a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103

available at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/aca

On line photochemically induced

excitation–emission-kinetic four-way data
Analytical application for the determination of folic acid
and its two main metabolites in serum by U-PLS
and N-PLS/residual trilinearization (RTL) calibration

A. Jiménez Girón a , I. Durán-Merás a , A. Espinosa-Mansilla a , A. Muñoz de la Peña a,∗ ,

F. Cañada Cañada a , A.C. Olivieri b
aDepartment of Analytical Chemistry, University of Extremadura, 06071 Badajoz, Spain
bDepartamento de Quı́mica Analı́tica, Facultad de Ciencias Bioquı́micas y Farmacéuticas, Universidad Nacional de Rosario, Instituto de
Quı́mica de Rosario (CONICET), Suipacha 531, S2002LRK Rosario, Argentina

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

Article history: The determination of folic acid and its two main serum metabolites, 5-methyltetrahydrofolic
Received 25 March 2008 acid and tetrahydrofolic acid, has been accomplished using four-way data modelled by
Accepted 27 May 2008 the third-order multivariate calibration methods unfolded and N-dimensional partial least-
Published on line 12 June 2008 squares (U-PLS and N-PLS), in combination with the separate procedure known as residual
trilinearization (RTL). The four-way data were acquired by following the photochemical reac-
Keywords: tion of these compounds by on line irradiation with a UV lamp. The excitation–emission
Folic acid matrices (EEMs) were recorded as a function of the irradiation time, using a fast scanning
Tetrahydrofolic acid spectrofluorimeter. The method achieves selectivity from the different rates at which the
5-Methyltetrahydrofolic acid corresponding photoproducts of the folic acid derivatives are formed and degraded. Sev-
Photochemically induced eral N-dimensional chemometric algorithms were used and the method was applied to
fluorescence the determination of these compounds in serum samples. The best algorithms to perform
Four-way data the multivariate calibration were U-PLS and N-PLS in combination with the separate resid-
Unfolded partial ual trilinearization procedure, achieving the second-order advantage. The approach allows
least-squares/residual minimizing or eliminating traditionally time-consuming sample pre-treatments and can
trilinearization facilitate quantifying an analyte in its native environment.
N-Dimensional partial © 2008 Elsevier B.V. All rights reserved.
least-squares/residual
trilinearization

1. Introduction the biosynthesis of amino and nucleic acids. Folate defi-

Folate derivatives act as co-enzymes in several enzymatic
reactions given rise to methyl and formyl transfer in
ciency may be linked to several pathological alterations as
pregnancy complications [1,2], neural tube defects [3] and
other numerous well-established pathologies. Folic acid (FA)
and some coenzymes, particularly calcium 5-formyl-5,6,7,8-
tetrahydrofolate, are administered to counter the toxic effects

∗
Corresponding author.
E-mail address: arsenio@unex.es (A. Muñoz de la Peña).
0003-2670/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2008.05.079
 a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103
of antitumoral drugs as methotrexate [4]. Tetrahydrofolic
(THF) and 5-methyltetrahydrofolic acid (5-MTF) are the most
important FA metabolites involved in several biochemical
routes in the human organism.
FA is a photosensitive compound and is degraded in aque-
ous solution by the sun light, ultraviolet and visible light,
to various photoproducts. Researchers studying the photo-
chemistry of folates and other biologically active derivatives of
pterin have payed most attention to the activity of the pterin
heterocycle. It has been demonstrated that the excited het-
erocycle is involved in the photochemical reactions and that
this is accompanied by a change on its reduction degree [5,6].
Notable changes of the fluorescence quantum yield may be
associated to the reduction degree of the pterin rings. It is
known that formyl, methyl or other one-carbon substituents
in tetrahydrofolic acid affect the properties of the excited
molecules of folate, and that the photolysis of folic acid, folates
and non-conjugated pterins, involves chemical bonds in the
pteridine heterocycle and other positions of the molecule.
Photolysis is also affected by the molecular environment
[7]. Although the conventional way to generate fluorescent
pteridin rings from tetrahydro- and dihydro-folates has been
by chemical oxidation [8,9] the photoirradiation could be a
more simple and efficient mode of developing differential
analytical methodologies, by using chemometric tools in con-
junction with kinetic spectral information. The variation on
the luminescent properties of a folate derivative, monitored as
an excitation–emission fluorescent matrix during the photoir-
radiation process (third-order signal), may generate a selective
information for both substituted and non-substituted folates.
Introducing an extra dimension in the data leads to higher-
order data, in which case the mathematical object obtained by
grouping third-order data for several samples into the fourth
dimension is known as a four-way array.
Third-order data forming four-way arrays have been
scarcely proposed to date for the resolution of mixtures
of components in complex samples. However, they provide
the opportunity of introducing an additional dimension to
second-order (three-way) data sets, allowing for a theoreti-
cal increase in the predictive ability of the models. They also
exploit, in some cases, the second-order advantage. In this
context, two recent reviews on multiway calibration [10,11]
described advances and applications in this field, in specific
areas of analytical chemistry.
Although third-order data may be acquired in several ways,
in most of the reported analytical applications, the third
dimension is obtained by recording the time evolution of
the analytical signal. For example, acetylsalicylic and ascor-
bic acid were analysed using UV spectrophotometric-time–pH
data processed with N-dimensional partial least-squares
(N-PLS), in this case with no need of the second-order
advantage [12]. In most cases, however, the kinetic evolution
of excitation–emission matrices (EEMs) is followed. This is
because of the availability of spectrofluorimeter and the easi-
ness of acquisition of the EEMs in a single instrument. In this
context, Tan et al. resolved four-way data arrays by parallel fac-
tor analysis (PARAFAC), in a kinetic system that involved the
simultaneous degradation of spinach-extracted chlorophylls
a and b, initiated by treatment with an acid buffer [13]. A com-
mon problem for obtaining the kinetic evolution of EEMs is the
95
time needed to record the complete EEM of a sample, because
this may lead to trilinearity losses. In the above referenced
case, no problems arose from the time needed to obtain the
EEM, as the reaction was very slow.
The quantification of the catecholamines adrenaline and
noradrenaline has also been reported by using PARAFAC and
N-PLS, modelling four-way data recorded by following the
kinetic evolution of excitation–emission fluorescence matri-
ces (EEMs). The recording of the EEMs was performed within
a second with a laboratory-constructed fluorimeter equipped
with a charge-coupling device (CCD) camera, previously
described by Muroski et al. [14]. By employing the imaging
CCD, an entire EEM spectrum was collected in a single mea-
surement in this application example, avoiding the problem of
acquiring the EEM with a conventional scanning fluorimeter.
In this latter case, the second-order advantage was not neces-
sary, although the authors postulated its requirement if future
urine samples were analysed [15].
Quantitative photochemically induced EMMs of several
non-fluorescent pesticides, including fenvalerate, difluben-
zuron, lufenuron, deltamthrin and carbaryl, were recorded
and a photolysis-based determination of fenvalerate was also
performed, employing a four-way PARAFAC model, with excel-
lent figures of merit [16].
Similar data, based on following the kinetic evolution
of EEMs, have been recorded in order to determine 5-
formyl-5,6,7,8-tetrahydrofolate (leucovorin) and methotrexate
in human urine samples, by applying PARAFAC, trilinear least-
squares (TLLS), an extension of bilinear least-squares (BLLS)
to the third dimension, and unfolded-PLS (U-PLS) [17]. In this
case, a commercial fast-scanning fluorimeter was used, for the
fist time, for the recording of the EEMs-kinetic data. Interest-
ingly, the time needed to acquire the complete EEM with this
instrument was of the same order of magnitude of the CCD
detector, taking into account the integration time needed in
this later case [15].
These latter proposed methodologies, TLLS and U-PLS, con-
veniently combined with the separate procedure of residual
trilinearization (RTL), an extension of residual bilinearization
(RBL) to the third dimension, have shown to adequately exploit
the second-order advantage, allowing analyte concentrations
to be estimated even in the presence of an unexpected fluo-
rescent background in the test samples [18].
Very recently, similar data were employed for the deter-
mination of the antineoplastic combination of folic acid and
methotrexate in urine samples. N-PLS and PARAFAC were
employed, and both methods gave similar predictive results,
indicative that samples did not present unexpected con-
stituents in the particular case of the urine matrix background
[19]. Later on, the same system was satisfactorily applied for
the determination of these two compounds in human serum
samples, where significant unexpected components and/or
inner filter effects may occur, by U-PLS coupled to RTL [20].
Kim et al. [21] described the analysis of photocatalytically
enhanced fluorescence EEMs with PARAFAC. The time-
dependent photocatalysis degradation of polycyclic aromatic
hydrocarbons (benzo[a]anthracene, benzo[k]fluoranthene,
dibenzo[a,h]-anthracene) was employed to create an addi-
tional analytical dimension. It was demonstrated that the
subsequent four-dimensional degradation-EEM data arrays
96 a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103
have greater selectivity for each hydrocarbon than do
three-dimension EEM data alone.
Goicoechea et al. [22] reported the first application of
PARAFAC to higher-order instrumental data generated from
Shpol’skii matrices at liquid helium temperature. Third-order
data arrays, consisting of excitation-modulated emission
wavelength-delay time matrices, were collected with the aid
of a cryogenic fiber-optic probe, a tunable dye laser, and a mul-
tichannel system for phosphorescence detection. These data
were applied to the analysis of 2,3,7,8-tetrachloro-dibenzo-p-
dioxin in water samples. The feasibility to directly determine
parts-per-trillion concentration levels of the target compound
was demonstrated with heavily contaminated samples of
unknown composition.
Finally, a new algorithm, N-PLS coupled to RTL, was
recently proposed by our group and applied to the determi-
nation of procaine and its metabolite, p-aminobenzoic acid, in
equine serum, by analysing four-way data based in the record-
ing of kinetic fluorescence excitation–emission four-way data
[23].
In the present work, the simultaneous determina-
tion of folic acid and its two main metabolites, 5-
methyltetrahydrofolic acid and tetrahydrofolic acid, is pro-
posed and applied to serum samples. The third dimension
is obtained by following the kinetic evolution of the EEMs,
after on line UV–vis photoirradiation, in a similar way to the
approach reported by other authors [16,21]. The analytical
performances of several N-dimensional algorithms were com-
pared, and the best results were found with the U-PLS and
N-PLS/RTL combinations.
2. Experimental
2.1. Apparatus and software
Fluorescence spectral measurements were performed on
a Varian Cary Eclipse fluorescence spectrophotometer,
equipped with two Czerny–Turner monochromators and a
xenon flash lamp, and connected to a PC microcomputer via
an IEEE 488 (GPIB) serial interface. The Cary Eclipse software
was used for data acquisition.
The flow system used to implement the irradiation-
continuous system is described in Fig. 1. The sample stream
was pumped by a Gilson Minipuls-3 peristaltic pump. The
pump rate was 10 rpm and the reagents circulated in
poly(tetrafluoroethylene) (PTFE) flow tubes (Tygon, 0.5 mm
i.d., acid-resistant) until the UV-reactor where the sam-
ples were irradiated. The length of the reactor was 1.5 m.
Excitation–emission-time data arrays were recorded in a 1-
cm quartz flow cell, in the following ranges: excitation,
250–316 nm, each 6 nm, emission, 330–504 nm, each 6 nm, and
time 0–12 min, each 1 min. Thus, a third-order data array for
a given sample was of size 12 × 30 × 13, consisting of a total
of 4680 data points. The rapid-scanning instrument allows
the acquisition of a complete EEM in 0.2 min, at a wavelength
scanning speed of 24,000 nm min−1 . In this work, a scanning
speed of 14,400 nm min−1 was optimized, allowing the record-
ing of the EEM in a time considerably smaller than the time
between successive measurements. It should be noticed that,
Fig. 1 – Manifold for the on-line photoirradiation of the
folates.
if reaction significantly evolves during spectral acquisition, the
data sample would not be strictly quadrilinear. In other words,
since each scan is taken at a different time, in a dynamic con-
text, the individual spectra do not truly represent the same
state of a given sample. In our case, we did not find this prob-
lem to be important, as the effect is minimized by the fast
scanning recording. The cell was thermostated at 15 ◦ C, by use
of a thermostatic cell holder and a Selecta thermostatic bath.
All calculations were done using MatLab 5.3, using dif-
ferent routines and graphical interfaces: MVC3 (MultiVariate
Calibration for third-order), an integrated Matlab toolbox for
third-order calibration, developed by Olivieri et al. [17], which
allows performing third-order calibration with different mod-
elling methods, including PARAFAC [24], U-PLS and N-PLS,
both coupled to RTL [18,23], and TLLS coupled to RTL [18].
2.2. Reagents
All experiments were performed with analytical reagent
grade chemicals. Folic acid, 5-methyltetrahydrofolic acid and
tetrahydrofolic acid were obtained from Sigma. Ultra pure
water was obtained from a Milli-Q system (Waters Millipore).
Stock standard solutions were prepared by dissolving 0.010 g
of each compound in 100 mL of alkalinized ultra pure grade
water. Exposure to direct sunlight was avoided. Working FA,
5-MTF and THF solutions of different concentrations, were
prepared by dilution of stock solutions with ultra pure water.
A buffer solution (pH 4; Ct = 0.5 M) was prepared from acetic
acid and sodium acetate (Panreac).
2.3. Calibration and test sets
In this work, the method of external calibration was employed.
For this purpose, a calibration set was constructed, using a
central composite design with the central point replicated
three times. The levels correspond to values in the range
0–200 ␮g L−1 for FA, 5-MTF and THF.
Reagents were mixed in a 10-mL volumetric flask, by
pipetting an appropriate volume of FA, 5-MTF and THF solu-
tion, 0.8 mL of 0.5 M acetic/acetate buffer solution (pH 4) and
 a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103 97

Table 1 – Results obtained when applying N-PLS in the analysis of synthetic samples
Test FA THF 5-MTF

Actual Predicted Rec (%) Actual Predicted Rec (%) Actual Predicted Rec (%)
(␮g L−1 ) (␮g L−1 ) (␮g L−1 ) (␮g L−1 ) (␮g L−1 ) (␮g L−1 )

1 100 89 89 0 1.4 – 0 2.30 –

2 95 84 89 78 78 100 60 54 90
3 110 106 96 100 88 88 35 39 111
4 75 77 103 90 94 104 100 91 91
5 85 86 101 75 73 97 40 37 92
6 0 2.4 – 60 65 108 70 69 98
7 100 86 86 100 114 114 60 60 100
8 150 146 97 150 157 105 50 58 117
9 150 122 81 150 152 101 150 118 79
10 100 91 91 100 81 81 40 39 98

Reca ± S.D. 92 ± 7 100 ± 10 97 ± 11

RMSEPb 11.6 8.9 11.1
REPc (%) 12.0 9.9 18.4

a
Rec: average recovery.
b
RMSEP: root mean square error prediction.
c
REP: relative error of prediction.

deionised water to complete 10 mL. The instrument was set
up as follows: monochromators bandpassex/em(nm/nm) = 5/10,
detector voltage 750 V and 15 ◦ C of temperature. A volume of
reagents of 2 mL was necessary in order to fill the flow system.
Two test sets were used. The first one, composed of ten
synthetic samples (Tables 1 and 2), was prepared in the same
way as those for calibration, but using a random design, i.e.,
selecting the target concentrations of these analytes, at ran-
dom, from the calibration range for each analyte. The second
one was composed of 12 serum samples.
2.3.1. Serum samples
Volumes of 100 ␮L of serum samples (taken from a pool of sera
from healthy individuals) were used, after spiking them with
concentrations of the analytes selected at random from their
corresponding calibration ranges. The serum samples were
treated with 1 mL of methanol and centrifuged during 5 min
for deproteinization. Then, 0.5 mL of the supernatant were
placed in a 10 mL flask, and treated as described for the cali-
bration set. Finally, after the above described pre-treatment,
the samples contained a 4.2% of methanol, which was not
significant in the fluorescence of the samples.
2.4. Theory
2.4.1. U-PLS/RTL
The theory corresponding to the U-PLS algorithm, in combi-
nation with RBL, was recently published [25]. The U-PLS/RBL
model constitutes a second-order multivariate calibration
method capable of achieving the second-order advantage

Table 2 – Results obtained when applying U-PLS in the analysis of synthetic samples
Test FA THF 5-MTF

Actual Predicted Rec (%) Actual Predicted Rec (%) Actual Predicted Rec (%)
(␮g L−1 ) (␮g L−1 ) (␮g L−1 ) (␮g L−1 ) (␮g L−1 ) (␮g L−1 )

1 100 87 87 0 4.5 – 0 6 –
2 95 94 99 78 83 106 60 53 88
3 110 113 103 100 89 89 35 46 131
4 75 80 107 90 92 102 100 92 92
5 85 85 100 75 77 103 40 40 100
6 0 5 – 60 58 97 70 69 99
7 100 97 97 100 106 106 60 68 113
8 150 154 103 150 166 111 50 54 108
9 150 128 85 150 160 107 150 125 83
10 100 98 98 100 70 70 40 41 103

Reca ± S.D. 98 ± 7 99 ± 13 102 ± 14

RMSEPb 8.6 12.1 9.8
REPc (%) 8.9 13.4 16.3

a
Rec: average recovery.
b
RMSEP: root mean square error prediction.
c
REP: relative error of prediction.
98 a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103
[25–27]. For four-way calibration, U-PLS combined with RTL
constitutes an extension of U-PLS/RBL one further dimen-
sion [18], and will be briefly described in this section. When
analyzing four-way data with the U-PLS method, the origi-
nal matrix data is transformed into uni-dimensional arrays
(vectors) by concatenating (unfolding) the original three-
dimensional information, and concentration information is
first employed into the calibration step (without including data
for the unknown sample) [28]. The I calibration third-order
arrays Xc,i (size J × K × L, where J, K and L are the number of
channels in each dimension) are vectorized (unfolded), and a
usual U-PLS model is calibrated with these data and the vec-
tor of calibration concentrations y (I × 1, where I is the number
of calibration samples). This provides a set of loadings P and
weight loadings W (both of size JKL × A, where A is the num-
ber of latent factors), as well as regression coefficients v (size
A × 1). The parameter A can be selected by techniques such as
leave-one-out cross-validation [29]. If no unsuspected interfer-
ences occur in the test sample, v can be employed to estimate
the analyte concentration:
yu = tT
uv
where tu (size A × 1) is the test sample score, obtained by pro-
jection of the (unfolded) data for the test sample Xu [vec(Xu ),
size JKL × 1] onto the space of the A latent factors:
−1
tu = (WT P) WT vec(Xu )
when uncalibrated constituents occur in Xu , the sample scores
given by Eq. (2) are not suitable for analyte prediction using
Eq. (1). In this case, the residuals of the U-PLS prediction step
will be abnormally large in comparison with the typical instru-
mental noise, assessed by replicate measurements:
−1
||vec(Ep )|| ||vec(Xu ) − P(WT P) WT vec(Xu )||
sp = = Tucker3 models having one or two loadings in each dimen-
1/2 1/2
(JKL − A) (JKL − A)
||vec(Xu ) − Ptu ||
= (3)
1/2
(JKL − A)
where ||·|| indicates the Euclidean norm, and JKL − A corre-
sponds to the degrees of freedom (number of variables minus
number of adjustable parameters).
If interferent components occur in the test sample, the sit-
uation can be handled by a separate procedure called residual
trilinearization, based on a Tucker3 decomposition, that mod-
els the interferent effects, as already described [18]. RTL aims
at minimizing the norm of the residual vector eu , computed
while fitting the sample data to the sum of the relevant con-
tributions to the sample signal. For a single interferent, the
relevant expression is:
vec(Xu ) = Ptu + gint (dint ⊗ cint ⊗ bint ) + eu
where bint , cint and dint are normalized profiles in the three
modes for the interference and gint is the first core element,
obtained by Tucker3 analysis of Ep in the following way:
(gint , bint , cint , dint ) = Tucker3(Ep )
During this RTL procedure, P is kept constant at the calibration
values and tu is varied until ||eu || is minimized. The mini-
mization can been carried out using either a Gauss–Newton
(GN) procedure or an iterative least-squares algorithm, in both
cases starting with tu from Eq. (2). Once ||eu || is minimized in
Eq. (4), the analyte concentrations are provided by Eq. (1), by
introducing the final tu vector found by the RTL procedure.
The number of interferents Ni can be assessed by compar-
ing the final residuals su with the instrumental noise level,
with su given by:
||eu ||
su = (6)
1/2
[JKL − (Nc + Ni )]
where eu is from Eq. (4) and Nc is the number of calibrated
analytes. Typically, a plot of su computed for trial number of
components will show decreasing values, starting at sp when
the number of components is equal to A (the number of latent
variables used to described the calibration data), until it sta-
bilizes at a value compatible with the experimental noise,
allowing to locate the correct number of components.
(1) To analyse the presently discussed data, the Tucker3 model
in Eq. (5) is constructed by restricting the loadings to be orthog-
onal, and with no special constraints on the core elements.
For a single unexpected component, this analysis is straight-
forward, and provides the corresponding interferent profiles in
the three dimensions. For additional unexpected constituents,
(2) however, the retrieved profiles no longer resemble true spectra
(or time profiles). Moreover, in this latter case, several different
Tucker3 models could, in principle, be constructed, because
the number of loadings may be different in each dimension.
We notice that the aim which guides the RTL procedure is
the minimization of the residual error term su of Eq. (6), to
a level compatible with the degree of noise present in the
measured signals. Therefore, if two unexpected components
are considered, for example, one should explore the possible
sion, and select the simplest model giving a residual value
of su , which is not statistically different than the minimum
one. For more unexpected components, a similar procedure
is recommended. The final Tucker3 model, selected to model
the unexpected effects, is the simplest one which provides a
value of su which is not statistically different than the noise
level.
We note that two different residual parameters appear in
the above discussion, which should not be confused: sp [Eq. (3)]
corresponds to the difference between the test sample signal
and that model by U-PLS before the RTL procedure, while su
[Eq. (6)] arises from the difference after the RTL modeling of the
interferent effects. Hence it is the latter one which should be
comparable to the instrumental noise level if RTL is successful.
2.4.2. N-PLS/RTL
(4) In the N-PLS method applied to third-order data, concentra-
tion information is employed in the calibration step, without
including data for the unknown sample. The I calibration data
arrays, together with the vector of calibration concentrations
y (size I × 1) are employed to obtain sets of loadings Wj , Wk
and Wl (of sizes J × A, K × A and L × A, where A is the num-
(5) ber of latent factors), as well as regression coefficients b (size
 a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103
A × 1) [30]. The parameter A can be selected by techniques such
as leave-one-out cross-validation [29]. If no unexpected com-
ponents occurred in the test sample, b could be employed to
estimate the analyte concentration according to:
yu = t T
ub
where tu is the test sample score vector, obtained by
appropriate projection of the test data onto the calibration
loading matrices. When unexpected constituents occur in the
unknown sample, the latter scores are unsuitable for analyte
prediction through Eq. (7). In this case, it is useful to consider
the residuals of the N-PLS modeling of the test sample signal
[sp , see Eq. (8) below] before prediction is made. These resid-
uals will be abnormally large in comparison with the typical
instrumental noise level:
sp = ||ep || ||vec(Xu ) − vec(X̂u )||
= (8)
(JKL − A)
1/2
(JKL − A)
1/2
where X̂u is the sample three-way data array (Xu ) recon-
structed by the N-PLS model and ||·|| indicates the Euclidean
norm.
This situation can be handled by residual trilinearization
based on a Tucker3 model of the unexpected effects, as dis-
cussed above for U-PLS/RTL. In the case of N-PLS/RTL, the
analogous expression to Eq. (4) is:
Xu = reshape{tu [(Wj | ⊗ |Wk )| ⊗ |Wl ]} + Tucker3(X̂u − Xu ) + Eu
where ‘reshape’ indicates transforming a JKL × 1 vector into
a J × K × L three-way array, and |⊗| indicates the Kathri-Rao
operator [30]. During this RTL procedure, the weight loadings
Wj , Wk and Wl are kept constant at the calibration values, and
tu is varied until the final RTL residual error su is minimized
using a Gauss–Newton procedure, with su given by:
||Eu ||
su =
[(JKL − (Nc + Ni )]
1/2
where Eu is from Eq. (9). Once this is done, the analyte con-
centrations are provided by Eq. (7), by introducing the final tu
vector found by the RTL procedure. The considerations dis-
cussed above concerning the Tucker3 model of Eq. (9) do also
apply to N-PLS/RTL.
3. Results and discussion
3.1. Kinetic-fluorimetric study of the analytes
FA is a weakly fluorescent compound; moreover, the excita-
tion and emission spectra of FA and its metabolites, 5-MTF
and THF, are strongly overlapped. In order to increase both the
fluorescence of FA and the spectral differentiation among the
three compounds, the effect of UV irradiation on the fluores-
cent properties of the analytes has been studied. In a previous
paper [31], the simultaneous determination of folic acid and
99
5-methyltetrahydrofolic acid in human plasma has been pro-
posed, based on the recording of the kinetic evolution of the
emission spectra, exciting at a single excitation wavelength,
and modelling with multivariate second-order calibration
methods. The aim of this work is the differentiation between
(7) the photoproducts formed from folic acid and its two main
metabolites. Taking advantage of this transformation, it is
hoped to increase the sensitivity of the determination, mainly
in the case of the FA, and to be able to perform the simulta-
neous determination of the three species, taking advantage of
the difference in the kinetics of each of the photoreactions as
a discriminatory parameter.
When an FA solution is irradiated, an increase of the fluo-
rescence signal occurs, without displacement of the maxima
of excitation (275 and 350 nm) and emission (445 nm). In the
case of a THF solution, new excitation and emission maxima
appear, located at 270 and 445 nm, respectively. The original
excitation maximum (293 nm) disappears and the intensity of
the emission maximum at 362 nm slightly decreases. Finally,
when a 5-MTF solution is irradiated, the fluorescence signal
decreases without displacement of the excitation (290 nm) and
emission (360 nm) maxima.
As a precedent on the use of photochemically induced
four-way arrays of data, Kin et al. [21] described the time
dependent photocatalysis degradation of several polycyclic
aromatic hydrocarbons, employed to create an additional ana-
lytical dimension. It was demonstrated that the subsequent
four-dimensional degradation-EEM data arrays have greater
selectivity for each PAH than do three-dimension EEM data
alone.
(9) The experimental parameters of the corresponding pho-
toreactions were studied and optimized, in order to obtain the
appropriate kinetics and fluorescence properties of the prod-
ucts originated. The above-described irradiation-continuous
system was employed, and instrumental parameters were
optimized using the surface response methodology. The
pH, buffer concentration and temperature were optimized
sequentially, selecting an optimum pH value of 4. An
acetic/acetate buffer was employed, selecting an optimum
concentration of 0.04 M in the final solution. The influence of
(10)
temperature was studied between 10 and 40 ◦ C. The reaction
rates increased exponentially with temperature, and the fluo-
rescence of the photoproducts decreased, because exist more
probability of deactivation of the excited state by non-radiative
mechanisms. Considering both effects, 15 ◦ C was selected as
a compromise value.
Second- and third-order methods were investigated and,
owing to the complexity of the problem to be solved, third-
order calibration was necessary to resolve the mixture. This is
because the mixture is really complex as two of the analytes,
FA and THF, develop during the photoirradiation, to origi-
nate the same final compound, and the difference is related
to the kinetic of formation of the corresponding photoprod-
ucts, that it is faster in the case of THF. On the other hand,
the third mixture component, 5-MTF, is photodecomposing,
which produces a diminishing of the fluorescence emission
with time. The four-way data used were obtained by recording
the evolution with the time of EEM fluorescence measure-
ments (Fig. 2) and PARAFAC [24], TLLS coupled to RTL [18],
unfolded-PLS and multiway-PLS coupled to RTL [18,23] chemo-
100 a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103

Fig. 2 – Evolution of the contour plot of the excitation–emission matrices with the irradiation time, of solutions of FA
(150 ng mL−1 ), THF (150 ng mL−1 ) and 5-MTF (150 ng mL−1 ).

metric calibration models were applied in synthetic and serum
samples.
It is necessary to remark that the fluorescence signals were
not additive. Thus, the fluorescence of the ternary mixture
(at different reactions times) was smaller than the sum of
the individual fluorescence of the analytes, probably due to
the effect of inner filter, because of the overlapping of the
emission and excitation fluorescence maxima among sample
components. Also, the time needed to obtain a complete EEM
may introduce small changes in the third dimension profiles.
Because of these reasons, the obtained data will not be strictly
quadrilinear.

Fig. 3 – EJCR (95% confidence level) for the slope and intercept of the regressions of the theoretical versus predicted
concentrations of FA, THF and 5-MTF using N-PLS and U-PLS in synthetic samples.
 a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103 101

3.2. Third-order calibration methods

Fig. 2 shows the contour plots corresponding to the EEMs for

three samples containing FA, THF and 5-MTF, respectively,
at different reactions times. For calibration and prediction
purposes, the Rayleigh scattering and diffraction grating har-
monics were avoided to set the recording range for the
employed EEMs.
It can be appreciated that the fluorescence intensity of AF
and THF considerably increases as a function of reaction time,
whereas the fluorescence of 5-MTF decreases and, as can be
seen in Fig. 2, FA and THF are weakly fluorescent, but their irra-
diation with UV light in acidic medium generates in both cases
a strongly fluorescent compound. For both compounds, the
photoproducts have identical excitation and emission wave-
lengths, 270 and 445 nm, respectively.
The set of 10 test ternary samples (Tables 1 and 2)
was investigated with the aid of N-PLS, U-PLS, PARAFAC
and TLLS, employing the MVC3 interface. The number of
latent variables was established in N-PLS by resorting to
the well-known cross-validation leave-one-sample-out pro-
cedure, according to the criterion of Haaland and Thomas
Fig. 4 – Plot of the prediction residual (su ) as a function of
[29]. The optimum number of factors was estimated by
the number of interferents (Ni ) for serum sample 4,
calculating the ratios F(A) = PRESS(A < A*)/PRESS(A) (where
containing 117 ng mL−1 FA, 169 ng mL−1 of THF and 
PRESS = (Ci,act − Ci,pred )2 , A is a trial number of factors and A*
87 ng mL−1 of 5-MTF. The noise level in this system is ca. 25
corresponds to the minimum PRESS) and selecting the num-
relative fluorescence intensity units (— - — - —). For values
ber of factors leading to a probability of less than 75% that
of Ni larger than one, several Tucker3 models were
F > 1. This analysis led to the conclusion that the later number
examined, selecting for the present plot the value of su
was 3 for the three calibration models, corresponding to the
corresponding to the simplest model giving a value which
existence of the three analytes.
is not statistically different than the minimum one.
As the fluorescence signals of the ternary mixture were
not additive, suggesting that inner filter effects may occur,
both PARAFAC and TLLS fail in giving adequate prediction
results. This was the reason of investigating in this work
two new approaches, U-PLS and N-PLS in combination with

Table 3 – Results obtained when applying N-PLS/RTL in the analysis of serum samples
Test FA THF 5-MTF

Actual Predicted Rec (%) Actual Predicted Rec (%) Actual Predicted Rec (%)
(ng mL−1 ) (ng mL−1 ) (ng mL−1 ) (ng mL−1 ) (ng mL−1 ) (ng mL−1 )

1 0 – – 0 – – 91 101 101
2 32 24 75 168 189 113 28 33 118
3 36 29 81 34 30 88 0 – –
4 117 108 92 169 167 99 87 80 92
5 77 71 92 168 187 111 76 72 95
6 0 – – 0 – – 82 93 113
7 72 62 86 0 – – 138 129 93
8 71 74 104 167 174 104 0 – –
9 93 100 108 92 115 125 47 55 117
10 52 45 87 162 140 86 150 121 81
11 16 26 163 156 165 106 61 66 108
12 52 58 112 213 233 109 58 45 78

Reca ± S.D. 100 ± 25 105 ± 12 100 ± 14

RMSEPb 7.6 16.2 12.1
REPc (%) 12.2 11.0 14.9

a
Rec: average recovery.
b
RMSEP: root mean square error prediction.
c
REP: relative error of prediction.
102 a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103

Table 4 – Results obtained when applying U-PLS/RTL in the analysis of serum samples
Test FA THF 5-MTF

Actual Predicted Rec (%) Actual Predicted Rec (%) Actual Predicted Rec (%)
(ng mL−1 ) (ng mL−1 ) (ng mL−1 ) (ng mL−1 ) (ng mL−1 ) (ng mL−1 )

1 0 – – 0 – – 91 99 109
2 32 26 80 168 140 83 28 37 137
3 36 40 111 34 42 123 0 – –
4 117 129 110 169 174 103 87 89 102
5 77 75 97 168 148 88 76 86 113
6 0 – – 0 – – 82 81 99
7 72 86 119 0 – – 138 112 81
8 71 75 106 167 176 105 0 – –
9 93 113 105 92 127 137 47 62 133
10 52 48 79 162 146 90 150 119 79
11 16 25 156 156 148 95 61 57 93
12 52 49 95 213 219 103 58 59 98

Reca ± S.D. 106 ± 22 103 ± 17 104 ± 19

RMSEPb 9.6 18.0 14.6
REPc (%) 15.5 12.2 17.8

a
Rec: average recovery.
b
RMSEP: root mean square error prediction.
c
REP: relative error of prediction.

RTL, that have demonstrated their utility in similar situations
[20,23].
The statistical parameters and the results, obtained in
the analysis of the test set of synthetic samples, are col-
lected in Tables 1 and 2, respectively. The results obtained
for the three analytes are satisfactory for the two methods
employed.
The EJCR of the regression [32,33] of predicted versus nom-
inal concentrations in the test set was studied for the two
third-order calibration methods. The corresponding plots are
shown in Fig. 3. All confidence regions contain the ideal point
of unit slope and zero intercept (indicating accuracy), and the
elliptic sizes obtained with N-PLS are slightly smaller, suggest-
ing that this chemometric methodology show better predictive
ability than U-PLS, mainly for THF predictions.
3.3. Serum samples
The set of serum samples was investigated with the aid
of N-PLS and U-PLS combined with RTL, constituting third-
order multivariate calibration methods capable of achieving
the second-order advantage. When N-PLS and U-PLS were
applied, three latent variables were estimated by means of
cross-validation [29]. Subsequently, one additional component
was considered to be necessary in the RTL procedure when
the serum samples were analysed, suggesting one unexpected
component in the serum samples. Fig. 4 shows the varia-
tion of prediction residuals (su ) as a function of trial values
of the number of interferents (Ni ). The plot shows that, when
the analysed sample is the one corresponding to the test set
(sample 4), the necessary number of factors for interferences

Fig. 5 – EJCR (95% confidence level) for the slope and intercept of the regressions of the theoretical versus predicted
concentrations of FA, THF and 5-MTF using N-PLS/RTL and U-PLS/RTL in serum samples.
 a n a l y t i c a c h i m i c a a c t a 6 2 2 ( 2 0 0 8 ) 94–103
(Ni ) reaches 1. As can be seen, this model shows a predic-
tion residual comparable to the instrumental noise level (ca.
25 fluorescence units). Predictions using the N-PLS/RTL and
U-PLS/RTL models are collected in Tables 3 and 4. Predictions
using the N-PLS/RTL model give relative errors of prediction
(REP%) among 10 and 15%. The results were similar when
using the U-PLS/RTL model. All predictions are seen to be rea-
sonable for samples of the complexity of human serum.
In order to get further insight into the accuracy and pre-
cision of the algorithms analysed, nominal versus found
concentration values were compared by application of the
EJCR test. Fig. 5 shows the EJCR plots for N-PLS/RTL and U-
PLS/RTL. As can be seen, the ellipses contain the theoretical
(a = 0, b = 1) point, what means that no significant differences
were found for the confidence level proposed (95%).
4. Conclusions
The determination of folic acid in the presence of its two main
metabolites, tetrahydrofolic acid and 5-methyltetrahydrofolic
acid, using four-way data in combination with unfolded
and multiway partial least-squares/residual trilinearization is
reported. The four-way data were acquired by following the
photochemical reaction of these compounds, by on line irra-
diation with a UV lamp. The EEMs were recorded as a function
of the irradiation time, using a fast scanning spectrofluorime-
ter. Several N-dimensional chemometric algorithms were used
and compared, and the method was applied to the determina-
tion of these compounds in serum samples, overcoming the
limitations of conventional fluorimetric analysis. Because the
quadrilinearity conditions are not adequately accomplished,
PARAFAC, and TLLS do not provide satisfactorily results. How-
ever, N-PLS and U-PLS, in combination with the separate
procedure RTL, give rise to satisfactory AF, THF and 5-MTF
resolution, in the presence of non-modelled serum interfer-
ences. This is because of the more flexible structure of these
advanced multiway spectral deconvolution and calibration
algorithms in comparison with PARAFAC, which make them
a more appropriate approach for processing four-way data,
which are not strictly quadrilinear.
Acknowledgements
The Ministerio de Educación y Ciencia of Spain (Project
CTQ2005-02389) and AECI (Programa Intercampus Iberoamer-
ica/PCI Project A/6576/06), are gratefully acknowledged for
financial support. A.J.G. thanks the Ministerio de Educación
y Ciencia of Spain for a fellowship.
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