THE TITLE PAGE
Cyromazine
GRM029.01A
DRAFT COPY
Cyromazine (CGA72662) - Analytical Method for the
Determination of Residues of Cyromazine and its Metabolite
Melamine (C1803) in High water Content Crops.
Final Determination by LC-MS/MS
Analytical Method
AUTHORS:
S L Hargreaves
EFFECTIVE DATE:
DATA REQUIREMENT:
EPA OPPTS 860.1340
EC Guidance Document SANCO/3029/99 rev 4
EC Guidance Document SANCO/825/00 rev 7
PERFORMING LABORATORY: Syngenta Ltd.
Jealott’s Hill International Research Centre
Bracknell, Berkshire, RG42 6EY, UK
www.syngenta.com
SPONSOR:
Syngenta Ltd.
Jealott’s Hill International Research Centre
Bracknell, Berkshire, RG42 6EY, UK
www.syngenta.com
Summary of revisions to previous version
Version
Summary of Revisions
GRM029.01A This confirmatory method supersedes method REM174.02 (Reference 1)
for analysis of high water content crops. The method includes a cation
exchange SPE procedure and final determination is by LC-MS/MS with a
confirmatory transition.
Authorisation
Authorised by
:
DRAFT GRM029.01A
S Hadfield
Technical Manager Date
Product
Metabolism
Page 2 of 57
Abbreviations and symbols
Abbreviation
A
Å
a.i.
amt
amu
C
CAS
CFR
cm
DA[#]A
EPA
EU
FIFRA
ft
g
gal
GC
GLP
GRM
ha
HPLC
i.d.
ID
in
IUPAC
kg
L
lb
LC
LC-MS/MS
LOD
LOQ
m
Definition
acre
angstrom (10-8 m)
active ingredient
amount
atomic mass unit
Celsius or centigrade
Chemical Abstract Services
Code of Federal Regulations
centimetre
days after application, [#] = 1, 2, 3 etc., if there are multiple applications
Environmental Protection Agency (U.S.)
European Union
Federal Insecticide, Fungicide and Rodenticide Act (U.S.)
foot (feet)
gram
gallon
gas chromatography
Good Laboratory Practice
Global Residue Method
hectare
high performance liquid chromatography
inside diameter
identification
inch
International Union of Pure and Applied Chemistry
kilogram
litre
pound
liquid chromatography
liquid chromatography - tandem mass spectrometry/mass spectrometry
limit of detection
limit of quantification
meter/metre
DRAFT GRM029.01A
Page 3 of 57
Abbreviations and symbols (continued)
Abbreviation
µg
µL
µm
mbar
MDL
mg
min
mL
mm
mmol
mol
ms
MS
MS/MS
mV
MW
m/z
na
nd
ng
No.
oz
PMRA
ppb
ppm
pg
psi
QAU
2
R (or r2)
RSD
RT
s
SD
SPE
Definition
microgram
microliter
micrometer
millibar
method detection limit
milligram
minute
millilitre
millimetre
millimole
mole
millisecond
mass spectrometry
tandem mass spectrometry/mass spectrometry
millivolt
molecular weight
mass to charge ratio
not applicable
not detectable (below limit of detection)
nanogram
number
ounce
Pest Management Regulatory Agency (Canada)
parts per billion or micrograms per kilogram
parts per million or microgram per gram or milligrams per kilogram
picogram
pounds per square inch
quality assurance unit
square of correlation coefficient
relative standard deviation
retention time
second
standard deviation
solid phase extraction
DRAFT GRM029.01A
Page 4 of 57
Abbreviations and symbols (continued)
Abbreviation
USDA
UV
V
vol
v/v
wt
w/v
Definition
United States Department of Agriculture
ultraviolet
volt
volume
volume/volume
weight
weight/volume
DRAFT GRM029.01A
Page 5 of 57
TABLE OF CONTENTS
THE TITLE PAGE
1
TABLE OF CONTENTS
6
1.0
INTRODUCTION
9
1.1
1.2
Scope and chemical structures ................................................................9
Method summary ..................................................................................10
2.0
MATERIALS AND APPARATUS
2.1
2.2
2.3
2.3.1
2.3.2
2.4
Apparatus ..............................................................................................10
Reagents ................................................................................................10
Preparation of analytical standard solutions .........................................10
Fortification solutions ...........................................................................11
Standard solution storage and expiration ..............................................11
Safety precautions and hazards .............................................................11
3.0
ANALYTICAL PROCEDURE
3.1
3.2
3.3
3.4
3.5
3.6
3.7
Modifications and potential problems ...................................................13
Sample preparation................................................................................13
Extraction ..............................................................................................13
Sample Clean-up ...................................................................................14
Time required for analysis ....................................................................15
Method stopping points .........................................................................15
Preparation of calibration standards for LC-MS/MS ............................16
4.0
FINAL DETERMINATION
4.1
4.2
4.3
Instrument description ...........................................................................16
Chromatography conditions ..................................................................17
Mass spectrometer conditions ...............................................................17
5.0
CALCULATION OF RESULTS
5.1
5.2
Single point calibration procedure ........................................................18
Multi point calibration procedure..........................................................19
6.0
CONTROL AND RECOVERY SAMPLES
20
7.0
SPECIFICITY
20
7.1
Matrix interference ................................................................................21
DRAFT GRM029.01A
10
13
16
18
Page 6 of 57
7.2
7.3
Reagent and solvent interference ..........................................................21
Labware interference .............................................................................21
8.0
METHOD VALIDATION
8.1
8.2
8.3
8.4
8.5
Recovery data and repeatability ............................................................21
Limit of quantification (LOQ) ..............................................................21
Limit of detection (LOD) ......................................................................21
Detector linearity ...................................................................................22
Extract stability .....................................................................................22
9.0
LIMITATIONS
22
10.0
CONCLUSIONS
22
11.0
REFERENCES
23
APPENDICES SECTION
APPENDIX 1
APPENDIX 2
APPENDIX 3
APPENDIX 4
APPENDIX 5
APPENDIX 6
APPENDIX 7
APPENDIX 8
21
24
APPARATUS .......................................................................................25
REAGENTS ..........................................................................................28
METHOD VALIDATION DATA ........................................................30
REPRESENTATIVE CHROMATOGRAMS ......................................36
DETECTOR LINEARITY GRAPHS ...................................................50
API4000 MS/MS TUNING PROCEDURE..........................................54
MS/MS PRODUCT ION SPECTRA ....................................................55
METHOD FLOWCHART ....................................................................57
DRAFT GRM029.01A
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1.0
INTRODUCTION
1.1
Scope and chemical structures
Analytical method GRM029.01A is suitable for the determination of cyromazine and its
metabolite melamine (Figures 1 & 2) in high water content crop matrices. The limit of
quantitation (LOQ) of the method has been established at 0.05 mg/kg.
This method satisfies EU guidelines SANCO/3029/99 rev. 4, SANCO/825/00 rev. 7 and
US EPA guideline OPPTS 850.7100.
Figure 1
Common Name
:
Compound Code Number :
CAS Number
:
IUPAC Name
:
Molecular Formula
:
Molecular Mass
:
Cyromazine
CGA72662
66215-27-8
N-cyclopropyl-1,3,5-triazine-2,4,6-triamine
C6H10N6
166.19
H
N
H2N
N
N
N
NH2
Figure 2
Common Name
Compound Code Number
CAS Number
IUPAC Name
Molecular Formula
Molecular Mass
:
:
:
:
:
:
Melamine
C1803
108-78-1
1,3,5-triazine-2,4,6-triamine
C3H6N6
126.12
NH2
H2N
N
N
N
NH2
DRAFT GRM029.01A
Page 9 of 57
1.2
Method summary
High water content crop samples are extracted by homogenisation with 0.025 M KH2PO4:
in 0.04 M H3PO4 and by mechanical shaking following the addition of methanol. After
centrifugation and filtration, aliquots equivalent to 0.2 g are taken through an Oasis MCX
solid phase extraction (SPE) procedure. Final determination is by high performance
liquid chromatography with triple quadrupole mass spectrometry detection (LC-MS/MS).
The limit of quantification of the method is 0.05 mg/kg.
2.0
MATERIALS AND APPARATUS
2.1
Apparatus
The recommended equipment and apparatus are listed in Appendix 1. Equipment with
equivalent performance specifications may be substituted.
2.2
Reagents
All solvents and other reagents must be of high purity, e.g. glass distilled/HPLC grade
solvents and analytical grade reagents. Particular care must be taken to avoid
contamination of the reagents used. Reagents of comparable purity may be substituted as
long as acceptable performance is demonstrated. A list of reagents used in this method
along with details of preparation of solutions is included in Appendix 2.
2.3
Preparation of analytical standard solutions
It is recommended that the following precautions should be taken when weighing the
analytical materials.
1.
2.
3.
4.
Ensure good ventilation.
Wear gloves and laboratory coat.
Prevent inhalation and contact with mouth.
Wash any contaminated area immediately.
Prepare individual 200 µg/mL stock solutions for cyromazine and melamine by one of the
following methods.
Weigh out accurately, using a five figure balance, sufficient cyromazine and melamine
analytical standard into separate amber “Class A” volumetric flasks (50 mL). Dilute to
the mark with acetonitrile for cyromazine and melamine to give separate 200 µg/mL
stock solutions.
Alternatively, the appropriate volume of solvent to add to a known amount of standard
material may be determined using the equation below. The standard concentration is
corrected for its chemical purity.
DRAFT GRM029.01A
Page 10 of 57
W P
 1000
C
P
= Standard purity in decimal form (P(%)/100)
V
= Volume of acetonitrile required
W
= Weight, in mg, of the solid analytical standard
C
= Desired concentration of the final solution, (g/mL)
1000 = Unit conversion factor
V 
In this case, the standard material is weighed directly into an appropriate storage vessel.
2.3.1 Fortification solutions
The stock solutions should then be diluted by serial dilution to 0.01 g/mL in acetonitrile
and used for fortification and for preparation of calibration standards for LC-MS/MS
analysis. Mixed cyromazine and melamine standards may be prepared similarly. It is
recommended that the following solutions are prepared as required: 1.0 µg/mL,
0.1 µg/mL and 0.01 µg/mL. The preparation of LC-MS/MS calibration standards is
discussed in Section 3.7.
2.3.2 Standard solution storage and expiration
All standard solutions should be stored in a refrigerator or freezer when not in use to
prevent decomposition and/or concentration of the standard. Standard solutions should be
allowed to equilibrate to room temperature before use.
An expiry date of six months is recommended unless additional data are generated to
support a longer expiry date.
2.4
Safety precautions and hazards
The following information is included as an indication to the analyst of the nature and
hazards of the reagents used in this procedure. If in any doubt, consult the appropriate
MSDS or a monograph such as ‘Hazards in the Chemical Laboratory’, edited by S G
Luxon, The Chemical Society, London (Reference 2).
DRAFT GRM029.01A
Page 11 of 57
Solvent and Reagent hazards
Methanol
Acetonitrile
KH2PO4
Harmful Vapour



Highly Flammable



Harmful by Skin Absorption



Irritant to respiratory system
and eyes



Causes severe burns



Syngenta Hazard Category
SHC-C, S
SHC-C, S
N/A*
OES Short Term (mg/m3)
310
105
N/A
260
70
N/A
3
OES Long Term (mg/m )
o-phosphoric
acid
Ammonium
hydroxide
(30% w/v)
Acetic acid
Harmful Vapour



Highly Flammable



Harmful by Skin Absorption



Irritant to respiratory system
and eyes



Causes severe burns



Syngenta Hazard Category
SHC-C, S
SHC-C, S
SHC-C, S
OES Short Term (mg/m3)
N/A
24
37
N/A
17
25
3
OES Long Term (mg/m )
N/A - Not known.
N/A* - Not known. Assume Syngenta Hazard Category SHC-C
In all cases avoid breathing vapour. Avoid contact with eyes and skin.
Cyromazine has been designated as Syngenta Hazard Category SHC-A and melamine as
SHC-C. The hazard category scale rates highly toxic chemicals as SHC-E and non-toxic
chemicals as SHC-A. An additional hazard category of S indicates the compound is a
severe skin and eye irritant.
DRAFT GRM029.01A
Page 12 of 57
3.0
ANALYTICAL PROCEDURE
3.1
Modifications and potential problems
a)
Melamine is widely used in a number of applications and is found as a
contaminant in wastewater (Reference 3). Melamine is a ubiquitous compound
and may be present at significant concentrations in soil even where cyromazine
has not been applied. It is recommended that prior to sample preparation, soil is
removed as far as possible from crops which have had contact with soil e.g. potato
crops.
b)
Spot contamination may also be an issue and it is recommended that equipment
such as the macerator heads especially, vacuum manifold inlets and needles and
sample concentrator needles are taken apart and cleaned thoroughly with 0.1 M
HCl, ultra pure water and methanol before use. Disposable labware should be
used where possible.
c)
Bottled HPLC grade ultra pure water is used to prepare the LC mobile phase,
which produces a lower background noise in the MS/MS chromatograms than
water taken from a laboratory water purification system.
d)
The condition of the LC-MS/MS instrument is also important to ensure optimum
chromatography and sensitivity. Where sensitivity is poor, it is recommended that
the mass spectrometer front plate and ionisation source be cleaned.
3.2
Sample preparation
Samples should be prepared using an approved method of sample preparation for residue
analysis. It is recommended that prior to sample preparation, soil is removed as far as
possible from crops which have had contact with soil e.g. potato crops.
3.3
Extraction
In order to verify method performance and allow recovery corrections to be made (if
appropriate), fortified control samples should be included with each sample set. To each
pre-weighed control crop sample, add the appropriate amount of standard solution
containing cyromazine and melamine in acetonitrile. Let each sample stand for at least
five minutes after fortification to allow the spiking solution to soak into the matrix before
proceeding with the extraction procedure. At least one untreated control and two fortified
control samples should be analysed with each sample set.
The method is summarized in flow chart form in Appendix 8.
The following extraction procedure is based on that given in REM179.02 (Reference 1).
a)
Accurately weigh a representative amount of crop (10 g) into separate plastic
centrifuge bottles (250 mL size) and fortify recovery samples as required.
DRAFT GRM029.01A
Page 13 of 57
b)
Add 0.025 M KH2PO4 in 0.04 M H3PO4 (50 mL) (50 mL minus the water content
of the samples). Homogenise at high speed for 1 minute.
Note: Estimate the percentage water content in each matrix type and hence the
total volume of water in the 10 g sub-sample. E.g. for a 10 g sub-sample with 90%
natural water content add 50 mL – (10 x 90/100) mL = 41 mL extraction solution.
It is sufficient to round the natural water content to the nearest ten percent value.
Any volume contraction due to mixing organic solvents with water and
evaporation loss during extraction is considered to be negligible.
The water content of matrices can be obtained from published sources. The
relevant information can be obtained from the following USDA web site:
http://www.nal.usda.gov/fnic/cgi-bin/nut_search.pl
Alternatively, where information is not available from such sources, it may be
necessary to determine the moisture content experimentally, following a suitable
moisture content determination procedure.
c)
Rinse the macerator head with methanol (53 mL) to remove as much residual
sample as possible into the sample container.
Note: 53 mL methanol is the volume of methanol required to prepare a 100 mL
mixture of 50:50 v/v methanol water, due to the contraction of solvents on mixing.
d)
Secure the lids and shake the samples on a flat bed mechanical shaker at a speed
which visibly agitates the contents for 30 mins at room temperature.
e)
Centrifuge samples at a speed which separates the soil from the supernatant
(e.g.3500 rpm for 5 minutes). The sample concentration is now 0.1 g/mL and
2 mL of extract is equivalent to 0.2 g crop.
f)
Using a disposable plastic syringe (10 mL size) carefully draw approximately
5-10 mL of the extract from Section 3.3 (e) above into the syringe barrel. Attach a
disposable 25 mm 4.5 m PTFE syringe filter for aqueous samples to the tip of the
syringe. Carefully push the extract through the filter, collecting the filtrate into a
clean disposable glass test tube.
3.4
Sample Clean-up
a)
Take one Oasis MCX SPE cartridge (30 mg size, with 3 mL reservoir) for each
sample to be analysed and place on a suitable vacuum manifold (e.g. IST
Vacmaster). Add methanol (2 mL) and allow to percolate through under
gravity or draw through under vacuum to the level of the top frit at a rate of
approximately 1 mL/min, discarding the column eluate. Do not allow the
cartridges to become dry. Add 0.1 M hydrochloric acid (2 mL) to the top of
each cartridge and allow to percolate through under gravity or draw through
DRAFT GRM029.01A
Page 14 of 57
under vacuum to the level of the top frit at the same rate, again discarding the
column eluates. Do not allow the cartridges to become dry.
b)
Using e.g. a Gilson p5000 pipetteman , accurately transfer a 2 mL aliquot of
the sample from Section 3.3 (f) onto the cartridge and allow to percolate
through under gravity or draw through under vacuum to the level of the top frit
at a rate of approximately 1 mL/min. Discard the column eluate.
c)
Sequentially add 0.1 M HCl (aq) (2 mL), acetonitrile (2 mL) and methanol
(2 mL) to the top of each cartridge and allow to percolate through under gravity
or draw through under vacuum to the level of the top frit at a rate of
approximately 1 mL/min. Discard the column eluates.
d)
Place suitable disposable glass test tubes (e.g. 15 mL size) under each port, as
required, in the manifold rack. Add 95:5 methanol:30% ammonium hydroxide
solution (2 mL) to the top of each cartridge and allow to percolate through
under gravity or draw through under vacuum to the level of the top frit at a rate
of approximately 1 mL/min. Collect the column eluate.
e)
Close the manifold taps. Add a further portion of 95:5 methanol:30% ammonia
solution (2 mL) to the top of each cartridge and leave to stand for at least 5.0
minutes to allow equilibration of the slow ionic interactions to take place.
Open the manifold tap and collect the column eluate.
f)
Evaporate the samples to dryness under a stream of air, in a sample
concentrator at 35 oC.
g)
Add ultra pure water (5 mL) and ultrasonicate samples briefly to mix
thoroughly. The final sample concentration is now 0.04 g/mL.
h)
Transfer aliquots into suitable autosampler vials for analysis by LC-MS/MS.
Note: The above SPE procedures have been developed using columns from
the stated manufacturer; however, it is possible to carry out the procedure
using similar columns from other manufacturers. In all cases it is strongly
recommended that the elution profile is checked prior to commencing analysis.
This will rule out any variation between manufacturers’ products and between
batches. Each batch of cartridges should be checked for interference prior to
use.
3.5
Time required for analysis
The methodology is normally performed with a batch of up to 15 samples. One person
can complete the analysis of up to 15 samples in 1 day (8 working hour period).
3.6
Method stopping points
The analytical procedure can be stopped at various points for overnight and weekend
breaks unless otherwise specified in the analytical procedure. Acceptable method
DRAFT GRM029.01A
Page 15 of 57
recoveries will validate any work flow interruptions. Samples should be stored
refrigerated in sealed containers where the analysis cannot be completed in a single day.
3.7
Preparation of calibration standards for LC-MS/MS
Some matrix suppression of the instrument response for cyromazine and melamine has
been observed in the crops tested using the above procedure in this laboratory (Table 5,
Appendix 3). Matrix matched standards may be used to compensate for these effects, at
the discretion of the study director. It is recommended however, that samples are
quantified using reagent matrix matched calibration standards.
Calibration standards for LC-MS/MS analysis are prepared from the fortification
standards described in Section 2.3.1.
To prepare e.g. a 0.002 g/mL mixed cyromazine and melamine reagent-matched
calibration standard, a method blank (2 mL of 50:50 v/v methanol:0.025 M KH2PO4 in
0.4 M H3PO4) is taken through the SPE procedure as described in Section 3.4 to point 3.4
(f). Add 100 L of 0.1 g/mL mixed cyromazine and melamine in acetonitrile followed
by 4.9 mL ultra-pure water. Mix thoroughly by ultra-sonicating the sample briefly and
transfer to a suitable autosampler vial for final determination by LC-MS/MS.
A calibration curve may also be generated to quantify cyromazine and melamine residues.
Standards over an appropriate concentration range should be prepared as described above,
using the requisite volumes of mixed cyromazine and melamine standard in acetonitrile.
4.0
FINAL DETERMINATION
The following instrumentation and conditions have been found to be suitable for this
analysis. Other instrumentation can also be used, though optimisation may be required to
achieve the desired separation and sensitivity. The operating manuals for the instruments
should always be consulted to ensure safe and optimum use. The method has been
developed for use on the Applied Biosystems API 4000 LC-MS/MS.
Final determination by LC-MS/MS with 2 transitions is considered to be highly specific
and no further confirmatory conditions are included.
4.1
Instrument description
Pump
: Agilent 1100 series quaternary pump model
number G1311A
Degasser
: Agilent 1100 series model number G1322A
Column Oven
: Agilent 1100 series model number G1316A
fitted with column switching valve
Detector
: Applied Biosystems API 4000 triple
quadrupole mass spectrometer with Analyst™
DRAFT GRM029.01A
Page 16 of 57
software version 1.4.1
Autosampler
: Agilent 1100 series model number G1313A
Gas Supply
: Peak Scientific NM20ZA gas station
4.2
Chromatography conditions
Column
Column Oven Temperature
Injection volume
Stop Time
Injection protocol
Mobile phase
:
:
:
:
:
Phenomenex Luna CN 5 µm 150 x 2.0 mm i.d.
40C
10 L
5.5 minutes
Analyse calibration standard after 3 to 4 sample
injections
: Solvent 1 = Acetonitrile
Solvent 2 = Acetic acid (0.2%, v/v) in ultra pure water
Isocratic Mobile Phase
Time (min)
0.0
5.5
% Solvent 1
30
30
% Solvent 2
70
70
Flow (mL/min)
0.5
0.5
Under these conditions the retention times of melamine and cyromazine are
approximately 2.0 and 2.3 minutes respectively. The retention time of both cyromazine
and melamine can vary over a period of time, depending on the condition of the HPLC
column.
4.3
Mass spectrometer conditions
Interface
: TurboIonSpray
Polarity
: Positive
Curtain gas (CUR)
: Nitrogen set at 17 (arbitrary units)
Temperature (TEM)
: 500C
Ionspray voltage
: 4500V
Collision gas setting (CAD)
: Nitrogen set at 4 (arbitrary units)
Gas 1 (GS1)
: Air set at 60 (arbitrary units)
Gas 2 (GS2)
: Air set at 60 (arbitrary units)
Interface heater (ihe)
: On
Scan type
: Multiple reaction monitoring (MRM)
DRAFT GRM029.01A
Page 17 of 57
Cyromazine
(primary)
MRM Conditions
Cyromazine
Melamine
(confirmatory) (primary)
Melamine
(confirmatory)
Q1 m/z
:
167
167
127
127
Q3 m/z
:
85
68
85
68
Dwell Time
:
150 ms
150 ms
150 ms
150 ms
Resolution Q1
:
Unit
Unit
Unit
Unit
Resolution Q3
:
Unit
Unit
Unit
Unit
Declustering Potential (DP) :
66 V
66 V
66 V
66 V
Entrance Potential (EP)
:
10 V
10 V
10 V
10 V
Collision Energy (CE)
:
29 V
49 V
27 V
43 V
Collision Cell Exit Potential :
(CXP)
4V
2V
4V
4V
Note: Either transition for cyromazine and melamine may be used for quantification.
Typical chromatograms are shown in Appendix 4.
5.0
CALCULATION OF RESULTS
5.1
Single point calibration procedure
Residues may be calculated in mg/kg for each sample using a mean standard response
from each of the injections bracketing the sample as follows.
a) Make repeated injections of a mixed standard containing cyromazine and melamine at
an appropriate concentration into the LC-MS/MS operated under conditions as
described in Section 4. When a consistent response is obtained, measure the peak
areas obtained for the analytes.
b) Make an injection of each sample solution and measure the areas of the peaks
corresponding to the analytes.
c) Re-inject the standard solution after a maximum of four injections of sample
solutions.
d) Calculate the residues in the sample, expressed as mg/kg, using a mean standard
response from each of the injections bracketing the sample as follows.
DRAFT GRM029.01A
Page 18 of 57
Residue (mg/kg ) 
PK area (SA)
PK area (STD)
Standard Conc.
Sample Conc.
=
=
=
=
PK area (SA) Standard Conc.

PK area (STD) Sample Conc.
Peak response for sample
Average peak response for bracketing standards
Concentration of standard (g/mL)
Sample concentration (g/mL)
If residues need to be corrected for average percentage recovery e.g. for storage stability
studies, then the equation below should be used.
Corrected Residue =
Residue 100
(mg/kg)
Average percentage Recovery
Although single point calibration may be used to quantify residues it is recommended that
a calibration curve is generated with each analytical run to demonstrate the linearity of
instrument response (Reference 4).
5.2
Multi point calibration procedure
Residues may be calculated in mg/kg for each sample as follows.
a)
Prepare mixed standard solutions of cyromazine and melamine over a
concentration range appropriate to the expected residues in the samples (for
example, 50% LOQ to 10 x LOQ). An appropriate number of different
concentrations within this range should be prepared (at least four).
b)
Make an injection of each sample solution and measure the areas of the peaks
corresponding to the two analytes. Calibration standard solutions should be
interspersed throughout the analysis, after a maximum of four injections of sample
solutions.
c)
Generate calibration curve parameters using an appropriate regression package.
d)
The following equation can be rearranged and used to calculate residues as
follows:
y  mx  c
Where y is the instrument response value, x is the standard concentration, m is the
gradient of the line of best fit (“X-variable 1” in MS Excel) and c is the intercept
value. An example of this equation generated using the experimental values of m
and c should be included in the raw data, as should the “R-Squared” value for the
regression.
Re-arrangement for x gives
DRAFT GRM029.01A
Page 19 of 57
yc
m
Alternatively (depending on the regression analysis software available) a quadratic
equation may be used to fit the data. In this case the following general equation
should be re-arranged and used to calculate residues:
x
e)
y  a  bx  cx 2
Where y is the instrument response value, x is the standard concentration and a, b,
c are constants.
f)
Calculate the residues of cyromazine and melamine in the sample, expressed as
mg kg-1, as follows
Analyte found ( g/mL )
Residue (mg/kg ) 
Sample conc. (g/mL )
Where analyte found (g/mL) is calculated from the standard calibration curve
and sample conc. is the final sample concentration in g/mL.
If residues need to be corrected for average percentage recovery e.g. for storage
stability studies, then the equation below should be used.
Corrected Residue =
6.0
Residue 100
(mg/kg)
Average percentage Recovery
CONTROL AND RECOVERY SAMPLES
Control samples should be analysed with each set of samples to verify that the sample
used to prepare recovery samples is free from contamination. A minimum of one control
should be analysed with each batch of samples.
At least two recovery samples (control samples accurately fortified with known amounts
of cyromazine and melamine in acetonitrile) should also be analysed alongside each set of
samples. Provided the recovery values are acceptable they may be used to correct any
residues found. The fortification levels should be appropriate to the residue levels
expected.
Recovery efficiency is generally considered acceptable when the mean values are
between 70% and 110% and with a relative standard deviation of <20%.
7.0
SPECIFICITY
It is recommended that two method blanks should be analysed with each batch of
samples and that prior to sample preparation, soil is removed as far as possible from crops
which have had contact with soil e.g. potato crops. Melamine is a ubiquitous compound
DRAFT GRM029.01A
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and may be present at significant concentrations in soil even where cyromazine has not
been applied. Additionally, untreated control samples should be screened for melamine
residues prior to use in any recovery experiments.
7.1
Matrix interference
LC-MS/MS is a highly specific detection technique. Interference arising from the
matrices tested has not been observed.
7.2
Reagent and solvent interference
Using high purity solvents and reagents no interference has been found.
7.3
Labware interference
This method uses disposable labware. All reusable glassware should be detergent washed
and then rinsed with HPLC-grade methanol, acetone or acetonitrile prior to use.
8.0
METHOD VALIDATION
8.1
Recovery data and repeatability
Method validation has been carried out on the procedures described in Section 3. The
method validation data are reported in ??? (Reference 5), and a summary is included in
Appendix 3.
8.2
Limit of quantification (LOQ)
The limit of quantification of the method is defined as the lowest analyte concentration in
a sample at which the methodology has been validated and a mean recovery of 70-110%
with a relative standard deviation of  20% has been obtained. Generally, for accurate
quantification, the response for an analyte peak should be no lower than four times the
mean amplitude of the background noise in an untreated sample at the corresponding
retention time.
The limit of quantification has been set at 0.05 mg/kg for cyromazine and melamine.
8.3
Limit of detection (LOD)
The limit of detection of the method is defined as the lowest analyte concentration
detectable above the mean amplitude of the background noise in an untreated sample at
the corresponding retention time. An estimate of the LOD can be taken as three times
background noise. Note that the LOD may vary between runs and from instrument to
instrument.
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8.4
Detector linearity
For accurate quantification of residue concentrations, analyses should be carried out
within the linear range of the detector. Detector linearity graphs are given in Appendix 5.
The linearity of the LC-MS/MS detector response for cyromazine and melamine was
tested in the range from 0.001 g/mL to 0.05 g/mL (equivalent to 10 pg to 500 pg
injected on column when using a 10 L injection volume) and was found to be linear for
both transitions in cyromazine and melamine. If a residue beyond the tested
concentration range is expected, dilute the sample appropriately to bring it within the
tested linear range prior to quantitation.
Standards at ? different concentration levels (n = ?) were injected and the response plotted
against amount injected on column, using Microsoft Excel 2003.
Detector linearity graphs are given in Appendix 5.
8.5
Extract stability
Cyromazine and melamine were proven to be stable in the final extracts for a period of up
to ? days when stored at a temperature of 7°C. Storage stability data are presented in
Appendix 3.
9.0
LIMITATIONS
The method has been tested on representative high water content crop matrices. It can
reasonably be assumed that the method can be applied to other crop matrices not tested in
this study, provided successful recovery tests at the relevant levels validate the suitability
of the method.
10.0 CONCLUSIONS
Method GRM029.01A has been demonstrated to be a reliable and accurate procedure for
the determination of cyromazine and melamine in high water content crop matrices, using
commercially available laboratory equipment and reagents. The limit of quantification of
the method is 0.05 mg/kg for cyromazine and melamine.
This method complies with EU guidelines SANCO/3029/99 rev. 4, SANCO/825/00 rev. 7
and US EPA guideline OPPTS 860.1340.
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11.0 REFERENCES
1.
Cyromazine (CGA72662): REM174.02 Residue Analytical Method for the
Determination of Cyromazine and its Metabolite Melamine (C1803) in Crops by
High Performance Liquid Chromatography.
2.
Luxon S G (1992): Hazards in the Chemical Laboratory 5th Edition. The Royal
Society of Chemistry. Thomas Graham House, The Science Park, Cambridge,
CB4 4WF, UK. ISBN 0-85186-229-2.
3.
Yokley, R et al : Analytical Method for the Determination of Cyromazine and
Melamine Residues in Soil Using LC-UV and GC-MSD. Journal of Agricultural
and Food Chemistry (2000), 48(8), 3352-3358
4.
Cardone M J, Palermo P J and Sybrand L B: Potential error in single point ratio
calculations based on linear calibration curves with a significant intercept. Anal
Chem., 52 pp 1187-1191, 1980
5.
? (2007): Cyromazine (CGA72662): Validation of a Residue Method for the
Determination of Cyromazine (CGA72662) and Melamine in High Water Content
Crop. Report No. xxxxx.
DRAFT GRM029.01A
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APPENDICES SECTION
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APPENDIX 1
APPARATUS
UK suppliers
Equipment for the initial preparation of samples e.g. Robot Coupe R20 bowl chopper
available from Lockhart Catering Equipment, Lockhart House, Brunel Road, Theale,
Reading, Berkshire, RG7 4XE.
General glassware, available from Fisher Scientific UK, Bishop Meadow Road,
Loughborough, Leicestershire LE11 5RG
Plastic centrifuge bottles, 250 mL size, available from Fisher Scientific UK, Bishop
Meadow Road, Loughborough, Leicestershire LE11 5RG.
High speed homogeniser for extraction of samples e.g. Janke and Kunkel Ultra Turrax
T25, available from Fisher Scientific UK, Bishop Meadow Road, Loughborough,
Leicestershire LE11 5RG.
Mechanical shaker, available from Fisher Scientific UK, Bishop Meadow Road,
Loughborough, Leicestershire LE11 5RG.
Laboratory centrifuge e.g. MSE Mistral 1000 series, available from Fisher Scientific UK,
Bishop Meadow Road, Loughborough, Leicestershire LE11 5RG, UK.
Disposable borosilicate glass test tubes, available from Fisher Scientific UK, Bishop
Meadow Road, Loughborough, Leicestershire, LE11 5RG.
Plastic disposable pipettes, available from Fisher Scientific UK, Bishop Meadow Road,
Loughborough, Leicestershire, LE11 5RG.
Isolute Vacmaster-20™ sample processing station, available from Biotage, Tir-y-Berth
Industrial Estate, New Road, Hengoed, Mid Glamorgan, CF8 8AU.
Oasis MCX solid phase extraction columns, 3 mL 60 mg size, available from Waters
Ltd., 730-740 Centennial Court, Centennial Park, Elstree, Hertfordshire, WD6 3SZ.
Techne Dri-block 3D sample concentrator, available from Fisher Scientific UK, Bishop
Meadow Road, Loughborough, Leicestershire, LE11 5RG.
Ultrasonic bath e.g. Ultrawave U300/D, available from Fisher Scientific UK, Bishop
Meadow Road, Loughborough, Leicestershire, LE11 5RG
Crimp cap autosampler vials and caps, available from Agilent Technologies UK Limited,
Chemical Analysis Group, Lakeside Heath, Cheadle Royal Business Park, Stockport,
Cheshire, SK8 3GR.
API 4000 LC-MS/MS system equipped with a TurboIonSpray source, available from
Applied Biosystems, 120 Birchwood Boulevard, Warrington, Cheshire, WA3 7PB.
Agilent 1100 HPLC system equipped with quaternary pump, vacuum degasser and
column compartment with column switching valve, available from Agilent Technologies
DRAFT GRM029.01A
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UK Limited, Chemical Analysis Group, Lakeside Heath, Cheadle Royal Business Park,
Stockport, Cheshire, SK8 3GR.
CTC HTS PAL autosampler, available from Presearch Ltd, System House, 59-61
Knowlpiece, Hitchin, Herts, SG4 0TY.
HPLC column, Phenomenex Luna CN 5 m 150 mm  2.0 mm i.d., available from
Phenomenex, Queens Avenue, Hurdsfield Ind. Est., Macclesfield, Cheshire, SK10 2BN.
Peak Scientific NM20ZA gas station, available from Peak Scientific Instruments Ltd.,
Fountain Crescent, Inchinnan Business Park, Inchinnan, Renfrew, PA9 4RE
US suppliers
Equipment for the initial preparation of samples e.g. Tecator homogeniser available from
Perstorp Analytical inc., 12101 Tech Road, Silver Spring, Maryland 20904.
General glassware, available from Fisher Scientific UK, Liberty Lane, Hampton, NH
03842.
Plastic centrifuge bottles, 250 mL size, available from Fisher Scientific UK, Liberty Lane,
Hampton, NH 03842.
High speed homogeniser for extraction of samples e.g. Janke and Kunkel Ultra Turrax
T25, available from Fisher Scientific, Liberty Lane, Hampton, NH 03842.
Mechanical shaker, available from Fisher Scientific, Liberty Lane, Hampton NH 03842
Laboratory centrifuge e.g. Heraeus Instruments model 17RS, available from Heraeus
Instruments, 111-A Corporate Blvd, South Plainfield, NJ 07080.
Disposable borosilicate glass test tubes, available from Fisher Scientific, Liberty Lane,
Hampton, NH 03842.
Plastic disposable pipettes, available from Fisher Scientific, Liberty Lane, Hampton, NH
03842.
Isolute Vacmaster-20™ sample processing station, available from Biotage Ltd., PO Box
280 329, Lakewood, Colorado, 8022-0329.
Oasis™ MCX solid phase extraction columns, 3 mL 60 mg size, available from Waters
Corporation, 34 Maple Street, Milford, Massachusetts, 01757-3696
Techne Dri-block 3D sample concentrator, available from Fisher Scientific, Liberty Lane,
Hampton, NH 03842.
Ultrasonic bath available from Fisher Scientific, Liberty Lane, Hampton, NH 03842.
Crimp cap auto sampler vials and caps, available from Agilent Technologies, 395 Page
Mill Road, Palo Alto, CA 94304.
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API 4000 LC-MS/MS system equipped with a TurboIonSpray source, available from
Applied Biosystems, 850 Lincoln Center, Foster City, CA 94404-1128.
Agilent 1100 HPLC system equipped with quaternary pump, vacuum degasser and
column compartment with column switching valve, available from Agilent Technologies,
395 Page Mill Road, Palo Alto, CA 94304.
CTC HTS PAL autosampler, available from LEAP Technologies Inc., P.O. Box 969,
Carrboro, NC 27510.
HPLC column, Phenomenex Luna CN 5 m 150 mm  2.0 mm i.d., available from
Phenomenex, 411 Madrid Avenue, Torrance, CA 90501-1430.
Peak Scientific NM20ZA gas station, available from Peak Scientific Instruments, 1300
West Belmont Ave., Chicago, IL 60657.
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APPENDIX 2
REAGENTS
UK suppliers
Solvents: Ultra pure water (HPLC grade), methanol and acetonitrile available from
Rathburn Chemicals Ltd., Walkerburn, EH43 6AU.
Analytical grade potassium dihydrogen phosphate (KH2PO4), o-phosphoric acid,
hydrochloric acid, ammonium hydroxide solution (30% w/v) and acetic acid available
from Sigma-Aldrich, The Old Brickyard, New Road, Gillingham, Dorset, SP8 4XT or
www.sigmaaldrich.com
Cyromazine and melamine analytical standards, available from Syngenta Crop Protection,
GLP Testing Facility WMU, CH-4333, Munchwilen, Switzerland.
US suppliers
Solvents: Analytical grade methanol, acetonitrile and acetone available from B & J Brand
Solvents, from Scientific Products Division of Baxter Healthcare Corporation.
Ultra pure HPLC grade water from e.g. Fluka via Sigma-Aldrich www.sigmaaldrich.com
Analytical grade Analytical grade potassium dihydrogen phosphate KH2PO4, ophosphoric acid, hydrochloric acid, ammonium hydroxide solution (30% w/v) and acetic
acid available from www.sigmaaldrich.com
Cyromazine and melamine analytical standards, available from Syngenta Crop Protection
Inc., P.O. Box 18300, Greensboro, NC 27419-8300.
Preparation of Reagents
a) 1 M phosphoric acid:
Carefully add 11.5 g H3PO4 (85% w/w) to ultra pure water (~ 50 mL) in a 100 mL
volumetric flask. Adjust to the mark with ultra pure water. Stopper flask securely
and mix thoroughly by shaking.
b) 0.025 M potassium dihydrogen phosphate in 0.04M phosphoric acid:
Dissolve 3.41 g KH2PO4 in ultra pure water (~200 mL) in a 1 L volumetric flask.
Add 1 M H3PO4 (40 mL). Adjust to the mark with ultra pure water. Stopper flask
securely and mix thoroughly by shaking.
c) 0.1 M Hydrochloric acid
Carefully add 8.4 g concentrated HCl to ultra pure water (~ 500 mL) in a 1 L
volumetric flask. Adjust to the mark with ultra pure water. Stopper flask securely
and mix thoroughly by shaking.
d) 95:5 v/v methanol:30% ammonium hydroxide
Carefully add 5 mL ammonium hydroxide solution (30% w/v) to methanol (~50 mL)
DRAFT GRM029.01A
Page 28 of 57
in a 100 mL volumetric flask. Adjust to the mark with methanol. Stopper flask
securely and mix thoroughly by shaking.
e) 0.2% v/v glacial acetic acid in ultra pure water:
Carefully add 2 mL acetic acid to ultra pure water (~500 mL) in a 1 L volumetric
flask. Adjust to the mark with ultra pure water. Stopper flask securely and mix
thoroughly by shaking.
DRAFT GRM029.01A
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APPENDIX 3
Table 1. :
Matrix
*
**
METHOD VALIDATION DATA
Cyromazine recovery data obtained during method
validation. Primary transition m/z 167  68
Fortification Level
(mg/kg)
Recovery (%)**
n
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
Mean
(%)
RSD
(%)
Range
(%)
Limit of quantitation, defined by the lowest validated fortification level.
Two control samples and a reagent blank were analysed with each analytical batch. No residues were
measured at or above 30% of the LOQ in any of the samples. All recovery data were generated using
reagent matched standards.
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Table 2. :
Matrix
*
**
Cyromazine recovery data obtained during method
validation. Confirmatory transition m/z 167  85
Fortification Level
(mg/kg)
Recovery (%)**
n
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
Mean
(%)
RSD
(%)
Range
(%)
Limit of quantitation, defined by the lowest validated fortification level.
Two control samples and a reagent blank were analysed with each analytical batch. No residues were
measured at or above 30% of the LOQ in any of the samples.
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Table 3. :
Matrix
*
**
Melamine recovery data obtained during method validation
Primary transition m/z 127  68
Fortification Level
(mg/kg)
Recovery (%)**
n
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
Mean
(%)
RSD
(%)
Range
(%)
Limit of quantitation, defined by the lowest validated fortification level.
Two control samples and a reagent blank were analysed with each analytical batch. No residues were
measured at or above 30% of the LOQ in any of the samples.
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Table 4. :
Matrix
*
**
Melamine recovery data obtained during method validation
Primary transition m/z 127.0  85.0
Fortification Level
(mg/kg)
Recovery (%)**
n
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
0.05*
5
0.5
5
Overall
10
Mean
(%)
RSD
(%)
Range
(%)
Limit of quantitation, defined by the lowest validated fortification level.
Two control samples and a reagent blank were analysed with each analytical batch. No residues were
measured at or above 30% of the LOQ in any of the samples.
.
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Table 5. :
Determination of LC-MS/MS Matrix Effects
The effect of crop matrices on the LC-MS/MS response was assessed by preparing
standards in the presence of matrix and comparing the peak areas of cyromazine and
melamine against reagent-matrix standards at an equivalent concentration. Matrix and
reagent matched standards were prepared as described in Section 3.7.
Matrix effects (enhancement or suppression) on the instrument response were considered
not to be significant and reagent-matched calibration standards should be used. Matrix
matched standards may be used for calibration however, to compensate for these effects
at the discretion of the study director.
Matrix
Table 6. :
Matrix
Matrix Effect
Cyromazine
Transition
m/z 167  68
Matrix Effect
Cyromazine
Transition
m/z 167  85
Matrix Effect
Melamine
Transition
m/z 127  68
Matrix Effect
Melamine
Transition
m/z 127  85
Cyromazine recovery data obtained after ? days storage
Calculated using the primary transition m/z 167  68
Fortification
Level
(mg/kg)
Recovery (%)**
Mean
(%)
RSD
(%)
Range
(%)
0.05*
0.5
0.05*
0.5
0.05*
0.5
0.05
0.5
*Limit of quantification, defined by the lowest validated fortification level
**Residues in control samples and reagent blanks were less than 30% of the LOQ.
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Table 7. :
Matrix
Melamine recovery data obtained after ? days storage
Calculated using the primary transition m/z 127  68
Fortification
Level
(mg/kg)
Recovery (%)**
Mean
(%)
RSD
(%)
Range
(%)
0.05*
0.5
0.05*
0.5
0.05*
0.5
0.05
0.5
*Limit of quantification, defined by the lowest validated fortification level
**Residues in control samples and reagent blanks were less than 30% of the LOQ.
DRAFT GRM029.01A
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APPENDIX 4
REPRESENTATIVE CHROMATOGRAMS
Figure 3:
0.002 g/mL Cyromazine Standard
m/z 167 68
Figure 4:
0.002 g/mL Cyromazine Standard
m/z 167  85
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Figure 5:
0.002 g/mL Melamine Standard
m/z 127  68
Figure 6:
0.002 g/mL Melamine Standard
m/z 127  85
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Figure 7:
Control Crop 1 Sample Concentration 0.04 g/mL,
Cyromazine Residue <LOQ
m/z 167  68
Figure 8:
Control Crop 1 Sample Concentration 0.04 g/mL,
Cyromazine Residue <LOQ
m/z 167  85
.
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Figure 9:
Control Crop 1 Sample Concentration 0.04 g/mL,
Melamine Residue <LOQ
m/z 127  68
Figure 10:
Control Crop 1 Sample Concentration 0.04 g/mL,
Melamine Residue <LOQ
m/z 127  85
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Figure 11:
Control Crop 1 Fortified with 0.05 mg/kg Cyromazine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 167  68
Figure 12:
Control Crop 1 Fortified with 0.05 mg/kg Cyromazine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 167  85
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Figure 13:
Control Crop 1 Fortified with 0.05 mg/kg Melamine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 127  68
Figure 14:
Control Crop 1 Fortified with 0.05 mg/kg Melamine
Sample Concentration 0.04 g mL-1, Recovery = ?
m/z 127  85
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Figure 15:
Control Crop 1 Fortified with 0.5 mg/kg Cyromazine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 167  68
Figure 16:
Control Crop 1 Fortified with 0.5 mg/kg Cyromazine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 167  85
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Figure 17:
Control Crop 1 Fortified with 0.5 mg/kg Melamine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 127  68
Figure 18:
Control Crop 1 Fortified with 0.5 mg/kg Melamine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 127  85
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Figure 19:
Control Crop 2 Sample Concentration 0.04 g/mL,
Cyromazine Residue <LOQ
m/z 167  68
Figure 20:
Control Crop 2 Sample Concentration 0.04 g/mL
Cyromazine Residue <LOQ
m/z 167  85
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Figure 21:
Control Crop 1 Sample Concentration 0.04 g/mL,
Melamine Residue <LOQ
m/z 127  68
Figure 22:
Control Crop 1 Sample Concentration 0.04 g/mL,
Melamine Residue <LOQ
m/z 127  85
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Figure 23:
Control Crop 2 Fortified with 0.05 mg/kg Cyromazine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 167  68
Figure 24:
Control Crop 2 Fortified with 0.05 mg/kg Cyromazine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 167  85
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Figure 25:
Control Crop 2 Fortified with 0.05 mg/kg Melamine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 127  68
Figure 26:
Control Crop 2 Fortified with 0.05 mg/kg Melamine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 127  85
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Figure 27:
Control Crop 2 Fortified with 0.5 mg/kg Cyromazine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 167  68
Figure 28:
Control Crop 2 Fortified with 0.5 mg/kg Cyromazine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 167  85
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Figure 29:
Control Crop 2 Fortified with 0.5 mg/kg Melamine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 127  68
Figure 30:
Control Crop 2 Fortified with 0.5 mg/kg Melamine
Sample Concentration 0.04 g/mL, Recovery = ?
m/z 127  85
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APPENDIX 5
Figure 31:
DETECTOR LINEARITY GRAPHS
LC-MS/MS Calibration Graph for Cyromazine (No Intercept Set)
m/z 167  68
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Figure 32:
LC-MS/MS Calibration Graph for Cyromazine (No Intercept Set)
m/z 167  85
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Figure 33:
LC-MS/MS Calibration Graph for Melamine (No Intercept Set)
m/z 127  68
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Figure 34:
LC-MS/MS Calibration Graph for Melamine (No Intercept Set)
m/z 127  85
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APPENDIX 6
API4000 MS/MS TUNING PROCEDURE
Calibration of instrument
The instrument must be mass-calibrated on a regular basis using polypropylene glycol
(PPG) solutions according to the manufacturer’s instructions. Calibrate both massresolving quadrupoles (Q1 and Q3).
The instrument must be mass calibrated on a regular basis using polypropylene glycol
(PPG) solutions according to the manufacturer’s instructions. Calibrate both mass
resolving quadrupoles (Q1 and Q3).
Tuning instrument for Cyromazine and Melamine
Infuse separate standard solutions of cyromazine and melamine (0.001 to 0.1 µg/mL) in
mobile phase (see section 4.2) directly into the mass spectrometer interface at a rate at of
approximately 10-20 µL/min. Roughly adjust interface parameters (sprayer position,
spray, heater/auxiliary gas flows, as well as voltages of spray, orifice and focusing ring)
for a sufficiently high parent ion signal at m/z = 167 for cyromazine and m/z = 127 for
melamine
Using the Analyst software quantitative optimisation routine, tune the instrument for
cyromazine and melamine, ensuring that the correct ions are selected. If desired, manual
tuning of the ion optics and collision energy can be carried out to ensure maximum
sensitivity.
Finally, connect the LC-pump via the autosampler directly to the MS/MS instrument.
Perform repetitive flow injection of a mixed cyromazine and melamine standard using
mobile phase at the flow rate to be used. Tune the interface parameters (sprayer position,
spray and heater gas flows, spray, orifice and focusing ring voltages) and the collision gas
flow for maximum sensitivity.
In positive ionisation mode, the protonated molecular ions generated in the ion source
(m/z = 167 for cyromazine and m/z = 127 for melamine) are selected and subjected to
further fragmentation by collisional activation. The two most sensitive daughter ions
(m/z = 85 and m/z = 68 for cyromazine and melamine are then selected and used for
quantitative analysis.
The most sensitive daughter ion (m/z = 85) corresponds to the loss of a cyanamine group
from the 1, 3, 5-trazine-2, 4, 6-triamine molecular ion. A second transition (m/z = 68)
corresponding protonated imidazole may be used to confirm any cyromazine and
melamine residues detected.
Final determination by LC-MS/MS with two transitions is considered to be highly
specific; hence no further confirmatory conditions are included.
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APPENDIX 7
MS/MS PRODUCT ION SPECTRA
Figure 35:
Initial product ion Scan for Cyromazine (positive ionisation
mode).
File:Cyromazine_20070208081733
This scan was not produced as part of the validation study
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Figure 36:
Initial product ion Scan for melamine (positive ionisation
mode).
File:melamine_20070208082010
This scan was not produced as part of the validation study
DRAFT GRM029.01A
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APPENDIX 8
METHOD FLOWCHART
Extract crop sample (10 g) with by maceration with 0.025 M KH2PO4 in 0.025 M H3PO4

Add methanol to sample and shake for 30 mins using a mechanical shaker

Take aliquot (0.2 g) through cation exchange SPE clean-up procedure

Collect and evaporate column eluate to dryness

Redissolve in ultra pure water

Final determination by LC-MS/MS
DRAFT GRM029.01A
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