TEST PROCEDURE
Determination of Nitroimidazoles in Egg, Plasma, Muscle and Honey by
means of LC/MS-MS:
Valid in
LSV/THKS/HVTA
Valid for
all employees
Managed by
Purpose
Parameter(s)
tested
Matrix
LSV/THKS/ HVTA
The test procedure describes the method for extracting nitroimidazoles from egg,
plasma, honey and muscle as well as their analytical detection using LC/MS-MS.
Nitroimidazoles were used as chemotherapy substances to treat illnesses caused by
to protozoa, but have now been prohibited (Commission Regulation (EU) No
37/2010, Table 2). Therefore, detecting metabolites as indicators for prohibited use
is also of interest. If residues occur in foodstuffs, they may have a harmful effect
on health when consumed. Nitroimidazoles and their metabolites are suspected to
be carcinogenic and mutagenic.
Dimetridazole
HMMNI (= Dimetridazolhydroxide; 2-hydroxymethyl-1-methyl-5-nitroimidazole)
Ipronidazole
Hydroxyipronidazole
Metronidazole
Hydroxymetronidazole
Ronidazole
Ternidazole
Carnidazole
Ornidazole
Secnidazole
Tinidazole
Eggs
Blood plasma of different animal species
Muscle of different animal species
Honey
1 Previous version
Determination of nitroimidazoles in egg and plasma using LC/MS-MS (PV_CC_VIE_TAHO_228_01)
1.1 Changes since the previous version
-
Expanding the test procedure to include the muscle and honey matrices
Section 8.3: Specification of the instrumental measurement method
Sectopm 9.1: Specification of the file name of the Microsoft Excel template used for interim calculations
Formal changes through transfer of the content into the current document format
TESTING PROCEDURE
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02
1/14
2 Method
2.1 Principle
The substances to be tested are extracted from eggs, plasma, honey or muscle and concentrated and
determined using mass spectroscopic detection after HPLC separation.
2.2 Brief description of the method
-
Extraction/Protein precipitation with trichloracetic acid
Extract purification using liquid/liquid extraction
HPLC separation with mass spectroscopic detection (LC/MS-MS, HESI positive)
3 Terms, abbreviations and symbols used
ISTD: Internal standard
rpm: rotations per minute
HESI: heated electrospray ionization
4 Warnings and safety instructions
Organic solvents are potentially dangerous. All operations must be carried out such that no inhalation of
the vapors or contact with the skin occurs.
Follow the safety data sheets!
Acetonitrile
Harmful to health
Highly flammable
Methanol
n-hexane
Toxic
Harmful to health
Highly flammable
Highly flammable
Ethyl acetate
Irritant
Highly flammable
Hazardous to environment
Hazardous to environment
The corrosive potential of acids and alkalis must be kept in mind when handling them. Significant changes in pH or neutralizations must be conducted slowly to avoid a violent reaction.
Hydrochloric acid
Trichloracetic acid
Sodium hydroxide
Corrosive
Corrosive
Corrosive
Follow the safety data sheets!
5 Equipment and apparatus
The company names are given mainly as indicative of the quality of the products. Products from other
manufacturers can be used if they meet the requirements.
In addition to common laboratory equipment, the following instruments and apparatus are required:
5.1 Equipment
-
Micropipettes
Multipette Plus
TESTING PROCEDURE
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02
2/14
-
Analytical balance
pH meter
Centrifuge with cooling
Dilutor
Shaker (IKA MS1 Minishaker)
Overhead shaker
Drying device with water bath and inert gas connection (in-house construction)
Ultrasonic bath (Transsonic Digital S, Elma)
HPLC system (Accela by Thermo or Agilent 1200 by Agilent)
Mass spectrometer (TSQ Quantum Ultra by Thermo or API 4000 QTrap by Applied Biosystems)
5.2 Apparatus
-
Extraction tubes (15 ml Greiner vials)
HPLC vials
HPLC column: ODS Hypersil, 200 x 2.1 mm; 5 µm (Thermo, UK)
Precolumn: ODS Hypersil, 10 x 2.1 mm; 5 µm (Thermo, UK)
6 Reagents, solutions and test organisms
The requirements in SVA_CC_VIE_TAHO_003 (Ordering, labeling, storing and handling of chemicals and
reference substances) must be followed.
6.1 Standard and reference substances
The suppliers indicated guarantee the required quality of the standards. Standards from other manufacturers can also be used if they have the same minimum quality.
Substance
Abbreviation
CAS no.
Supply source
-
DMZ
HMMNI
551-92-8
936-05-0
Sigma D4025
EU-RL Berlin
IPZ
IPZ-OH
MNZ
MNZ-OH
RNZ
TNZ
SNZ
TDZ
ONZ
CNZ
d3-DMZ
14885-29-1
35175-14-5
443-48-1
4812-40-2
7681-76-7
1077-93-6
3366-95-8
19387-91-8
16773-42-5
42116-76-7
64678-69-9
EU-RL Berlin
EU-RL Berlin
EU-RL Berlin
EU-RL Berlin
Sigma R7635
EU-RL Berlin
Chemos
EU-RL Berlin
LGC
EU-RL Berlin
EU-RL Berlin
d3-HMMNI
1015855-78-3
EU-RL Berlin
1015855-87-4
EU-RL
EU-RL
Sigma
EU-RL
EU-RL
-
Dimetridazole
2-hydroxymethyl-1-methyl5-nitroimidazole
Ipronidazole
Hydroxyipronidazole
Metronidazole
Hydroxymetronidazole
Ronidazole
Ternidazole
Secnidazole
Tinidazole
Ornidazole
Carnidazole
d3-dimetridazole
d3-2-hydroxymethyl-1methyl-5-nitroimidazole
d3-ipronidazole
d3-hydroxyipronidazole
d3-metronidazole
d2-hydroxymetronidazole
d3-ronidazole
TESTING PROCEDURE
d3-IPZ
d3-IPZ
d3-MNZ
MNZ-OH
d3-RNZ
Berlin
Berlin
M-9036
Berlin
Berlin
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02
3/14
6.2 Chemicals and reagents
The company names are given mainly as indicative of the quality of the products. Substances and products from other manufacturers can be used if they fulfill the required criteria.
-
Methanol, LiChrosolv
Hydrochloric acid 1M, Titrisol
25% hydrochloric acid p.a.
Trichloracetic acid p.a
Ammonium acetate
Water: ultrapure water
Ethyl acetate p.a.
Glycerin anhydrous
Merck 1.06018
Merck 1.09970
Merck 1.00316
Merck 1.00807.1000
Merck 1.16103
purified with Modulab 2020
Merck 1.09623.2500
Fluka 497700
6.3 Gases
From the central gas supply
- Nitrogen 5.0 (for LCMS)
- Argon 5.0 (for LCMS)
- Compressed air for nitrogen generator
6.4 Solutions/Culture media
Unless otherwise specified, a portion or a multiple of the given preparation for the solutions and culture
media can be made with the required accuracy.
The requirements in SVA_CC_VIE_TAHO_004 (Handling of solutions) must be observed.
The following list of preparations for standards is only a recommendation.
The requirements in SVA_CC_VIE_TAHO_006 (Guide to the handling of standard substances) must be
observed.
Remarks: “water” always means deionized water (ultrapure water).
For all steps in which water is used, this must be ultrapure water.
6.4.1
Sodium chloride/Potassium dihydrogen phosphate solution
Dissolve 5.84 g sodium chloride and 13.61 potassium dihydrogen phosphate in 950 mL ultrapure water,
adjust to a pH of 3 using 25% HCl and fill up to the mark with ultrapure water in a 1000 mL volumetric
flask.
6.4.2
Trichloracetic acid solution (10%)
Weigh out 10 g of trichloracetic acid into a 100 mL volumetric flask and fill up with ultrapure water.
6.4.3
Tris buffer (ph: 9.0)
Dissolve 1.21 g Tris (hydroxmethyl) aminomethane in 90 mL ultrapure water and adjust with 1 M HCl to
a pH of 9.0, fill up with ultrapure water to 100 mL.
6.4.4
H2O-saturated ethyl acetate
A bottle of ethyl acetate is opened and filled up entirely with ultrapure water and briefly shaken.
6.4.5
Nitroimidazole stock solutions (500 µg/mL)
Weigh out 5 mg each of the substances (6.1) [convert to pure substance, if necessary!] in one 10 mL
volumetric flask each, fill with methanol to the mark and mix.
If stored in the dark at -20 °C, the solutions have a shelf life of 5 years.
TESTING PROCEDURE
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02
4/14
6.4.6
Nitroimidazole spiking solutions (.06 µg/mL –.015 µg/mL)
From the stock solutions of the non-deuterated substances – DMZ, HMMNI, IPZ, IPZ-OH, MNZ, MNZ-OH,
RNZ, TNZ, SNZ, TDZ, ONZ and CNZ – use a dilutor to prepare spiking standard mixture and from the
stock solutions of the deuterated substances – d3-DMZ, d3-HMMNI, d3-IPZ, d3-IPZ-OH, d3-MNZ, d2MNZ-OH, d3-RNZ, prepare ISTD spiking mixture. The following dilutions may be used for this (working
solutions 1, 2 and 3).
Working solutions
Working solution 1:
Working solution 2:
Working solution 3:
Substance
(Abbreviation)
DMZ
HMMNI
IPZ
IPZ-OH
MNZ
MNZ-OH
RNZ
TNZ
SNZ
TDZ
ONZ
CNZ
DMZ
HMMNI
IPZ
IPZ-OH
MNZ
MNZ-OH
RNZ
TNZ
SNZ
TDZ
ONZ
CNZ
d3-DMZ
d3-HMMNI
d3-IPZ
d3-IPZ
d3-MNZ
d2-MNZ-OH
d3-RNZ
Spiking standard solutions
Substance
(Abbreviation)
Spiking standard mixture:
DNZ
HMMNI
IPZ
IPZ-OH
MNZ
MNZ-OH
RNZ
TESTING PROCEDURE
Concentration
(µg/mL)
5
30
10
10
5
20
10
5
5
20
20
10
0.5
3
1
1
0.5
2
1
0.5
0.5
2
2
1
10
10
10
10
10
10
10
Concentration
(µg/mL)
0.015
0.09
0.03
0.03
0.015
0.06
0.03
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02
5/14
TNZ
SNZ
TDZ
ONZ
CNZ
d3-DMZ
d3-HMMNI
d3-IPZ
d3-IPZ
d3-MNZ
d2-MNZ-OH
d3-RNZ
Spiking ISTD mixture:
0.015
0.015
0.06
0.06
0.03
0.300
0.300
0.300
0.300
0.300
0.300
0.300
When stored in the dark at -20 °C, the solutions have a shelf life of 5 years.
6.4.7
Calibration standards
Prepare the following calibration standards for creating a calibration curve corresponding to the quantification range from the working solutions (6.4.6), for example, as follows:
Calibration
standard
Standard
Standard
Standard
Standard
Standard
Standard
Standard
equals
(µg/kg)*
1
2
3
4
5
6
7
0.1
0.2
0.5
1.0
2.0
5.0
10.0
Working solution 2
30 µL
60 µL
150 µL
Amount of
Working solution 1
30 µL
60 µL
150 µL
300 µL
Working solution 3
60 µL
60 µL
60 µL
60 µL
60 µL
60 µL
60 µL
dilute to
10 mL each
with ultrapure
water
*) applies to IPZ, IPZ-OH, RNZ and CNZ
Multiply values by factor f=2 for MNZ-OH, TDZ and ONZ
Multiply values by factor f=3 for HMMNI
Multiply values by factor f=0.5 for DMZ, MNZ, TNZ and SNZ
If stored in the dark at +4 °C, solutions have a shelf life of approx. 4 months.
6.4.8
HPLC eluent A (10 mM methanolic ammonium acetate solution)
Dissolve 771 mg ammonium acetate in methanol, transfer into 1000 mL volumetric flask and fill up to
mark.
6.4.9
HPLC eluent B (10 mM aqueous ammonium acetate solution)
Dissolve 771 mg ammonium acetate in ultrapure water, transfer into 1000 mL volumetric flask and fill up
to mark.
6.4.10 10% Glycerin solution
Pipette 10 mL glycerin into a 100 mL volumetric flask and fill up with methanol up to the mark.
6.4.11 Hydrochloric acid 1 mol/L
Empty the Titrisol ampoule as required into a 1000 mL volumetric flask, rinse out the ampoule with water
and fill the volumetric flask up to the mark.
6.4.12 Hydrochloric acid 0.1 mol/L
Pipette 100 mL hydrochloric acid solution into a 1000 mL volumetric flask and fill up with water up to the
mark.
TESTING PROCEDURE
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02
6/14
6.5 Disposal
Organic solvents must be disposed of in the solvent waste canisters provided in the laboratories. The
further details of the disposal system are established separately at the commercial site.
7 Sampling, sample preparation, and handling
Sample collection is not performed by the testing laboratory.
Weighings:
- Egg:
- Plasma:
- Honey:
- Muscle:
Weigh out exactly 3 g homogenized
Pipette 3 ml of plasma into a 15 mL
Weigh out exactly 3 g homogenized
Weigh out exactly 3 g homogenized
sample into a 15 mL Greiner vial to 0.01 g
Greiner vial
sample into a 15 mL Greiner vial to 0.01 g
sample into a 15 mL Greiner vial to 0.01 g
Store all samples in a freezer until prepared for testing; bring these to room temperature prior to analysis.
For further information on sample preparation and storage, see SVA_CC_VIE_TAHO_002 (Sample receipt
for the CC TAHO).
8 Implementation
The samples are routinely analyzed via simple determination (screening).
A positive screening result is confirmed at a minimum by conducting a determination in duplicate.
At least one positive control (spiked sample) is included for each analysis sequence.
Control samples serve to ascertain the current retention time, to determine the ion ratios, to calculate the
recovery rates and as a quality control for the analysis process using control charts.
The requirements in SVA_CC_VIE_TAHO_005 (Preparation of control samples) must be observed.
8.1 Preparatory work
8.1.1
-
Preparing control samples
Treat two analyte-free pool samples (egg, plasma, honey or muscle) with 100 or 200 µL of spiking
standard mixture (6.4.6) (+dot1, dot2; positive controls).
The spiking levels of the positive controls therefore are:
IPZ, IPZ-OH, RNZ, CNZ
MNZ-OH, TDZ, ONZ
HMMNI
DMZ, MNZ, TNZ, SNZ
-
dot1
(100 µL of spiking standard
mixture
)
1 µg/kg or 1 µg/L
2 µg/kg or 2 µg/L
3 µg/kg or 3 µg/L
0.5 µg/L or 0.5 µg/kg
dot2
(200 µL of spiking standard
mixture)
2
4
6
1
µg/kg
µg/kg
µg/kg
µg/kg
or
or
or
or
2
4
6
1
µg/L
µg/L
µg/L
µg/L
The third analyte-free pool sample is not spiked (= pure; negative control).
TESTING PROCEDURE
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02
7/14
8.1.2
-
Adding the internal standards
Add 20 µL of the spiking ISTD mixture (6.4.6) to all samples (including control samples)
[likewise 2 µg/kg egg, honey, muscle or 2 µg/mL plasma]
8.2 Preparing samples
8.2.1
-
Extraction/Protein precipitation
Add 3 mL of trichloracetic acid (6.4.2) to samples and control samples
Remarks: For honey, if necessary (viscous or crystalline), treat samples until they are fully dissolved:
treat in a 40 °C shaking water bath
-
Treat for 30 min in an overhead shaker
Then centrifuge at 4°C/4000 rpm/15 min
Decant supernatant in a 15 mL Greiner vial, discard residue
8.2.2
-
Add 3 mL Tris buffer (6.4.3) and bring to pH of 9.00 (± 0.3) with 5M NaOH
Add 5 mL ethyl acetate (6.4.4) and extract for 30 min in overhead shaker
Then centrifuge at 4°C/4000 rpm/15 min
Transfer organic phase (top) into a 12 mL tube using a Pasteur pipette
Add another 5 mL of ethyl acetate (6.4.4) to aqueous residue (bottom) and shake for 30 min in the
overhead shaker
Centrifuge at 4°C/4000 rpm/15 min
Suction off organic phase (top) with Pasteur pipette and combine with the first
Discard aqueous phase
Pipette 100 µL glycerin solution (6.4.10) into the organic extract
Then reduce by evaporation with a water bath at 40 °C using nitrogen
8.2.3
-
Liquid-liquid-extraction (extract purification)
Preparing for LC/MS-MS measurement
Add 100 µL mobile phase (9% eluent A (6.4.8) + 93% eluent B (6.4.9) to residue)
Mix in the vortexer and then dissolve in ultrasonic bath
Centrifuge (13000 rpm/10 minutes)
Fill supernatant into HPLC vials with insert (measurement solution)
8.3 Measurement/testing
Analyze the measurement solution in the HPLC vials (8.2.3) under the following conditions:
Chromatography column:
Precolumn:
Eluent:
Gradient:
Injection volume:
HPLC flow:
Oven temperature:
TESTING PROCEDURE
ODS Hypersil 200 x 2.1 mm; 5 µm (Thermo, UK)
ODS Hypersil, 10 x 2.1 mm; 5 µm (Thermo, UK)
A: 10 mM methanolic ammonium acetate
B: 10 mM aqueous ammonium acetate (6.4.9)
9% A for 10 min isocratic,
then to 60% A and back to 9% A in 6 min
then 9% A isocratic for 4 min.
20 µL
0.25 mL/min
40 °C
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02
8/14
MS detection TSQ Quantum Ultra
Ionization mode: HESI positive
Analyte
RT (min)
Precursor (m/z)
Dimetridazole
d3-dimetridazole
d3-ipronidazole
Ipronidazole
d3-hydroxyipronidazole
Hydroxyipronidazole
d3-metronidazole
Metronidazole
Hydroxymetronidazole
d2-hydroxymetronidazole
Ternidazole
Ronidazole
d3-ronidazole
HMMNI
d3-HMMNI
Secnidazole
Tinidazole
Ornidazole
Carnidazole
11.60
11.60
13.20
13.20
12.50
12.50
8.80
8.80
5.90
5.80
11.70
8.15
8.10
7.20
7.10
14.80
13.40
17.60
21.20
142
145
173
170
189
186
175
172
188
190
186
201
204
158
161
186
186
220
245
Fragment ions (m/z)
95, 96
99
127
109, 124
171
122, 168
85
82, 128
123, 126
128
82, 128
55, 140
143
112, 140
143
82, 128
82, 128
82, 128
75, 118
API 4000 QTrap MS detection
Ionization mode: HESI positive
Analyte
Precursor (m/z)
Dimetridazole
d3-dimetridazole
d3-ipronidazole
Ipronidazole
d3-hydroxyipronidazole
Hydroxyipronidazole
d3-metronidazole
Metronidazole
Hydroxymetronidazole
Ternidazole
Ronidazole
d3-ronidazole
HMMNI
d3-HMMNI
RT (min)
12.1
11.9
21.0
21.1
19.2
19.3
7,5
7.6
4.6
13.4
6.9
6.8
5.9
142
145
173
170
189
186
175
172
188
186
201
204
161
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Fragment ions (m/z)
81, 96
99
127
109, 124
125
122, 168
131
128, 82
123, 126
128
140, 55
143
143
MS experiment
MS experiment
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
Q1/Q3
*) retention time (RT) only serves as a reference point since it depends on a number of parameters (particularly of
the column age and column).
The measuring method is called NIM-XXX, where XXX is a consecutive number. The method with the
highest number is the current and therefore the measuring method to be used.
TESTING PROCEDURE
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02
9/14
9 Evaluation
9.1 Evaluation/Calculation
Evaluate the measurement results obtained with the appropriate software (Xcalibur or Analyst):
- Identify the signals (peaks) using the retention times and ion fragments (m/z) and attribute them to
the corresponding substances;
- Integrate the peaks to determine the area values;
- Create calibration curve from the results of the calibration standards, and calculate the associated
concentrations. Save the results.
- Export the raw data from the instrument software into Microsoft Excel.
Perform the following further calculations using Microsoft Excel:
- Relative retention times and retention time deviations of the corresponding fragment ions
- Ion ratios of the corresponding fragment ions for identification according to EU criteria (9.1.2)
- Recovery rates (WFRs)
- Analyte content in the sample (corrected for WFR)
Remarks: Ensure that sample weights and any dilution factors are reflected in the calculations of analyte content.
Use the master template “Berechnungsvorlage_LCMSMS_XX.xlt“, found under
L:\taho\Analysendaten\ as a starting point for carrying out these calculations. Here, XX is a consecutive number, and the file with the highest number XX must be used.
9.1.1
a)
Assessment screening
Check the retention times of standard solutions, control samples and samples:
aa) The retention time of the analyte in question must agree with the mean retention time of the
spiked control sample within a tolerance of ± 5%.
ab) The retention times of the corresponding fragment ions must agree within a tolerance of 0.2
min.
ac) The relationship of the retention time for the analyte in question and that of an appropriate internal standard (i.e., relative retention time) must correspond to that of the standard solutions
or the spiked control samples within a tolerance of ±5%.
Analytes that do not meet these criteria are considered as suspected positives. Otherwise, the analyte is “not detectable”.
b)
Check the relative ion intensities:
The relative ion intensities of the corresponding fragment ions are calculated for all samples (calibration standards, control samples, samples) based on the ion with the highest intensity (greatest peak
area). An analyte is then considered to be a “suspected positive” if the relative ion intensity of the
sample lies within the following tolerance ranges, which are based on the mean ion ratios of the calibration standards and control samples:
Relative intensity of the corresponding fragment ions
> 50%
> 20% - 50%
> 10% - 20%
 10%
TESTING PROCEDURE
Maximum permissible relative deviation
(tolerance range)
 40%
 50%
 60%
 80%
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02 10/14
If the relative ion intensities are outside of these computed ranges, the analyte is considered “not
detectable.”
c)
Check the internal standards:
If one of the internal standard substances entrained in all the samples is found to be less than 20%
of the mean concentration of the accompanying control samples, or the ISTD is not detectable at all,
the sample is classified as “not evaluable" and must be repeated. If in doubt, consult with the responsible supervisor.
d)
Compare the analyte content (corrected for recovery rate) in the sample with the corresponding
detection limit:
If the analyte content is above the detection limit, the sample is considered as a “suspected positive,”, otherwise, the analyte is considered “not detectable.”
e)
Check the analyte content with respect to the calibration range:
If the analyte content is higher than the highest calibration standard, the measurement must be repeated with a suitable dilution. If necessary, a new partial sample must be extracted and spiked with
a correspondingly higher amount of internal standards so that the concentration is consistent with
the required dilution.
If in doubt, consult the responsible supervisor.
If the presence of a questionable substance in a sample is established during screening (“suspected positive”), the extraction must be repeated for confirmation with at least two new aliquots of the sample in
question, to validate the initial finding. The preparation and analysis of the parallel samples are done
according to Section 8, and the assessment proceeds according to Section 9.1.2.
If a sample is considered “not evaluable”, a new aliquot of the sample must be weighed out and the
analysis repeated.
If in doubt, the specific procedure should be clarified with the responsible supervisor.
9.1.2
a)
Assessment confirmation
Check the retention times of standard solutions, control samples and samples:
aa) The retention time of the analyte in question must agree with the mean retention time of the
spiked control samples within a tolerance of ± 5%.
ab) The retention time of the corresponding fragment ions must agree within a tolerance of 0.2 min.
ac) The relationship of the retention time for the analyte in question and that of an appropriate internal standard (i.e., relative retention time) must correspond to that of the standard solutions
or the spiked control samples within a tolerance of ±2.5%.
b)
Check the relative ion intensities:
For the correct identification of an analyte, the ion ratios of the corresponding fragment ions must
be compared with those of the standards or control samples and lie within an established tolerance
limit. The following tolerances are permissible according to the Commission Decision of August 12,
2002 (2002/657/EC):
Relative intensity of the qualifier to the
target ion
> 50%
> 20% - 50%
> 10% - 20%
 10%
TESTING PROCEDURE
Maximum permissible relative deviations
(EU tolerances)
 20%
 25%
 30%
 50%
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02 11/14
c)
Check the internal standards:
The concentration of the internal standard corresponding to the analyte in question must be 20%
higher than the mean concentration of the accompanying control samples.
d)
The analyte content in the sample must be above the detection limit (see Section 10).
e)
The value measured must be within the calibration range.
A substance is considered to be confirmed when all of the listed criteria are met. The mean value for
analyte content in parallel samples is used in the presentation of quantitative results.
If one of the three criteria specified under a), b), and d) is not met, then the substance is considered
“not detectable” providing that criteria c) and e) are met.
Conversely, if criterion c) is not met, further procedures must be clarified with the responsible supervisor.
If criterion e) is not met, a suitable dilution of the measurement solution must be carried out. If necessary, a new partial sample must be extracted and spiked with a correspondingly higher amount of an
internal standard so that the concentration is consistent with the required dilution.
If in doubt, consult the responsible supervisor.
9.2 Documentation
At
-
a minimum, the Test Report must contain:
Preparer, analyst (name or initials);
Date of the sample preparation and measurement;
Sample identifier (LISA number);
Statement of the standards and control samples used, with dates and the concentrations employed;
Results from the samples;
Any deviations from the test procedure.
9.3 Presentation of results
The results are presented in the Test Report based on the values determined in Section 9.1:
-
Analyte not detectable.
Egg:
“ND (LOD: ... μg/kg)“
Plasma: “ND (LOD: ... μg/kg)“
Muscle: “ND (LOD: ... μg/kg)“
Honey: “ND (LOD: ... μg/kg)“
-
Analyte detectable, but below the quantitation level
Egg:
“detectable < LOQ (LOD: ... μg/kg; LOQ ... μg/kg)“
Plasma: “detectable < LOQ (LOD: ... μg/L; LOQ ... μg/L)“
Muscle: “detectable < LOQ (LOD: ... μg/kg; LOQ ... μg/kg)“
Honey : “detectable < LOQ (LOD: ... μg/kg; LOQ ... μg/kg)“
“
-
Content of the detected analyte is quantifiable:
Egg:
"xx.y (µg/kg (LOD: ... (µg/kg; LOQ: ... (µg/kg)"
Plasma: "xx.y (µg/kg (LOD: ... (µg/l; LOQ: ... (µg/l)"
Muscle: "xx.y (µg/kg (LOD: ... (µg/kg; LOQ: ... (µg/kg)"
Honey: "xx.y (µg/kg (LOD: ... (µg/kg; LOQ: ... (µg/kg)"
Remarks: The detection and quantitation limits can be taken from the current LISA method under the
“Limits” tab.
TESTING PROCEDURE
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02 12/14
10 Validation
The results from the validation are available in electronic form at:
L:\taho\QM\Validierung\NITROIMIDAZOLE-ei_lcmsms(quantum ultra) or
L:\taho\QM\Validierung\NITROIMIDAZOLE-plasma_lcmsms(quantum ultra) or
L:\taho\QM\Validierung\NITROIMIDAZOLE-honig_lcmsms(quantum ultra) or
L:\taho\QM\Validierung\NITROIMIDAZOLE-muskel_lcmsms(quantum ultra).
11 References
11.1 QM documents and document templates
-
SVA_CC_VIE_TAHO_002 (Sample receipt for CC TAHO)
SVA_CC_VIE_TAHO_003 (Ordering, labeling, storing and handling chemicals and reference substances)
- SVA_CC_VIE_TAHO_004 (Handling of solutions)
- SVA_CC_VIE_TAHO_005 (Preparation of control samples)
- SVA_CC_VIE_TAHO_006 (Guide to handling of standard substances)
- DOT_PQO_PV
Location: DOXIS
11.2 Standards, laws and guidelines
-
Council Regulation (EC) no. 470/2009 from May 6, 2009 laying down Community procedure for the
establishment of maximum residue limits of pharmacologically active substances in foodstuffs of animal origin
-
Council Regulation (EU) 37/2010 of the Commission on 22 December, 2009, on pharmacologically
active substances and their classification regarding maximum residue limits in foodstuffs of animal
origin
- Commission Decision from August 12, 2002 (2002/657/EC) implementing Council Directive 96/23/EC
concerning the performance of analytical methods and the interpretation of results
Location: in digital form at L:\taho\QM\Gesetzliche Grundlagen
Remarks: Additional statutory provisions (e.g., amendments, supplements, changes) are stored electronically at the above-mentioned location.
11.3 Scientific literature
-
Manual: Workshop “Nitroimidazoles in Residue Analysis“ 7-9 July 1999, Berlin.
Manual: CRL Workshop “Analysis of Nitroimidazoles: Screening, Validation, Proficiency Test” 16-19
April 2002, Berlin.
Both workshop manuals from the EU reference laboratory for residues of veterinary medicines and
contaminants in food of animal origin BVL Berlin
(formerly BgVV)
Location: B/2.33
12 Appendices
None
TESTING PROCEDURE
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02 13/14
Created/revised
Technical check
QM check
Release
Name
Johann Burger
Georg Mayerhofer
Irina SchwaigerNemirova
Thomas Kuhn
Date
November 19, 2011
May 12, 2011
May 12, 2011
May 12, 2011
Signature
signed
signed
signed
signed
DOT_PQO_PV_03
TESTING PROCEDURE
Valid from Dec. 7, 2011
PV_CC_VIE_TAHO_228_02 14/14