Fresenius' Journal of
Fresenius J Anal Chem (1994) 350:692-701
Multicomponent determination of the pesticide naptalam
and its metabolites in river water, by applying partial
least squares calibration to the derivative spectrophotometric signals
T. Galeano Diaz, M. I. Acedo Valenzuela, F. Salinas
Department of Analytical Chemistry, University of Extremadura, E-06071 Badajoz, Spain
Received: 21 December 1993/Revised: 23 April 1994
Abstract. A multivariate calibration method, partial least
squares (types PLS-1 and PLS-2), was applied to the
simultaneous determination of naptalam (N-(1-naphthyl)
phthalamic acid) and its metabolites N-(1-naphthyl)
phthalimide and 1-naphthylamine in mixtures by UV-
visible absorption spectrophotometry. The absorption
and first-derivative absorption spectra of mixtures were
used to perform the optimization of the calibration ma-
trices by the PLS method. Two different experimental
designs for the three-component mixtures are assayed and
the results are discussed. The proposed method with the
derivative spectra was applied to the determination of
these analytes in river water at the ppb level.
Introduction
This paper reports on the resolution of ternary mixtures
of the pesticide naptalam and its metabolites 1-naph-
thylamine and N-(1-naphthyl) phthalimide. Naptalam is
a growth-regulator [-1] which inhibits seed germination
and is recommended as a pre-emergency herbicide for use
in cucurbits, groundnuts and soyabeans. It is fixed in the
ground by clay and to be active it needs some degree of
moisture. It is estimated to be of low toxicity (acute oral
LDso for rats 8200 mg a.i./kg). However, the possible
presence of 1-naphthylamine, one of the degradation
products of naptalam, whose cytotoxic and genotoxic
effects are well known [2, 3] must be taken into considera-
tion when using this pesticide.
Another degradation product of naptalam is N-(1-
naphthyl) phthalimide, which is obtained from naptalam
at high temperature. In a previous paper, we have investi-
gated [4] the degradation process of naptalam and have
proved that at 25 °C in weakly acid medium, this com-
pound gives rise to 1-naphthylamine and N-(1-naphthyl)
phthalimide by different reactions. We have also pro-
posed a method for determining naptalam and N-(1-
naphthyl) phthalimide in river water by HPLC. In the
Correspondence to: M. I. A. Valenzuela
© Springer-Verlag1994
present paper we report on the resolution of ternary
mixtures of naptalam and its metabolites 1-naph-
thylamine and N-(1-naphthyl) phthalimide using a multi-
variate method (PLS) and its application to their analysis
in river water at the ppb level.
In literature we did not find any reference about the
determination of N-(1-naphthyl) phthalimide; there are
however, numerous references about 1-naphthylamine de-
termination in diverse matrices (sea water, fish, urine,
tobacco smoke, etc) by means of spectrophotometric [-5,
6] spectrofluorometric [7] or chromatographic tech-
niques [,8-14].
As to the determination of naptalam, the major num-
ber of methods reported in literature are based on its
hydrolysis in strong basic medium and 1-naphthylamine
detection [,15-19].
With computer-controlled instrumentation, derivative
techniques and multivariate calibration methods are play-
ing a very important role in the multicomponent analysis
of mixtures by UV/visible molecular absorption spectro-
photometry [-20 22]. Both approaches are useful for the
resolution of band overlapping in quantitative analysis.
Multivariate calibration has been found to be the method
of choice for complex mixtures [-23-26]. Although they
can present difficulties if the maxima are near to each
other or if e values differ strongly, their possibilities can be
increased by combination with the use of derivative
spectra. The optimal choice of the calibration method
depends on the particular experimental conditions.
Among the full-spectrum calibration methods (classical
least squares, CLS; principal component regression, PCR;
partial least squares, PLS), with the factor-based methods
PCR and PLS all overlapping spectral components do
not have to be known, nor has the spectral base line to be
explicitly modelled, unlike with CLS. On the other hand,
PCR and PLS calibrations are very similar. The major
difference between these two methods is that PLS seems
to predict better than PCR in the cases when there are
random linear base lines or independently varying major
spectral components which overlap the spectral features
of the analyte [25]. Therefore in our case, the PLS method
seems to have major predictive ability. The basic concept

of PLS regression was originally developed by Wold [27],
and the use of the PLS method for chemical applications
was pioneered by Wold and co-workers [28, 29]. There
are two types of PLS methods (PLS-1 and PLS-2). They
differ in that type 1 is used to perform the decomposition
and regression for only one component at a time, whereas
type 2 is used to calculate the "loading" on the basis of all
of the concentrations simultaneously, and only one calib-
ration matrix is necessary; therefore PLS-2 is faster and
slightly simpler to use than PLS-1 [29].
Experimental
Apparatus
A Milton Roy Spectronic 3000 Array spectrophotometer
(PC-286 microcomputer), equipped with a sample auto-
matic introductor was used. A Milton Roy Rapid Scan
Software was used for acquisition of the spectra and
calculation of the first-derivative absorption spectra by
the Savitzky-Golay simplified least-squares method [30].
An Epson EL Plus microcomputer provided with an
80386SX microprocessor and an Intel 80387SX co-pro-
cessor was used, equipped with the GRAMS/386 software
[31] package (version 1.06A) with the PLS Plus version
2.1 G application software. The software allows statistical
treatment of the data and the application of the PLS-1,
PLS-2 and PCR methods.
The software incorporates matrix handling routines,
allowing manipulation of files in the Milton Roy Stan-
dard format.
Reagents
N-(1-naphthyl) phthalamic acid and N-(1-naphtyl)
phthalimide obtained from Chem Service were used. 1-
Naphthylamine was supplied by Merck. Standard solu-
tions of these compounds were prepared by dissolving the
appropriate amounts in ethanol (Carlo Erba, RPE).
Procedure for analysing mixtures of naptalam,
N-(1-naphthyl) phthalimide and 1-naphthylamine
Introduce an aliquot of the sample containing between
0 and 40 ~tg of naptalam; 0 and 20 gg of N-(1-naphthyl)
phthalimide and 0 and 30 gg of 1-naphthylamine into
a 10 ml calibrated flask, and dilute to the mark with
ethanol. Record the absorption spectrum between 200
and 400 nm against a blank of ethanol. Apply the opti-
mized calibration matrices, calculated by application of
the PLS-1 and/or PLS-2 methods, to analyse the spectra
of the samples and calculate the concentrations of nap-
talam, N-(1-naphthyl) phthalimide and 1-naphthylamine
in the mixture.
Perform also the optimization of the calibration ma-
trices by application of PLS-1 and PLS-2 methods using
the first-derivative spectra calculated with A2 = 5.25 nm
by the Savitzky-Golay procedure, apply it to the analysis
693
of the first-derivative spectra of the samples and calculate
the concentrations of naptalam, N-(1-naphthyl) phthalim-
ide and 1-naphthylamine.
Procedure for determining naptalam, N- (1-naphthyl)
phthalimide and 1-naphthylamine in river water
We have used the method proposed by Galeano et al. [4]
for the previous treatment of the samples and the precon-
centration of analytes by means of solid-liquid extraction
in Sep-pak plus C18 cartridges. However in our case some
modifications have been made:
Pipette aliquots of 100 ml of water previously filtered
through 0.45 gm cellulose acetate filters, and after adjust-
ing a pH value between 5.1 and 5.2 pass them through
a Sep-pak plus C18 cartridge previously conditioned with
acetonitrile and water at pH 5. Subsequently, pass vol-
umes of 20 ml of ethanol 5% (v/v) in water through the
Sep-pak cartridges for diminishing the background of
river water and finally elute the analytes with ethanol.
Record the absorption spectra of these solutions, calcu-
late the first-derivative system with A2 = 5.25 nm and
finally calculate the concentrations of naptalam, N-(1-
naphthyl) phthalimide and 1-naphthylamine by analysis
of the first-derivative spectra using the optimized PLS-1
calibration matrices.
Results and discussion
The three analytes studied are substances highly absorb-
ing in the UV region of the spectrum (Fig. 1). Naptalam
shows absorption maxima at 222.7 nm and 290 nm, N-
(1-naphthyl) phthalimide (NPhDA) at 221.9 nm and
280 nm, 1-naphthylamine (NNA) at 210.9, 242.7 and
320 nm. Because of the highly overlapping peaks, conven-
tional spectrophotometry cannot be satisfactorily applied
to the determination of these compounds.
With the aim of improving the analysis for the pesti-
cide naptalam and its metabolites in river water, two
chemometric approaches (PLS-1 and PLS-2) were evalu-
ated. Multivariate calibrations are useful in spectral ana-
lyses since the simultaneous inclusion of multiple spectral
intensities can greatly improve the precision and the pre-
dictive ability.
Because the relative performance of the methods are
often dependent on the particular data set being analysed,
it is very difficult to generalize about the superiority of
one method of multivariate calibration over another.
Experimental design of the calibration matrix
and selection of the spectral zone Jor the analysis
Two calibration matrix designs were used to statistically
maximize the information content of the spectra. At first,
a triangular design with a training set of 19 samples was
taken. The concentration of naptalam was varied between
0.6 and 6 ~tg ml -i, of NPhDA between 0.1 and 1 ~tg m1-1
and of NNA between 0.14 and 1.4 ggm1-1 by the
694
Table 1. Concentration data for the different mixtures used in the
calibration set corresponding to a triangular design of naptalam,
NPhDA and NNA

.4 /i Naptalam
Mixture
Naptalam
gg ml 1
NPhDA
gg ml 1
NNA
gg ml- 1

M1 0.58 1.09 1.43

M2 2.33 1.09 1.00
M3 4.08 1.09 0.57
M4 5.83 1.09 0.14
~ 0.2
M5
M6
M7
5.83
5.83
5.83
0.77
0.44
0.11
0.57
1.00
1.43
M8 4.08 0.44 1.43
M9 2.33 0.77 1.43
M10 1.75 0.98 1.29
Mll 3.50 0.98 0.86
M12 5.24 0.98 0.43
0,0 , ~ "~'-
M13 5.24 0.66 0.86
200.0 ~80.0 :]60.0 M14 5.24 0.33 1.29
M15 3.50 0.66 1.29
M16 2.91 0.87 1.15
0,015 M17 4.66 0.87 0.72
M18 4.66 0.55 1.15
M19 4.08 0.77 1.00

• r-4 ,/
\
0.005 /I Table 2. Concentration data for the different mixtures used in the
NN£ calibration set corresponding to an orthogonal design for the determina-
/"\ tion of naptalam, NPhDA and NNA
/ ~/ ~ .......... ~ - - -
,r,,4
,'\V. , Naptalam NPhDA NNA
Mixture gg ml- 1 ~tg ml- 1 gg ml- 1
"~3 - 0 . 0 0 5
I
/ \,.] M1 0 0 0
M2 2 0 0
~£~i~talam M3 4 0 0
I
,r-4 M4 0 1 0
M5 0 2 0
NPhDA M6 0 0 1.5
-0.015 * i i I M7 0 0 3
200.0 280.0 360.0 M8 2 1 0
Wavelength (nm) M9 4 1 0
M10 2 2 0
Fig. 1. Absorption spectra and first-derivative spectra of naptalam Mll 4 2 0
(2 ggml 1), N-(1-naphthyl) phthalimide (NPhDA, 1.0 ~tgml-1) and N- M12 0 1 1.5
1-naphthylamine (NNA, 1.5 ggml- 1) M13 0 1 3
M14 0 2 1.5
M15 0 2 3
M16 2 0 1.5
M17 4 0 1.5
c a l i b r a t i o n m a t r i x . I n T a b l e 1 the c o m p o s i t i o n of the M18 2 0 3
t e r n a r y m i x t u r e s used in the c a l i b r a t i o n m a t r i x is s u m - M19 4 0 3
m a r i z e d , a n d a d i a g r a m m a t i c r e p r e s e n t a t i o n of the mix- M20 2 1 1.5
M21 4 1 1.5
t u r e designs is s h o w n in Fig. 2. Secondly, a n o r t h o g o n a l
M22 2 2 1.5
d e s i g n with a t r a i n i n g set of 27 s a m p l e s was t a k e n . T h e M23 4 2 1.5
c o n c e n t r a t i o n of n a p t a l a m was 0, 2 or 4 g g m 1 - 1 , of M24 2 1 3
N P h D A 0, 1 or 2 p g m l 1 a n d of N N A 0, 1.5 or M25 4 1 3
3 gg m l - 1. T h e c o n c e n t r a t i o n s of the t e r n a r y m i x t u r e s are M26 2 2 3
M27 4 2 3
s u m m a r i z e d in T a b l e 2 a n d a d i a g r a m m a t i c r e p r e s e n t a -
t i o n is also s h o w n in Fig. 2.
T h e spectral r e g i o n b e t w e e n 199.7 a n d 400.1 n m ,
w h i c h implies w o r k i n g with 546 e x p e r i m e n t a l p o i n t s p e r
s p e c t r u m (as the s p e c t r a are digitized each 0.35 nm), was
Selection of the optimum number of factors
selected in b o t h cases for the a n a l y s i s of the a b s o r p t i o n
s p e c t r a a n d b e t w e e n 201.2 a n d 397.6 (535 e x p e r i m e n t a l F o r selecting the n u m b e r of factors in the P L S a l g o r i t h m
p o i n t s ) for the d e r i v a t e spectra, b e c a u s e this is the z o n e in o r d e r to m o d e l the s y s t e m w i t h o u t o v e r f i t t i n g the
w i t h the m a x i m u m spectral i n f o r m a t i o n a b o u t the m i x - c o n c e n t r a t i o n data, a c r o s s - v a l i d a t i o n m e t h o d was used,
ture c o m p o n e n t s . l e a v i n g o u t o n e s a m p l e at a t i m e [32].
 695

M7 NNA

M19 M20 M21

........ :,~ 2"- n

~ 6 M2,.6
M,~6.. " I !

• ! !
I ! I
1 i I I I
I N12
---'I-- ;~
MI3! -. - ,, _W_N14
I , . . . . . . . MI5

N16 d___ T . . . . . . . 1 ......

! I ! |
!
i, I
I I :i I
I M1 I J ~ N2 : M3
. . . . . _52 . . . . . ~ptalam

M7 i Hs"
519
M1 M2 M3 N4
NPhDA '
Naptalam

Fig. 2. Designs for the three-component mixtures used in the data set for the PLS-I and PLS-2 methods. The concentrations of the different mixtures
are given in Tables 1 and 2

For example, in the first-case, given the set of 19 calib-
ration spectra, PLS calibrations on 18 calibration spectra
were performed, and using this calibration, the concentra-
tions of the compounds in the sample left out during
calibration was predicted. This process was repeated 19
times until each calibration sample had been left out once.
The predicted concentrations of the compounds in each
sample were compared with the known concentrations of
the compounds in this reference sample and the prediction
error sum of squares (PRESS) was calculated. One reason-
able choice for the optimum number of factors would be
that number which yielded the minimum PRESS, however
this usually leads to some overfitting. A better criterion is
selecting the model with the fewest number of factors such
that PRESS for that model is not significantly greater than
PRESS from the model with h factors. The F statistic was
used to make the significance determination. Haaland and
Thomas [23] empirically determined that an F-ratio prob-
ability of 0.75 is a good choice.
We selected as optimum the number of factors for the
first PRESS value, the F-ratio probability of which drops
below 0.75.
For the triangular and orthogonal designs, the PRESS
obtained by optimizing the calibration matrices with the
absorption spectra and by optimizing the calibration ma-
trices with the first-derivative absorption spectra with
PLS-1, are shown in Figs. 3 and 4. It can be observed that
there are no significant differences in the selected opti-
mum number of factors and that a high number is neces-
sary. This behaviour is also observed when the PLS-2
method is used (see Tables 3, 4).
Statistical parameters
The expression of the root mean square difference
(RMSD), which is an indication of the average error in the
analysis, for each component is
RMSD = I1/N _~1 (Xi - Xi) 211/2
and the square of the correlation coefficient (R2), which is
an indication of the quality of fit of all the data to
a straight line, is
N
Z xt 2
82 _ i=l
N
F, ( x i - x) 2
i=1
where Xi is the true concentration of the analyte in the
sample i, ~[i represents the estimated concentration of
the analyte in the sample i, ~2 is the mean of the true
concentrations in the prediction set, and N is the total
number of samples used in the prediction set. These
statistical parameters were calculated in each instance
and the values found for the absorbance spectral data
and for the first-derivative spectral data in the triangular
design are summarized in Table 3. Those found in the
orthogonal design are summarized in Table 4. It can
be seen that there are no differences between the values
of RMSD and R 2 for each component, obtained with
the absorption and first-derivative absorption spectral
data, and these are also similar with PLS-1 as well as
with PLS-2 and in both matrices. With respect to the
values of R 2, they are similar in the different approaches
used and for the three components. In all instances the
obtained values of R 2 are satisfactory. By comparing
the results for the two assayed designs, slightly better
results for RMSD are observed in the triangular design
for the three compounds.
696

16.00
A B C
18.00 18.00

14.00 -
14.00 14.00

10.00
• I0.00 10.00
-

6 . 0 0 -
6.00 0.00

Optimum factor Optimum factor 2.00 - Optimum factor

3.00 8.00 [

-8.00 -2.00 -2.oo i

0.00 4.00 8.00 ~ 18.00 0.00 4.60 8.()0 12.00 0.00 ~.6o a.6o
Number of factors

1 8 . 0 0

D E F
14.00 18.00 -

14.00
14.00 -

I0.00
(/) 10.00
o,)
10.00 -

6 . 0 0

6.00
8.00 -

2.00 Optimum factor 2.00 Optimum factor

r Optimum factor 2 . 0 0 -

-2"0O.oo 4.6o 8.~o -2.0o.,o -2.00

4.60 8.60 12.bo 0.00 4.60 8.60 le.OO
Number of factors

Fig. 3A-F. Representation of PRESS values generated from the prediction of naptalam (A, D), N P h D A (B, E) and NNA (C, F) by the PLS-1 method,
as a function of the number of factors used in the calibration for: A, B and C absorbance, and for: D, E and F first-derivative spectral data. Triangular
design

Table 3. Statistical parameters of the PLS (types 1 and 2) method using the absorption spectral and first-derivative absorption spectral data sets.
Triangular design

Absorption spectral data First-derivative spectral data

PLS-1 PLS-2 PLS-1 PLS-2

Component RMSD R2 RMSD R2 RMSD R2 RMSD R2
Naptalam 0.0278(9)" 0.9996 0.0317(8) 0.9996 0.0309(10) 0.9996 0.0356(8) 0.9995
NPhDA 0.0111(8) 0.9984 0.0102(8) 0.9987 0.0192(4) 0.9953 0.0186(8) 0.9956
NNA 0.0109(5) 0.9991 0.0110(8) 0.9992 0.0153(5) 0.9983 0.0162(8) 0.9982

a The numbers in parenthesis are the selected numbers of factors

Table 4. Statistical parameters of the PLS (types 1 and 2) method using the absorption spectral and first-derivative absorption spectral data sets.
Orthogonal design

Absorption spectral data First-derivative spectral data

PLS-1 PLS-2 PLS-1 PLS-2

Component RMSD Rz RMSD R2 RMSD R2 RMSD R2
Naptalam 0.0847(7) ~ 0.9973 0.0799(8) 0.9976 0.0752(5) 0.9979 0.0760(6) 0.9978
NPhDA 0.0414(8) 0.9974 0.0451(8) 0.9970 0.0253(13) 0.9990 0.0393(6) 0.9977
NNA 0.0284(6) 0.9995 0.0260(8) 0.9995 0.0265(6) 0.9995 0.0369(6) 0.9991

"The numbers in parenthesis are the selected numbers of factors

 697

A B C
15.00 ~.50
1.50

oz 11.00
1.5o -~

7.00 0.50
Optimum facto~ 0.50
Optimum factor
1
3.00 [
Optimum factor

-1"°°.00- 4.60 8.6o 1 ~.0~0- -0.50o.oo

I -0.50
4.60 8.60 0.00 4.00
Number of factors

D E F
2.50 I
1.50
5.50

1.50 -
3.50
O.
0.50
Optimum factor
0.50 -
1.50 Optimum factor
Optimum factor I
r
-0'5°.00 4.60 8.60 12.00-°'5g.00 4.00 8.00 -0"%.00 4.~0 8.~0
Number of f a c t o r s

Fig. 4A-F. Representation of PRESS values generated from the prediction of naptalam (A, D) NPhDA (B, E) and NNA (C, F) by the PLS-1 method,
as a function of the number of factors used in the calibration for: A, B and C absorbance, and for: D, E and F first-derivative spectral data. Orthogonal
design

Application to artificial ternary samples
The proposed PLS-1 and PLS-2 methods, applied to the
absorption spectra or to the first-derivative absorption
spectra, allow the resolution of mixtures of the three
components prepared from standard solutions. In Tables
5 and 6, the composition of the ternary mixtures assayed
in the triangular and orthogonal designs, respectively,
are shown. In Figs. 5 and 6, we summarize the recovery
values obtained by the application of the PLS-1 and
PLS-2 methods and by using the absorbance and first-
derivative absorption spectral data, in the triangular
and the orthogonal designs respectively. It can be
seen that slightly better results are obtained with the
triangular design. Also, there are no significant differences
between the values found from the absorption spectra
and from the first-derivative spectra. However, with the
aim of comparing the ability of prediction of the two
mixture designs, these procedures have been also applied
to the resolution of artificial mixtures which cannot be
represented in the triangular design; but they are in the
assayed range of concentrations for each component. The
composition of these ternary mixtures (M1, M2 and M3)
and the % recoveries are shown in Table 7.

Table 5. Composition of the synthetic mixtures of naptalam, NPhDA Table 6. Composition of the synthetic mixtures of naptalam, NPhDA
and NNA for their resolution by PLS. Triangular design and NNA for their resolution by PLS. Orthogonal design

Naptalam NPhDA NNA Naptalam NPhDA NNA

Mixture pg ml- 1 gg ml 1 gg ml Mixture ~tgml- 1 pg ml 1 ~tgml- 1

P1 4.66 0.77 0.86 P1 0.80 0.40 0.45

P2 5.24 0.55 1.00 P2 1.50 0.80 0.75
P3 4.66 0.44 1.29 P3 3.00 1.50 3.00
P4 3.50 0.87 1.00 P4 0.50 0.50 1.50
P5 3.50 0.55 1.43 P5 1.50 1.00 0.75
698

J
100 100 100 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
looi~
80 . . . . . . 80
80 80

60 60 rio ..................

40 40 40 . . . . . . . . . . . . . . . . . . . . . . .

20 20 2O

pl ~2 p3 p4 p5
pl p2 p3 p4 p5 pl p2 p3 p4 p5 pl p2 p3 p4 p5
b
b

100 100 100

i ii
80 80 80

60 60 ooll 60

4-0 40 40 40

20 20 20 20

/1 p2 p3 p4 p5 pd p2 p3 p4 p5 ~1 p2 p3 p4 p5 pl p2 p3 p4 p5
d d

F
I
100 100 ............................ 100

80

60

40
I
80 ............ 80

60

40
100-
80 -11111106O
20 20 20 2O

pl p2 p3 p4 p5 pl p2 p3 p4 p5 pl p2 p3 p4 p5 pl p2 p3 p4 p5
e f f
I PLS- 1 ~ PLS-2 I PLS-1 1 ~ PLS-2

Fig. 5a f. Diagrammatic representation of the percentage recovery in
the analysis of five mixtures of: naptalam (a and d); NPhDA (b and
e); NNA (e and f) by PLS-1 and PLS-2 methods, using a-e
direct absorbance, (d-f) first-derivate spectral data. Triangular
design
Fig. 6a-L Diagrammatic representation of the percentage recovery in
the analysis of five mixtures of naptalam (a and d); NPhDA (b and
e); NNA (e and f) by PLS-1 and PLS-2 methods, using (a-e)
direct absorbance, (d f) first-derivative spectral data. Orthogonal
design

It can be seen that the use of the PLS method with
the calibration matrix obtained by the triangular design
does not resolve these ternary mixtures, while in the
orthogonal design any ternary mixtures can be represent-
ed and the use of the PLS method with the calibration
matrix by the orthogonal design resolves the ternary
mixtures which can be represented in the triangle
(M4, M5) as well as those that cannot be represented
(M1, M2, M3).
We have chosen the orthogonal design of the calib-
ration matrix for the resolution of ternary mixtures of
naptal~/m, NPhDA and NNA by applying the PLS
methods. Also we can appreciate that no differences ap-
pear when PLS-1 and PLS-2 methods are used.
Simultaneous determination of naptalam, NPhDA
and NNA in river water samples by the P L S method
We have appfied the proposed method described under
Experimental. In a previous work [4] we have found that
optimum retention of these analytes in the Sep-pak plus
C18 cartridges was obtained when a pH between 5.1
and 5.2 was adjusted in the river water. This pH is a
 699

Table 7. Recovery values (%) found in the analysis of ternary mixtures by the PLS method using the absorption spectra and first-derivative absorption
spectra. T.D.: Triangular design. O.D.: Orthogonal design

PLS-1 PLS-2
Absorption s p e c . First-derivative spec. Absorption spec. First-derivative spec.
T.D. O.D. T.D. O.D. T.D. O.D. T.D. O.D.

M1 97.8 96.3 100.6 93.1 99.3 96.2 95.9 94.2

M2 105.6 100.4 111.8 102.2 106.4 100.2 108.1 100.8
Naptalam M3 99.2 96.5 102.6 99.2 100.2 96.1 101.9 98.3
M4 100.5 99.7 99.9 92.0 100.5 99.3 100.1 94.3
M5 100.8 100.1 99.3 96.6 101.3 99.9 99.7 97.6

M1 181.5 100.4 196.2 92.1 186.1 97.5 187.8 108.1

M2 179.8 102.3 190.7 104.2 183.3 100.3 170.5 100.6
NPhDA M3 142.2 106.9 144.8 106.3 144.4 104.2 137.1 103.3
M4 100.1 104.2 99.8 98.9 100.2 99.1 100.1 I12.0
M5 99.7 99.6 99.1 92.5 99.9 96.2 100.8 105.9

M1 75.9 110.7 93.3 102.1 96.2 110.7 116.7 99.6

M2 72.4 98.6 85.9 97.7 87.3 96.4 104.3 95.6
NNA M3 86.8 102.5 97.1 103.6 95.3 100.3 106.0 102.8
M4 99.5 97.2 99.7 93.9 99.6 96.7 99.9 90.6
M5 99.2 97.8 98.8 91.4 99.7 96.2 99.3 88.4

Composition of ternary mixture: Naptalam; NPhDA; NNA; (in ~tgml- 1)

MI: 1.75; 0.82; 0.57 M2: 1.50; 0.80; 0.75 M3: 1.50; 1.00; 0.75; M4: 1.75; 0.98; 1.29 M5: 2.33; 1.09; 1.00

c o m p r o m i s e value because of the different a c i d - b a s e p r o p -

erties of the a n a l y t e s n a p t a l a m a n d 1 - n a p h t h y l a m i n e .
F i g u r e 7 shows the a b s o r p t i o n s p e c t r u m of a river
1.2
w a t e r s a m p l e s p i k e d with n a p t a l a m , N P h D A a n d N N A ,
t r e a t e d a c c o r d i n g to the p r o p o s e d p r o c e d u r e , t o g e t h e r
with the a b s o r p t i o n s p e c t r u m of an u n s p i k e d river w a t e r
s a m p l e o b t a i n e d u n d e r the same conditions. T h e o v e r l a p -
0 0.8 ping of the two spectra can be seen. T h e first-derivatives of
DO
b o t h s p e c t r a with A2 = 5.25 n m are also s h o w n in Fig. 7.
..<
W i t h the different a n d smaller A2 a s s a y e d (up to 1.75 nm)
p r a c t i c a l l y identical spectra are o b t a i n e d . The diminish-
0.4 ing of the interference degree of the river w a t e r b a c k -
g r o u n d can be observed. Therefore, we have a p p l i e d the
P L S m e t h o d to the first,derivative a b s o r p t i o n spectral
d a t a set for the r e s o l u t i o n o f t e r n a r y m i x t u r e s in river
0.0 , , ~ I ~ , ' I water. T h e b a c k g r o u n d interference p r o b l e m can be re-
200.0 ~80.0 gflO.O d u c e d if river water samples are t a k e n with k n o w n con-
c e n t r a t i o n s of the three analytes for o b t a i n i n g the calib-
0.04 r a t i o n set, b u t it is tedious, because it is necessary to
o b t a i n different c a l i b r a t i o n m a t r i c e s for waters of different
sources a n d the p r e p a r a t i o n of the samples is laborious.
T h e a p p l i c a t i o n of the P L S m e t h o d to the first-derivative
0,02
spectral d a t a set allows to d i m i n i s h the river w a t e r b a c k -
g r o u n d influence a n d it can be possible to use a single
0) c a l i b r a t i o n set p r e p a r e d from s t a n d a r d s o l u t i o n s of the
c o m p o u n d s for the analysis of the different waters. The
cO 0.00 p r e d i c t e d c o n c e n t r a t i o n s a n d the recovery values o b -
.m.I
~j t a i n e d by a p p l i c a t i o n of the P L S m e t h o d with the a b s o r p -
tion spectra a n d first-derivative a b s o r p t i o n s p e c t r a are
s u m m a r i z e d in T a b l e 8. It can be seen t h a t satisfactory
I
+a -o.o2

Fig. 7. Absorption spectra and first-derivative absorption spectra of

-0,04 1 I I a river water sample spiked with 23.3 gg of naptalam, 11.0 gg of
200.0 280.0 360.0 NPhDA and 10.0 ~tg of NNA (solid line), and a river water sample
Wavelength (nm) unspiked (dashed line)
700

Table 8. Recoveryof naptalam,

NPhDA and NNA in fiver water by Added Found
the PLS method using the absorption
spectra and the first-derivative PLS-1 PLS-2
absorption spectra (values given in
ngml- 1) (Recoveries (%) ± RSD") Abs. First Abs. First
spectrum Def. spectrum Oer.
80 218 ± 7.6 133.8 ± 6.4 237 ± 7.0 128.0 ± 8.4
Naptalam
100 170 + 3.4 109.3 ± 5.3 177.8 ± 2.9 106.9 + 5.8

40 Negativevalues 109.6 +_5.3 Negative values 68.5 ± 3.1

NPhDA
60 33.4 ± 6.2 108 ± 1.9 Negative values 74.8 ± 1.5

45 63.6 ± 3.4 96.2 ± 5.5 260 ± 5.9 118.9 ± 9.5

NNA
75 65.7 ± 4.2 89.3 ± 6.1 180.7 ± 1.9 102.0 ± 6.5

~RSD: relative standard deviation. Each value corresponds to the mean of three separate determinations.

recoveries have been obtained in all instances except for
naptalam in the lowest contamination degree, when the
PLS-1 method is applied with the first-derivative spectral
data set corresponding to the orthogonal design. The
PLS-2 method, however yields more unsatisfactory
results.
Conclusions
It is well known that the design of the calibration set
is important for the predictive ability of the multi-
variate calibration methods, but in most papers any
information about this topic is lacking. In our case,
we have proved that the experimental design is very
important for the resolution of the ternary mixtures
assayed.
When the overlapping of the spectral bands is very
important, as in our case (see Fig. 1), the commonly used
"zero-crossing" derivative method is poor because only
a single wavelength signal is used for the analysis [33].
The limitations of this method are greater for ternary
mixtures. However the multivariate calibration method
PLS allows the solution of these problems, as we have
proved in this work.
On the other hand, there are contradictory results
about the convenience of the use of derivative techniques as
a prior step in the application of multivariate calibration
methods [33, 34]. In our case, the background of river
water makes the derivation of the spectra necessary (see
Fig. 7) as it is demonstrated by the results shown in Table 8.
So it can be possible to use only one calibration set,
prepared from standard solutions of the compounds, for
the analysis of different waters. Also in this case, we have
obtained better results when we have applied the PLS-1
method to first-derivative spectral data instead of the
PLS-2 method (as occurs in other real samples [24]). Even
so, recoveries found for naptalam and N P h D A are high
with respect to those found by H P L C of similarly prepared
samples [4], because of the background of river water.
Acknowledgment.The authors acknowledgethe DGICYT of the Minis-
terio de Educaci6n y Ciencia of Spain (Project PB91-0856)for financial
support of this work.
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