Vibrational Spectroscopy 32 (2003) 129–136

Applications of FT-IR spectrometry to plasma contents

analysis and monitoring
Gérard Délérisa,*, Cyril Petiboisa,b
a
INSERM U443, Groupe de Chimie Bio-Organique, Université Victor Segalen Bordeaux 2,
146 Rue Léo Saignat, 33 076 Bordeaux, France
b
Faculté des Sciences du Sport et de l’Education Physique, Université Victor Segalen Bordeaux 2,
Avenue Camille Julian, 33 405 Talence, France

Abstract

Here, we review recent methodological developments for plasma analysis by Fourier-transform infrared (FT-IR) spectrometry
to provide both a high sensitivity and a global overview of its biomolecular contents along with the variations of these ones.
Transmittance FT-IR spectrometry has been used to analyze plasma micro samples (50 ml) using an iterative process. Results in
accordance with clinical data were obtained from a single FT-IR spectrum for the following biomolecules: amino acids, fatty
acids, albumin, glucose, fibrinogen, lactate, triglycerides, glycerol, urea, a1-antitrypsin, a2-macroglobulin, transferin, Apo-A1,
Apo-B, Apo-C3, IgA, IgD, IgG1, IgG2, IgG3, IgG4, IgM, haptoglobin, a1-acid glycoprotein, cholesterol, and cholesterol esters.
Therefore, as only micro samples are necessary, high frequency blood analysis become available. We also present a novel
application of this method for the monitoring of inflammatory processes related to given metabolic stresses in rugby players. We
show that an FT-IR spectrum constitutes a ‘‘metabolic photography’’ of the subject, allowing classification between metabolic
groups (pathologic or others). It was used on difference spectra in order to raise ‘‘signal-to-noise’’ ratio by elimination of the
unvarying spectral contribution. Among others, it allowed to uncover overtraining in high-level sportsmen several weeks before
any physiologic or clinical symptom occurred.
# 2003 Published by Elsevier B.V.

Keywords: FT-IR spectrometry; Plasma; Molecular analysis; Proteins; Microsample; Classification; Clinics; Method

1. Introduction as the most representative. Limited to 30 at maximum

A pertinent description of the metabolic response to
a physical or pathological stress and its evolutions
emphasizes the place of biochemical adaptations to
the metabolic stress induced by this perturbation. The
present methods allowing the study the evolution of
blood biochemical parameters require a previous arbi-
trary selection of those that clinical biology considers
*
Corresponding author. Tel.: þ33-5-5757-1002;
fax: þ33-5-5757-1002.
E-mail address: gerard.deleris@u-bordeaux2.fr (G. Déléris).
their number is highly insufficient to describe multi-
factorial physiological processes. For routine analyses
giving specific information about the metabolic
response to exercise, blood microsamples are widely
used to determine accessible biological markers of
fatigue stress, such as for glucose, lactate and ammo-
nium concentrations. This is because blood is easily
drawn at the fingertip and the current portable analy-
zers now offer the opportunity to obtain results in
accordance with clinical analytical methods. How-
ever, to date, these materials are not able to analyze
more than these few biochemical parameters while

0924-2031/$ – see front matter # 2003 Published by Elsevier B.V.

doi:10.1016/S0924-2031(03)00053-5
130 G. Déléris, C. Petibois / Vibrational Spectroscopy 32 (2003) 129–136
there are various processes to consider in the global
metabolic response to an exercise. Therefore, global
analytical methods are required to analyze all blood
organic content modifications and to obtain a global
vision of the biochemical processes involved in the
metabolic response to an exercise. There are only two
global physico-chemical techniques that offer the
opportunity to analyze pertinent information of bio-
logical sample contents; these are, nuclear magnetic
resonance (NMR) and Fourier-transform infrared
(FT-IR) spectrometries.
FT-IR spectrometry has proved to be a global,
sensitive and highly reproducible physico-chemical
analytical technique with which structural biomole-
cular moieties are characterized by their infrared
absorption [1]. Since a biomolecule is determined
by unique structure, unique FT-IR spectrum will be
exhibited by this biomolecule, representing its struc-
tural fingertip. Furthermore, every biomolecular
family present in the sample will exhibit almost
similar and overlapping FT-IR absorptions. FT-IR
analytical applications allowed blood contents deter-
mination using various materials and sample prepara-
tions. Concentrations of glucose [2–4], total proteins,
creatinine, urea, triglycerides [1], and cholesterol [5]
in blood, plasma or serum have been determined with
clinical accuracy. However, important sample pre-
parations, spectra manipulations, and mathematical
treatments were necessary to obtain such results [2].
Clinical analyzes require methods where sample
manipulations are minimized as every one may be
considered as a source of quantitative error affecting
the prediction performances of the method used.
With FT-IR spectrometry, water absorption fluctua-
tions due to environmental conditions (air tempera-
ture and dryness, atmospheric pressure variations. . .)
made sample and/or spectra manipulations necessary
[2–6].
2. Methodology: FT-IR spectra acquisition
Recently, we showed that reproducible FT-IR spec-
tra could be obtained from dried plasma samples
without the use of internal standards, baseline correc-
tion, and spectra normalization [7]. Capillary blood
samples (approximately 50 ml, two to three drops)
were taken for FT-IR analyses in a non-anticoagulated
gel separator tube (microtainer no. 365960, Becton–
Dickinson) using a standard lancet device (Softclix
Pro, Boehringer-Mannheim, Germany). Blood was
immediately centrifuged for 3 min at 15 000  g
(Heittich, Germany) to separate plasma from erythro-
cytes. Sample were then stored at 20 8C before
analyses. After samples returned to room temperature
(about 15 min at 20–25 8C), 20 ml aliquots were
diluted with 80 ml of water. The diluted samples were
homogenized with an agitator (Vortex Reax 2000,
Heidolf) at 1000 g for 10 s. Then, 35 ml of each
solution were exactly deposited within the cell limits
of a zinc selenide (ZnSe) wheel bearing 15 sample
cells (Bruker). The wheel was subsequently placed
into a drying vacuum (2 mmHg) to evaporate water
(45 min). A KBr cover was then screwed onto the
wheel to protect it. The wheel was finally put into the
analysis compartment of a Bruker IFS 28/B spectro-
meter equipped with a Globar (MIR) source (7 V), a
KBr beam splitter and a DTGS/B detector (18–28 8C).
Beam diameter at the sample location was 6 mm. In all
experiments, a 2.0 cm1 resolution was used and
acquisitions were performed using 32 scans. A Black-
man–Harris three-term apodization and a zero filling
of four were applied before FT. All analyses were
performed in triplicate on three successive wheels.
Between-run coefficient of variation was 1.5% [7]. An
example of a plasma FT-IR spectrum obtained from
plasma dried film is given in Fig. 1 and Table 1.
Typically, a signal-to-noise ratio 700 is obtain after
these plasma FT-IR spectra acquisitions.
Fig. 1. Plasma FT-IR spectrum obtained from dried plasma sample
(3).
 G. Déléris, C. Petibois / Vibrational Spectroscopy 32 (2003) 129–136
Table 1
Major plasma content assignments for plasma FT-IR spectra
absorption bands
Bands (cm1) Major assignments for plasma contents
3020–3000 n(CH): unsaturated fatty acids, cholesterol esters
2990–2950 nas(CH3): cholesterol esters, triglycerides
2950–2880 nas(CH2): long chain fatty acids, phospholipids
2880–2860 ns(CH3): cholesterol esters, triglycerides, glycerol
2870–2830 ns(CH2): long chain fatty acids, phospholipids
2996–2819 nas(CH3), ns(CH3), nas(CH2), ns(CH2): fatty acids,
phospholipids, triglycerides
1739–1732 n(C=O): lipids, cholesterol esters, triglycerides
1720–1600 n(C=O): (amide I) b-sheet: proteins, turns, coils
1630–1560 d(NH2): amino acids
1600–1480 d(N–H): (amide II) a-helix: proteins
1480–1430 das(CH3), das(CH2), ds(CH3), ds(CH2): fatty acids,
phospholipids, triglycerides
1430–1360 n(COO): amino acids
1300–900 n(C–O): saccharides, glucose, lactate, glycerol
n: stretching vibrations, d: bending (scissoring) vibrations, s:
symmetric, as: asymmetric.
3. Data: FT-IR determination of plasma
concentrations
Subsequent studies [7–11] were then performed to
determine a wide range of molecular concentrations
from single plasma FT-IR spectra. Our first aim was to
avoid the use of mathematical analyses (namely PLS
regression) to determine plasma concentrations. We
felt that such mathematical analyses did not necessa-
rily obey to the Beer–Lambert Law since chosen
absorbances of the analyzed molecule receive differ-
ent weights factors to obtain result ‘‘statistically’’ in
accordance with reference data. Indeed, we preferred
to analyze the correlativity between single molecular
absorbances found at the surface of plasma FT-IR
spectra and reference concentrations. Therefore, the
challenge was to find a spectral absorption sufficiently
characteristic of a molecule to allow its concentration
determination without the use of any other mathema-
tical treatment. In this way, the Beer–Lambert Law
could be respected. Another important methodologi-
cal point of our procedure was the spectral contribu-
tion subtraction of every determined molecular
concentration from plasma FT-IR spectrum. This sub-
traction allowed to find and analyze spectral absor-
bances that could be partly or totally overlapped by the
previous ones.
131
To respect the Beer–Lambert Law, we had to com-
pare absorbances of molecules regarding the organic
matter concentration found within plasma. Dried mat-
ter in plasma is ca. 100 g/l and this value suffers very
little inter-individual variability for homeostasis rea-
sons. We previously demonstrated that FT-IR analyzes
of plasma diluted with four volumes of water allowed
to obtain the higher signal-to-noise ratio (determined
in the 2100–2200 cm1 region, where no plasma
absorption is found) and signal reproducibility on
successive analyzes. These four-fold diluted solutions
contain about 20 g/l of dried matter. Therefore, five
standard solutions ranging from 12 to 28 g/l were
used to obtain pure product FT-IR spectra where
absorption level was comparable to that of plasma
FT-IR spectra. Pure products FT-IR spectra (98–99%,
Sigma–Aldrich) were used for reference FT-IR spectra
acquisitions and for determination of the most char-
acteristic absorption bands in every molecule ana-
lyzed. These spectra were further used for all
biomolecule absorption subtractions. For every bio-
molecule, signal detection linearity between these
concentrations was assessed by total area integration
(4000–500 cm1), representing the whole biomolecu-
lar FT-IR absorption (with P < 0:01). Signal-to-noise
ratio obtained on pure product spectra were always
600–800. The method for iterative biomolecular con-
centrations determination was assessed as follows:
1. Spectral windows (peaks, bands, shoulders) found
in surface of spectra were determined by Opus-
Ident 3.03 (Bruker, Germany) as follows: Starting
from the highest wave number exhibiting a non-
null absorption, a cord was adjusted in order it did
not cross the spectrum curve before the second limit
of the window was reached. From this point of the
spectrum, a second cord, i.e. a second spectral
window, was determined by the same way. This
process was achieved till the last possible spectral
window was reached, roughly at 530 cm1.
2. For each defined spectral window common to the
35 plasma FT-IR spectra of our experiments, area
found between the spectrum and the cord running
through the two limit points was integrated by this
software and expressed in its arbitrary digits (U).
3. A correlation matrix was used to compare series
of integration results with biomolecular con-
centrations measured with reference methods.
132 G. Déléris, C. Petibois / Vibrational Spectroscopy 32 (2003) 129–136

The spectral window that led to the highest

correlation between integration values and refer-
ence concentrations of one of the considered
biomolecules was selected for subsequent use.
4. A series of accurately determined solutions of this
biomolecule allowed to build a standard solution
series, which was used to integrate spectral area of
the pure compound under the previously deter-
mined spectral window (biomolecular concen-
tration expressed in g/l per spectral area digit:
g/(l U)).
5. For each FT-IR plasma spectrum, biomolecule
concentration was then calculated using this
absorption value and the spectral area integration
result. (Results for albumin are presented in Fig. 2, Fig. 3. Relationship between IgG3 concentration measured by
for IgG3 in Fig. 3, and Apo-C3 in Fig. 4.) reference and FT-IR spectrometry methods; n ¼ 35 subjects.
6. The contributing spectrum of the biomolecule was
calculated from its pure product spectrum and its
plasma concentration and was subsequently sub-
tracted from total plasma spectrum.
7. The resultant spectrum was then submitted to the
same iterative method (from first to sixth step) to
determine the subsequent biomolecule concentra-
tion, and so on. Illustration of this final step for
IgG1 is shown on Fig. 5.
It is noticeable that FT-IR spectra of biomolecules
belonging to the same families presented important
differences, which reflected their structural specifici-
ties. As an example, immunoglobulins exhibit very
different spectral signals between 1300 and 900 cm1

Fig. 4. Relationship between Apo-C3 concentration measured by

reference and FT-IR spectrometry methods; n ¼ 35 subjects.

(n(C–O) region), due to glucidic contents differences

Fig. 2. Relationship between albumin concentration measured by
reference and FT-IR spectrometry methods; n ¼ 35 subjects.
(Fig. 6). This was also observed for apolipoproteins
concentrations determination since three different
spectral regions were used. Finally, after 26 biomole-
cular subtractions [7–11], corresponding to 69  4 g/l
of plasma dried matter, mean total spectral area of
resultant spectra was 114:5  11:2 U, i.e. 24.2% of
initial plasma FT-IR spectral area. One can also
observe that resultant spectra were not ‘‘noisy’’ after
all these subtractions (Fig. 7), that was due the high
signal-to-noise ratio obtained on pure product spectra
and plasma spectra.
 G. Déléris, C. Petibois / Vibrational Spectroscopy 32 (2003) 129–136 133

Fig. 5. Subtraction of IgG1 absorption contribution from plasma FT-IR spectrum.

Global data obtained from 35 patients suffering This blind tests demonstrated the usefulness of this
from various metabolic diseases (i.e. diabetes, anemia, iterative method for plasma contents analysis [8].
severe infections, renal dysfunction . . .) are presented
in Table 2 [8]. This population was chose since plasma
4. Global analyses of plasma FT-IR spectral
protein concentration exhibited important concen-
information
tration variations, one- to five-folds out of the phy-
siological range. This sequence order of analysis was 4.1. The biomolecular response to exercise
subsequently tested for determining concentrations by
assaying plasma samples from 14 patients in which the Two FT-IR spectra of the same type of biological
concentrations of several proteins varied also widely. sample (plasma for example) may be subtracted to

Fig. 6. Absorbance of immunoglobulin G subclasses within the Fig. 7. Resultant plasma FT-IR spectrum after 26 successive
1850–500 cm1 spectral interval. molecular absorption subtractions.
134 G. Déléris, C. Petibois / Vibrational Spectroscopy 32 (2003) 129–136

Table 2
Statistical indices between reference and FT-IR spectrometry values [7–10]

Biomolecule FT-IR spectrometry data Statistical data

Left Right Absorption Area (U) Concentration r Sx/y Slope Intercept

(cm1) (cm1) (g/(l U)) (g/l)

Albumin 1600 1488 1.76 23.3  0.3 41.0  3.9 0.98 0.67 1.00 0.67
Glucose 1062 997 7.27* 0.71  0.05 5.22  0.17* 0.95 0.05* 0.99 0.04*
Fibrinogen 1428 1363 2.42 1.38  0.17 3.36  0.46 0.97 0.10 1.04 0.16
Immunoglobulin-G2 1652 1622 3.72 0.74  0.11 2.57  0.57 0.97 0.18 1.01 0.02
Lactate 1134 1115 65.46* 0.026  0.007 1.80  0.36* 0.94 0.11* 1.09 0.08*
Glycerol 882 837 8.81* 0.005  0.001 0.23  0.05* 0.97 0.03* 1.03 0.06*
Triglycerides 1122 1091 3.91* 0.015  0.004 0.84  0.17* 0.98 0.14* 0.98 0.02*
Cholesterol 1074 1033 8.21 0.11  0.08 0.89  0.69 0.97 0.14 0.97 0.05
Cholesterol esters 1186 1147 2.11 0.42  0.09 0.90  0.22 0.93 0.18 1.01 0.01
Immunoglobulin-G1 1419 1361 9.07 0.83  0.23 7.66  2.08 0.98 0.35 1.07 0.35
a1-Antitrypsin 712 691 48.4 0.059  0.015 2.89  0.68 0.96 0.18 1.04 0.14
a2-Macroglobulin 1116 983 1.98 1.05  0.26 2.11  0.51 0.98 0.922 1.01 0.03
Transferin 1338 1292 12.39 0.19  0.02 2.45  0.31 0.97 0.08 0.99 0.02
Apolipoprotein-A1 1484 1427 1.81 0.91  0.11 1.55  0.22 0.97 0.05 1.06 0.06
Urea 1652 1632 35.01* 0.17  0.04 6.25  1.39* 0.97 0.33* 1.07 0.25*
Apolipoprotein-B 2862 2835 6.95 0.13  0.01 0.96  0.17 0.96 0.05 0.96 0.07
Immunoglobulin-M 1428 1360 1.96 0.61  0.25 1.27  0.65 0.98 0.13 0.97 0.11
Apolipoprotein-C3 1676 1660 8.09 0.062  0.028 0.52  0.19 0.97 0.04 1.06 0.004
Immunoglobulin-A 1418 1372 5.31 0.36  0.11 1.88  0.70 0.97 0.16 0.99 0.003
Immunoglobulin-G4 1538 1505 6.89 0.075  0.031 0.54  0.26 0.95 0.08 0.93 0.08
Immunoglobulin-G3 1652 1628 4.29 0.14  0.05 0.63  0.23 0.95 0.07 1.03 0.01
Immunoglobulin-D 1479 1464 21.8 0.019  0.006 0.38  0.16 0.92 0.05 1.11 0.03
Haptoglobin 1428 1378 3.35 0.29  0.14 0.98  0.47 0.99 0.08 1.04 0.01
a1-Acid glycoprotein 1337 1279 2.77 0.33  0.08 0.82  0.23 0.96 0.07 0.99 0.02
Fatty acyl moieties 2996 2819 0.82* 10.4  1.11 8.54  0.91 0.94 0.11 0.97 0.22
Amino acids 1430 1360 0.062 5.43  0.79 0.33  0.08 0.95 0.04 0.93 0.26
P < 0.001 if r > 0:99; P < 0:005 if r > 0:95. All values expressed in g/l  S.D., except (*) in mmol/l  S.D. [7–11].

give a resulting FT-IR spectrum in which should

appear only differences of the molecular content
between the two samples [11,12]. Thus, the use of
difference FT-IR spectra obtained from two plasma
samples avoids unchanged molecular information, and
it is only the effects of a stress factor (therapeutics,
physical exercise, psychological stress . . .) on blood
organic contents that are analyzed. As an example, it
has been demonstrated that a physical exercise of
any intensity do not change more than 5–7% of the
plasma contents [12]. An illustration of a biomolecular
response to a physical stress is given in Fig. 8. This
difference FT-IR spectrum is the mean of 16 experi-
ments obtained from an intense physical exercise
performed by rugby players (Rugby study). From these Fig. 8. Difference plasma FT-IR spectrum (rest-plasma FT-IR spec-
subjects, eight entered a recovery program (Group A; trum subtracted from exercise-plasma FT-IR spectrum); n ¼ 16 rugby
massages, sauna, moderate exercises, stretching. . .) players.
 G. Déléris, C. Petibois / Vibrational Spectroscopy 32 (2003) 129–136
for 4 days, while the eight others (Group B) did not
performed any other activity. Eight other subjects were
used as controls and did not performed any physical
exercise (Group C). Rest and exercise (end of game)
plasma samples were drawn in the Groups A and B
subjects, and rest samples were drawn at the fasting
state every morning during 5 days for all subjects.
From the mean difference FT-IR spectrum obtained
from the 16 individual rest- and exercise-plasma
FT-IR spectra, the global metabolic response to exer-
cise could be described as follows: (1) absorbances of
nas(CH3), nas(CH2), ns(CH3), and ns(CH2) decreased,
which corresponded to a decrease of 0:31 
0:12 mmol/l of fatty acyl moieties (clinical data);
(2) absorbance of amide I and II (n(C=O) and
d(N–H)) increased, which corresponded to a 2:11 
0:45 g/l of proteins (clinical data); and (3) absorbance
of d(NH2) increased, which corresponded to a
0:24  0:06 g/l of amino acids (clinical data). Over
the determination of 26 individual molecular con-
centrations from plasma FT-IR spectra, these spectra
could also be used for estimating with high correlation
with clinical data, the concentration of families
of metabolites (fatty acyl moieties and amino acids),
which could not be studied individually due to the
diversity of the molecules considered and their low
concentrations [11].
Fig. 9. Classification of difference FT-IR spectra (4000–500 cm1) obtained from plasma sampled before and after exercise (n ¼ 24); (A)
rugby players with recovery program; (B) rugby players without recovery program; (C) controls.
135
4.2. Classification of FT-IR spectra
Another global analysis of plasma FT-IR informa-
tion may be obtained from classification of spectra
or selected spectral intervals [13]. These ones can be
classified to differentiate samples by clusters for-
mation using Ward’s algorithm [14] on Euclidian
distances between data. This algorithm attempts to
find the most homogeneous groups between spectra.
Each group is formed in order to lead to the smallest
growth in heterogeneity (H). Heterogeneity is roughly
the geometric distance in the multidimensional
space, computed for the spectra matrix (3630
points per spectrum) as follows: distance ðxi ; yi Þ ¼
fSi ðx  yÞ2 g1=2 . Since our method for spectra acquisi-
tion is a quantitative method, heterogeneity values
between groups of spectra will be proportional to their
absorption differences. To be significant, heterogeneity
value (we noted ‘‘H’’) between two clusters
must be above the sum of these clusters heterogeneity
(we noted ‘‘h’’) values. As an example, Fig. 9 shows
the classification of plasma FT-IR spectra obtained the
day after intense exercise for Groups A–C subjects
(Rugby study). Groups A and B subjects performed the
exercise and were clearly differentiated from controls.
In this case, the use of the 4000–500 cm1 spectral
interval is sufficient for discriminating exercising
136 G. Déléris, C. Petibois / Vibrational Spectroscopy 32 (2003) 129–136
Fig. 10. Classification of difference FT-IR spectra (1700–
1500 cm1) obtained from plasma sampled before exercise and
after 3 days of recovery (n ¼ 16); (A) rugby players with recovery
program; (B) rugby players without recovery program.
from non-exercising subjects. However, Groups A and
B subjects could not be clearly separated since the
recovery program had not begun. In Fig. 10, Groups A
and B subjects could be clearly differentiated after 3
days of the recovery program (for Group B). This
classification was performed using the 1700–
1400 cm1 spectral interval of plasma FT-IR spectra
obtained 3 days after exercise. This classification could
be related to changes in acute phase reactants concen-
tration, notably for a1-antitrypsin, a2-macroglobulin,
a1-acid glycoprotein, haptoglobin, and transferin.
5. Conclusions
Different applications of FT-IR spectrometry have
been developed to propose a useful tool for the
monitoring of plasma contents. They allow the deter-
mination of 26 plasma concentrations from a single
plasma FT-IR spectrum, the analysis of exercise-
induced changes in plasma contents from two blood
microsamples taken at the fingertip. Moreover they
allow to estimate concentration of biomolecular
families with high correlation with clinical data
and provide the opportunity to differentiate physio-
logical profiles from FT-IR spectra classifications.
Therefore, the methodology we proposed allows a
global overview of stress effects on the human body.
As an example, we had previously used with success
this methodology for monitoring the metabolic
response to exercise of rowers, allowing to reveal
discriminant parameters of overtraining occurrence
several weeks before its clinical diagnosis [12]. To
find these parameters, reproducible exercise-tests
were performed every training week over 1 year. Such
an experimental protocol may be envisaged only if
using blood microsamples.
Acknowledgements
The authors are indebted to the ‘‘Conseil Régional
d’Aquitaine’’ and the ‘‘Fédération Française de Rubgy;
Recherche’’ for financial support and technical assis-
tance.
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