Documente Academic
Documente Profesional
Documente Cultură
DOI 10.1007/s00216-002-1731-y
O R I G I N A L PA P E R
Received: 15 July 2002 / Revised: 8 November 2002 / Accepted: 27 November 2002 / Published online: 12 February 2003
Springer-Verlag 2003
Abstract Samples distributed in proficiency testing schemes (PTS) need to be homogeneous in order to be sure
that if a laboratory has a result different from the other
laboratories, its error can be attributed to its analysis
method and not to its sample. This control must be done
according to the ISO 13528 draft standard before sending
the samples to the laboratories. It can be done by determining homogeneity targets by sub-contracting to accredited laboratories using reference methods, but this engenders logistic and financial problems. That is why a homogeneity check using Near Infrared Spectroscopy (NIR)
has been developed for agricultural and food industries
samples prepared for PTS at Bipea (Bureau Interprofessionnel dEtudes Analytiques). To evaluate the homogeneity among samples, this procedure involves a comparison
of NIR spectra, the determination of global homogeneity
criteria and the use of control charts. The method of control developed and carried out at Bipea allows the rapid
and easy monitoring of the performance of the sample
preparation.
Keywords Homogeneity Near infrared spectroscopy
Spectra comparison Distances Control charts
Samples for PTS
M. E. Lafargue ()
Bipea (Bureau Interprofessionnel dtudes Analytiques),
6/14 avenue Louis Roche, 92230 Gennevilliers, France
e-mail: bipea@wanadoo.fr
M. H. Feinberg D. N. Rutledge
Institut National Agronomique Paris-Grignon,
UMR INAPG-INRA
Ingnirie Analytique pour la Qualit des Aliments,
16 rue Claude Bernard, 75005 Paris, France
J.-J. Daudin
Institut National Agronomique Paris-Grignon,
UMR INAPG-INRA Biomtrie,
16 rue Claude Bernard, 75005 Paris, France
Introduction
Laboratories participate in proficiency testing schemes
(PTS) to control the accuracy of their results. PTS can be
defined as regularly organised interlaboratory comparisons to determine the testing performance of laboratories,
which test the same or similar items. The procedure for a
PTS is as follows: each participant in a given PT scheme
receives a sample prepared by the organiser and has to determine a list of analytes, the results are sent back to the
organiser, data are analysed and conventional true values
and tolerance values are calculated according to a reference procedure. Finally, the participating laboratories can
check their accuracy and detect eventual bias.
The Bureau Interprofessionnel dEtudes Analytiques
(Bipea) is a PTS organiser for agricultural, agro-food and
environmental fields, and counts more than 900 laboratories participating in more than 40 schemes. The requirements for PTS organisers are described in ISO guide 43
[1] and in a new standard under preparation, ISO 13528
draft norm [2].
One of the most important requirements of a PT organiser is to provide homogenous samples. In that way, one
can be sure that if a laboratory has a result different from
that of the other laboratories, its error can be attributed to
its analysis method and not to the particular sample. Complete homogeneity is the state of a batch of a material of
which all the elements are rigorously identical whereas
heterogeneity is the state of a batch of material of which
all the elements are not rigorously identical. Two degrees
of heterogeneity can be distinguished: heterogeneity of
constitution (it is inherent and depends on the nature of
the material) and heterogeneity of distribution (it can be
reduced by homogenisation or increased by segregation)
[3]. From a batch of raw material, various steps of homogenisation and division with the correct equipment allow the preparation of N homogeneous samples.
The aim of the homogeneity control is to check if the
samples are identical before sending them to the laboratories. Therefore, in our context, the homogeneity aimed for
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and gives global information about the chemical composition of the product (not limited to just one analyte). NIRS
is widely applied for quantitative analysis of chemical
constituents such as protein content, moisture and fats in
cereals, animal feeds, fats, meat and milk, as well as carbohydrates in fruit juices and alcohol in beverages [7, 8].
The aim of this paper is to present the methodology for
the NIRS control of homogeneity of agricultural and food
products.
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Table 1 Products controlled by the near infrared technique
Analysis by:
Family of products
Products
Reflection
Cereals
Oilseeds
Animal feeds
Food pulses
Human Food
Transmission
Alcoholic beverages
Molasses
1111 to 2631 nm) for the reflection mode and 10,500 to 5200 cm1
(or 952 to 1923 nm) for the transmission mode. The resolution is
fixed at 8 cm1 and each spectrum is the average of 32 scans of the
sub-sample. The sample holder is continuously rotated in the reflection mode, whereas in transmission mode the sample is static.
To take into account possible differences between the superficial layer which interacts with the infrared beam, and the rest of
the product, three spectra are acquired for each sub-sample after
remixing. These three repetitions are not done successively, neither are the two sub-samples.
is necessary to perform mathematical pretreatment such as Standard Normal Deviate (SNV) already presented by many authors
[11, 12]. In our application, the aim is to verify that the samples are
homogenous both with respect to their chemical composition and
to their granulometry. That is why both pretreated and raw spectra
are analysed in the study. To illustrate this, Figs. 1 and 2 present
spectra of wheat flour and of alfalfa. For spectra acquired by transmission, scattering is not a problem because of the nature of the
products (liquids and pasty). Spectral processing is done using the
Opus (Bruker) and JMP (SAS Institute) software.
Comparison of spectra
For classical near infrared applications, chemical parameters such
as the protein or moisture content of wheat or flour are determined.
In our application, instead of determining these parameters, which
involves lengthy calibration and validation steps, the idea is to
compare the entire spectrum of each samplea spectrum being
considered as the physical and chemical fingerprint of the product,
and for this reason, much richer in information than a single calculated parameter. The assessment of the homogeneity among samples is therefore done by comparing their spectra. However, this
comparison of entire spectra requires the definition of a similarity
criterion. Two metrics were selected for testing:
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Fig. 1 Wheat flour NIR spectra. (A) Raw spectra. (B) SNV pretreated spectra
Dmax calculated from the Euclidian distance between each spectrum and the average spectrum (d2 (i,m)=[SijSmj]2), computed
for each series of ten control samples. Dmax is the maximal distance (Dmax=maxd2 [i,m]) and is considered as a measure of the
dispersion of the samples;
the Standard deviation spectrum (S spectrum) calculated from
the ten spectra obtained for each series of ten control samples.
The S spectrum shows the variability between the control samples at each absorbency and is also considered as a measurement
of the homogeneity between samples.
In order to decide if the difference between samples is or is not significant, it is necessary to have threshold limits: if the Dmax and the
S spectrum are within the defined limits then it can be concluded
that the differences among samples is not significant. Using set of
homogeneous samples from Bipeas PTS, these thresholds were
estimated. To detect outlier samples, control charts like those used
for quality control were applied as described below.
Application of the control chart method to homogeneity checking
The theory of control charts has been extensively presented by several authors [13, 14, 15, 16, 17] and will not be addressed here.
Two types of control charts were used in this study, usually called
the mean chart (X bar chart) and the standard deviation chart
(S chart). Basically a control chart consists of a Central Line (CL)
for the target value, close to which the measurements are expected
to remain, and the upper and intermediate limits for control and
warning, respectively.
For the homogeneity control, the target value is set to zero as
the parameter measured is a degree of homogeneity but the Central
Line is plotted nevertheless because it represents the average degree of homogeneity. Only the upper control limit is plotted because the lower limit is the zero (which cannot be reached because
perfect homogeneity does not exist in nature...).
Fig. 2 Alfalfa NIR spectra. (A) Raw spectra. (B) SNV pretreated
spectra
The standard formulae used to calculate the limits are:
For the X bar chart:
Upper Control Limit UCL = CL + 3s/n
Upper Warning Limit UWL = CL + 2s/n
where s is equal to the standard deviation of the process under control and n the number of replicates.
For the S chart:
Upper control limit: Fs (F depending on the number of replicates, values of F found in Ref. [17]) with s as the standard deviation of the process under control.
For these calculations, data distribution is assumed to be Normal,
which needs to be checked. If the data are not Normally distributed, the risk of wrongly rejecting a point outside of the limits will
change. Normality was tested with KolmogorovSmirnov test and
Normal probability plot [17].
Two parameters are required to set up a mean chart and define
the limitsa centre line defined by the average level of homogeneity and a standard deviation that measures the accepted variability between homogeneous samples. These parameters must be
determined when the process, here the preparation of the samples,
is under control, that is, the process is producing homogeneous
samples. The parameters are estimated for each kind of product using sets of control samples from preparations judged homogeneous
based on the results of PT Schemes.
To illustrate the setting up of control charts, the example of the
Flour/Alveograph PTS is taken:
eighteen sets of ten wheat flour control samples from eighteen
different preparations were available,
analysis of each set under conditions of repeatability (each set
was analysed three times),
calculation of the three values of Dmax for each set, corresponding to the three repetitions,
calculation of the average Dmax (or central line of the chart) with
the 54 (318) values of Dmax,
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Table 2 Flour. Results of the setting up of the Dmax chart (on raw
spectra)
Number of sets of control samples
Number of replicates
Average Dmax (central line)
Standard deviation of Dmax
Upper warning limit (UWL)
Upper control limit (UCL)
18
3
0.16
0.03
0.19
0.21
calculation of the variance within each set (with the three Dmax
values calculated) and then the average of the eighteen variances
to obtain the final variance. Its square root gives an estimate of
the final standard deviation,
calculation of the warning and control limits.
The results are presented in Table 2 for raw data and can be used to
build Dmax charts for raw data. These steps are repeated for the data
pretreated by normalization and SNV. Such charts are established
for each kind of product.
It was determined from the Normality tests that the Dmax plotted on the control charts are not distributed Normally but follow a
Gamma probability distribution function. As the limits are based
on the assumption of Normality, it is necessary to evaluate the risk
of wrongly rejecting a data (Type I error).
With Normally distributed data and a classical control chart:
The risk of having a data point wrongly between the warning
limit and the control limit is equal to 2.3% and not 4.6% (probability=95.4 when k=2) because only the upper limit is used
(one-sided risk),
The risk of having a data point wrongly below the control limit
is equal to 0.135% and not 0.27% (probability=99.73 when k=3)
because only the upper limit is used (one- sided risk).
With data distributed according to a Gamma law, the computations
gave:
results. In order to validate this homogeneity check by NIRS, determination of the homogeneity targets by the reference methods
have been done for each PTS. These analyses were subcontracted
to accredited laboratories, except of course when an accreditation
program for a given analysis did not exist. Analyses of variance
were performed on the results to estimate whether the differences
among samples were statistically significant and to calculate the
between-samples standard deviation (Se). The Se value must represent less than 30% of the total variability of the given PTS results.
Results
Examples of control charts for wheat flour and alfalfa
The setting up of the control charts with definition of the
limits was done with the control samples of the 1999/2000
and 2000/2001 campaigns. A campaign begins in July and
ends in June and generally there is a proficiency test each
month for each scheme, except in July and August. During the 2001/2002 campaign, the control charts were used.
Each month, the control samples were analysed by NIRS,
and Dmax and S spectra were calculated from the spectra.
Figures 3 and 4 show D max charts for 2 different schemes
and 2 products in particular: Flour from the Flour/Alveograph PTS and alfalfa from the Forages PTS. Each month
between September 2001 and May 2002, a D max is plotted. For both products, the Dmax are within the control lim-
501
Fig. 4 Dmax charts for the Forages PTS on alfalfa, campaign 2001/
2002. (A) Raw spectra. (B) SNV pretreated spectra
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uniformly distributed among the samples to give representative samples of the product. This is summarised in
Fig. 9 where two wheat samples containing six barley
seeds are sampled to be analysed. The surface of the prod-
503
Table 3 Flour and alfalfa. Results of the control by reference method with protein content (expressed in g/100 g) as
homogeneity target
Sample
January 2001
Wheat Flour
1
2
3
4
5
6
7
8
9
10
Mean
Repeatability standard deviation, Sr
Between samples standard deviation, Ss
Assigned value of the PTS
Robust standard deviation of the PTS,
(Ss/)100
F value
Critical value of F
February 2001
Alfalfa Pellets
Rep. 1
Rep. 2
Rep. 1
Rep. 2
9.52
9.51
9.48
9.52
9.51
9.51
9.48
9.48
9.47
9.51
9.49
9.52
9.57
9.5
9.53
9.54
9.48
9.48
9.46
9.51
9.50
0.02
0.01
9.52
0.14
9.4
0.23
3.02
24.4
24.0
24.7
24.4
24.5
24.1
24.8
24.4
24.8
24.0
24.9
24.5
24.7
24.3
24.4
24.4
24.2
24.2
24.6
24.0
24.4
0.2
0.1
23.6
0.6
16.9
1.99
3.02
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this means that only heterogeneity due to chemical constituents such as moisture or protein content can be easily
detected. The heterogeneity due to compounds present as
traces (vitamins, minerals...) and contaminants cannot be
detected by the technique. If the steps of preparation of
the samples give homogeneous samples for all the major
chemical constituents, then it is reasonable to think the
other constituents should be homogeneous too. This hypothesis could be verified for some trace compounds for a
limited number of products, but it is difficult to do so for
all the products and all traces. The choice of homogeneity
targets is sometimes difficult and the comparison of the
complete spectra could be a solution to avoid this, even if
it is not perfect (i.e. small quantity of barley in wheat).
To demonstrate the performance of NIRS to detect
small differences between samples, studies of heterogeneous samples, artificially manufactured, are currently being carried out at Bipea.
In the future, the method of homogeneity checking by
NIRS will be extended to other products such as oils and
fats. It will be interesting to apply the procedure to samples prepared for besatz (impurities) determination PTS,
in order to see the capacity of the technique to detect impurities, such as damaged grains, foreign seeds...
References
1. ISO Guide 431 (1997) Proficiency testing by interlaboratory
comparisons Part 1: Development and operation of proficiency
testing schemes. International Standardisation Organisation,
Geneva, Switzerland