ISO

8466-1

INTERNATIONAL
STANDARD

First edition
1990-03-01

and evaluation
Water quality - Calibration
analytical
methods and estimation
of
Performance
characteristics
Part 1:
Statistical
Qualith de lea
des caractkes
Partie 1: haha

evaluation
rEtalonnage
de Performance

tion s ta tistique

of the linear calibration

et Evaluation
de Ia fonction

des mhthodes

of

function

danalyse et estimation

lin&aire dh talonnage

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Reference number
ISO 8466-1 : 1990 (EI

ISO 8466-1 : 1990 (EI

Contents

Page

Foreword

.. .
Ill

............................................................

Scope ..............................................................

Definitions..

Symbols.........................................................~

Performance

........................................................
..

........................................................

4.1

Choice of working

4.2

Calibration

4.3

Assessment

range ..........................................

and characteristics

of the method

........................

range ..........................................

5.2

Calibration

and characteristics

5.3

Evaluation

......................................................

Annex

Bibliography

of the method

.................................................

Example ............................................................
Choice of working

....................................................

5.1

........................

6
7
7
8

0
ISO 1990
All rights reserved. No patt of this publication may be reproduced or utilized in any form or by any
means, electronie or mechanical, including photocopying and microfilm, without Permission in
writing from the publisher.
International Organization for Standardization
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ii

ISO 8466-1 : 1990 (E)

Foreword
ISO (the International
Organization for Standardization)
is a worldwide
federation of
national Standards bodies (ISO member bedies). The work of preparing International
Standards is normally carried out through ISO technical committees.
Esch member
body interested in a subject for which a technical committee has been established has
the right to be represented
on that committee.
International
organizations,
governmental and non-governmental,
in liaison with ISO, also take patt in the work. ISO
collaborates
closely with the International
Electrotechnical
Commission
(IEC) on all
matters of electrotechnical
standardization.
Draft International
Standards adopted by the technical committees
are circulated to
the member bodies for approval before their acceptance as International
Standards by
the ISO Council. They are approved in accordance with ISO procedures requiring at
least 75 % approval by the member bodies voting.
International
Standard
Water quaiity.

ISO 8466-1 was prepared

by Technical

Committee

ISO 8466 consists of the following

Parts, under the general title
Calibration
and evaluation
of analytical methods
and estimation
charac teris tics :
-

Part 7: Statisticaf

evaluation

Part 2: Calibration

strategy

Part 3: Method

- Part 4: Estimation
basis method.
Annex

of Standard

of the linear calibration

for non-linear

calibration

ISO/TC

147,

Water gua/ity of Performance

function
functions

addition

of Limit of detection

and limit of determination

A of this part ISO 8466 is for information

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only.

of an analytical

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INTERNATIONAL

STANDARD

ISO 8466-1 : 1990 (E)

and evaluation
of analytical
Water quality - Calibration
methods and estimation
of Performance
characteristics
Part 1:
Statistical
1

evaluation

of the linear calibration

Scope

This part of ISO 8466 describes the Steps to be taken in

evaluating the statistical characteristics
of the linear calibration
function. lt is applicable to methods requiring a calibration. Further Parts of ttis lnternationa~ Standard will cover the determina$.i!! ok IGr%t of detection and FEmit of determination,
the
effect of Ln$erferences and other performante
chara,cteristics

function

Originai

MeasUri!g

lt is intended especially for the evaluation of the pure analytical

method and for the calculation of Performance characteristics
of the calibration function.

Sample

;X~~nstructi;

measuring instructions

In Order to derive comparable analytical results and as a basis

for analytical quality control the calibration and evaluation of
analytical methods have to be performed uniformly.

Measur;

value

r-YG&q

calibrating and evaluating instructions

Definitions

For the purposes

tions apply.

of this part of ISO 8466, the following

2.1

method:

analytical

of procedural,
measuring,
tions (see figure 1).

defini-

An analytical method is composed

calibrating and evaluating instruc-

Figure 1 - The analytical

method

23

Whereas the procedural and measuring instructions depend on

the method, and are therefore the Object of standardization
of
the respective method, the calibrating and evaluating instructions are valid for any analytical method requiring calibration.

2.2

calibrating
instruction : Describes the approach to
determine the calibration function from information
values, yi,
obtained by measuring given Standard concentrations,
xi. The
slope of the calibration function, b, as a measure of sensitivity
of the analytical method and the Standard deviation of the
which
method, sXO, are figures of merit and characteristics
result from the calibration experiment.

The Standard deviation, sXO, allows the comparison

dent analytical methods.

of indepen-

evaluating
instruction : A calculation
guide for the
computation
of concentrations
from the measured values by
the use of the calibration function. Additionally,
the confidence
range permits an objective assessment of the imprecision of the
analytical result[*J.
2.4

measured

values : The concentration-dependent

values (e.g. extinction)

of a measuring

initial

System.

NOTE - Information value and measured volume are synonymous.

25

residual Standard deviation, s : The residual Standard

deviation describes the scatter of the information
values about
the calculated regression line. lt is a figure of merit, describing
the precision of the calibration.

For the purpose of this Standard, the Standard deviation of the

method means the Standard of deviation of the calibration proFor the user of the method,
these characteristics
present
cedure.
criteria for the internal laboratory quality control.
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ISO 8466-1 : 1990 (EI

Yi

Information
value of the Standard concentration Xi calculated from the calibration function.

s2
i

Variante
of the information
analyses of Standard samples,
centration Xi.

For the purpose of this Standard, the Standard deviation of the

method means the Standard of deviation of the calibration procedure.

fi

Degrees of freedom for the calculation

variance (f i = tli- 1).

2.7 coefficient

Calculated blank (Ordinate intercept

bration straight line).

See also note to 2.5 and 2.6.

Sensitivity
of the method (slope of the calibration line; coefficient
of regression

Mean of the Standard

concentrations
resulting from the calibration experiment.

Mean of the information

values Yit resulting
from the calibration experiment.

2.6 Standard deviation

of the method
sXO: The ratio of
the residual Standard deviation, s,,, to the sensitivity of the
calibration
function,
b. lt is a figure of merit for the performance of the analytical method, and is valid within the working
range (sec equation 13).

of Variation of the method, VXO: The ratio

of the Standard deviation of the method sXOto the appertaining
mean, F, which is the centre of the working range.

2.8 working

range (of an analytical method): The interval,

being experimentally
established and statistically proved by the
calibration
of the method, between the lowest and highest
quantity or mass concentration.
The lowest possible limit of a
working range is the limit of detection of an analytical method.

2.9

homogeneity
of variances: Homogeneity of variances
of pooled data, such as those resulting from replicate analyses
at different
levels, is confirmed
if these variances are not
significantly
correlated to their appertaining
concentrations.

2.10

sensitivity

of the analytical

the calibration function of the complete

clusive of all procedural
Steps, within
question.

The slope of
analytical method, inthe working
range in

measuring Sample (reaction Sample) : A Sample which

tan be directly submitted to the measurement of the determinand. A measuring Sample is normally obtained by adding the
required reagents to the analytical Sample. Obviously, measuring and analytical Sample are identical if no reagents have to be
added to the analytical Sample.

Symbols

Xi

Concentration

Subscript

i=
N

the

concentration

levels,

where

by linear

sY2

Residual Standard deviation

linear regression calculation.

obtained

by non-

DS2

Differente

Information

Number
Sample.

Mean of information
replicates.

Concentration
sf the
analytical
Sample,
calculated from the i nformation
value Ym

Concentration
of the
analytical
Sample,
calculated from the mean of the information
values 7.

of variances.
value of an analysed
of replicates

on the same analysed

values,

resulting

from

levels (for this part of

tV;,

Concen tration of the Standard Sample at the lower

level of the working range ( 1st Standard Sample).

XI0

Concentration
of the Standard Sample at the
upper level of the working range (10th Standard
Sample).

Yi,j

jth information

Subscript

value for the concentration

value of the t-distribution

with fl =
and a confidence
level of (1 - a) (f-factor of Students distribution).

Tabled

l-a)

N - 2 degrees of freedom

FV;,f2,1

-a)

Tabled value of the F-distribution

(FisherSnedecor) with fl and f2 degrees of freedom
and a confidence
level of (1 - a).

Xi.
%O

Standard

deviation

of the method.

VXO

Coefficient

of Variation

VB (x,

Confidence

interval for the concentration

of the replicate j of level i, where j = 1,

2, . . . . ni.
of replicates

per level Xi.

Mean of the information

values Yi,j of Standard
Confidence interval
Xi.
samples, having the concentration
centration.
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Sample.

Number of concentration
ISO 8466, N = IO).

Number

deviation.
obtained

1,2, . . . . N.

Xl

of the cali-

Residual Standard deviation

regression calcula tion.

of the Ph Standard Sample.

of

of the

sY1

method:

2.11

Residual Standard

sY

values for the

having the con-

of the method.
.?.

of the mean Z?of the con-

ISO 8466-1 :, 1990 IE)

Performance

4.1

Choice

4.1.2

of working

a)

range depends

the practice-related

with

the choice

10

of a

(Y i t j - El*

t:
j=l

S*i =

on
objective

of the variances

Both data sets of the concentrations

x1 and xlo are used t~
calculate the variances sf and s$, as given in equation (1) :

range

Esch calibration
experiment
is started
preliminary working rangeL3].
The working

Test for homogeneity

Ili -1

. . .

(11

. . .

(2)

differentes

at

of the calibration.
with the mean

The working
range shall cover, as far as possible, the
application range for water, waste water, and sludge analysis. The most frequently
expected Sample concentration
should lie in the centre of the working range.

10

Y-,J *

c
j=l

.-

Yj

fori

= 1 ori

= 10

yli
b)

feasibilities

of technical

realizability.

The measured values obtained must be linearly correlated to

the concentrations.
This requires that the measured values
obtained near the lower limit of the working range tan be
distinguished
from the blanks of the method. The lower
limit of the working range should therefore be equal to or
greater than the limit of detection of the method. Dilution
and concentrating
Steps should be feasible without the risk
of bias.
c) the variance of the information
dent of the concentration.

values must be indepen-

The variances are tested (F-test) for significant

the limits of the working rangeL5t 6].
The test value
equation (3).

PG = 2

PG

forsyo

is

determined

for

the

> sf

F-test

from

. . (3)

1
S*

PG = --$

for s: > sf0

10

The independence
linearity16t *l.

is verified

by a statistical

test on the

PG is compared

with the tabled values of the F-distribution[?

Decision :

4.1.1

Reparation

of the calibration
a)

After establishing
the preliminary
working
range, measured
values of at least five (recommended
N = 10) Standard
samples are determined.
The concentrations,
xj, of these standard samples shall be distributed equidistantly
over the working
range. In Order to check for the homogeneity
of the variances,
ten replicates of each of the lowest and the highest concentrations (x, and x,~) of the working range are determined. Ten information values, )pi jl result from these series of measurements
(see table 1).

Table 1 -

If PG

<

the
Ffl. I f2. I 099
I

differente

between

the

between

the

variances sf and SS is not significant.

b) If PG > q, ; j-2;

0,99 the

differente

variances sf and s$ is significant.

If the differente
between
the variances is significant,
the
preliminary working range should be made smaller until the difference between the variances is found to be random only.

Data sheet for the calibration

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lSO,8466-1 : 199OE)

4.13

Test for linearity[*J

# *]

The easiest test for the linearity is the graphical representation

of the calibration data with the calculated regression line. Any
unlinearity is evident (see figure 2).

The measurement against a blank is not allowed, since thereby

valuable information on the magnitude of the blank will be lost.
The comparison medium for zeroing the instrument is always, if
possible, a pure solvent (e.g. pure water).

. Table 2 -

5
B
c

06
I

.-0

051
04
03

+E
8
z-

Data sheet for simple

I
j

xj

-vj

x;

lI
I

linear regression
-V2i

Xjuj

--y-----------l----g:ppIIj
1

--

----

.,

----t---l----l

--L----1 t------t-m
I

6
7

,)c

.-----j----~----~--q-----j

5
~--

--L------

-t-

.--~~
--_.-

-----+------

---

12345-6789
Concentration

Figure 2 - Graphical

linearity

check

In the statistical linearity test the calibration data are used to

calculate a linear calibration function as weil as a non-tin.ear
calibration function,
both with the residual Standard deviation
Syl Or 52-

The differente

of the variances

(4) ..

OS = (N Degrees

OS* and the variance

: f = 1.
of the non-linear

calibration

function

to a F-test in Order to examine for significant

The test value

equation (5)
PG =-

from equation

. . . (4)

2) s;, - (IV - 3) sy,

of freedom

are submitted
ferences.

OS* is calculated

PG required

for the F-test

OS*

is calculated

sv2
dif-

from

. . . (5)

S*
Y*

The ten data Sets, consisting of the va2ues of Xi and Yj, are submitted to a linear regression analysis to obtain the coefficients a
and b of the calibration function whsich describe the linear correlation
between
the concentration
x as an independent
variable, and the measured value y as a dependent variable.
The calibration function as well as the characteristics
of the
method should result from data obtained from a working range
x1 to xIo, as received from the measurement and not corrected
for blanks. Generally, no blank value (concentration
x = 0) is
to be included in the calibration experiment and, consequently,
in the least-squares fit of the regression.
The linear calibration

(6)

. . .

Y =U+bX

a) If PG 4 F: The non-linear calibration function does not

lead to a significantly
better adjustment,
e.g. the calibration
function is linear.

(6)

N
c

b) If PG > F: The working range should be reduced as far

as possible to receive a linear calibration function;
otherwise
the information
values of analyzed
samples must be
evaluated using the non-linear calibration function.

Calibration

is given by equation

The coefficients are obtained from equations (7) for sensitivity

(slope of the calibration
function)
and (8) for the Ordinate
intercept (calculated blank)

Decision :

4.2

function

and characteristics

of the method

(Xj

XI

'

(Yj

i=l

b = p--_Ip--m_-

Y)

. . . (7)

(x; - XI*
Y
M
.
I=

a = r--b%

. . .

(8)

After the final working

range is established,
ten Standard
The coefficients provide an estimate of the true function, which
samples are analyzed in accordance with all the Steps of the
is limited by the unavoidable procedural scatter. The precision
analytical method in Order to obtain ten (IV = 10) measured
of the estimate is quantified by the residual Standard deviation,
values Yj (see table 2).
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ISO 8466-1 : 1990 (EI

sv, which is a measure of the scatter of the information

values
about the calibration line and is given by equation (9).

Fromsthe law of error propagation

it follows that, for each value
X, a confidence
interval for the true value y exists whose
limiting Points are on two hyperbolic
paths bracketing
the
calibration line. Between these paths the true calibration function tan be expected with a significance level of a (fl : N - 2,
confidence level = 1 - a), determined by Students t-factor.

[Y j - (a + bx,)l*

i=l

(9)

N-2
4.3

The confidence
intervals for analytical results, calculated from
the calibration function, are given by the intersections with the
respective hyperbolic paths in figure 3. The estimation of the
confidence intervals are given by equation (12) [71

Assessment

The concentration
a)

of an analyzed

from the measured

Y
x =-

valuey,

Sample is obtained

7,,* = % + VB (.%,

to give ?

-a

. . .

.a
Y -a
.2,,2 =
b

(IO)

(jf- Fl2

or
b)

from the mean of a series of replicates,

the same original

7, petformed

on

Sample, to give ?

g=-

(Xi -

b*x

.
Z=

7-a

. . .

(11)

(12)

NOTE - If i; = 1, F,,* =

As to the uncertainty
of an analytical result, keep in mind that
the analytical error is a combination
of the uncertainty
of the
determination
of the measured value, and the uncertainty
of
the estimation of the regression coeff icients[*!

Equation
brackets
statistical
VB(g) is

X1,2-

(12) indicates that the confidence

interval VB(?)
the true analytical value with a range governed by the
security of Students distribution.
The magnitude of
mainly determined by the number of replicates n^ and

7
0

a /

1
I
I
I

- 44

vl3m

I
I
I
I
I
I
I
I
I
I
X

10

Working range

Figure 3 - Working

range x, to xlo, calibration line with confidence band and a Single analytical
appertaining
confidence interval
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result with its

ISO 8466-1 : 1990 (E)

5.1

their results, the mean of the information

values 7, as weil as
the characteristics
of the method, the residual Standard deviation s,,, and the sensitivity b.

. . .

of working

range

For the analysis of drinking water and surface water,

range of 0,05 mg to 0,5 mg (NO;)/1 is appropriate.

The quality of the analytical procedure increases therefore with

increasing sensitivity and decreasing residual Standard deviation.
The Standard deviation of the method sXO[see equation (1311 is
the characteristic
which allows the analyst to check the quality
of his own work.

sXO =2

Choice

5.1.1

Testing

the hornogeneity

v xo = s~xloo

(13)

analytical
expressed

(3)

= 2,9

4,67 x 10-6

F(9,9;

0,991 = 5,35

The comparison of the calculated value PC with the tabled one

indicates a random differente
between the variances under
examination.
As the variances are homogeneous
a simple
regression analysis may be petformed.

The photometric
determination
of nitrite is used to demonstrate
the calibration and the subsequent estimation of the statistical
characteristics
of the method and their influence on the final
results of the evaluation.

Table 3 - Data sheet for the calibration

mgll

xi

Y*l,l

Yi,2

Yi,3

Y-1,4

Y-lt5

0,05

0,140

0,143

0,143

0,146

0,lO

0,281

0,15

0,405

0,20

0,535

0,25

0,662

0,30

0,789

0,35

0,916

0,40

1,058

0,45

1,173

0,50

1,303

1,302

1,300

1,304

10 =N

from equation

Consulting the F-tablesL5] forfi

= f2 = YE- 1 = 9 degrees of
freedom for the variances ST and S$ gives

(14)

Example

is calculated

13,54 x IO-6

S2

PG=s=
. . .

sf the variancesl)

According
to the approach outlined in 4.1 .l, the variances ~2
of the information
values obtained from the Standard concen:
trations at the lower or upper limit of the working range respectively, were determined (sec table 4).

The test value PG for the F-test

For the comparison

of different
standardized
methods, the coefficient of Variation of the method,
as a percentage,
is given by equation (14)

a working

of NO;

Yfj
i,

Y-1,7

i,

0,144

0,145

0,344

'

1,300

1,296

1,295

R,a

&,!3

Yi, 10

0,146

0,145

0,148

1,301

1,296

1,306

1
j
I
/
I
)
I
1
I
1
1

Table 4 -

Data sheet for the analysis

sf variance.

Object:

nitrite

i
1
I

10

xi

Y.

Yi,2

Yi,3

Yi, 10

Ext.*)

Ext.*)

Ext.*)

Ext. *)

Y*
EX:, )

Yi, 9

Ex%

Y*
Ex;.:)

yi,8

mgll

Ext.,

Ext. *)

Ext. *)

mg& 12

0,05

0,140

0,143

0,143

0,146

0,144

0,145

0,144

0,146

0,145

0,148

4,67. IO-6

0,50

1 1,303

1,302

1 1,300

1 1,304

1 1,300

1,295

1 1,301

1 1,296

1 1,306

Yi, 5

yi,6

1,296

s?

11356 . lW6 1

I *c) Ext. : (extinction)

1) Forthe sake oftransparency,

to the result.

alldimensions have intentionally been omitted in allequations, withoutambiguity.

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The dimensions are finallyadded

5.1.2

Testing

A non-linear

linearity

regression

The Standard
equation ( 13)
function

may be derived

0,005
sy
b2,575

5.2

Calibration

xo

5.3

0,002o

as a

= 0,73

0,275

Single determination

The analysis of an unknown

Sample, performed in the same
way as the analysis of the Standards, gave an information value
jk
0,641 (extinction).
The analytical result was obtained using
equation (12) with a confidence interva195 %, t(8; 0,951 = 2,31

0,641 - 0,018

Since the prerequisites for the Performance

of a simple linear
regression
are fulfilled,
the calibration
function
and the
characteristics
of the method tan be calculated using equations
(71, (8) and (13). The results are shown in table 5.

%,2

+
is calculated

x 100

expressed

Evaluation

5.3.1

of the method

The slope, b, as a measure for the sensitivity,

equation (7)

from

= 0,002 0

Sxo
V xo = x x loo=

procedure)

are equal, the differente

(411 does not need to be
function does not lead to
the calibration function is

and characteristics

is calculated

= 0,005 2 mg/l.
sY*
As both residual Standard deviations
of the variances DS* [see equation
calculated. The non-linear calibration
a significantly better adjustment, e.g.
linear.

method

The coefficient
of Variation of the method,
percentage,
is given by equation (14)

The residual Standard deviations of the linear and the non-linear

calibration function, sY, and su2, are compared:
= 0,005 2 mg/1 (see 5.2 for calculation

of the

[*l as

Y = 0,013 5 + 2,62 x - 0,081 8 ~2, giving a residual standard deviation of sY2 = 0,005 2 mg/l.

sYl

deviation

2,575

0,002 0 x 2,31

+ -

(0,641 - 0,726 2)*

from

(2,575)*

x 0,206 25

= (0,242 + 0,005) mg/1

N

b =

(Xi

33

(yi

i=l

Thus the true value of the concentration

within the range 0,237 < x < 0,247 mg/l,
level of 0,95.

jd

= 2,575

tan be expected
with a confidence

(X i-

c
i=l

The Ordinate intercept,

equation (8)
a = v--

32

5.3.2

a, (calculated

For three replicate determinations,

the analytical
the information
values 0,641; 0,631 and 0,633.

blank) is calculated

from

Replicate

Calculation

b X = 0,726 2 - 2 1575 2 x 0 1275 = 0 I 018 Ext .

The residual
equation (9).

Standard

deviation,

sY, is

calculated

of the analytical
0,635

%,2

from

analysis

- 0,018

2,575
(0,635 - 0,726 2)*

(2,575)*

= 0,005 2 [Ext.l

N-2

The equation

for the straight

Y = 0,018

gave

result:

(Y i - yi)*

sy =

method

x 0,206 25

= (0,240 + 0,003) mg/l

line is given by equation

Thus the true value of the concentration

within the range 0,237 < x < 0,243 mg/I,
level of 0,95.

(4)

+ 2,575 2 x

Table 5 -

tan be expected
with a confidence

Data sheet for the regression

4

1 1 1 $i 1 ,66,

X = 0,275 mg /I

0,20

0,535

7 = 0,726 (Extinction)

0,30

0,789

0,35

0,916

0,40

1,058

dV*1

mg0

T--t-/
r
9

0,45

1,173

10

0,50

1,303

--

xi

c I 2f75l

i=l

7,262

I
l

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rSO8466-1 : 1990 (El

Annex A
(informative)
Bibliography

111 VONDERHEID, C., DAMMAN, V. , DRR, w., FUNK, W. and KRUTZ, H., Statistical
assessment

and comparison

of analytical

procedures.

141 FRANKE, J.P., dE

tion spectrometry

of quantitative

ZEEW,

analytical

procedures,

BI

SACHS, L., Methods

for statistkaI

DOERFFEL, K., Statistics

2nd Edition,

theory and methodology

in Chemical analysis,

a Ienergie

atomique,

in science and enghering,

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VEB- Verlag fr die Grundstoffindustrie,

series -

Presentation

of a Computer

Statistique appliquee & lexploitation

program,

(1966), Leipzig.

Vom Wasser 58, (1982), pp. 231-255.

des mesures, tome 2, Masson,

quality,

tests, Chemical analysis, calibration,

statistical analysis.

Price based on 8 pages

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Single user licence only, copying and networking prohibited

Springer Verlag, (1966),

J. Wiley & Sens, (1965), New York.

UDC 543.3 : 53.088

Descriptors:

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evaluation, 3rd Edition, Springer Vdag, (1971), Berlin, Heidelberg, New York.

191 WAGNER, R., Evaluation of BOD-dilution

no1 Commissariat

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method

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[71 BROWNLEE, K.A., Statistical

BI

for the

Z Ana!. Chem. 275, (1975), pp. 1-10.

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Vom Wasser 57, (1981), pp. 59-74.

PubL, J. Wiley & Sons, (1964), New York.

151 GRAF, U., HENNING, H.J. and STANGE, K., Formulae and tables of mathematical
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[Zl MANDEL, J., The statistical analysis of experimental

[31 GOTTSCHALK, G., Standardization

An approach

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