Workflow to perform quantification
by standard addition procedure
Standard addition is an effective way to compensate for matrix effects, e.g. in cases of “difficult” matrix-pesticide combinations or if no blank matrix is available for external matrix
matched calibration. Standard addition can be performed either to aliquots of the sample extract or direct to analytical portions prior to extraction. In the first case, it only corrects for matrix effects as well as for analyte degradation in the extract, the latter case, it also corrects for
recovery during extraction and clean-up. Because of linear extrapolation linearity of response
throughout the measured concentration range is paramount. Furthermore it should be considered, that added amounts are not too small in order to reduce the influence of measurement variability on the slope of the linear regression.
The following four steps should be followed:
1) The likely concentration of the analyte in the sample should be approximately estimated, e.g. by quantification using external calibration.
Example: Chlorpyrifos concentration of about 0.2 mg/kg
2) Calculate the absolute amount of the analyte in a representative volume of the sample
extract, for example 1 ml.
Example: Extract concentration 1 g/mL  about 0.2 µg in 1 mL sample extract.
3) Three addition levels should be prepared. The amounts to be added should be within
the range of approximately half of the estimated amount, the estimated amount and
1.5 times of the estimated amount. The linearity range allowing it, greater amount can
be added e.g. 1x, 2x and 3x the estimated amount). Prepare a standard solution of the
analyte with a concentration level in such a way that about 100 µL of the solution contains the lowest amount to be added.
Example: Addition levels 0.1 µg, 0.2 µg, 0.3 µg. Prepare a standard solution of
Chlorpyrifos with a concentration of 1 mg/L (µg/mL); 100 µL of this solution contains
0.1 µg.
4) Take 4 vials and fill 1 mL of sample extract in each vial. Spike the vials 2 - 4 with the
appropriate volume of the pesticide standard solution; in vial 1 the original sample remains without addition of standard solution. Adjust the volume by adding the corresponding solvent amounts.
This procedure should be repeated with a second extract of the same sample and with an
extract of a recovery sample, which was spiked with an adequate amount of the analyte.
EU Reference Laboratory for Pesticides Requiring Single Residue Methods
CVUA Stuttgart, Schaflandstr. 3/2, 70736 Fellbach, Germany
[email protected]
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Example 1: Residues of Chlorpyriphos in Sweet Pepper, concentration ~1 mg/kg
Standard addition TO EXTRACT ALIQUOTS (exemplary pipetting scheme)
Aliquot volume used
Sample amount represented in the aliquot
Expected absolute amount of pesticide in the in the aliquot
Conc. of chlorpyrifos std solution to be used for StAdd
no
addition
Volume of sample extract aliquots
Added volume of an appropriate dilution
of pesticide stock solution
Resulting mass of pesticide added to
each vial
Volume of solvent to be added
Final volume
1000 µl
_
0
300 µL
1300 µL
1 ml
1g
~0.2 µg
1 µg/mL
addition
level 1
Commodity: Sweet Pepper
Analyte: Chlorpyrifos
Expected Conc.: 0.2 mg/kg
addition
level 2
addition
level 3
1000 µl
1000 µl
1000 µl
100 µL
200 µL
300 µL
0.2 µg
0.3 µg
0.1 µg
200 µL
1300 µL
100 µL
- µL
1300 µL
1300 µL
Example 2: Residues of Methamidophos in tea, concentration ~2 mg/kg
(Extraction with QuEChERS-Method (2 g sample + 10 ml water+10 ml MeCN))
Standard addition TO EXTRACT ALIQUOTS (exemplary pipetting scheme)
Aliquot volume used
Sample amount represented in the aliquot
Expected absolute amount of pesticide in the in the aliquot
Conc. of methamidophos std solution to be used for StAdd
no
addition
Volume of sample extract aliquots
1 ml
0.2 g
~0.4 µg
2 µg/mL
addition
level 1
Commodity: Tea
Analyte: Methamidophos
Expected Conc.: 2 mg/kg
addition
level 2
addition
level 3
1000 µl
1000 µl
1000 µl
1000 µl
_
100 µL
200 µL
300 µL
0 µg
0.2 µg
0.4 µg
0.6 µg
Volume of solvent to be added
300 µL
200 µL
100 µL
- µL
Final volume
1300 µL
1300 µL
1300 µL
1300 µL
Added volume of an appropriate dilution
of pesticide stock solution
Resulting mass of pesticide added to
each vial
EU Reference Laboratory for Pesticides Requiring Single Residue Methods
CVUA Stuttgart, Schaflandstr. 3/2, 70736 Fellbach, Germany
[email protected]
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Example 3: Residues of Cyromazine in Lettuce, conc. determined in initial analysis
0.06 mg/kg (expected recovery rate ~ 30%), thus expected conc. in sample ~ 0.2 mg/kg
Standard addition TO SAMPLE PORTIONS (exemplary pipetting scheme)
Aliquot volume used
Sample amount represented in the aliquot
Expected absolute amount of pesticide in the in the aliquot
Conc. of Cyromazine std solution to be used for StAdd
no
addition
Volume of sample extract aliquots
Added volume of an appropriate dilution
of pesticide stock solution
Resulting mass of pesticide added to
each vial
Volume of solvent to be added
Final volume
1000 µl
_
0
300 µL
1300 µL
1 ml
1g
~ 0.2 µg
1 µg/mL
addition
level 1
Commodity: Lettuce
Analyte: Cyromazine
Expected Conc.: 1 mg/kg
addition
level 2
addition
level 3
1000 µl
1000 µl
1000 µl
100 µL
200 µL
300 µL
0.2 µg
0.3 µg
0.1 µg
200 µL
1300 µL
100 µL
- µL
1300 µL
1300 µL
Quantification by internal calibration using the procedure of standard addition,
schematically
Key
Y
X
Ratio of peak area of analyte divided by peak area of ISTD
add
Added absolute mass of analyte m std
in µg
pest
|x|
absolute amount of analyte in the sample portion (in µg) before standard addition (y = 0)
x
y  in tercept (c)
slope of the curve (b)
EU Reference Laboratory for Pesticides Requiring Single Residue Methods
CVUA Stuttgart, Schaflandstr. 3/2, 70736 Fellbach, Germany
[email protected]
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