SIST EN 16924:2017

SLOVENSKI STANDARD
oSIST prEN 16924:2015
01-december-2015
äLYLOD'RORþHYDQMH]HDUDOHQRQDYMHGLOQLKUDVWOLQVNLKROMLK]/&)/'DOL/&0606
Foodstuffs - Determination of zearalenone in edible vegetable oils by LC-FLD or LC-
MS/MS
Lebensmittel - Bestimmung von Zearalenon in pflanzlichen Speiseölen mit LC-FLD oder
LC-MS/MS
Denrées alimentaires - Dosage de la zéaralénone dans les huiles végétales alimentaires
par CL-FLD ou CL-SM/SM
Ta slovenski standard je istoveten z: prEN 16924
ICS:
67.200.10 5DVWOLQVNHLQåLYDOVNH Animal and vegetable fats
PDãþREHLQROMD and oils
oSIST prEN 16924:2015 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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oSIST prEN 16924:2015

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oSIST prEN 16924:2015


DRAFT
EUROPEAN STANDARD
prEN 16924
NORME EUROPÉENNE

EUROPÄISCHE NORM

October 2015
ICS 67.200.10
English Version

Foodstuffs - Determination of zearalenone in edible
vegetable oils by LC-FLD or LC-MS/MS
 Lebensmittel - Bestimmung von Zearalenon in
pflanzlichen Speiseölen mit HPLC-FLD oder LC-MS/MS
This draft European Standard is submitted to CEN members for enquiry. It has been drawn up by the Technical Committee
CEN/TC 275.

If this draft becomes a European Standard, CEN members are bound to comply with the CEN/CENELEC Internal Regulations
which stipulate the conditions for giving this European Standard the status of a national standard without any alteration.

This draft European Standard was established by CEN in three official versions (English, French, German). A version in any other
language made by translation under the responsibility of a CEN member into its own language and notified to the CEN-CENELEC
Management Centre has the same status as the official versions.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and
United Kingdom.

Recipients of this draft are invited to submit, with their comments, notification of any relevant patent rights of which they are
aware and to provide supporting documentation.

Warning : This document is not a European Standard. It is distributed for review and comments. It is subject to change without
notice and shall not be referred to as a European Standard.


EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. prEN 16924:2015 E
worldwide for CEN national Members.

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Contents Page
European foreword . 3
Introduction . 4
1 Scope . 5
2 Normative references . 5
3 Principle . 5
4 Reagents . 5
5 Instruments and equipment . 8
6 Procedure. 10
6.1 Extraction of zearalenone from the sample . 10
6.1.1 General . 10
6.1.2 Preparation for LC-FLD . 10
6.1.3 Preparation for LC-MS/MS . 10
6.2 LC-FLD analysis . 10
6.3 LC-MS/MS analysis . 11
7 Evaluation . 12
7.1 General . 12
7.2 Calculation with external standard (FLD measurement) . 12
7.3 Calculation with external standard (LC-MS/MS measurement) . 13
7.4 Calculation with the internal standard zearalanone (ISTD 1) . 13
8 Precision . 14
8.1 General . 14
8.2 Repeatability . 14
8.3 Reproducibility . 14
9 Test report . 15
Annex A (informative) Typical chromatograms. 16
A.1 Example chromatograms for LC-FLD method . 16
A.2 Example chromatograms for LC-MS/MS method . 17
Annex B (informative) Example conditions for suitable LC-MS/MS systems . 20
B.1 System settings for Waters Quattro Premier . 20
™
B.2 System settings for AB Sciex API 3000 . 21
™
B.3 Detector parameters for AB Sciex API 4000 Scan type: MRM . 22
™
B.4 Detector parameters for Sciex Triple Quad 5500 System . 23
Annex C (informative) Precision data . 24
Bibliography . 27

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European foreword
This document (prEN 16294:2015) has been prepared by Technical Committee CEN/TC 275 “Food
analysis - Horizontal methods”, the secretariat of which is held by DIN.
This document is currently submitted to the CEN Enquiry.
This document has been prepared under a mandate given to CEN by the European Commission and the
European Free Trade Association.
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Introduction
The mycotoxin zearalenone is a resorcylic acid derivative, which is produced by several species of the
fungi genus Fusarium, in particular by Fusarium roseum var. graminearum. Especially cereals like maize
and wheat are affected, so that zearalenone can also be detected in the oils produced from them.
WARNING — Suitable precaution and protection measures need to be taken when carrying out
working steps with harmful chemicals. The hazardous substances ordinance (EU) 1907/2006,
[3] should be taken into account as well as appropriate National statements e.g. such as in
Bibliographical Reference [4].
WARNING — The use of this document can involve hazardous materials, operations and
equipment. This document does not purport to address all the safety problems associated with
its use. It is the responsibility of the user of this document to establish appropriate safety and
health practices and determine the applicability of regulatory limitations prior to use.
WARNING — Zearalenone is known to have strong oestrogenic effects.
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1 Scope
This European Standard describes a procedure for the determination of the zearalenone content in
edible vegetable oils specifically maize germ oil by either of the following techniques: High performance
liquid chromatography with fluorescence detection (LC-FLD) or high performance liquid
chromatography with tandem mass spectrometry (LC-MS/MS) after basic extraction of the diluted oil.
The method has been validated for zearalenone in naturally contaminated maize germ oil at levels of
61,2 µg/kg to 515 µg/kg [5].
Laboratory experiences [6] have shown that this method is also applicable to vegetable oils such as
wheat germ oil (n = 4), sunflower oil (n = 5), pumpkin seed oil (n = 1), soybean oil (n = 5), hemp seed oil
(n = 5), rape seed oil (n = 11), and mixed oils including maize germ oils (n = 3). However occasionally,
samples can result in interferences in the FLD-chromatograms. In this case, the detection with MS/MS is
recommended.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
EN ISO 3696, Water for analytical laboratory use — Specification and test methods (ISO 3696)
3 Principle
After diluting the edible oil with dilution solvent, it is extracted by shaking with an alkaline methanol -
ammonium hydrogen carbonate mixture.
For the determination by LC-FLD, an aliquot of the centrifuged methanolic-alkaline extract is
evaporated to dryness, then the residue is diluted in acidified LC-eluent and the zearalenone content is
determined by LC-FLD.
For the determination by LC-MS/MS, an aliquot of the centrifuged methanolic-alkaline extract is used
directly for analysis.
4 Reagents
Use only reagents of recognized analytical grade and water complying with grade 1 of EN ISO 3696,
unless otherwise specified. Solvents shall be of quality for LC analysis, unless otherwise specified.
4.1 Methanol, p. a. (pro analysis) for extraction.
4.2 Dilution solvent (n-hexane or, alternatively, n-heptane), p. a.
n-Heptane may be used instead of n-hexane, however, only n-hexane was used in the interlaboratory
test.
4.3 Acetonitrile, LC quality.
4.4 Ammonium hydrogen carbonate (NH HCO ).
4 3
4.5 Ammonium hydrogen carbonate solution, mass concentration ρ = 10 g/l.
Weigh in 1 g of ammonium hydrogen carbonate (4.4) into a 100 ml volumetric flask and fill up to the
mark with water. Prepare a fresh solution each day of analysis.
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4.6 Alkaline methanol extraction solution.
Mix 9 volumes of methanol (4.1) with 1 volume of ammonium hydrogen carbonate solution (4.5).
Prepare a fresh alkaline methanol extraction solution each day of analysis.
4.7 Zearalenone, CAS-No: 17924-92-4, e.g. crystalline, as a film or as certified standard solution with
100 µg/ml.
4.8 Stock solution of zearalenone, mass concentration ρ = 100 µg/ml.
Weigh in 10 mg of crystalline zearalenone (4.7) to the nearest 0,1 mg into a 100 ml volumetric flask and
fill up to the mark with acetonitrile (4.3). Alternatively, transfer the complete content of the standard
vial containing zearalenone as a film with acetonitrile quantitatively into a 100 ml volumetric flask. The
stock solution is stable for at least 4 weeks to 12 weeks if deep frozen at < −18 °C [7].
This step can be omitted when using the certified standard solution. The certified standard solution
then serves as stock solution.
4.9 Standard solution of zearalenone, ρ = 10 µg/ml, and determination of concentration.
Pipette 1 ml of the stock solution (4.8) into a 10 ml volumetric flask and fill up to the mark with
acetonitrile (4.3). Determine the exact concentration of zearalenone in this standard solution
spectrometrically.
For this purpose, record the absorption curve of the standard solution from 190 nm to 350 nm against
acetonitrile. To check the concentration of the stock solution of zearalenone, calculate the mass
concentration of zearalenone in the standard solution ρ in µg/ml according to Formula (1):
st
EM⋅⋅100
max
ρ = (1)
st
δε⋅
where
E is the maximum extinction value determined from the absorption curve (here: 274 nm);
max
2
ε is the molar extinction coefficient in acetonitrile: 1262,3 m /mol;
M is the molar mass of zearalenone = 318,4 g/mol;
δ is the layer thickness of the cuvette in cm.
4.10 Reagents, mobile phases and calibration solutions for LC-FLD analysis.
4.10.1 Glacial acetic acid for LC-FLD, volume fraction φ (CH COOH) approximately 99 %.
3
4.10.2 Mobile phase A for LC-FLD: Acetonitrile/water/glacial acetic acid (47+52+1, v+v+v).
Mix 47 volumes of acetonitrile (4.3) with 52 volumes of water and 1 volume of glacial acetic acid
(4.10.1).
For the stability of the chromatographic separation it is necessary to acidify the mobile phase.
4.10.3 Mobile phase B for LC-FLD: Acetonitrile (100 %) (4.3).
4.10.4 Calibration solutions of zearalenone for LC-FLD.
Prepare a series of calibration solutions from the standard solution (4.9) of zearalenone. According to
the pipetting scheme in Table 1, pipette the corresponding volumes of standard solution into
volumetric flasks, evaporate the solvent with nitrogen at approximately 40 °C to dryness and dissolve
the residue in mobile phase A (4.10.2). Dissolve by means of laboratory shaker (5.4) or ultrasonic bath
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(5.3) at room temperature. Then fill up to the mark. The calibration range shall be adapted to the
desired working range. The calibration solutions can be stored in the refrigerator at 4 °C for
approximately 1 month.
Table 1 — Examples of suitable calibration solutions for LC-FLD
Calibration Volume of Volume of Mass concentration of
solution volumetric flask standard solution calibration solution
ml (4.9) ng/ml
µl
1 10 25 25
2 10 50 50
3 10 100 100
4 10 150 150
5 10 200 200
6 10 250 250

4.11 Reagents, mobile phases, and additional standards and standard solutions for LC-MS/MS
analysis:
4.11.1 Methanol, LC quality.
4.11.2 Ammonium hydrogen carbonate (NH HCO ), for LC-MS, molecular weight: 79 g/mol.
4 3
4.11.3 Mobile phase A for LC-MS/MS: 1 mmol/l NH HCO in water/methanol (95+5, v+v).
4 3
Dissolve 79 mg of ammonium hydrogen carbonate (4.11.2) in 950 ml of water and mix with 50 ml of
methanol (4.11.1).
4.11.4 Mobile phase B for LC-MS/MS: 1 mmol/l NH HCO in water/methanol (5+95, v+v).
4 3
Dissolve 79 mg of ammonium hydrogen carbonate (4.11.2) in 50 ml of water and mix with 950 ml of
methanol (4.11.1).
4.11.5 Working solution of zearalenone for calibration, ρ = 200 ng/ml.
Transfer 0,5 ml of the standard solution (4.9) into a 25 ml volumetric flask and fill up to the calibration
mark with methanol (4.11.1).
4.11.6 Zearalanone, internal standard (ISTD 1) CAS-No: 5975-78-0 for LC-MS/MS, e.g. crystalline or
as certified standard solution with 10 µg/ml.
4.11.7 Zearalenone, isotopic labelled internal Standard (ISTD 2) for LC-MS/MS, as optional
13
C-labelled (fully).
internal standard, e.g. zearalenone
4.11.8 Stock solution of zearalanone (ISTD 1), ρ = 100 µg/ml.
Weigh in 10 mg of zearalanone (ISTD 1) (4.11.6) to the nearest 0,1 mg into a 100 ml volumetric flask
and fill up to the mark with acetonitrile (4.3). Alternatively, transfer the complete content of the
standard vial containing zearalanone (ISTD 1) as a film with acetonitrile (4.3) quantitatively into a
100 ml volumetric flask.
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The ISTD 1 stock solution is stable for at least 4 weeks to 12 weeks if deep-frozen at < −18 °C, [7].
4.11.9 Internal standard solution of zearalanone (ISTD 1), ρ = 10 µg/ml.
Transfer 1 ml of the stock solution of zearalanone (4.11.8) into a 10 ml volumetric flask and fill up to the
calibration mark with methanol (4.11.1).
4.11.10 Working solution of internal standard zearalanone (ISTD 1) for calibration,
ρ = 200 ng/ml.
Transfer 0,5 ml of the internal standard solution of zearalanone (ISTD 1) (4.11.9) into a 25 ml
volumetric flask and fill up to the calibration mark with methanol (4.11.1).
However, it is also possible to use isotopic labelled zearalenone as ISTD 2 (4.11.7) instead of
zearalanone ISTD 1, which is sufficient in most cases.
4.11.11 Calibration solutions with zearalenone and zearalanone (ISTD 1) for LC-MS/MS.
Prepare a series of calibration solutions from the working solutions of zearalenone (4.11.5) and
zearalanone (ISTD 1) (4.11.10). For this purpose, pipette the corresponding volumes of the working
solutions into a LC vial, evaporate the solvent with nitrogen at approximately 40 °C to dryness and
dissolve the residue in the alkaline methanol extraction solution (4.6).
The mass concentration of the ISTDs and the calibration range shall be adapted to the desired working
range. Taking into account the dilution steps, the calibration can be carried out as described in Table 2.
The calibration solutions can be stored in the refrigerator at 4 °C for approximately 1 month.
Table 2 — Examples for suitable calibration solutions for LC-MS/MS
Volume of working Mass concentration of
solutions calibration solution
(200 ng/ml) ng/ml
Final
Calibration
volume
ISTD 1
solution
Zearalenone
ml
Zearalanone ISTD 1
(4.11.5) Zearalenone
(4.11.10) Zearalanone
µl
µl
1 25 100 1 5 20
2 50 100 1 10 20
3 100 100 1 20 20
4 150 100 1 30 20
5 200 100 1 40 20
6 250 100 1 50 20
To control possible reciprocal interferences of zearalenone and zearalanone (ISTD 1) at the chosen
measurement conditions, produce one calibration standard with the highest level of zearalenone only
and one calibration standard with zearalanone only.
5 Instruments and equipment
Usual laboratory apparatus and, in particular, the following:
5.1 Laboratory balance, accuracy: 0,01 g.
5.2 Analytical balance, accuracy: 0,1 mg.
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5.3 Ultrasonic bath.
5.4 Laboratory shaker for test tubes.
5.5 Manual dispensers, microlitre syringes or microlitre pipettes for 10 µl to 2 ml.
5.6 Dispenser, suitable for 20 ml.
5.7 Solvent evaporator with heating module.
1)
5.8 Centrifuge, suitable for 3 000 g .
5.9 Microcentrifuge, suitable for 14 000 g.
5.10 Centrifugation tubes, 50 ml, made of polypropylene with screw cap.
5.11 LC vials.
5.12 UV-spectrometer with quartz cuvettes.
5.13 LC-FLD system with the following components:
5.13.1 LC system, including:
5.13.2 LC pump, suitable for gradient elution;
5.13.3 Injection system;
5.13.4 Column thermostat;
5.13.5 Fluorescence detector, with variable wavelengths;
5.13.6 Data evaluation system;
® ®2)
5.13.7 LC column, e.g. LiChroCART 250-4 filled with LiChrospher 100 RP-18 250 mm x 4 mm,
particle size 5 µm and corresponding pre-column.
5.14 LC-MS/MS system with the following components:
5.14.1 LC system, including:
5.14.2 LC pump, suitable for gradient elution;
5.14.3 Injection system;
5.14.4 Column thermostat;
5.14.5 Data evaluation system;

−2
1) g = 9,81 m ⋅ s
® ®
2) LiChroCART 250-4 filled with LiChrospher are trade names of products supplied by Merck, XBridge® is a trade name
of a product supplied by Waters. Gemini® is a trade name of a product supplied by Phenomenex. This information is given for
the convenience of users of this European Standard and does not constitute an endorsement by CEN of the products named.
Equivalent products may be used if they can be shown to lead to the same results.
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®2)
5.14.6 LC column, suitable for chromatography under alkaline conditions, e.g. XBridge C18 or
®2)
Shield RP18 100 mm × 3 mm; particle size 3,5 µm, or Gemini C18 150 mm × 3 mm; particle size
5 µm and corresponding pre-column;
5.14.7 Tandem mass spectrometer (MS/MS):
Interface: electrospray ionization (ESI)
Ion mode: multiple reaction monitoring (MRM)
6 Procedure
6.1 Extraction of zearalenone from the sample
6.1.1 General
Weigh in 2 g of the sample into a 50 ml centrifugation tube (5.10) to the nearest 0,01 g, add 2,0 ml of
dilution solvent (4.2) and 20,0 ml of alkaline methanol extraction solution (4.6).
If the sample is measured by LC-MS/MS, add 40 µl of zearalanone internal standard solution (ISTD 1)
(4.11.9) to the sample (corresponds to a mass fraction of 200 µg/kg zearalanone in the sample).
To determine the recovery rate, add 40 µl of the standard solution of zearalenone (4.9) to the sample
prior to dilution (corresponds to a mass fraction of 200 µg/kg zearalenone in the sample) and leave for
approximately 5 min at room temperature.
Extract the mixture for approximately 20 min using a laboratory shaker (5.4). To separate the phases,
centrifuge for 10 min at approximately 3000 g.
6.1.2 Preparation for LC-FLD
Evaporate 5,0 ml of the clear, alkaline methanol supernatant of the extract to dryness at approximately
40 °C and dissolve in 1,0 ml of mobile phase A (4.10.2).
Dissolve the residue using an ultrasonic bath. If necessary, centrifuge the sample solution again in a
microcentrifuge (5.9) for 2 min at 14 000 g and transfer into a LC vial.
6.1.3 Preparation for LC-MS/MS
Transfer a part of the clear, alkaline methanol supernatant into a LC vial.
If further dilutions of the extract are prepared – e.g. in order to be able to use isotopic labelled
zearalenone as ISTD 2 (4.11.7) to take account of the matrix effect – the methanol content in the diluted
extract shall still be 90 %.
6.2 LC-FLD analysis
Inject a suitable amount of the sample test solution into the LC-FLD system.
When using the column specified under 5.13.7, the following parameters and gradient programme as in
Table 3 are recommended:
Flow rate: 0,9 ml/min
Injection volume: 20 µl to 50 µl
Column temperature: 30 °C
Excitation 274 nm
wavelength:
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Emission 446 nm
wavelength:
Gradient: see Table 3
Table 3 — Example of a gradient programme
Percentage of Percentage of
Time
mobile phase A (4.10.2) mobile phase B (4.10.3)
min % %
0 100 0
17 100 0
20 20 80
27 20 80
30 100 0
40 100 0

The LC parameters listed in Table 3 are regarded as guidance values. Depending on the column used
(RP-18 packaging material, different column dimensions), the eluent ratio and/or the flow should be
adapted in such a way as to achieve the best separation between the analyte and other extract
components of the matrix. Example chromatograms are listed in Annex A.
If difficulties (e.g. chromatographic interferences) occur during the analysis, it is recommended to use
LC-MS/MS for the detection and confirmation step.
6.3 LC-MS/MS analysis
Inject a suitable amount of the sample test solution into the LC-MS/MS system.
The separation and detection parameters for the determination of the analytes shall be optimized for
each measurement system. Suitable example systems are listed in Annex B.
When using the example columns specified in 5.14.6 and the mobile phases specified in 4.11, the
following parameters have been shown to be applicable for LC:
Flow: 0,5 ml/min
Oven 35 °C
temperature:
Injection volume: 5 µl
Gradient: see Table 4
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Table 4 — Example of a gradient programme
Time Percentage of Percentage of
mobile phase A (4.11.3) mobile phase B (4.11.4)
(min) % %
0 70 30
10 20 80
13 5 95
18 5 95
19 70 30
25 70 30

Zearalenone and zearalanone (ISTD 1) can be measured with positive ionization as well as with
negative ionization. The best measurement conditions for quantifier and qualifier shall therefore be
determined depending on the respective instrument setup. The mass transition with the largest signal-
to-noise ratio shall be selected as the quantifier.
The measurement and chromatography conditions shall be chosen in such a way that the zearalanone
(ISTD 1) does not produce an interfering signal at zearalenone.
Example chromatograms are given in Annex A (gathered with instrument settings B.2).
7 Evaluation
7.1 General
Identify zearalenone by comparing retention times of the calibration solutions with that of the sample
test solution.
Carry out a multi-point calibration with calibration solutions, for example according to Table 1 in 4.10.4
and Table 2 in 4.11.11.
7.2 Calculation with external standard (FLD measurement)
According to the external standard method, calculate the mass fraction w of zearalenone in the sample,
expressed in µg/kg, according to Formula (2)
ρ⋅⋅VV
e final
w= (2)
m ⋅V
s c
where
ρ is the mass concentration of zearalenone in the injection solution calculated by linear
regression, in ng/ml;
V is the extraction volume, in ml (here: 20,0 ml);
e
V is the volume to which the residue is diluted after evaporation, in ml (here: 1,0 ml);
final
m is the mass of the test portion, in g (here: 2,0 g);
s
V is the volume of evaporated extract, in ml (here: 5,0 ml).
c
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7.3 Calculation with external standard (LC-MS/MS measurement)
According to the external standard method, calculate the mass fraction w of zearalenone in the sample,
expressed in µg/kg, according to Formula (3):
ρ⋅V
e
w= (3)
m
s
where
ρ is the mass concentration of zearalenone in the injection solution calculated by linear
regression, in ng/ml;
V is the extraction volume, in ml (here: 20,0 ml);
e
m is the mass of the test portion, in g (here: 2,0 g).
s
7.4 Calculation with the internal standard zearalanone (ISTD 1)
Carry out a multi-point calibration with a standard solution, for example according to Table 2 in
4.11.11.
For the linear regression plot the peak area ratio from the analyte against the corresponding
concentration ratio. The mass concentration of zearalenone in the injection solution is derived from
linear regression in ng/ml, see Formula (4).
A ρ
a a
a + b (4)
Cal Cal
A ρ
ISTD ISTD
where
A is the peak area of the analyte in the calibration solution (4.11.11);
a
A is the peak area of the internal standard in the calibration solution (4.11.11);
ISTD
ρ is the mass concentration of the analyte in the calibration solutions (4.11.11), in ng/ml;
a
ρ is the internal standard mass concentration in the calibration solution (4.11.11) in ng/ml;
ISTD
a is the slope of the calibration determined by the calibration solutions (4.11.11);
Cal
b is the axis intercept of the calibration determined by the calibration solutions (4.11.11).
Cal
s
Calculate the mass concentration ρ of the analyte in the sample test solution, in ng/ml by Formula (5).
a
s
A
a
− b
Cal
s
A
s ISTD s
ρρ= (5)
a ISTD
a
Cal
where
s
is the peak area of the analyte in the sample test solution;

A
a
b is the axis intercept of the calibration determined by the calibration solutions (4.11.11);
Cal
s
is the peak area of the internal standard in the sample test solution;

A
ISTD
13
=

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a is the slope of the calibration determined by the calibration solutions (4.11.11);
Cal
s
is the mass concentration of the internal standard in the sample test solution, in ng/ml.
ρ
ISTD
Calculate the mass fraction w , of the ana
...