there is not lack of fit (p-value 0.90).
The mean of the absolute value of the relative errors is 0.9%
in calibration (N = 14) and 4.9% when predicting the test samples
of series 2–4 (without the blanks and the outliers). However, the
absolute value of the relative errors for the samples in series 5 is
189.45%.
The regression lines “predicted concentration of ciprofloxacin
versus added concentration” for series 2, 3, 4 and 5 show a sig-
nificant bias for the test samples. The four regression models are
significant and there is not lack of fit, but their intercepts (31.9,
18.8, 13.7 and 319.7 respectively) are significantly non-null in the
four regression lines. That means that model 2 introduces a constant
bias in the estimated concentrations, which is different depending
on the matrix where the ciprofloxacin is added. Nevertheless, the
slopes of the four regression lines (0.853, 0.880, 0.911 and 0.857)
are similar to the ones obtained with model 1 (Cases 1 and 2).
When comparing the three calibration lines fitted in Cases 1, 2
and 3 with the same 14 spiked samples, the intercepts are signifi-
cantly equal (p-value 0.12) and also the three slopes (p-value 0.92).
202 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205
Fig. 7. Loadings in the sample profile of the PARAFAC decomposition for Case 2 (a)
second factor loadings, linked to ciprofloxacin, represented with blue circles ();
(b) first factor loadings, linked to urine, represented with green crosses (+); (c) third
factor loadings, linked to mesalazine, represented with red squares (). Letters a, b,
c, d and e correspond to the blank sample of each series. (For interpretation of the
references to color in this figure legend, the reader is referred to the web version of
the article.)
Therefore, including the test samples for the PARAFAC decomposi-
tion does not modify the calibration line. This was expected because
of the uniqueness of PARAFAC decomposition.
The conclusion is thus that the bias is caused by the way the
loadings of the test samples were computed (model 1 or model 2) to
predict their concentration. In any case, the Q and T2 statistics may
detect the kind of problems that these samples present because, in
Fig. 8. Loadings in the sample profile of the PARAFAC decomposition for Case 3.
Letters a, b, c, d and e correspond to the blank sample of each series.
practice, the concentration of the test samples is unknown. Fig. 9(a)
shows both statistics with the corresponding critical values at 95%
confidence level for the 15 calibration samples. Note the position of
the blank (point a) which has the minimum values. In Fig. 9(b), we
can see that the samples of series 2, 3 and 4 have values of Q much
greater than the critical value, different among series and almost
constant within each series. This indicates the existence of residu-
als in the PARAFAC decomposition for the test samples anomalous
in relation to the calibration samples. These ‘anomalous’ residu-
als are caused by the different types of urine. Fig. 9(b) also shows
that the series appear sorted from greatest to lowest values of Q.
The ‘steps’ in the graph coincide with the magnitude of the bias
in the determination of the concentration of ciprofloxacin in the
corresponding series, and evidence the same effect previously seen
in the intercepts of the regression lines “computed ciprofloxacin
versus added concentration”. The blanks (points b, c and d) follow
the same pattern as the loadings of the samples of the correspond-
ing series. However, the three series have values of T2 similar to
those of the calibration samples (Fig. 9(a). Finally, Fig. 9(c) high-
lights that the samples of series 5, with mesalazine, exceed the
critical values for both T2 (except for the blank) and Q (more than
2.5 × 104 times larger than the threshold value at 95%). Therefore,
the PARAFAC decomposition cannot be accepted for these sam-
ples.
The comparison of the results obtained with model 1 (Cases 1
and 2) and model 2 (Case 3) show that: (i) the resulting calibration
line does not depend on whether the test samples are or not in the
PARAFAC decomposition. (ii) The errors when predicting the test
samples are similar in both models for the series 2, 3 and 4 (about
5%), but very different for series 5 (189% for model 2), that is, the
prediction error depends on the analytical matrix. (iii) Because the
intercepts are significant non-null for the series 2, 3, 4 and 5 with
model 2, there is a significant bias related to the type of urine and
to the mesalazine. (iv) For the model 2 the loadings of the blanks of
the test samples are greater than those of calibration samples. (v)
With model 2, only the samples that contain mesalazine have values
of T2 and Q greater than the critical values and their loadings are
outside the calibration range. Therefore, the calibration line should
not been used for them.
According to the usual criteria, the samples of series 2–4 will not
be considered outliers in relation to the PARAFAC model because
they only show residuals (Q statistic) greater than those of the cali-
bration samples. Nevertheless, this is an indication of the presence
of other analytes that were not modelled in the PARAFAC decom-
position.
 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205
Fig. 9. Case 3, values of the T2 and Q statistics for each sample in the sample profile
of the PARAFAC decomposition. Dashed lines are the corresponding critical values
at 95% confidence level. (a) Calibration samples, ciprofloxacin added in water; (b)
test samples, ciprofloxacin added in urine from different people; (c) test samples,
ciprofloxacin added to the urine from a patient who was taking mesalazine.
Consequently, for now on, only model 1 will be used for these
data.
Finally, for comparative purposes, a 3-PLS calibration model is
built with half of the samples of series 1–4 randomly chosen. The
mean of the relative errors of this model is 3.6% in calibration (34
samples) and 3.7% in prediction with the remaining 34 samples.
203
However, the mean of the relative errors obtained when the model
is used to predict the samples of series 5 (N = 17) was 737%. This
large error is due to the fact that for PLS the second-order property
does not apply and these urine samples have mesalazine which
was not calibrated. In any case, again the values of the T2 and Q
statistics for the test samples with mesalazine are greater than the
critical values, in such a way that the incorrect application of a 3-PLS
calibration model would be avoided. In addition, half of the urine
samples have been used as calibration samples and not as test sam-
ples, which implies the need of having calibration samples with the
same interferents expected in the test samples, interferents that, in
practice, are unknown.
4.4. Performance characteristics
For the calibration models computed in the preceding cases
the multivariate sensitivity or capability of discrimination, as is
described in Section 2.3, is established.
The regression lines between the true concentration and the cor-
responding concentrations calculated with the calibration models
are in Table 3. All of them have correlation coefficients greater than
0.99.
Also Table 3 contains the p-values of the hypothesis tests used
to decide whether the intercept and the slope were equal or not
to zero and one respectively. As can be seen, in all the cases, we
do not have experimental evidence for rejecting the corresponding
null hypothesis at 5% of significance level because the p-value is
greater than 0.05. This means, among other things, that the con-
centration predicted with the methods is statistically equal to the
true concentration, that is, there is no bias.
On the other hand, the precision of each method measured as the
ˆ of the regression line [53] shows similar values, around 2 ␮g L−1 .
Finally, the multivariate sensitivity of the PARAFAC-based cali-
bration models for ˛ = ˇ = 0.05, and at x0 = 200 ␮g L−1 as reference
value is 8.2 ␮g L−1 for Case 1, 8.5 ␮g L−1 for Case 2 and 8.1 ␮g L−1 for
Case 3. These quantities are similar to those obtained by HPLC [38],
but in our case without a chromatographic technique.
Therefore, from Table 3 we can conclude that the presence of a
new interferent in Case 2 does not significantly worsen the sensi-
tivity of the procedure, nor either the use of model 1 or model 2 for
the PARAFAC decomposition.
4.5. Case 4
In this case, the aim is the determination of ciprofloxacin in
urine samples of a patient that was being treated with BayCip, Bayer
(ciprofloxacin).
The experimental procedure is similar to that of the previous
cases. For computing the PARAFAC decomposition, series 6 and 7
of Table 1 were used. The first 17 samples, series 6, were spiked
with ciprofloxacin in water. The test samples were measured three
times and, then, three days later another three times, thus the 6
samples in series 7. Therefore, the tensor X that was decomposed
is 23 × 201 × 15.
After the decomposition two factors were selected (CORCONDIA
equal to 99.9% and explained variance of 99.8%) and subsequently
identified as the ciprofloxacin and the urine. Fig. 10 shows the load-
ings in the sample profile, the first 17 samples have null loadings for
the urine (green crosses) whereas the loadings of the ciprofloxacin
show the pattern of the quantity added (blue circles,).
The last six samples have non-null and almost constant loadings
for urine. However, there are the loadings of ciprofloxacin which
allow determining, after applying the corresponding dilution factor,
the concentration of ciprofloxacin in the urine of the patient. The
predicted concentration, applying the recovery factor, is 434 and
467 mg L−1 for the sample that was measured three days later.
204 M.C. Ortiz et al. / Analytica Chimica Acta 642 (2009) 193–205
Table 3
Regression lines fit with the concentration calculated by means of PARAFAC-based calibration methods versus the true concentration.
Fitted regression line
Correlation coefficient
Residual standard deviation ()
ˆ 1.9714 ␮g L−1
p-value of the hypothesis test:
H0 : The intercept is zero
Ha : It is not
p-value of the hypothesis test:
H0 : The slope is one
Ha : It is not
Multivariate sensitivity at 200 ␮g L−1 (˛ = ˇ = 0.05, R = 1)
Fig. 10. Loadings in the sample profile of the PARAFAC decomposition in Case 4.
Blue circles () represent those of ciprofloxacin and green crosses (+) those of the
urine. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of the article.)
As in the previous cases the trueness of the method has been
verified by fitting the regression line “concentration calculated
with PARAFAC loadings” versus “true concentration” and statisti-
cally testing that the slope and intercept were 1 and 0 respectively
at 5% significance level.
5. Conclusions
Modelling second-order fluorescence signals through parallel
factorial analyses (PARAFAC) makes it possible to quickly and pre-
cisely identify and quantify ciprofloxacin present in human urine
with independence of the individual and other fluorescent sub-
stances that can be present in the matrix. Only standard samples
with ciprofloxacin added in water are necessary. However, it is nec-
essary to compute the ‘joint’ PARAFAC decomposition, that is, with
the calibration and test samples (model 1). If only calibration sam-
ples are used (model 2), significant biased estimations are obtained
when predicting the concentration of ciprofloxacin of the urine
samples, and the mean of the absolute value of the relative errors
is 189.5% for the urine samples that also contain mesalazine. Nev-
ertheless, the calibration line is the same with model 2 and model
1.
The multivariate sensitivity of this procedure for a nominal value
of 200 ␮g L−1 in all the cases is around 8 ␮g L−1 when the probabil-
ity of false non-compliance and false compliance are fixed at 0.05.
These values show the suitability of the fluorescence with PARAFAC-
based calibration to determine ciprofloxacin, at trace level, in urine
samples without prior treatment.
Case 1 Case 2 Case 3
y = 0.0289 + 1.0001x y = 0.0128 + 1.0001x y = 0.0021 + 0.9999x
0.999 0.998 0.999
2.0322 ␮g L−1 1.9460 ␮g L−1
0.989 0.995 0.999
0.994 0.985 0.999
8.2 ␮g L−1 8.5 ␮g L−1 8.1 ␮g L−1
The analysis of the results obtained show that the recovery fac-
tor of the ciprofloxacin in urine is 85.5% independently of the kind
of urine and of the presence of mesalazine. By applying this recov-
ery factor to the test samples, the mean of the absolute value of
the relative errors is 5.8%. Urine test samples without mesalazine
let us obtained 88.3% of recovery and a mean of the absolute value
of the relative errors equals to 4.2% when ciprofloxacine is deter-
mined.
Acknowledgements
The authors acknowledge the support by project CTQ2008-
02264/BQU of Ministerio de Ciencia e Innovación and project
BU024A07 of Junta Castilla y León, both co-financed with European
FEDER funds.
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